U.S. patent application number 12/441045 was filed with the patent office on 2010-07-29 for undercarboxylated/uncarboxylated osteocalcin increases beta-cell proliferation, insulin secretion, insulin sensitivity, glucose tolerance and decreases fat mass.
This patent application is currently assigned to THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY IF. Invention is credited to Patricia F. Ducy, Gerard Karsenty.
Application Number | 20100190697 12/441045 |
Document ID | / |
Family ID | 39184387 |
Filed Date | 2010-07-29 |
United States Patent
Application |
20100190697 |
Kind Code |
A1 |
Karsenty; Gerard ; et
al. |
July 29, 2010 |
UNDERCARBOXYLATED/UNCARBOXYLATED OSTEOCALCIN INCREASES BETA-CELL
PROLIFERATION, INSULIN SECRETION, INSULIN SENSITIVITY, GLUCOSE
TOLERANCE AND DECREASES FAT MASS
Abstract
The present invention relates to methods and compositions for
treating and diagnosing disorders related to energy metabolism and
the OST-PTP signaling pathway involving gamma-carboxylase,
osteocalcin and adiponectin. Such disorders include, but are not
limited to, metabolic syndrome, glucose intolerance, diabetes types
1 and 2, atherosclerosis and obesity.
Inventors: |
Karsenty; Gerard; (New York,
NY) ; Ducy; Patricia F.; (New York, NY) |
Correspondence
Address: |
KENYON & KENYON LLP
ONE BROADWAY
NEW YORK
NY
10004
US
|
Assignee: |
THE TRUSTEES OF COLUMBIA UNIVERSITY
IN THE CITY IF
New York
NY
|
Family ID: |
39184387 |
Appl. No.: |
12/441045 |
Filed: |
September 13, 2007 |
PCT Filed: |
September 13, 2007 |
PCT NO: |
PCT/US07/20029 |
371 Date: |
November 20, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60844203 |
Sep 13, 2006 |
|
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60870604 |
Dec 18, 2006 |
|
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60909712 |
Apr 2, 2007 |
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60945081 |
Jun 19, 2007 |
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Current U.S.
Class: |
514/1.1 ; 436/86;
514/6.9; 530/350 |
Current CPC
Class: |
A61P 3/06 20180101; A61P
43/00 20180101; G01N 33/5088 20130101; A61P 19/08 20180101; A61P
3/04 20180101; A61P 9/10 20180101; A61P 9/14 20180101; A61P 5/50
20180101; G01N 2333/916 20130101; A61K 47/60 20170801; A61P 3/10
20180101; G01N 33/54306 20130101; A61K 38/16 20130101; G01N 33/502
20130101; G01N 33/5008 20130101; G01N 2333/78 20130101; A61P 5/48
20180101; G01N 33/5023 20130101; A61P 19/10 20180101; G01N 33/5038
20130101; A61P 29/00 20180101; A61P 3/14 20180101; A61K 38/39
20130101; G01N 2333/988 20130101; A61P 3/00 20180101 |
Class at
Publication: |
514/12 ; 530/350;
436/86 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 3/10 20060101 A61P003/10; A61P 3/04 20060101
A61P003/04; A61P 3/14 20060101 A61P003/14; A61P 3/00 20060101
A61P003/00; C07K 14/435 20060101 C07K014/435; G01N 33/68 20060101
G01N033/68 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. PHS 398/2590 (Rev. 09/04, Reissued 4/2006). The Government has
certain rights in the invention.
Claims
1-122. (canceled)
123. A method for treating or preventing a disorder selected from
the group consisting of diabetes, metabolic syndrome, glucose
intolerance, and obesity in a mammal, said method comprising
administering an effective amount of
uncarboxylated/undercarboxylated osteocalcin or a fragment
thereof.
124. The method of claim 123, wherein the disorder is type 1
diabetes.
125. The method of claim 123, wherein the disorder is type 2
diabetes.
126. The method of claim 123, wherein the disorder is obesity.
127. The method of claim 123, wherein the disorder is metabolic
syndrome.
128. The method of claim 123, wherein the
uncarboxylated/undercarboxylated osteocalcin is administered in an
amount effective to produce an effect selected from the group
consisting of an increase in glucose tolerance, an increase in
insulin production, an increase insulin sensitivity, an increase in
pancreatic beta-cell proliferation, and an increase in adiponectin
serum level.
129. The method of claim 123, wherein the mammal is a human.
130. The method of claim 123, wherein the
uncarboxylated/undercarboxylated osteocalcin is administered in
combination with an agent used to prevent bone loss.
131. The method of claim 123, wherein at least one of the glutamic
acids in the undercarboxylated/uncarboxylated osteocalcin at the
positions corresponding to positions 17, 21, and 24 of mature human
osteocalcin is not carboxylated.
132. The method of claim 123, wherein all three of the glutamic
acids in the undercarboxylated/uncarboxylated osteocalcin at the
positions corresponding to positions 17, 21, and 24 of mature human
osteocalcin are not carboxylated.
133. The method of claim 123, wherein the
undercarboxylated/uncarboxylated osteocalcin is a preparation of
undercarboxylated/uncarboxylated osteocalcin in which more than
about 20% of the total Glu residues at the positions corresponding
to positions 17, 21, and 24 of mature human mature human
osteocalcin in the preparation are not carboxylated.
134. The method of claim 123, wherein the
undercarboxylated/uncarboxylated osteocalcin shares at least 80%
amino acid sequence identity with mature human osteocalcin when the
undercarboxylated/uncarboxylated osteocalcin and mature human
osteocalcin are aligned for maximum sequence homology.
135. The method of claim 123, wherein the
undercarboxylated/uncarboxylated osteocalcin comprises the mature
human osteocalcin protein.
136. The method of claim 123, wherein the
undercarboxylated/uncarboxylated osteocalcin is a polypeptide
selected from the group consisting of: (a) a fragment comprising
mature human osteocalcin missing the first 10 amino acids from the
N-terminal end; (b) a fragment comprising mature human osteocalcin
missing the last 10 amino acids from the C-terminal end; (c) a
fragment comprising amino acids 62-90 of SEQ ID NO:2; (d) a
fragment comprising amino acids 1-36 of mature human osteocalcin;
(e) a fragment comprising amino acids 13-26 of mature human
osteocalcin; (f) a fragment comprising amino acids 13-46 of mature
human osteocalcin; and (g) variants of the above.
137. The method of claim 136, wherein the
undercarboxylated/uncarboxylated osteocalcin is a fragment
comprising mature human osteocalcin missing the first 10 amino
acids from the N-terminal end and missing the last 10 amino acids
from the C-terminal end of mature human osteocalcin.
138. The method of claim 136, wherein the
undercarboxylated/uncarboxylated osteocalcin is a fragment
consisting of amino acids 1-36 of mature human osteocalcin.
139. The method of claim 136, wherein the
undercarboxylated/uncarboxylated osteocalcin is a fragment
consisting of amino acids 13-26 of mature human osteocalcin.
140. The method of claim 136, wherein the
undercarboxylated/uncarboxylated osteocalcin is a fragment
consisting of amino acids 13-46 of mature human osteocalcin.
141. The method of claim 123, wherein the
undercarboxylated/uncarboxylated osteocalcin has reduced binding to
hydroxyapatite compared to carboxylated osteocalcin.
142. A method for treating or preventing a vascular disorder in a
mammal comprising administering an effective amount of
uncarboxylated/undercarboxylated osteocalcin.
143. A method of diagnosing a patient at risk of developing a
disease related to energy metabolism and the OST-PTP signaling
pathway comprising (i) determining the ratio of
undercarboxylated/uncarboxylated osteocalcin to total osteocalcin
in a biological sample from the patient; and (ii) comparing the
ratio to a standard ratio; wherein, if the patient ratio is lower
than the standard ratio, the patient is at risk of developing a
disease related to the OST-PTP signaling pathway.
144. A method for increasing insulin sensitivity, increasing
insulin production, increasing glucose tolerance, increasing
pancreatic beta-cell proliferation, or increasing adiponectin serum
level in a mammal comprising administering an effect amount of
uncarboxylated/undercarboxylated osteocalcin.
145. A pharmaceutical composition for treating or preventing a
disorder selected from the group consisting of diabetes, metabolic
syndrome, glucose intolerance, and obesity in a mammal, said
pharmaceutical composition comprising an effective amount of
uncarboxylated/undercarboxylated osteocalcin.
146. The pharmaceutical composition of claim 145, wherein the
uncarboxylated/undercarboxylated osteocalcin is in an amount
effective to produce an effect selected from the group consisting
of an increase in glucose tolerance, an increase in insulin
production, an increase insulin sensitivity, an increase in
pancreatic beta-cell proliferation, and an increase in adiponectin
serum level.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Applications Nos. 60/844,203 filed Sep. 13, 2006; 60/870,604
filed Dec. 18, 2006; 60/909,712 filed Apr. 2, 2007; and 60/945,081
filed Jun. 19, 2007; the contents of which are hereby incorporated
by reference as if set forth fully herein.
FIELD OF THE INVENTION
[0003] The present invention relates to methods and compositions
for treating, preventing and diagnosing disorders related to energy
metabolism and the OST-PTP signaling pathway, which involves
gamma-carboxylase, osteocalcin and adiponectin. Such disorders
include, but are not limited to, metabolic syndrome, glucose
intolerance, diabetes types 1 and 2, atherosclerosis and
obesity.
BACKGROUND OF THE INVENTION
[0004] The prevailing research paradigm in bone biology is that
differentiation and functions of the two bone-specific cell types,
osteoblasts and osteoclasts, are determined by secreted molecules
that can either be cytokines acting locally, or hormones acting
systemically (Harada and Rodan, 2003; Takayanagi, 2006; Teitelbaum
and Ross, 2003). Applicants have discovered a previously unknown
genetic pathway related to energy metabolism and occurring in
osteoblasts in which decreased activity of OST-PTP leads to
decreased activity of gamma-carboxylase, which in turn leads to
increased secretion of undercarboxylated/uncarboxylated osteocalcin
from the osteoblasts, with beneficial effects on glucose
homeostasis.
[0005] OST-PTP is the protein encoded by the Esp gene. The Esp gene
was originally named for embryonic stem (ES) cell phosphatase and
it has also been called the Ptprv gene in mice. (Lee et al, 1996,
Mech Dev 59: 153-164). OST-PTP is a receptor-like protein
osteotesticular protein tyrosine phosphatase as well as fragments
and variants thereof. OST-PTP is a large, 1711 amino-acid long
protein that includes three distinct domains. OST-PTP has a 1068
amino-acid long extracellular domain containing multiple
fibronectin type III repeats.
[0006] Esp expression is restricted to ES cells, the gonads and the
skeleton. In the gonads, Esp is specifically expressed in Sertoli
cells of the testis and coelomic epithelial cells of the ovaries.
During development, Esp is initially expressed in the apical
ectodermal ridge of the limbs. Later during embryonic development
and after birth, its expression becomes restricted to
pre-osteoblasts and osteoblasts (i.e., Runx2-positive cells) of the
perichondrium and periosteum. Because of its bone and testicular
localization, the gene product of Esp is often referred to as
osteoblast testicular protein tyrosine phosphatase (OST-PTP).
[0007] Osteocalcin, one of the very few osteoblast-specific
proteins, has several features of a hormone. Ducy et. al.
demonstrated that mineralized bone from aging osteocalcin-deficient
mice was two times thicker than that of wild-type. It was shown
that the absence of osteocalcin led to an increase in bone
formation without impairing bone resorption and did not affect
mineralization. Multiple immunoreactive forms of human osteocalcin
have been discovered in circulation (Garnero et al. J Bone Miner
Res 1994; 9:255-4) and also in urine (Taylor et al. J. Clin.
Endocrin. Metab. 1990; 70:467-72). Fragments of human osteocalcin
can be produced either during osteoclastic degradation of bone
matrix or as the result of the catabolic breakdown of the
circulating protein after synthesis by osteoblasts.
[0008] Metabolic syndrome is a combination of medical disorders
that increase the risk of cardiovascular disease and diabetes. Some
of the symptoms of metabolic syndrome include: fasting
hyperglycemia, high blood pressure, decreased HDL cholesterol,
elevated triglycerides, and elevated uric acid levels.
[0009] The experiments described herein provide the first evidence
that the skeleton is an endocrine regulator of energy metabolism
and thereby determines, in part, the onset and severity of
metabolic syndrome or type 2 diabetes, as well as the risk of
developing these disorders. The experiments described herein
establish that the skeleton makes and secretes undercarboxylated
osteocalcin which acts as a hormone regulating energy metabolism.
Described herein is a previously unknown genetic pathway relating
to energy metabolism and occurring in osteoblasts in which
decreased activity of OST-PTP leads to decreased activity of
gamma-carboxylase, which in turn leads to increased secretion of
undercarboxylated osteocalcin from the osteoblasts, with beneficial
effects on glucose homeostasis.
SUMMARY OF THE INVENTION
[0010] The present invention provides pharmaceutical compositions
comprising an agent that modulates energy metabolism and the
OST-PTP signaling pathway, wherein the agent reduces OST-PTP
phosphorylase activity, reduces gamma-carboxylase activity, or
increases undercarboxylated/uncarboxylated osteocalcin, wherein the
pharmaceutical compositions comprise the agent in an amount that
produces an effect selected from the group consisting of an
increase in glucose tolerance, an increase in insulin production,
an increase insulin sensitivity, an increase in pancreatic
beta-cell proliferation, an increase in adiponectin serum level, a
reduction of oxidized phospholipids, a regression of
atherosclerotic plaques, a decrease in inflammatory protein
biosynthesis, a reduction in plasma cholesterol, a reduction in
vascular smooth muscle cell (VSMC) proliferation and number, and a
decrease in the thickness of arterial plaque.
[0011] In certain embodiments, the pharmaceutical compositions
comprise the agent in an amount effective to treat or prevent a
disorder selected from the group consisting of metabolic syndrome,
glucose intolerance, type 1 diabetes, type 2 diabetes,
atherosclerosis, and obesity. In certain embodiments, the agent
inhibits the expression or activity of OST-PTP or
gamma-carboxylase, inhibits phosphorylation of gamma-carboxylase,
increases the level of undercarboxylated or uncarboxylated
osteocalcin, inhibits carboxylation of osteocalcin, decarboxylates
osteocalcin. In certain embodiments, the agent is selected from the
group consisting of a small molecule, an antibody, a nucleic acid
and a biologically active fragment or variant thereof.
[0012] In certain embodiments, the agent is
undercarboxylated/uncarboxylated osteocalcin. In certain
embodiments, at least one of the glutamic acids in the
undercarboxylated/uncarboxylated osteocalcin at the positions
corresponding to positions 17, 21 and 24 of mature human
osteocalcin is not carboxylated. In certain embodiments, all three
of the glutamic acids in the undercarboxylated/uncarboxylated
osteocalcin at the positions corresponding to positions 17, 21 and
24 of mature human osteocalcin are not carboxylated.
[0013] In certain embodiments, the undercarboxylated/uncarboxylated
osteocalcin is a preparation of undercarboxylated/uncarboxylated
osteocalcin in which more than about 20% of the total Glu residues
at the positions corresponding to positions 17, 21 and 24 of mature
human mature human osteocalcin in the preparation are not
carboxylated. In certain embodiments, the
undercarboxylated/uncarboxylated osteocalcin shares at least 80%
amino acid sequence identity with mature human osteocalcin when the
undercarboxylated/uncarboxylated osteocalcin and mature human
osteocalcin are aligned for maximum sequence homology.
[0014] In certain embodiments, the undercarboxylated/uncarboxylated
osteocalcin is a polypeptide selected from the group consisting of:
[0015] (a) a fragment comprising mature human osteocalcin missing
the last 10 amino acids from the C-terminal end; [0016] (b) a
fragment comprising mature human osteocalcin missing the first 10
amino acids from the N-terminal end; [0017] (c) a fragment
comprising amino acids 62-90 of SEQ ID NO:2; [0018] (d) a fragment
comprising amino acids 1-36 of mature human osteocalcin; and [0019]
(e) variants of the above.
[0020] In certain embodiments, the pharmaceutical composition
comprises a small molecule selected from the group consisting of
warfarin, beta-blockers, statins, vitamin K inhibitors and
biologically active fragments or variants thereof. In a preferred
embodiment, the small molecule is warfarin. In another preferred
embodiment, the agent is a small molecule that increases the
activity or expression of osteocalcin or adiponectin.
[0021] In certain embodiments, the pharmaceutical composition
comprises an antibody or antibody fragment that binds to OST-PTP or
gamma-carboxylase. Preferably, the antibody or antibody fragment is
a monoclonal antibody. In certain embodiments, the antibody or
antibody fragment binds to the extracellular domain of OST-PTP. In
preferred embodiments, the OST-PTP is human OST-PTP. In certain
embodiments, the OST-PTP is the mouse OST-PTP of SEQ ID NO:19 or an
OST-PTP having an amino acid sequence that is substantially
homologous or identical to SEQ ID NO:19. In certain embodiments,
the OST-PTP is an OST-PTP having an amino acid sequence that is at
least 70% homologous or identical to SEQ ID NO:19.
[0022] In certain embodiments, the pharmaceutical composition
comprises a nucleic acid that inhibits the expression or activity
of OST-PTP or gamma-carboxylase. In certain embodiments, the
nucleic acid is an antisense oligonucleotide or a siRNA. In certain
embodiments, the nucleic acid is an isolated nucleic acid that is
selected from the group consisting of an antisense DNA, antisense
RNA, and small interfering RNA, which nucleic acid is sufficiently
complementary to SEQ ID NO:18 or a sequence that is substantially
homologous or identical to SEQ ID NO:18 to permit specific
hybridization to SEQ ID NO:18 or the sequence that is substantially
homologous or identical to SEQ ID NO:18, and wherein the
hybridization prevents or reduces expression of OST-PTP in
osteoblasts.
[0023] In certain embodiments, the pharmaceutical composition
comprises an agent that increases the level of serum insulin.
[0024] In certain embodiments, the pharmaceutical composition
comprises about 1 mg to about 750 mg of the agent. In certain
embodiments, the pharmaceutical composition comprises an agent that
is formulated into a controlled release preparation. In certain
embodiments, the pharmaceutical composition comprises an agent that
is chemically modified to prolong its half life in the human
body.
[0025] In certain embodiments, the pharmaceutical composition
comprises an anti-coagulant, a vasodilator, a drug used to treat
atherosclerosis, a drug used to treat diabetes, a vitamin K
inhibitor, a statin, or a beta blocker.
[0026] The present invention also provides a pharmaceutical
composition comprising an undercarboxylated osteocalcin polypeptide
comprising an amino acid sequence
TABLE-US-00001 (SEQ ID NO: 23)
YLYQWLGAPVPYPDPLX.sub.1PRRX.sub.2VCX.sub.3LNPDCDELADHIGFQEAYRRFYG
PV
wherein [0027] X.sub.1, X.sub.2 and X.sub.3 are each independently
selected from an amino acid or amino acid analog, with the proviso
that if X.sub.1, X.sub.2 and X.sub.3 are each glutamic acid, then
X.sub.1 is not carboxylated, or less than 50 percent of X.sub.2 is
carboxylated, and/or less than 50 percent of X.sub.3 is
carboxylated, [0028] or said osteocalcin polypeptide comprises an
amino acid sequence that is different from SEQ. ID. NO:23 at 1 to 7
positions other than X.sub.1, X.sub.2 and X.sub.3; and [0029]
wherein said amino acid sequence can include one amide backbone
substitutions.
[0030] In certain embodiments, the osteocalcin polypeptide of SEQ.
ID. NO:23 is a fusion protein. In certain embodiments, the arginine
at position 43 of SEQ. ID. NO:23 is replaced with an amino acid or
amino acid analog that reduces susceptibility of the osteocalcin
polypeptide to proteolytic degradation. In certain embodiments, the
arginine at position 44 of SEQ. ID. NO:23 is replaced with
f3-dimethyl-arginine. In certain embodiments, the osteocalcin
polypeptide is a retroenantiomer of uncarboxylated human
osteocalcin (1-49).
[0031] The present invention also provides a method of modulating a
pathway related to energy metabolism and the OST-PTP signaling
pathway comprising administering an agent that reduces OST-PTP
phosphorylase activity, reduces gamma-carboxylase activity, or
increases undercarboxylated/uncarboxylated osteocalcin, wherein the
agent is administered in an amount that produces an effect selected
from the group consisting of an increase in glucose tolerance, an
increase in insulin production, an increase insulin sensitivity, an
increase in pancreatic beta-cell proliferation, an increase in
adiponectin serum level, a reduction of oxidized phospholipids, a
regression of atherosclerotic plaques, a decrease in inflammatory
protein biosynthesis, a reduction in plasma cholesterol, a
reduction in vascular smooth muscle cell (VSMC) proliferation and
number, and a decrease in the thickness of arterial plaque. In
certain embodiments, the agent is administered in an amount
effective to treat or prevent a disorder selected from the group
consisting of metabolic syndrome, glucose intolerance, type 1
diabetes, type 2 diabetes, atherosclerosis, and obesity.
[0032] In certain embodiments, the method comprises administering
the agent in an amount effective to treat or prevent a disorder
selected from the group consisting of metabolic syndrome, glucose
intolerance, type 1 diabetes, type 2 diabetes, atherosclerosis, and
obesity. In certain embodiments, the agent inhibits the expression
or activity of OST-PTP or gamma-carboxylase, inhibits
phosphorylation of gamma-carboxylase, increases the level of
undercarboxylated or uncarboxylated osteocalcin, inhibits
carboxylation of osteocalcin, decarboxylates osteocalcin. In
certain embodiments, the agent is selected from the group
consisting of a small molecule, an antibody, a nucleic acid and a
biologically active fragment or variant thereof.
[0033] In certain embodiments, the agent is
undercarboxylated/uncarboxylated osteocalcin. In certain
embodiments, at least one of the glutamic acids in the
undercarboxylated/uncarboxylated osteocalcin at the positions
corresponding to positions 17, 21 and 24 of mature human
osteocalcin is not carboxylated. In certain embodiments, all three
of the glutamic acids in the undercarboxylated/uncarboxylated
osteocalcin at the positions corresponding to positions 17, 21 and
24 of mature human osteocalcin are not carboxylated. In certain
embodiments, the present invention provides methods of
administering undercarboxylated/uncarboxylated osteocalcin to
increase insulin production.
[0034] In certain embodiments, the undercarboxylated/uncarboxylated
osteocalcin is a preparation of undercarboxylated/uncarboxylated
osteocalcin in which more than about 20% of the total Glu residues
at the positions corresponding to positions 17, 21 and 24 of mature
human mature human osteocalcin in the preparation are not
carboxylated. In certain embodiments, the
undercarboxylated/uncarboxylated osteocalcin shares at least 80%
amino acid sequence identity with mature human osteocalcin when the
undercarboxylated/uncarboxylated osteocalcin and mature human
osteocalcin are aligned for maximum sequence homology.
[0035] In certain embodiments, the undercarboxylated/uncarboxylated
osteocalcin is a polypeptide selected from the group consisting of:
[0036] (a) a fragment comprising mature human osteocalcin missing
the last 10 amino acids from the C-terminal end; [0037] (b) a
fragment comprising mature human osteocalcin missing the first 10
amino acids from the N-terminal end; [0038] (c) a fragment
comprising amino acids 62-90 of SEQ ID NO:2; [0039] (d) a fragment
comprising amino acids 1-36 of mature human osteocalcin; and [0040]
(e) variants of the above.
[0041] In certain embodiments, the agent is a small molecule that
inhibits the expression or activity of OST-PTP or
gamma-carboxylase. In certain embodiments, the agent is a small
molecule selected from the group consisting of warfarin,
beta-blockers, statins, vitamin K inhibitors and biologically
active fragments or variants thereof. In a preferred embodiment,
the small molecule is warfarin. In another preferred embodiment,
the agent is a small molecule that increases the activity or
expression of osteocalcin or adiponectin.
[0042] In certain embodiments, agent is an antibody or antibody
fragment that binds to OST-PTP or gamma-carboxylase. Preferably,
the antibody or antibody fragment is a monoclonal antibody. In
certain embodiments, the antibody or antibody fragment binds to the
extracellular domain of OST-PTP. In preferred embodiments, the
OST-PTP is human OST-PTP. In certain embodiments, the OST-PTP is
the mouse OST-PTP of SEQ ID NO:19 or an OST-PTP having an amino
acid sequence that is substantially homologous or identical to SEQ
ID NO:19. In certain embodiments, the OST-PTP is an OST-PTP having
an amino acid sequence that is at least 70% homologous or identical
to SEQ ID NO:19.
[0043] In certain embodiments, the agent is a nucleic acid that
inhibits the expression or activity of OST-PTP or
gamma-carboxylase. In certain embodiments, the nucleic acid is an
antisense oligonucleotide or a siRNA. In certain embodiments, the
nucleic acid is an isolated nucleic acid that is selected from the
group consisting of an antisense DNA, antisense RNA, and small
interfering RNA, which nucleic acid is sufficiently complementary
to SEQ ID NO:18 or a sequence that is substantially homologous or
identical to SEQ ID NO:18 to permit specific hybridization to SEQ
ID NO:18 or the sequence that is substantially homologous or
identical to SEQ ID NO:18, and wherein the hybridization prevents
or reduces expression of OST-PTP in osteoblasts.
[0044] In certain embodiments, the methods of the present invention
are carried out by administering about 1 mg to about 750 mg of the
agent. In certain embodiments, the agent that is formulated into a
controlled release preparation. In certain embodiments, the agent
is chemically modified to prolong its half life in the human body.
In certain embodiments, the agent is co-administered with an
anti-coagulant, a vasodilator, a drug used to treat
atherosclerosis, a drug used to treat diabetes, a vitamin K
inhibitor, a statin, or a beta blocker.
[0045] The present invention also provides a method of diagnosing a
patient at risk of developing a disease related to energy
metabolism and the OST-PTP signaling pathway comprising (i)
determining the ratio of undercarboxylated/uncarboxylated
osteocalcin to total osteocalcin in a biological sample from the
patient; and (ii) comparing the ratio to a standard ratio; wherein,
if the patient ratio is lower than the standard ratio, the patient
is at risk of developing a disease related to the OST-PTP signaling
pathway.
[0046] In certain embodiments, the disease related to the OST-PTP
signaling pathway is selected from the group consisting of
metabolic syndrome, glucose intolerance, type 1 diabetes, type 2
diabetes, atherosclerosis, and obesity. In certain embodiments,
disease related to the OST-PTP signaling is characterized by
decreased insulin production, decreased insulin sensitivity,
decreased glucose tolerance and/or increased fat mass.
[0047] In certain embodiments, the biological sample is blood.
[0048] In certain embodiments of the diagnostic method described
above, the standard ratio is 5%-10%, 10%-15%, 15%-20%, 20%-25%,
25%-30%, or 30%-35.
[0049] The present invention provides a use of an agent that
modulates energy metabolism and the OST-PTP signaling pathway and
reduces OST-PTP phosphorylase activity, reduces gamma-carboxylase
activity, or increases undercarboxylated/uncarboxylated
osteocalcin, in an amount that produces an effect selected from the
group consisting of an increase in glucose tolerance, an increase
in insulin production, an increase insulin sensitivity, an increase
in pancreatic beta-cell proliferation, an increase in adiponectin
serum level, a reduction of oxidized phospholipids, a regression of
atherosclerotic plaques, a decrease in inflammatory protein
biosynthesis, a reduction in plasma cholesterol, a reduction in
vascular smooth muscle cell (VSMC) proliferation and number, and a
decrease in the thickness of arterial plaque, as a medicament.
[0050] In certain embodiments, the agent is used to treat or
prevent a disorder selected from the group consisting of metabolic
syndrome, glucose intolerance, type 1 diabetes, type 2 diabetes,
atherosclerosis, and obesity.
[0051] In certain embodiments, the agent inhibits phosphorylation
of gamma-carboxylase. In certain embodiments, the agent increases
the level of uncarboxylated osteocalcin. In certain embodiments,
the agent inhibits carboxylation of osteocalcin. In certain
embodiments, the agent decarboxylates osteocalcin.
[0052] In certain embodiments, the agent is
undercarboxylated/uncarboxylated osteocalcin. In certain
embodiments, the undercarboxylated/uncarboxylated osteocalcin
increases insulin production. In certain embodiments, at least one
of the glutamic acids in the undercarboxylated/uncarboxylated
osteocalcin at the positions corresponding to positions 17, 21 and
24 of mature human osteocalcin is not carboxylated. In certain
embodiments, all three of the glutamic acids in the
undercarboxylated/uncarboxylated osteocalcin at the positions
corresponding to positions 17, 21 and 24 of mature human
osteocalcin are not carboxylated. In certain embodiments, the
undercarboxylated/uncarboxylated osteocalcin is a preparation of
undercarboxylated/uncarboxylated osteocalcin in which more than
about 20% of the total Glu residues at the positions corresponding
to positions 17, 21 and 24 of mature human mature human osteocalcin
in the preparation are not carboxylated. In certain embodiments,
the undercarboxylated/uncarboxylated osteocalcin shares at least
80% amino acid sequence identity with mature human osteocalcin when
the undercarboxylated/uncarboxylated osteocalcin and mature human
osteocalcin are aligned for maximum sequence homology.
[0053] In certain embodiments, the undercarboxylated/uncarboxylated
osteocalcin is a polypeptide selected from the group consisting of:
[0054] (a) a fragment comprising mature human osteocalcin missing
the last 10 amino acids from the C-terminal end; [0055] (b) a
fragment comprising mature human osteocalcin missing the first 10
amino acids from the N-terminal end; [0056] (c) a fragment
comprising amino acids 62-90 of SEQ ID NO:2; [0057] (d) a fragment
comprising amino acids 1-36 of mature human osteocalcin; and [0058]
(e) variants of the above.
[0059] In certain embodiments, the agent is selected from the group
consisting of a small molecule, an antibody, a nucleic, acid and a
biologically active fragment or variant thereof.
[0060] In certain embodiments, the agent is a small molecule that
inhibits the expression or activity of OST-PTP or
gamma-carboxylase. In certain embodiments, the agent is a small
molecule selected from the group consisting of warfarin,
beta-blockers, statins, vitamin K inhibitors and biologically
active fragments or variants thereof. In a preferred embodiment,
the small molecule is warfarin. In another preferred embodiment,
the agent is a small molecule that increases the activity or
expression of osteocalcin or adiponectin.
[0061] In certain embodiments, agent is an antibody or antibody
fragment that binds to OST-PTP or gamma-carboxylase. Preferably,
the antibody or antibody fragment is a monoclonal antibody. In
certain embodiments, the antibody or antibody fragment binds to the
extracellular domain of OST-PTP. In preferred embodiments, the
OST-PTP is human OST-PTP. In certain embodiments, the OST-PTP is
the mouse OST-PTP of SEQ ID NO:19 or an OST-PTP having an amino
acid sequence that is substantially homologous or identical to SEQ
ID NO:19. In certain embodiments, the OST-PTP is an OST-PTP having
an amino acid sequence that is at least 70% homologous or identical
to SEQ ID NO:19.
[0062] In certain embodiments, the agent is a nucleic acid that
inhibits the expression or activity of OST-PTP or
gamma-carboxylase. In certain embodiments, the nucleic acid is an
antisense oligonucleotide or a siRNA. In certain embodiments, the
nucleic acid is an isolated nucleic acid that is selected from the
group consisting of an antisense DNA, antisense RNA, and small
interfering RNA, which nucleic acid is sufficiently complementary
to SEQ ID NO:18 or a sequence that is substantially homologous or
identical to SEQ ID NO:18 to permit specific hybridization to SEQ
ID NO:18 or the sequence that is substantially homologous or
identical to SEQ ID NO:18, and wherein the hybridization prevents
or reduces expression of OST-PTP in osteoblasts.
[0063] In certain embodiments, about 750 mg of the agent is used as
a medicament. In certain embodiments, the agent is formulated into
a controlled release preparation. In certain embodiments, the agent
is chemically modified to prolong its half life in the human body.
In certain embodiments, the agent is co-administered with an
anti-coagulant, a vasodilator, a drug used to treat
atherosclerosis, a drug used to treat diabetes, a vitamin K
inhibitor, a statin, or a beta blocker.
[0064] The present invention provides the use of an
undercarboxylated osteocalcin polypeptide, or mimetic thereof, in
the manufacture of a medicament for treatment of a metabolic
condition.
[0065] The present invention also provides the use of an agent that
modulates energy metabolism and the OST-PTP signaling pathway and
reduces OST-PTP phosphorylase activity, reduces gamma-carboxylase
activity, or increases undercarboxylated/uncarboxylated
osteocalcin, in an amount that produces an effect selected from the
group consisting of an increase in glucose tolerance, an increase
in insulin production, an increase insulin sensitivity, an increase
in pancreatic beta-cell proliferation, an increase in adiponectin
serum level, a reduction of oxidized phospholipids, a regression of
atherosclerotic plaques, a decrease in inflammatory protein
biosynthesis, a reduction in plasma cholesterol, a reduction in
vascular smooth muscle cell (VSMC) proliferation and number, and a
decrease in the thickness of arterial plaque, for the manufacture
of a medicament for the treatment or prevention of a disorder
selected from the group consisting of metabolic syndrome, glucose
intolerance, type 1 diabetes, type 2 diabetes, atherosclerosis, and
obesity.
BRIEF DESCRIPTION OF THE FIGURES
[0066] FIG. 1. Increased insulin secretion and beta-cell
proliferation in Esp-/- mice. (A) LacZ-stained tissues from newborn
Esp-/- mice demonstrating Esp locus activity in bone and testis but
not in pancreas or fat pads. (B) Expression of Esp in osteoblasts,
adipocytes, and pancreatic islets by real-time PCR in 1-month-old
mice. (C) Southern blot analysis showing efficient recombination at
the Esp locus in osteoblasts of Esposb-/- mice. (D) Using real-time
PCR Esp expression is 90% decreased in osteoblasts but not altered
in testis of Esposb-/- mice. (E) Decreased percentage at weaning of
Esp-/- pups born from crosses between Esp+/- mice. (F) Lower
survival at birth and at weaning of Esp-/- pups born from Esp+/-
and Esp-/- mothers. (G and H) Blood glucose levels (G) and serum
insulin levels (H) in WT and Esp-/- newborn before feeding (P0) or
after random feeding at indicated ages. (I-J) GSIS (I) and GTT (J)
test in 1-month-old WT and Esp-/- mice. (K) H&E staining,
insulin immunostaining, and insulin/Ki67 double immunostaining
showing larger islets and increased beta-cell proliferation in
pancreas of WT and 1-month-old Esp-/- mice. Arrowheads indicate
islets, and arrows point at Ki67-positive cells. Scale bars are 100
mm except in upper panels, where they are 800 mm. Histomorphometric
comparisons of islet number, size, and beta-cell mass between
1-month-old WT and Esp-/- mice (lowest panel). (L) Pancreas insulin
content in 1-month-old WT and Esp-/- mice. (M) Quantification of
the number of Ki67-immunoreactive cells in pancreatic islets of P5
and 1-month-old WT and Esp-/- mice. All panels except (I) and (J),
.degree. p<0.05 and *p<0.01 versus WT (Student's t test). (I
and J) .degree. p<0.05 versus WT and *p % 0.001 versus WT (ANOVA
followed by post hoc analysis).
[0067] FIG. 2. Increased Insulin Sensitivity and Adiponectin
Expression in Esp-/- Mice. All experiments compare 1-month-old mice
WT and Esp-/- unless otherwise indicated. (A) ITT. (B) Glucose
infusion rate during hyperinsulinemic-euglycemic clamp. (C)
Expression of markers of insulin sensitivity in skeletal muscle
measured by real-time PCR. (D) Electron microscopy images (upper
panel, 20,0003) and corresponding quantification (lower panel) of
mitochondrial area in gastrocnemius muscle. Scale bars are 1 mm.
(E) Decreased number of lipid droplets on Oil red O stained liver
sections (upper panel) and modified expression of insulin target
genes by real-time PCR (lower panel) in Esposb-/- mice. Scale bars
are 50 mm. (F) Fat pad mass (fat pad weight over body weight). (G)
Energy expenditure. (H) Serum triglyceride levels after an
overnight fast. (I) H&E staining of adipose tissues of WT and
Esp-/- mice (upper panel) and respective distribution of diameters
for 100 measured adipocytes per slide (lower panel). Scale bars are
50 mm. (J) Expression of markers of adipogenesis, lipogenesis, fat
uptake, and lipolysis in fat. (K) Serum free fatty acid (FFA) in
fed and overnight-fasted mice. (L) Expression of Leptin, Resistin,
and Adiponectin in fat. (M) Serum levels of adiponectin in newborn
mice before feeding (P0) and after random feeding at other
indicated ages. (N) Expression of adiponectin target genes, in
tissues of WT and Esp-/- mice. In (A), .degree. p<0.05 versus WT
and *p % 0.001 versus WT (ANOVA followed by post hoc analysis); in
(B)-(N), *p<0.01 versus WT (Student's t test).
[0068] FIG. 3. Esp-/- Mice Are Protected from Obesity and Glucose
Intolerance A-F) Food intake per day (A), body weight curve (B),
fat pad mass (C), serum triglyceride levels (D), GTT (E), and ITT
(F) in 4-month-old WT and Esp-/- mice 3 months after GTG or vehicle
injection. (G-I) Body weight curve (G), GTT (H), and ITT (I) in 3
month-old WT and Esp-/- mice fed a high fat diet for 6 weeks. (J
and K) Serum insulin levels (J) and pancreatic insulin content (K)
in 1-month-old WT and Esp-/- mice 8 days after STZ or vehicle
injection. (L and M) Survival of mice (L) and change of blood
glucose levels (M) in 1-month-old WT and Esp-/- mice during the 8
days following STZ injection. (N) Urinary glucose assays in
1-month-old WT and Esp-/- mice 8 days after STZ injection. In
(A)-(F), (J), and (K): a, WT versus Esp-/-; b,WT+GTG (or STZ)
versus WT+vehicle; c,WT+GTG (or STZ) versus Esp-/-+GTG (or STZ); d,
Esp-/-+GTG (or STZ) versus Esp-/-+vehicle. In (G)-(I) and (M),
*p<0.05 WT versus Esp-/-. In (A), (C), (D), (J), and (K),
Student's t test, p<0.05 for a-d; in (B), (E)-(I), (L), and (M),
ANOVA followed by post hoc analysis when number of groups >2, p
% 0.001 for a-d.
[0069] FIG. 4. Osteoblasts Secrete a Factor Regulating Insulin and
Adiponectin Expression. A-E) All experiments compare 1-month-oldWT
and al(I)-Esp mice. (A) Insulin immunostaining (upper panel) and
histomorphometric comparisons of islet number, size, beta-cell
mass, and Ki67-immunoreactive cells in pancreas (lower panel).
Scale bars are 100 mm. (B) Blood glucose and serum
insulin/adiponectin levels. (C) GSIS test. (D) GTT. (E) ITT. (F)
Expression of Insulin and Glucagon in WT islets cocultured with
fibroblasts or osteoblasts. (G) Expression of Adiponectin and
Leptin in WT adipocytes cocultured with fibroblasts or osteoblasts.
(H) Expression of Insulin and Adiponectin in Esp-/- indicated cells
cocultured with fibroblasts or osteoblasts. (I and J) Expression of
Insulin (I) and Adiponectin (J) in WT indicated cells cocultured
with or without osteoblasts in presence of a filter preventing
cell-cell contact or in presence of conditioned medium (CM)
collected from osteoblast cultures. (A, B, and F-J) *p<0.05
versus WT (Student's t test); (C-E) .degree. p<0.05 versus WT
and *p % 0.001 versus WT (ANOVA).
[0070] FIG. 5. Osteocalcin Regulates Beta-Cell Proliferation,
Insulin Secretion, and Insulin sensitivity. All experiments compare
3-month-old WT and Oc-/- mice unless otherwise indicated. (A) Blood
glucose levels after random feeding. (B) Insulin levels. (C) GSIS
test. (D) GTT. (E) ITT. (F) Glucose infusion rate during
hyperinsulinemic-euglycemic clamp. (G) Energy expenditure. (H)
Expression of insulin target genes by real-time PCR. (I)
Histomorphometric comparisons of islet numbers, islet size,
beta-cell mass, insulin content in pancreas, and Ki67
immunoreactive cells in pancreatic islets. P5, 5-day-old pups; 3M,
3-month-old mice. (J) Fat pad mass (fat pad weight over body
weight). (K) Serum triglyceride levels after an overnight fast. (L
and M) serum levels (L) and gene expression (M) of adiponectin. (N)
Expression of adiponectin target genes by real time PCR. (O)
Expression of Insulin and Glucagon in WT pancreatic islets
co-cultured with osteoblasts of indicated genotypes. (P) Expression
of Adiponectin and Leptin in WT adipocytes co-cultured with
osteoblasts of indicated genotypes. (Q) Expression of Insulin and
Adiponectin in WT indicated cells cultured in presence of
conditioned media from COS cells transfected with an Osteocalcin
expression vector or its empty counterpart. (R) Expression of
Insulin and Adiponectin in WT islets and adipocytes co-cultured
with fibroblasts in presence of recombinant osteocalcin (3 ng/ml)
or vehicle, or with osteoblasts expressing (5d) or not (1d)
Osteocalcin. (S and T) Dynamic of glucose (S) and insulin levels
(T) in Ocn-/- mice injected simultaneously with glucose and 20 ng
of recombinant osteocalcin or vehicle. Panels A, B, F-R: *p<0.05
vs WT (Student's t test); panels C-E, S and T, .degree. p<0.01
vs WT and *p<0.001 vs WT (ANOVA). Results are given as
means.+-.SD except in FIG. 5F where means.+-.SEM are shown.
[0071] FIG. 6. Osteocalcin Regulates Insulin Sensitivity via
Adiponectin. (A-E) Comparison between 6-week-old WT, Adiponectin+/-
(Adipo+/-), Osteocalcin+/- (Ocn+/-), and Ocn+/-; Adipo+/- mice. (A)
ITT. (B) Insulin serum levels. (C) Blood glucose levels. (D) GSIS
test. (E) Adiponectin serum levels. In (A) and (D), *p % 0.001
versus WT (ANOVA followed by post hoc analysis); in (B), (C), and
(E), *p<0.05 versus WT (Student's t test).
[0072] FIG. 7. Esp-/- Mice Are a Model of Increased Osteocalcin
Bioactivity. (A-G) Comparison between 6-week-old WT, Esp-/, Ocn+/-,
and Esp-/-; Ocn+/- mice. (A) Blood glucose levels. (B) Serum
insulin levels. (C) Serum adiponectin levels. (D) GTT. (E) ITT. (F)
GSIS test. (G) Quantification of the number of Ki67-immunoreactive
cells in pancreatic islets. (H and I) Quantification of the
percentage of osteocalcin bound to hydroxyapatite (HA) resin after
a 15min incubation of serum of 1-month-old mice of indicated
genotypes (H) or of conditioned medium from osteoblast cultures
treated with warfarin or vehicle (I). (J) Expression of Adiponectin
in WT adipocytes cocultured with osteoblasts treated with warfarin
or vehicle. (K) Expression of Adiponectin in WT adipocytes cultured
in presence of vehicle or of 1 ng/ml of commercially available
carboxylated osteocalcin (Immunotopics) or bacterially produced
uncarboxylated osteocalcin. (L) Expression of Insulin and Cyclin D1
in WT islets cultured in presence of 0.3 ng/ml of bacterially
produced uncarboxylated osteocalcin or vehicle. (M) Metabolic
parameters and total serum osteocalcin levels in control and obese
patients. (N-O) Quantification of carboxylated osteocalcin
(HA-bound osteocalcin) in control and obese patients. In (A)-(C)
and (G)-(L): *p<0.05 versus WT (Student's t test); in (D)-(F),
.degree. p<0.05 versus WT and *p % 0.001 versus WT (ANOVA
followed by post hoc analysis).
[0073] FIG. 8. OST-PTP was mutated in COS cells using site directed
mutagenesis using a PCR method and a commercially available kit.
Mutated OST-PTP (GST-PTP CA) interacts with insulin receptor (InsR)
in COS cells (left upper panel) and ROS cells (right upper panel)
(third lane) whereas WT OST-PTP (GST-PTP WT) does not interact
(second lane). The same amount of GST fusion proteins were used for
substrate trapping (lower panels). GST=Recombinant bacterially
produced glutathione S-Transferase protein.
[0074] FIG. 9. Mutant enzyme OST-PTP.sup.D1316A traps
gamma-carboxylase, thereby demonstrating that gamma-carboxylase is
a substrate of OST-PTP. This, however, does not mean that
gamma-carboxylase is the only substrate of OST-PTP. There was no
binding in the GST lane because there is no PTP transfected. It is
a control to show that if there is trapping, it is not due to the
GST part of any GST fusion protein. There was also no trapping with
GST-PTP.sup.WT because this form dephosphorylated the substrate
gamma-carboxylase, which is then released. A band is clearly seen
in the lane having the mutant OST-PTP (GST-PTP.sup.D1316A) because
the mutation engineered a defect in OST-PTP phosphatase activity
that allowed the substrate to irreversibly bind to and be retained
by the enzyme.
[0075] FIG. 10. Osteocalcin subcutaneous infusion decreases
glycemia in wt mice. Indicated doses of recombinant osteocalcin or
PBS were infused subcutaneously for 28 days in wt mice. Blood
glucose was measured at indicated days.
[0076] FIG. 11. Osteocalcin subcutaneous infusion increases glucose
tolerance in wt mice. Wt mice were infused subcutaneously with
indicated doses of recombinant osteocalcin or PBS for 14 days
before receiving a single injection of glucose. Blood glucose was
measured thereafter at indicated times.
[0077] FIG. 12. Osteocalcin subcutaneous infusion increases insulin
sensitivity in wt mice. Wt mice were infused subcutaneously with
indicated doses of recombinant osteocalcin or PBS for 18 days
before receiving a single injection of insulin. Blood glucose was
measured thereafter at indicated times.
[0078] FIG. 13. Osteocalcin subcutaneous infusion decreases fat
mass in wt mice. 1) Indicated doses of recombinant osteocalcin or
PBS were infused subcutaneously for 28 days in wt mice. Body weight
was recorded at indicated days. (2) Gonadal fat pad mass was
measured after 28 days.
[0079] FIG. 14. Osteocalcin subcutaneous infusion prevents
GTG-induced obesity in wt mice. Wt mice were injected with gold
thioglucose (GTG) or vehicle to induce hyperphagia and obesity. Two
weeks later they were implanted with subcutaneous osmotic pumps
infusing 1 n/hr of recombinant osteocalcin or PBS for 28 days
before. Body weight gain was recorded thereafter at indicated
days.
[0080] FIG. 15. A fragment of osteocalcin (1-36) is as potent as
native osteocalcin in inducing adiponectin expression in vitro. Wt
adipocytes were treated for 4 hours with recombinant full-length
osteocalcin (1-46) or a truncated form (1-36) or vehicle.
Adiponectin expression was then quantified by real time PCR.
[0081] FIG. 16. Shows the effect of applying isoproterenol
(enhancing SNS activity) to wild type osteoblasts on the expression
of ptprv=Esp/OST-PTP, Ggcx=gamma-carboxylase, Vkor=recycling of
vitamin K/necessary for ggcx activity, and Bgp=osteocalcin. mRNA
levels in wt osteoblasts were measured using quantitative PCR.
[0082] FIG. 17. The level of serum insulin in one week old mice
having various genotypes: WT mice, ob-/+ mice (heterozygous for
obesity), ob/ob mice, Bgp -/+ (heterozygous for osteocalcin),
BGP-/- mice, and ob/ob/mice that are also Bgp-/- (Oc
deficient).
[0083] FIG. 18. Ostecalcin-deficient mice develop atherogenic
lesions after 6 weeks on the western diet. Histological analysis of
the entrance aorta at the level of the aortic valves clearly shows
that atherogenic lesions are present.
[0084] FIG. 19. Absence of the Esp gene that encodes OST-PTP
protects ApoE-/- mice from developing atherosclerotic lesions.
ApoE-/- mice develop atherosclerotic lesions after being fed a
western diet for 6 weeks, while double mutant ApoE-/-; Esp-/- mice
on the same diet do not.
[0085] FIG. 20. The pathway for regulation of osteocalcin
production by bone. (Upper panel) OST-PTP, the protein encoded by
Esp favors the .gamma.-carboxylation of osteocalcin thus lowering
the pool of uncarboxylated osteocalcin, which is the active form of
this hormone. As a result .beta.-cell proliferation, Insulin
expression and Adiponectin expression are normally stimulated.
(Lower panel) In absence of OST-PTP .gamma.-carboxylation of
osteocalcin is hampered, there is more uncarboxylated osteocalcin
and, as a result, .beta.-cell proliferation, Insulin expression and
Adiponectin expression are increased. This results in improved
glucose handling and decreased fat mass.
[0086] FIG. 21. A diagram showing the high degree of conserved
amino acid sequence homology of osteocalcin among species.
[0087] FIG. 22. Generation of Esposb-/- mice and normal bone
formation in Esp-/- animals. (A) Targeting construct for
conditional inactivation of Esp. White boxes, exons encoding the
phosphatase domain of OST-PTP; grey triangles, LoxP sites; black
bars, 5' and 3' external probes; S, Sacl; EV, EcoRV. (B) PCR
genotyping of Esposb-/- mice. WT and floxed allele (F1) yield
280-bp and 350-bp products, respectively. 1(I) collagen-Cre
(1(I)-Cre)) transgenic mice (TG) harbor a transgene-specific band.
(C-D) Similar body weight gain (C) and linear growth (D) in WT and
Esp-/- mice. P0, newborn; 1M, 1 month (E) Alizarin red/alcian blue
staining of skeletons of newborn WT and Esp-/- mice. Mice were
dissected, fixed in 95% ethanol, and stained in alcian blue and
alizarin red as previously described (Duey et al., 1996). There is
no overt defect in mineralization that could explain the perinatal
death of mutant pups. (F) Bone histomorphometry of 2 month-old WT
and Esp-/- mice. BV/TV, bone volume per Total tissue volume (%);
N.ob/B.Pm, number of osteoblasts per bone perimeter (mm-1);
Dpd/Creat. Relative levels of deoxypyridinolone crosslinks, a
marker of bone resorption.
[0088] FIG. 23. (A-E) Comparison of 1 month-old WT and Esp-/- mice.
Serum levels of C-peptide (A), serum Glucagon level (left) and
glucagons content in pancreas (right) (B) and serum levels of IGF-1
(C), PYY (D), and amylin (E) in Esp-/- mice. (F) Ratio of muscle
mass over body mass calculated by proton magnetic resonance
spectroscopy (1H-MRS) in 10 week-old WT and Esp-/- mice. (G)
Representative images of proton 1H-MRS of 10 week-old WT and Esp-/-
mice. (H) Food intake per day in 1 month- and 3 month-old Esp-/-
and WT mice. (I and J) Comparison of expression level by real time
PCR (I) and of serum levels (J) of TNF- (left) and IL-6 (right) in
1 month-old Esp-/- and WT mice. (K) Serum leptin (left) and
resistin (right) levels in 1 month-old Esp-/- and WT mice. In all
panels data represent the means.+-.SD of experiments. *, P<0.01
(t-test).
[0089] FIG. 24. Destruction of VMH nuclei by GTG. Cresyl violet
staining of section from hypothalamus of WT and Esp-/- mice
injected with GTG or vehicle. Arcuate nuclei are circled in blue,
VMH in red.
[0090] FIG. 25. Absence of cell transdifferentiation during the
co-culture assays. (A-D) Analysis of Runx2 (A), Osteocalcin (B),
adiponectin (C), and Leptin (D) expression by real time PCR in
indicated cells 4h after co-culture of osteoblasts with adipocytes
or islets.
[0091] FIG. 26. Bone-specific expression of osteocalcin. (A) In
situ hybridization analysis of osteocalcin and Esp expression in
pancreas of 18.5 dpc embryos. Neither gene is expressed in
pancreas. Insulin expression was used as a positive control.
Hematoxylin-eosin staining of adjacent sections was used to assess
tissue integrity. (B) Real time PCR analysis of osteocalcin
expression in osteoblasts, adipocytes, and pancreatic islets
collected from 1 month-old WT mice. Osteocalcin is not expressed in
adipocytes or islets.
[0092] FIG. 27. Normal osteocalcin expression and serum level in
Esp-/- and alpha1(I) Esp mice. (A-B) Real-time PCR analysis of
osteocalcin expression (A) and osteocalcin serum levels (B) in 1
month-old WT, Esp-/- and alpha1(I) Esp mice. (C) Analysis of the
purity of bacterially produced osteocalcin by SDS-PAGE stained with
Coomassie blue.
[0093] FIG. 28. Overexpression of adiponectin in transgenic mice
decreases fat mass and increases insulin sensitivity.(A) Schematic
representation of the transgene used to overexpress adiponectin
(Adipo) in mice. (B) PCR genotyping of SAP-Adipo transgenic mice.
(C) Adiponectin serum levels in 3 distinct SAP-Adipo transgenic
lines at 1 month of age. (D) Fat pad mass in WT and SAP-Adipo
transgenic mice at 3 month of age. (E) Serum insulin levels in WT
and SAP-Adipo transgenic mice at 3 month of age. (F) Insulin
tolerance test in WT and SAP-Adipo transgenic mice at 3 month of
age.
[0094] FIG. 29: Overexpression of insulin in transgenic mice
decreases fat mass and increases glucose tolerance. (A-B) Blood
glucose levels after fasting (A) or random feeding (B) in
SAP-insulin transgenic and wt mice. (C) Serum levels of
triglycerides in WT and SAP-insulin transgenic mice. (D) Serum
levels of free fatty acids in WT and SAP-insulin transgenic mice.
(E) Fat pad mass in WT and SAP-insulin transgenic mice. (F) Glucose
tolerance test in WT and SAP-insulin transgenic mice. (G) Food
intake in
[0095] WT and SAP-insulin transgenic mice.
DETAILED DESCRIPTION OF THE INVENTION
[0096] The present invention is based in part on the discovery that
undercarboxylated/uncarboxylated osteocalcin secreted by
osteoblasts in bone is responsible for regulating various aspects
of energy metabolism. For example, it increases pancreatic
beta-cell proliferation, insulin secretion, insulin sensitivity,
glucose tolerance, and serum adiponectin and decreases weight gain
and fat mass. It also reduces the pathological effects of
atherosclerosis. Therefore, certain aspects of the invention are
directed to the therapeutic use of undercarboxylated/uncarboxylated
osteocalcin, fragments and variants thereof, to treat or prevent
metabolic syndrome, type 1 and type 2 diabetes, atherosclerosis and
obesity.
[0097] The present invention is also based on the discovery that
gamma-carboxylase carboxylates osteocalcin, thereby inactivating
osteocalcin. Such inactivation of osteocalcin decreases pancreatic
beta-cell proliferation, insulin secretion, insulin sensitivity,
glucose tolerance, and serum adiponectin and increases weight gain
and fat mass. It also increases the pathological effects of
atherosclerosis. Therefore, certain aspects of the invention are
directed to the therapeutic use of agents that inhibit the activity
of gamma-carboxylase, to treat or prevent metabolic syndrome, type
1 and type 2 diabetes, atherosclerosis and obesity.
[0098] The present invention is further based on the discovery that
OST-PTP activates gamma-carboxylase through dephosphorylation. As
indicated above, activation of gamma-carboxylase leads to
inactivation of osteocalcin. Such inactivation of osteocalcin
decreases pancreatic beta-cell proliferation, insulin secretion,
insulin sensitivity, glucose tolerance, and serum adiponectin and
increases weight gain and fat mass. It also it increases the
pathological effects of atherosclerosis. Therefore certain aspects
of the invention are directed to the therapeutic use of agents that
inhibit the activity of OST-PTP, to treat or prevent metabolic
syndrome, type 1 and type 2 diabetes, atherosclerosis and
obesity.
[0099] The present invention is also based on the discovery that
undercarboxylated/uncarboxylated osteocalcin increases the level of
adiponectin expression, resulting in increased insulin sensitivity,
glucose tolerance, and decreased weight gain and fat mass. It also
reduces the pathological effects of atherosclerosis. Therefore,
certain aspects of the invention are directed to the therapeutic
use of undercarboxylated/uncarboxylated osteocalcin to regulate the
expression of adiponectin, to treat or prevent metabolic syndrome,
type 1 and type 2 diabetes, atherosclerosis and obesity.
[0100] The present invention thus relates to methods and
compositions for treating and diagnosing disorders related to the
OST-PTP signaling pathway involving gamma-carboxylase, osteocalcin
and adiponectin. Such disorders include, but are not limited to,
metabolic syndrome, glucose intolerance, diabetes types 1 and 2,
atherosclerosis and obesity. The invention is based on the
discovery that OST-PTP dephosphorylates gamma-carboxylase, thereby
leading to activation of gamma-carboxylase. Activation of
gamma-carboxylase results in carboxylation of osteocalcin, which,
as demonstrated herein, leads to symptoms associated with metabolic
syndrome, diabetes types 1 and 2, atherosclerosis and obesity.
[0101] Other aspects of the invention are directed to diagnostic
methods based on detection of decreased levels of
undercarboxylated/uncarboxylated osteocalcin, which is found to be
associated with metabolic syndrome, type 1 and type 2 diabetes,
atherosclerosis and obesity. In one aspect, the method of
diagnosing a disease, including metabolic syndrome, diabetes types
1 and 2, atherosclerosis and obesity, in a patient comprises (i)
determining a patient level of undercarboxylated/uncarboxylated
osteocalcin in a biological sample taken from the patient (ii)
comparing the patient level of undercarboxylated/uncarboxylated
osteocalcin and a control level of undercarboxylated/uncarboxylated
osteocalcin, and (iii) if the patient level is significantly lower
than the control level, then the patient is diagnosed as having, or
being at risk for, the disease, including metabolic syndrome,
diabetes types 1 and 2, atherosclerosis and obesity.
[0102] Other aspects of the invention are directed to diagnostic
methods based on detection of decreased ratios of
undercarboxylated/uncarboxylated vs carboxylated osteocalcin. Such
ratios are found to be associated with metabolic syndrome, type 1
and type 2 diabetes, atherosclerosis and obesity. In one aspect,
the method of diagnosing a disease, including metabolic syndrome,
diabetes types 1 and 2, atherosclerosis and obesity, in a patient
comprises (i) determining a patient ratio of
undercarboxylated/uncarboxylated vs carboxylated osteocalcin in a
biological sample taken from the patient (ii) comparing the patient
ratio of undercarboxylated/uncarboxylated vs carboxylated
osteocalcin and a control ratio of undercarboxylated/uncarboxylated
vs carboxylated osteocalcin, and (iii) if the patient ratio is
significantly lower than the control ratio, then the patient is
diagnosed has having, or being at risk for, the disease, including
metabolic syndrome, diabetes types 1 and 2, atherosclerosis and
obesity.
[0103] Other aspects of the invention are directed to methods for
diagnosing a patient at risk of developing a disease, including
metabolic syndrome, glucose intolerance, impaired pancreatic
beta-cell proliferation, impaired insulin secretion, impaired
insulin sensitivity, atherosclerosis and obesity, by determining
the patient level of, or level of activity of, OST-PTP and/or
gamma-carboxylase, wherein increases in said levels, as compared to
controls, indicates a patient is at risk of developing the
disease.
PHARMACEUTICAL COMPOSITIONS OF THE INVENTION
[0104] The present invention provides pharmaceutical compositions
comprising an agent for modulating the OST-PTP signaling pathway,
which as disclosed herein involves gamma-carboxylase and
osteocalcin, or for treating or preventing disorders related to the
OST-PTP signaling pathway. In particular embodiments, the agent
inhibits OST-PTP phosphorylase activity, reduces gamma-carboxylase
activity, and/or increases undercarboxylated/uncarboxylated
osteocalcin. In particular embodiments, the agent decarboxylates
osteocalcin. The agent may be selected from the group consisting of
small molecules, polypeptides, antibodies, and nucleic acids. The
pharmaceutical compositions of the invention provide an amount of
the agent effective to treat or prevent a disorder associated with
the OST-PTP signaling pathway. In certain embodiments, a
pharmaceutical composition provides an amount of the agent
effective to treat or prevent metabolic syndrome or a component
thereof, diabetes type 1, diabetes type 2, atherosclerosis, or
obesity in a subject. In other embodiments, the composition
provides an amount of the agent effective to treat or prevent a
disease characterized by decreased insulin production, decreased
insulin sensitivity, and decreased glucose tolerance or increased
fat mass
[0105] The pharmaceutical compositions of the invention may
function to increase serum osteocalcin levels (preferably
undercarboxylated or uncarboxylated osteocalcin), serum adiponectin
levels and/or serum insulin levels. The pharmaceutical compositions
may also increase glucose tolerance, increase insulin sensitivity,
and/or increase pancreatic beta-cell proliferation. Other
beneficial effects may include a reduction of oxidized
phospholipids, a regression of atherosclerotic plaques, a decrease
in inflammatory protein biosynthesis, a reduction in plasma
cholesterol, a reduction in vascular smooth muscle cell (VSMC)
proliferation and number, a decrease in the thickness of arterial
plaque, a reduction in clinical events such as heart attack,
angina, or stroke, and a decrease in hypertension.
[0106] In particular embodiments of the invention, therapeutic
agents that may be administered include undercarboxylated
osteocalcin; uncarboxylated osteocalcin; or inhibitors that reduce
the expression or activity of gamma-carboxylase or OST-PTP (e.g.,
antibodies, small molecules, antisense nucleic acids or siRNA). The
pharmaceutical agents may also include agents that decarboxylate
osteocalcin.
[0107] The therapeutic agents are generally administered in an
amount sufficient to treat or prevent metabolic syndrome, obesity,
diabetes type 1 and 2 and atherosclerosis in a subject. The
therapeutic agents may also be administered to reduce fat mass in a
subject.
[0108] Biologically active fragments or variants of the therapeutic
agents are also within the scope of the present invention. By
"biologically active" is meant capable of modulating the OST-PTP
signaling pathway involving gamma-carboxylase, osteocalcin and
adiponectin. As described herein, "biologically active" means
reducing the expression of OST-PTP or its ability to
dephosphorylate gamma-carboxylase and reducing the expression of
gamma-carboxylase or its ability to carboxylate osteocalcin, or
decarboxylating carboxylated osteocalcin thereby leading to
increased levels of undercarboxylated/uncarboxylated osteocalcin,
insulin and adiponectin. "Biologically active" also means capable
of causing at least one effect selected from the group consisting
of increasing pancreatic beta-cell proliferation, increasing
insulin secretion, increasing insulin sensitivity, increasing
glucose tolerance, decreasing weight gain, decreasing fat mass,
weight loss, increasing serum adiponectin, a reduction of oxidized
phospholipids, a regression of atherosclerotic plaques, a decrease
in inflammatory protein biosynthesis, a reduction in plasma
cholesterol, a reduction in vascular smooth muscle cell (VSMC)
proliferation and number, a decrease in the thickness of arterial
plaque, a reduction in clinical events such as heart attack,
angina, or stroke, and a decrease in hypertension. Fragments and
variants are described below. The screens or assays described below
may be used to identify, or assay, biologically active fragments
and variants of the therapeutic agents of the invention, as well as
other agents.
Compositions Comprising Undercarboxylated Osteocalan
[0109] In a specific embodiment of the invention, pharmaceutical
compositions comprising osteocalcin, particularly undercarboxylated
or uncarboxylated osteocalcin, are provided.
[0110] Osteocalcin, one of the very few osteoblast-specific
proteins, has several features of a hormone. For instance, it is
synthesized as a pre-pro-molecule and is secreted in the general
circulation (Hauschka et al., 1989; Price, 1989). Because of their
exquisite cell-specific expression, the osteocalcin genes have been
intensively studied to identify osteoblast-specific transcription
factors and to define the molecular bases of bone physiology (Ducy
et al., 2000b; Harada and Rodan, 2003).
[0111] Osteocalcin is the most abundant non-collagenous protein
found associated with the mineralized bone matrix and it is
currently being used as a biological marker for clinical assessment
of bone turnover. Osteocalcin is a small (46-50 residue) bone
specific protein that contains 3 gamma-carboxylated glutamic acid
residues in its primary structure. The name osteocalcin (osteo,
Greek for bone; Calc, Latin for lime salts; in, protein) derives
from the protein's ability to bind Ca.sup.2+ and its abundance in
bone. Osteocalcin undergoes a peculiar post-translational
modification whereby glutamic acid residues are carboxylated to
form gamma-carboxyglutamic acid (Gla) residues; hence osteocalcin's
other name, bone Gla protein (Hauschka et al., 1989). Gla residues
usually confer on proteins high affinity for mineral ions, yet
loss- and gain--of function experiments until now have failed to
identify a function for osteocalcin in extracellular matrix
mineralization in vivo (Ducy et al., 1996; Murshed et al.,
2004).
[0112] Osteocalcin is a vitamin K-dependent calcium binding protein
(Price et al. (1976) Proc. Natl. Acad. Sci. 73:3373-375). Mature
human osteocalcin contains 49 amino acids with a predicted
molecular mass of 5,800 kDa (Poser et al. (1980) The Journal of
Biological Chemistry, Vol 255, No. 18, pp. 8685-8691). Osteocalcin
is synthesized primarily by osteoblasts and ondontoblasts and
comprises 15 to 20% of the non-collagenous protein of bone. Poser
et al. (1980) J. Biol. Chem. 255:8685-8691 showed that mature
osteocalcin contains three carboxyglutamic acid residues which are
formed by post-translational vitamin K-dependent modification of
glutamic acid residues. The carboxylated Gla residues are at
positions 17, 21 and 24 of mouse and human mature osteocalcin. Some
human osteocalcin has been shown to contain only 2 Gla residues.
Poser, J. W. & Price, P. A. (1979) A Method for Decarboxylation
of .gamma.-Carboxyglutamic Acid in Proteins. J. Biol. Chem. 254,
431-436.
[0113] The conformation of decarboxylated (or uncarboxylated or
undercarboxylated) osteocalcin lies somewhere between the random
coil and helical form. Thus, in solution the peptide occurs as a
flexible structure and a single conformation cannot be defined for
it (Atkinson et al. Eur. J. Biochem. 1995; 232:515-21). Peptide
bonds between arginine residues 19 and 20 and between residues 43
and 44 are susceptible to tryptic hydrolysis, leading to peptides
1-19, 20-43, 45-49, 1-43, and 20-49 which may be the main products
of human osteocalcin breakdown in the circulation (Farrugia and
Melick, Calcif Tissue Int 1986; 39:234-8, Hellman et al. J Bone
Miner Res 1996; 11:1165-75 and Garnero et al. J Bone Miner Res
1994; 9:255-4).
[0114] Conformational study of osteocalcin by circular dichroism
(CD) has shown the existence of alpha-helical conformation in
osteocalcin and that addition of Ca.sup.2+ induces higher helical
content. Two-dimensional nuclear magnetic resonance (NMR) studies
of osteocalcin in solution, while structurally inconclusive,
revealed that the calcium-free protein was effectively unstructured
except for the turn required by the disulfide bridge between Cys23
and Cys29. All the proline residues (Hyp9, Pro11, Pro13, Pro15, and
Pro27) were in the trans conformation. Beta-turns are present in
the region of Tyr12, Asp14 and Asn26. The hydrophobic core of the
molecule is composed of the side chains of Leu2, Leu32, Va136 and
Tyr42. The calcium-induced helix is extremely rigid due to, in
part, the hydrophobic stabilization of the helical domain by the
C-terminal domain.
[0115] Osteocalcin in solution binds Ca.sup.2+ with a dissociation
constant ranging from 0.5 to 3 mM, with a stoichiometry of between
2 and 5 mol Ca.sup.2+/mol protein, and to hydroxyapatite (Kd.
approximately equal to 10.sup.-7 M). It appears that the Gla
residues in osteocalcin are important for its affinity toward
Ca.sup.2+. Binding of Ca.sup.2+ induces normal osteocalcin to adopt
the alpha-helical conformation; however, thermally decarboxylated
osteocalcin showed higher alpha-helical content than normal
osteocalcin and the calcium induced alpha-helical formation was
lost. Decarboxylated osteocalcin also lost its specific binding to
hydroxyapatite, which is consistent with the results showing that
uncarboxylated osteocalcin is the secreted bone hormone. When bound
to hydroxyapatite, the Gla residues are protected from thermal
decarboxylation. Furthermore, osteocalcin synthesized in animals
treated with warfarin, which inhibits the formation of Gla, failed
to bind to bone. Furthermore, hydroxyapatite competition studies
demonstrated that prothrombin (10 Gla/molecule) and decarboxylated
osteocalcin fail to compete with .sup.125I-labeled osteocalcin
bound to hydroxyapatite. Combining all the information discussed
above, a structural model has been constructed. This model consists
of two antiparallel alpha-helical domains. The Gla residues are
spaced about 5.4 angstroms apart on one of the helices, which is
similar to the interatomic lattice spacing of Ca.sup.2+ in the x-y
plane of hydroxyapatite. It was therefore predicted that the Gla
residues in osteocalcin bind to the (001) plane of the
hydroxyapatite lattice.
[0116] "Osteocalcin" also known as Bone Gla Protein or BGP, refers
to a small vitamin K-dependent calcium binding protein (Price et
al. (1976) Proc. Natl. Acad. Sci. 73:3373-5) which is highly
conserved among animal species. "Osteocalcin" includes both
carboxylated, uncarboxylated and undercarboxylated forms as well as
fragments and variants thereof as described herein.
[0117] "Undercarboxylated osteocalcin" means osteocalcin in which
one or more of the Glu residues at positions Glu17, Glu21 and Glu24
of the amino acid sequence of the mature human osteocalcin having
49 amino acids, or at the positions corresponding to Glu17, Glu21
and Glu24 in other forms of osteocalcin, are not carboxylated.
Undercarboxylated osteocalcin includes uncarboxylated osteocalcin,
i.e., osteocalcin in which all three of the glutamic acid residues
at positions 17, 21, and 24 are not carboxylated. Recombinant
osteocalcin expressed in bacteria is uncarboxylated because
bacteria do not have gamma-carboxylase. Preparations of osteocalcin
are considered to be "undercarboxylated osteocalcin" if more than
about 10% of the total Glu residues at positions Glu17, Glu21 and
Glu24 (taken together) in mature osteocalcin (or the corresponding
Glu residues in other forms) of the preparation are not
carboxylated. In particular preparations of undercarboxylated
osteocalcin, more than about 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, or 100% of the total Glu residues at positions Glu17,
Glu21 and Glu24 in mature osteocalcin (or the corresponding Glu
residues in other forms) of the preparation are not carboxylated.
In particularly preferred embodiments, essentially all of the Glu
residues at positions Glu17, Glu21 and Glu24 in mature osteocalcin
(or the corresponding Glu residues in other forms) of the
preparation are not carboxylated.
[0118] Human osteocalcin cDNA (SEQ ID NO:1) encodes a mature
osteocalcin protein represented by the last 49 amino acids of SEQ
ID NO:2 (i.e., positions 52-100) with a predicted molecular mass of
5,800 kDa (Poser et al., (1980) The Journal of Biological
Chemistry, Vol 255, No. 18, pp. 8685-8691). SEQ ID NO:2 is the
pre-pro-sequence of human osteocalcin and mature human osteocalcin
is the processed product of SEQ ID NO:2. In this application, the
amino acid positions of mature human osteocalcin are referred to.
It will be understood that the amino acid positions of mature human
osteocalcin correspond to those of SEQ ID NO:2 as follows: position
1 of mature human osteocalcin corresponds to position 52 of SEQ ID
NO:2; position 2 of mature human osteocalcin corresponds to
position 53 of SEQ ID NO:2, etc. In particular, positions 17, 21,
and 24 of mature human osteocalcin correspond to positions 68, 72,
and 75, respectively, of SEQ ID NO:2.
[0119] When positions in two amino acid sequences correspond, it is
meant that the two positions align with each other when the two
amino acid sequences are aligned with one another to provide
maximum homology between them. This same concept of correspondence
also applies to nucleic acids.
[0120] For example, in the two amino acid sequences AGLYSTVLMGRPS
and GLVSTVLMGN, positions 2-11 of the first sequence correspond to
positions 1-10 of the second sequence, respectively. Thus, position
2 of the first sequence corresponds to position 1 of the second
sequence; position 4 of the first sequence corresponds to position
3 of the second sequence; etc. It should be noted that a position
in one sequence may correspond to a position in another sequence,
even if the positions in the two sequence are not occupied by the
same amino acid.
[0121] Osteocalcin is synthesized primarily by osteoblasts and
ondontoblasts. "Osteocalcin" includes the mature protein and
further includes biologically active fragments derived from
full-length osteocalcin (SEQ ID NO:2) or the mature protein,
including various domains, as well as variants as described
herein.
[0122] In an embodiment of the invention, the pharmaceutical
compositions of the invention comprise a mammalian uncarboxylated
osteocalcin. In a preferred embodiment of the invention, the
compositions of the invention comprise human osteocalcin having the
amino acid sequence of SEQ ID NO:2, or portions thereof, and
encoded for by the nucleic acid of SEQ ID NO:1, or portions
thereof, or the compositions of the invention may comprise one or
more of the human osteocalcin fragments described herein.
[0123] In a specific embodiment, the present invention provides
pharmaceutical compositions comprising human undercarboxylated
osteocalcin which does not contain a carboxylated glutamic acid at
one or more of positions corresponding to positions 17, 21 and 24
of mature human osteocalcin. A preferred form of osteocalcin for
use in the present invention is mature human osteocalcin wherein at
least one of the glutamic acid residues at positions 17, 21, and 24
is not carboxylated. Preferably, all three of the glutamic acid
residues at positions 17, 21, and 24 are not carboxylated. The
amino acid sequence of mature human osteocalcin is shown in SEQ.
ID. NO: 25.
[0124] The invention also includes the use of polypeptide fragments
of osteocalcin. Fragments can be derived from the full-length,
naturally occurring amino acid sequence of osteocalcin (e.g., SEQ.
ID. NO:2). Fragments may also be derived from mature osteocalcin.
The invention also encompasses fragments of the variants of
osteocalcin described herein. A fragment can comprise an amino acid
sequence of any length that is biologically active.
[0125] Preferred fragments of osteocalcin include fragments
containing Glu17, Glu21 and Glu24 of the mature protein. Also
preferred are fragments of the mature protein missing the last 10
amino acids from the C-terminal end of the mature protein. Also
preferred are fragments missing the first 10 amino acids from the
N-terminal end of the mature protein. Also preferred is a fragment
of the mature protein missing both the last 10 amino acids from the
C-terminal end and the first 10 amino acids from the N-terminal
end. Such a fragment comprises amino acids 62-90 of SEQ ID
NO:2.
[0126] Other preferred fragments of osteocalcin for the
pharmaceutical compositions of the invention described herein
include polypeptides comprising, consisting of, or consisting
essentially of, the following sequences of amino acids:
[0127] positions 1-19 of mature human osteocalcin
[0128] positions 20-43 of mature human osteocalcin
[0129] positions 20-49 of mature human osteocalcin
[0130] positions 1-43 of mature human osteocalcin
[0131] positions 1-42 of mature human osteocalcin
[0132] positions 1-41 of mature human osteocalcin
[0133] positions 1-40 of mature human osteocalcin
[0134] positions 1-39 of mature human osteocalcin
[0135] positions 1-38 of mature human osteocalcin
[0136] positions 1-37 of mature human osteocalcin
[0137] positions 1-36 of mature human osteocalcin
[0138] positions 1-35 of mature human osteocalcin
[0139] positions 1-34 of mature human osteocalcin
[0140] positions 1-33 of mature human osteocalcin
[0141] positions 1-32 of mature human osteocalcin
[0142] positions 1-31 of mature human osteocalcin
[0143] positions 1-30 of mature human osteocalcin
[0144] positions 1-29 of mature human osteocalcin
[0145] positions 2-49 of mature human osteocalcin
[0146] positions 2-45 of mature human osteocalcin
[0147] positions 2-40 of mature human osteocalcin
[0148] positions 2-35 of mature human osteocalcin
[0149] positions 2-30 of mature human osteocalcin
[0150] positions 2-25 of mature human osteocalcin
[0151] positions 2-20 of mature human osteocalcin
[0152] positions 4-49 of mature human osteocalcin
[0153] positions 4-45 of mature human osteocalcin
[0154] positions 4-40 of mature human osteocalcin
[0155] positions 4-35 of mature human osteocalcin
[0156] positions 4-30 of mature human osteocalcin
[0157] positions 4-25 of mature human osteocalcin
[0158] positions 4-20 of mature human osteocalcin
[0159] positions 8-49 of mature human osteocalcin
[0160] positions 8-45 of mature human osteocalcin
[0161] positions 8-40 of mature human osteocalcin
[0162] positions 8-35 of mature human osteocalcin
[0163] positions 8-30 of mature human osteocalcin
[0164] positions 8-25 of mature human osteocalcin
[0165] positions 8-20 of mature human osteocalcin
[0166] positions 10-49 of mature human osteocalcin
[0167] positions 10-45 of mature human osteocalcin
[0168] positions 10-40 of mature human osteocalcin
[0169] positions 10-35 of mature human osteocalcin
[0170] positions 10-30 of mature human osteocalcin
[0171] positions 10-25 of mature human osteocalcin
[0172] positions 10-20 of mature human osteocalcin
[0173] positions 7-30 of mature human osteocalcin
[0174] positions 7-25 of mature human osteocalcin
[0175] positions 7-23 of mature human osteocalcin
[0176] positions 7-21 of mature human osteocalcin
[0177] positions 7-19 of mature human osteocalcin
[0178] positions 7-17 of mature human osteocalcin
[0179] positions 8-30 of mature human osteocalcin
[0180] positions 8-25 of mature human osteocalcin
[0181] positions 8-23 of mature human osteocalcin
[0182] positions 8-21 of mature human osteocalcin
[0183] positions 8-19 of mature human osteocalcin
[0184] positions 8-17 of mature human osteocalcin
[0185] positions 9-30 of mature human osteocalcin
[0186] positions 9-25 of mature human osteocalcin
[0187] positions 9-23 of mature human osteocalcin
[0188] positions 9-21 of mature human osteocalcin
[0189] positions 9-19 of mature human osteocalcin
[0190] positions 9-17 of mature human osteocalcin
[0191] Especially preferred is a fragment comprising positions 1-36
of mature human osteocalcin. Another preferred fragment is a
fragment comprising positions 20-49 of mature human osteocalcin.
Other fragments can be designed to contain Pro13 to Tyr76 or Pro 13
to Asn26 of mature human osteocalcin. Additionally, fragments
containing the cysteine residues at positions 23 and 29 of mature
human osteocalcin, and capable of forming a disulfide bond between
those two cysteines, are useful.
[0192] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment, a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the osteocalcin fragment and/or an
additional region fused to the carboxyl terminus of the
fragment.
[0193] Also provided for use in the compositions and methods of the
present invention are variants of the osteocalcin and osteocalcin
fragments described above. "Variants" refers to osteocalcin
peptides that contain modifications in their amino acid sequences
such as one or more amino acid substitutions, additions, deletions
and/or insertions but that are still biologically active. In some
instances, the antigenic and/or immunogenic properties of the
variants are not substantially altered, relative to the
corresponding peptide from which the variant was derived. Such
modifications may be readily introduced using standard mutagenesis
techniques, such as oligonucleotide directed site-specific
mutagenesis as taught, for example, by Adelman et al. (DNA, 2:183,
1983) or by chemical synthesis. Variants and fragments are not
mutally exclusive terms. Fragments also include peptides that may
contain one or more amino acid substitutions, additions, deletions
and/or insertions such that the fragments are still biologically
active.
[0194] One particular type of variant that is within the scope of
the present invention is a variant in which one of more of the
positions corresponding to positions 17, 21 and 24 of mature human
osteocalcin is occupied by an amino acid that is not glutamic acid.
In some embodiments, the amino acid that is not glutamic acid is
also not aspartic acid. Such variants are versions of
undercarboxylated osteocalcin because at least one of the three
positions corresponding to positions 17, 21 and 24 of mature human
osteocalcin is not carboxylated glutamic acid, since at least one
of those positions is not occupied by glutamic acid.
[0195] In particular embodiments, the present invention provides
osteocalcin variants comprising the amino acid sequence
TABLE-US-00002 (SEQ. ID. NO: 23) YLYQWLGAPV PYPDPLX.sub.1PRR
X.sub.2VCX.sub.3LNPDCD ELADHIGFQE AYRRFYGPV
wherein [0196] X.sub.1, X.sub.2 and X.sub.3 are each independently
selected from an amino acid or amino acid analog, with the proviso
that if X.sub.1, X.sub.2 and X.sub.3 are each glutamic acid, then
X.sub.1 is not carboxylated, or less than 50 percent of X.sub.2 is
carboxylated, and/or less than 50 percent of X.sub.3 is
carboxylated.
[0197] In certain embodiments, the osteocalcin variants comprise an
amino acid sequence that is different from SEQ. ID. NO: 23 at 1 to
7 positions other than X.sub.1, X.sub.2 and X.sub.3.
[0198] In other embodiments, the osteocalcin variants comprise an
amino acid sequence that includes one or more amide backbone
substitutions.
[0199] Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitutions of similar amino acids, which results in no change,
or an insignificant change, in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree. The activity of such functional osteocalcin variants can be
determined using assays such as those described herein.
[0200] Variants can be naturally-occurring or can be made by
recombinant means, or chemical synthesis, to provide useful and
novel characteristics for undercarboxylated/uncarboxylated
osteocalcin. For example, the variant osteocalcin polypeptides may
have reduced immunogenicity, increased serum half-life, increase
bioavailability and/or increased potency. In particular
embodiments, serum half-life is increased by substituting one or
more of the native Arg residues at positions 19, 20, 43, and 44 of
mature osteocalcin with another amino acid or an amino acid analog,
e.g., .beta.-dimethyl-arginine. Such substitutions can be combined
with the other changes in the native amino acid sequence of
osteocalcin described herein.
[0201] Provided for use in the pharmaceutical compositions and
methods of the present invention are variants that are also
derivatives of the osteocalcin and osteocalcin fragments described
above. Derivatization is a technique used in chemistry which
transforms a chemical compound into a product of similar chemical
structure, called derivative. Generally, a specific functional
group of the compound participates in the derivatization reaction
and transforms the educt to a derivate of deviating reactivity,
solubility, boiling point, melting point, aggregate state,
functional activity, or chemical composition. Resulting new
chemical properties can be used for quantification or separation of
the educt or can be used to optimize the compound as a therapeutic
agent. The well-known techniques for derivatization can be applied
to the above-described osteocalcin and osteocalcin fragments. Thus,
derivatives of the osteocalcin and osteocalcin fragments described
above will contain amino acids that have been chemically modified
in some way so that they differ from the natural amino acids.
[0202] Provided also are osteocalcin mimetics. "Mimetic" refers to
a synthetic chemical compound that has substantially the same
structural and functional characteristics of a naturally or
non-naturally occurring polypeptide, and includes, for instance,
polypeptide- and polynucleotide-like polymers having modified
backbones, side chains, and/or bases. Peptide mimetics are commonly
used in the pharmaceutical industry as non-peptide drugs with
properties analogous to those of the template peptide. Generally,
mimetics are structurally similar (i.e., have the same shape) to a
paradigm polypeptide that has a biological or pharmacological
activity, but one or more polypeptide linkages are replaced. The
mimetic can be either entirely composed of synthetic, non-natural
analogues of amino acids, or, is a chimeric molecule of partly
natural peptide amino acids and partly non-natural analogs of amino
acids. The mimetic can also incorporate any amount of natural amino
acid conservative substitutions as long as such substitutions also
do not substantially alter the mimetic's structure and/or
activity.
[0203] By way of example, example, Cho et al., 1993, Science
261:1303-5 discloses an "unnatural biopolymer" consisting of chiral
aminocarbonate monomers substituted with a variety of side chains,
synthesis of a library of such polymers, and screening for binding
affinity to a monoclonal antibody. Similarly, Cho et al, 1998, J.
Am. Chem. Soc. discloses libraries of linear and cyclic
oligocarbamate libraries and screening for binding to the integrin
GPIIb/IIIa. Simon et al., Proc. Natl. Acad. Sci. 89:9367-71
discloses a polymer consisting of N-substituted glycines
("peptoids") with diverse side chains. Schumacher et al, 1996,
Science 271:1854-7 discloses D-peptide ligands specific for Src
homology domain 3 (SH3 domain) by screening phage libraries of
L-peptides against a proteins (SH3) synthesized with D-amino acids
and then synthesizing a selected L-peptide using D-amino acids.
Brody et al., 1999, Mol. Diagn. 4: 381-8 describes generation and
screeing of hundreds to thousands of aptamers.
[0204] A particular type of osteocalcin variant within the scope of
the invention is an osteocalcin mimetic in which one or more
backbone amides is replaced by a different chemical structure or in
which one or more amino acids are replaced by an amino acid analog.
In a particular embodiment, the osteocalcin mimetic is a
retroenantiomer of uncarboxylated human osteocalcin.
[0205] Osteocalcin, as well as its fragments and variants, is
optionally produced by chemical synthesis or recombinant methods
and may be produced as a modified osteocalcin molecule (i.e.,
osteocalcin fragments or variants) as described herein. Osteocalcin
polypeptides can be produced by any conventional means (Houghten,
R. A. (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135). Simultaneous
multiple peptide synthesis is described in U.S. Pat. No. 4,631,211
and can also be used. When produced recombinantly, osteocalcin may
be produced as a fusion protein, e.g., a GST-osteocalcin fusion
protein.
[0206] Undercarboxylated/uncarboxylated osteocalcin molecules that
fall within the scope of the invention include proteins
substantially homologous to human osteocalcin including proteins
derived from another organism, i.e., an ortholog. One particular
ortholog is mouse osteocalcin. Mouse osteocalcin gene 1 cDNA is SEQ
ID NO:3; mouse osteocalcin gene 2 cDNA is SEQ ID NO:4; the amino
acid sequence of mouse osteocalcin gene 1 and gene 2 is SEQ ID
NO:5.
[0207] As used herein, two proteins are substantially homologous,
or identical, when their amino acid sequences are at least about
70-75%, typically at least about 80-85%, and most typically at
least about 90-95%, 97%, 98% or 99% or more homologous. "Homology"
between two amino acid sequences or nucleic acid sequences can be
determined by using the alogorithms disclosed herein. These
algorithms can also be used to determine percent identity between
two amino acid sequences or nucleic acid sequences.
[0208] In a specific embodiment of the invention, the
undercarboxylated/uncarboxylated osteocalcin is a osteocalcin
molecule sharing at least 80% homology with the human osteocalcin
of SEQ ID:2 or a portion of SEQ ID:2 that is at least 8 amino acids
long. In another embodiment of the invention, the
undercarboxylated/uncarboxylated osteocalcin is a osteocalcin
molecule sharing at least 80% amino acid sequence identity with the
human osteocalcin of SEQ ID:2 or a portion of SEQ ID:2 that is at
least 8 amino acids long. Homologous sequences include those
sequences that are substantially identical. In preferred
embodiments, the homology or identity is over the entire length of
mature human osteocalcin.
[0209] To determine the percent homology or percent identity of two
amino acid sequences or of two nucleic acid sequences, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in one or both of a first and a second amino acid
or nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). Preferably,
the length of a reference sequence aligned for comparison purposes
is at least 30%, preferably at least 40%, more preferably at least
50%, even more preferably at least 60%, and even more preferably at
least 70%, 80%, or 90% or more of the length of the sequence that
the reference sequence is compared to. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position. The percent identity between the
two sequences is a function of the number of identical positions
shared by the sequences, taking into account the number of gaps,
and the length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0210] The invention also encompasses polypeptides having a lower
degree of identity but which have sufficient similarity so as to
perform one or more of the same functions performed by
undercarboxylated/uncarboxylated osteocalcin. Similarity is
determined by considering conserved amino acid substitutions. Such
substitutions are those that substitute a given amino acid in a
polypeptide by another amino acid of like characteristics.
Conservative substitutions are likely to be phenotypically silent.
Guidance concerning which amino acid changes are likely to be
phenotypically silent is found in Bowie et al., Science
247:1306-1310 (1990).
[0211] Examples of conservative substitutions are the replacements,
one for another, among the hydrophobic amino acids Ala, Val, Leu,
and Ile; interchange of the hydroxyl residues Ser and Thr; exchange
of the acidic residues Asp and Glu; substitution between the amide
residues Asn and Gln; exchange of the basic residues Lys, His and
Arg; replacements among the aromatic residues Phe, Trp and Tyr;
exchange of the polar residues Gln and Asn; and exchange of the
small residues Ala, Ser, Thr, Met, and Gly.
[0212] The comparison of sequences and determination of percent
identity and homology between two osteocalcin polypeptides can be
accomplished using a mathematical algorithm. For example,
Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and
Griffin, H G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, van Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991. A non-limiting example of
such a mathematical algorithm is described in Karlin et al. (1993)
Proc. Natl. Acad. Sci. USA 90:5873-5877.
[0213] The percent identity or homology between two osteocalcin
amino acid sequences may be determined using the Needleman et al.
(1970) (.I Mol. Biol. 48:444-453) algorithm. Another non-limiting
example of a mathematical algorithm that may be utilized for the
comparison of sequences is the algorithm of Myers and Miller,
CABIOS (1989).
[0214] A substantially homologous osteocalcin, according to the
present invention, may also be a polypeptide encoded by a nucleic
acid sequence capable of hybridizing to the human osteocalcin
nucleic acid sequence under highly stringent conditions, e.g.,
hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% sodium
dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel F.M. et al., eds.,
1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing Associates, Inc., and John Wiley & sons, Inc., New
York, at p. 2.10.3) and encoding a functionally equivalent gene
product; or under less stringent conditions, such as moderately
stringent conditions, e.g., washing in 0.2.times.SSC/0.1% SDS at
42.degree. C. (Ausubel et al., 1989 supra), yet which still encodes
a biologically active undercarboxylated/uncarboxylated
osteocalcin.
[0215] A substantially homologous osteocalcin, according to the
present invention, may also be a polypeptide encoded by a nucleic
acid sequence capable of hybridizing to a sequence having at least
70-75%, typically at least about 80-85%, and most typically at
least about 90-95%, 97%, 98% or 99% identity to the human
osteocalcin nucleic acid sequence, under stringent conditions,
e.g., hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7%
sodium dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and
washing in 0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel F.M. et
al., eds., 1989, Current Protocols in Molecular Biology, Vol. I,
Green Publishing Associates, Inc., and John Wiley & sons, Inc.,
New York, at p. 2.10.3) and encoding a functionally equivalent gene
product; or under less stringent conditions, such as moderately
stringent conditions, e.g., washing in 0.2.times.SSC/0.1% SDS at
42.degree. C. (Ausubel et al., 1989 supra), yet which still encodes
a biologically active undercarboxylated/uncarboxylated
osteocalcin.
[0216] It will be understood that a biologically active fragment or
variant of human osteocalcin may contain a different number of
amino acids than native human osteocalcin. Accordingly, the
position number of the amino acid residues corresponding to
positions 17, 21 and 24 of mature human osteocalcin may differ in
the fragment or variant. One skilled in the art would easily
recognize such corresponding positions from a comparison of the
amino acid sequence of the fragment or variant with the amino acid
sequence of mature human osteocalcin.
[0217] Peptides corresponding to fusion proteins in which full
length osteocalcin, mature osteocalcin, or an osteocalcin fragment
or variant is fused to an unrelated protein or polypeptide are also
within the scope of the invention and can be designed on the basis
of the osteocalcin nucleotide and amino acid sequences disclosed
herein. Such fusion proteins include fusions to an enzyme,
fluorescent protein, or luminescent protein which provides a marker
function. In a preferred embodiment of the invention, the fusion
protein comprises fusion to a polypeptide capable of targeting the
osteocalcin to a particular target cell or location in the body.
For example, osteocalcin polypeptide sequences may be fused to a
ligand molecule capable of targeting the fusion protein to a cell
expressing the receptor for said ligand. Osteocalcin can also be
made as part of a chimeric protein for drug screening or use in
making recombinant protein. These comprise an osteocalcin peptide
sequence operatively linked to a heterologous peptide having an
amino acid sequence not substantially homologous to the
osteocalcin. "Operatively linked" in this context indicates that
the osteocalcin peptide and the heterologous peptide are fused
in-frame. The heterologous peptide can be fused to the N-terminus
or C-terminus of osteocalcin or can be internally located. In one
embodiment, the fusion protein does not affect osteocalcin
function. For example, the fusion protein can be a GST-fusion
protein in which the osteocalcin sequences are fused to the N- or
C-terminus of the GST sequences. Other types of fusion proteins
include, but are not limited to, enzymatic fusion proteins, for
example beta-galactosidase fusions, yeast two-hybrid GAL-4 fusions,
poly-His fusions and Ig fusions. Such fusion proteins, particularly
poly-His fusions, can facilitate the purification of recombinant
osteocalcin. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a protein can be increased by using
a heterologous signal sequence. Therefore, the fusion protein may
contain a heterologous signal sequence at its N-terminus.
[0218] EP-A 0 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions (Fc regions). The Fc
region is useful in therapy and diagnosis and thus results, for
example, in improved pharmacokinetic properties (EP-A 0 232 262).
In drug discovery, for example, human proteins have been fused with
Fc regions for the purpose of high-throughput screening assays to
identify antagonists (Bennett et al. (1995) J. Mol. Recog. 8:52-58
(1995) and Johanson et al. J. Biol. Chem. 270:9459-9471). Thus,
various embodiments of this invention also utilize soluble fusion
proteins containing an osteocalcin polypeptide and various portions
of the constant regions of heavy or light chains of immunoglobulins
of various subclasses (e.g., IgG, IgM, IgA, IgE, IgB). Preferred as
immunoglobulin is the constant part of the heavy chain of human
IgG, particularly IgG1, where fusion takes place at the hinge
region. For some uses, it is desirable to remove the Fc region
after the fusion protein has been used for its intended purpose,
e.g., when the fusion protein is to be used as antigen for
immunizations. In a particular embodiment, the Fc part can be
removed in a simple way by a cleavage sequence, which is also
incorporated and can be cleaved, e.g., with factor Xa.
[0219] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences can be ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). An osteocalcin-encoding nucleic acid
can be cloned into such an expression vector such that the fusion
moiety is linked in-frame to osteocalcin.
[0220] Chimeric osteocalcin proteins can be produced in which one
or more functional sites are derived from a different isoform, or
from another osteocalcin molecule from another species. Sites also
could be derived from osteocalcin-related proteins that occur in
the mammalian genome but which have not yet been discovered or
characterized.
[0221] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally-occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in polypeptides are described
below.
[0222] Accordingly, the osteocalcin polypeptides of the present
invention also encompass derivatives which contain a substituted
amino acid residue that is not one encoded by the genetic code, in
which a substituent group is included, in which the mature
polypeptide is fused with another compound, such as a compound to
increase the half-life of the polypeptide (for example,
polyethylene glycol), or in which the additional amino acids are
fused to the osteocalcin polypeptide, such as a leader or secretory
sequence or a sequence for purification of the osteocalcin
polypeptide or a pro-protein sequence.
[0223] Undercarboxylated/uncarboxylated osteocalcin can be modified
according to known methods in medicinal chemistry to increase its
stability, half-life, uptake or efficacy. Known modifications
include, but are not limited to, acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphatidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent crosslinks, formation of
cystine, formation of pyroglutamate, formylation, glycosylation,
GPI anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, proteolytic processing, phosphorylation,
prenylation, racemization, selenoylation, sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation,
and ubiquitination.
[0224] In a specific embodiment of the invention, modifications may
be made to the osteocalcin to reduce susceptibilty to proteolysis
at residue ARG43 as a means for increasing serum half life. Such
modifications include, for example, the use of retroenantio
isomers, D-amino acids, or other amino acid analogs.
[0225] Acylation of the N-terminal amino group can be accomplished
using a hydrophilic compound, such as hydroorotic acid or the like,
or by reaction with a suitable isocyanate, such as methylisocyanate
or isopropylisocyanate, to create a urea moiety at the N-terminus.
Other agents can also be N-terminally linked that will increase the
duration of action of the osteocalcin derivative as known in this
art.
[0226] Reductive amination is the process by which ammonia is
condensed with aldehydes or ketones to form imines which are
subsequently reduced to amines. Reductive amination is a useful
method for conjugating undercarboxylated/uncarboxylated osteocalcin
and its fragments or variants to PEG. Covalent linkage of
poly(ethylene glycol) (PEG) to undercarboxylated/uncarboxylated
osteocalcin and its fragments and variants may result in conjugates
with increased water solubility, altered bioavailability,
pharmacokinetics, immunogenic properties, and biological
activities. See, e.g., Bentley et al., J. Pharm. Sci. 1998
November; 87(11):1446-9.
[0227] Several particularly common modifications that may be
applied to undercarboxylated/uncarboxylated osteocalcin and its
fragments and variants such as glycosylation, lipid attachment,
sulfation, hydroxylation and ADP-ribosylation are described in most
basic texts, such as Proteins--Structure and Molecular Properties,
2nd ed., T. E. Creighton, W. H. Freeman and Company, New York
(1993). Many detailed reviews are available on this subject, such
as by Wold, F., Posttranslational Covalent Modification of
Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983);
Seifter et al. (1990) Meth. Enzymol. 182: 626-646) and Rattan et
al. (1992) Ann. NY: Acad. Sci. 663:48-62.
[0228] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[0229] Modifications can occur anywhere in the
undercarboxylated/uncarboxylated osteocalcin and its fragments and
variants, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. Blockage of the
amino or carboxyl group in a polypeptide, or both, by a covalent
modification, is common in naturally-occurring and synthetic
polypeptides and may be applied to the
undercarboxylated/uncarboxylated osteocalcin or its fragments and
variants of the present invention. For instance, the amino terminal
residue of polypeptides made in E. coli, prior to proteolytic
processing, almost invariably will be N-formylmethionine. Thus,
undercarboxylated/uncarboxylated osteocalcin and its fragments and
variants with N-formylmethionine as the amino terminal residue are
within the scope of the present invention.
[0230] A brief description of various protein modifications that
come within the scope of this invention are set forth in the table
below:
TABLE-US-00003 TABLE 1 Protein Modification Description Acetylation
Acetylation of N-terminus or .epsilon.-lysines. Introducing an
acetyl group into a protein, specifically, the substitution of an
acetyl group for an active hydrogen atom. A reaction involving the
replacement of the hydrogen atom of a hydroxyl group with an acetyl
group (CH.sub.3CO) yields a specific ester, the acetate. Acetic
anhydride is commonly used as an acetylating agent, which reacts
with free hydroxyl groups. Acylation may facilitate addition of
other functional groups. A common reaction is acylation of e.g.,
conserved lysine residues with a biotin appendage. ADP-ribosylation
Covalently linking proteins or other compounds via an
arginine-specific reaction. Alkylation Alkylation is the transfer
of an alkyl group from one molecule to another. The alkyl group may
be transferred as an alkyl carbocation, a free radical or a
carbanion (or their equivalents). Alkylation is accomplished by
using certain functional groups such as alkyl electrophiles, alkyl
nucleophiles or sometimes alkyl radicals or carbene acceptors. A
common example is methylation (usually at a lysine or arginine
residue). Amidation Reductive animation of the N-terminus. Methods
for amidation of insulin are described in U.S. Pat. No. 4,489,159.
Carbamylation Nigen et al. describes a method of carbamylating
hemoglobin. Citrullination Citrullination involves the addition of
citrulline amino acids to the arginine residues of a protein, which
is catalyzed by peptidylarginine deaminase enzymes (PADs). This
generally converts a positively charged arginine into a neutral
citrulline residue, which may affect the hydrophobicity of the
protein (and can lead to unfolding). Condensation of amines Such
reactions, may be used, e.g., to attach a peptide to other with
aspartate or glutamate proteins labels. Covalent attachment of
Flavin mononucleotide (FAD) may be covalently attached to flavin
serine and/or threonine residues. May be used, e.g., as a
light-activated tag. Covalent attachment of A heme moiety is
generally a prosthetic group that consists heme moiety of an iron
atom contained in the center of a large heterocyclic organic ring,
which is referred to as a porphyrin. The heme moiety may be used,
e.g., as a tag for the peptide. Attachment of a nucleotide May be
used as a tag or as a basis for further derivatising a or
nucleotide derivative peptide. Cross-linking Cross-linking is a
method of covalently joining two proteins. Cross-linkers contain
reactive ends to specific functional groups (primary amines,
sulfhydryls, etc.) on proteins or other molecules. Several chemical
groups may be targets for reactions in proteins and peptides. For
example, Ethylene glycol bis[succinimidylsuccinate, Bis[2-
(succinimidooxycarbonyloxy)ethyl]sulfone, and
Bis[sulfosuccinimidyl]suberate link amines to amines. Cyclization
For example, cyclization of amino acids to create optimized
delivery forms that are resistant to, e.g., aminopeptidases (e.g.,
formation of pyroglutamate, a cyclized form of glutamic acid).
Disulfide bond formation Disulfide bonds in proteins are formed by
thiol-disulfide exchange reactions, particularly between cysteine
residues (e.g., formation of cystine). Demethylation See, e.g.,
U.S. Pat. No. 4,250,088 (Process for demethylating lignin).
Formylation The addition of a formyl group to, e.g., the N-terminus
of a protein. See, e.g., U.S. Pat. Nos. 4,059,589, 4,801,742, and
6,350,902. Glycylation The covalent linkage of one to more than 40
glycine residues to the tubulin C-terminal tail. Glycosylation
Glycosylation may be used to add saccharides (or polysaccharides)
to the hydroxy oxygen atoms of serine and threonine side chains
(which is also known as O-linked Glycosylation). Glycosylation may
also be used to add saccharides (or polysaccharides) to the amide
nitrogen of asparagine side chains (which is also known as N-linked
Glycosylation), e.g., via oligosaccharyl transferase. GPI anchor
formation The addition of glycosylphosphatidylinositol to the C-
terminus of a protein. GPI anchor formation involves the addition
of a hydrophobic phosphatidylinositol group - linked through a
carbohydrate containing linker (e.g., glucosamine and mannose
linked to phosphoryl ethanolamine residue) - to the C-terminal
amino acid of a protein. Hydroxylation Chemical process that
introduces one or more hydroxyl groups (--OH) into a protein (or
radical). Hydroxylation reactions are typically catalyzed by
hydroxylases. Proline is the principal residue to be hydroxylated
in proteins, which occurs at the C.sup..gamma. atom, forming
hydroxyproline (Hyp). In some cases, proline may be hydroxylated at
its C.sup..beta. atom. Lysine may also be hydroxylated on its
C.sup..delta. atom, forming hydroxylysine (Hyl). These three
reactions are catalyzed by large, multi-subunit enzymes known as
prolyl 4-hydroxylase, prolyl 3-hydroxylase and lysyl 5-hydroxylase,
respectively. These reactions require iron (as well as molecular
oxygen and .alpha.-ketoglutarate) to carry out the oxidation, and
use ascorbic acid to return the iron to its reduced state.
Iodination See, e.g., U.S. Pat. No. 6,303,326 for a disclosure of
an enzyme that is capable of iodinating proteins. U.S. Pat. No.
4,448,764 discloses, e.g., a reagent that may be used to iodinate
proteins. ISGylation Covalently linking a peptide to the ISG15
(Interferon- Stimulated Gene 15) protein, for, e.g., modulating
immune response. Methylation Reductive methylation of protein amino
acids with formaldehyde and sodium cyanoborohydride has been shown
to provide up to 25% yield of N-cyanomethyl (--CH.sub.2CN) product.
The addition of metal ions, such as Ni.sup.2+, which complex with
free cyanide ions, improves reductive methylation yields by
suppressing by-product formation. The N-cyanomethyl group itself,
produced in good yield when cyanide ion replaces cyanoborohydride,
may have some value as a reversible modifier of amino groups in
proteins. (Gidley et al.) Methylation may occur at the arginine and
lysine residues of a protein, as well as the N- and C-terminus
thereof. Myristoylation Myristoylation involves the covalent
attachment of a myristoyl group (a derivative of myristic acid),
via an amide bond, to the alpha-amino group of an N-terminal
glycine residue. This addition is catalyzed by the N-
myristoyltransferase enzyme. Oxidation Oxidation of cysteines.
Oxidation of N-terminal Serine or Threonine residues (followed by
hydrazine or aminooxy condensations). Oxidation of glycosylations
(followed by hydrazine or aminooxy condensations). Palmitoylation
Palmitoylation is the attachment of fatty acids, such as palmitic
acid, to cysteine residues of proteins. Palmitoylation increases
the hydrophobicity of a protein. (Poly)glutamylation
Polyglutamylation occurs at the glutamate residues of a protein.
Specifically, the gamma-carboxy group of a glutamate will form a
peptide-like bond with the amino group of a free glutamate whose
alpha-carboxy group may be extended into a polyglutamate chain. The
glutamylation reaction is catalyzed by a glutamylase enzyme (or
removed by a deglutamylase enzyme). Polyglutamylation has been
carried out at the C-terminus of proteins to add up to about six
glutamate residues. Using such a reaction, Tubulin and other
proteins can be covalently linked to glutamic acid residues.
Phosphopantetheinylation The addition of a 4'-phosphopantetheinyl
group. Phosphorylation A process for phosphorylation of a protein
or peptide by contacting a protein or peptide with phosphoric acid
in the presence of a non-aqueous apolar organic solvent and
contacting the resultant solution with a dehydrating agent is
disclosed e.g., in U.S. Pat. No. 4,534,894. Insulin products are
described to be amenable to this process. See, e.g., U.S. Pat. No.
4,534,894. Typically, phosphorylation occurs at the serine,
threonine, and tyrosine residues of a protein. Prenylation
Prenylation (or isoprenylation or lipidation) is the addition of
hydrophobic molecules to a protein. Protein prenylation involves
the transfer of either a farnesyl (linear grouping of three
isoprene units) or a geranyl-geranyl moiety to C- terminal
cysteine(s) of the target protein. Proteolytic Processing
Processing, e.g., cleavage of a protein at a peptide bond.
Selenoylation The exchange of, e.g., a sulfur atom in the peptide
for selenium, using a selenium donor, such as selenophosphate.
Sulfation Processes for sulfating hydroxyl moieties, particularly
tertiary amines, are described in, e.g., U.S. Pat. No. 6,452,035. A
process for sulphation of a protein or peptide by contacting the
protein or peptide with sulphuric acid in the presence of a
non-aqueous apolar organic solvent and contacting the resultant
solution with a dehydrating agent is disclosed. Insulin products
are described to be amenable to this process. See, e.g., U.S. Pat.
No. 4,534,894. SUMOylation Covalently linking a peptide a SUMO
(small ubiquitin- related Modifier) protein, for, e.g., stabilizing
the peptide. Transglutamination Covalently linking other protein(s)
or chemical groups (e.g., PEG) via a bndge at glutamine residues
tRNA-mediated addition of For example, the site-specific
modification (insertion) of an amino acids (e.g., amino acid analog
into a peptide. arginylation) Ubiquitination The small peptide
ubiquitin is covalently linked to, e.g., lysine residues of a
protein. The ubiquitin-proteasome system can be used to carryout
such reaction. See, e.g., U.S. 2007-0059731.
[0231] To practice the methods of the invention, it may be
desirable to recombinantly express the osteocalcin protein. The
cDNA sequence and deduced amino acid sequence of human osteocalcin
is represented in SEQ ID NO:1 and SEQ ID NO:2. Osteocalcin
nucleotide sequences may be isolated using a variety of different
methods known to those skilled in the art. For example, a cDNA
library constructed using RNA from a tissue known to express
osteocalcin can be screened using a labeled osteocalcin probe.
Alternatively, a genomic library may be screened to derive nucleic
acid molecules encoding the osteocalcin protein. Further,
osteocalcin nucleic acid sequences may be derived by performing a
polymerase chain reaction (PCR) using two oligonucleotide primers
designed on the basis of known osteocalcin nucleotide sequences.
The template for the reaction may be cDNA obtained by reverse
transcription of mRNA prepared from cell lines or tissue known to
express osteocalcin.
[0232] While the osteocalcin polypeptides and peptides can be
chemically synthesized (e.g., see Creighton, 1983, Proteins:
Structures and Molecular Principles, W.H. Freeman & Co., N.Y.),
large polypeptides derived from osteocalcin and the full length
osteocalcin itself may be advantageously produced by recombinant
DNA technology using techniques well known in the art for
expressing a nucleic acid. Such methods can be used to construct
expression vectors containing the osteocalcin nucleotide sequences
and appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, for example, the techniques described in
Sambrook et al., 1989, supra, and Ausubel et al., 1989, supra).
[0233] A variety of host-expression vector systems may be utilized
to express the osteocalcin nucleotide sequences. In a preferred
embodiment, the osteocalcin peptide or polypeptide is secreted and
may be recovered from the culture media.
[0234] Appropriate expression systems can be chosen to ensure that
the correct modification, processing and subcellular localization
of the osteocalcin protein occurs. To this end, bacterial host
cells are preferred for expression of osteocalcin, as such cells
are unable to carboxylate osteocalcin.
[0235] The isolated osteocalcin can be purified from cells that
naturally express it, e.g., osteoblasts, or purified from cells
that naturally express it but have been modified to overproduce
osteocalcin, e.g., purified from cells that have been altered to
express it (recombinant), synthesized using known protein synthesis
methods, or by modifying cells that naturally encode osteocalcin to
express it. In a particular embodiment, a recombinant cell has been
manipulated to activate expression of the endogenous osteocalcin
gene. For example, WO 99/15650 and WO 00/49162 describe a method of
expressing endogenous genes termed random activation of gene
expression (RAGE), which can be used to activate or increase
expression of endogenous osteocalcin. The RAGE methodology involves
non-homologous recombination of a regulatory sequence to activate
expression of a downstream endogenous gene. Alternatively, WO
94/12650, WO 95/31560, WO 96/29411, U.S. Pat. No. 5,733,761 and
U.S. Pat. No. 6,270,985 describe a method of increasing expression
of an endogenous gene that involves homologous recombination of a
DNA construct that includes a targeting sequence, a regulatory
sequence, an exon, and a splice-donor site. Upon homologous
recombination, a downstream endogenous gene is expressed. The
methods of expressing endogenous genes described in the forgoing
patents are hereby expressly incorporated by reference.
[0236] Compositions Comprising Adiponectin and
Undercarboxylated/Uncarboxylated Osteocalcin
[0237] In a specific embodiment of the invention, pharmaceutical
compositions comprising adiponectin and
undercarboxylated/uncarboxylated osteocalcin or biologically active
undercarboxylated/uncarboxylated osteocalcin fragments or variants
may be administered to a patient in need of such administration. In
an embodiment of the invention, the adiponectin is a mammalian
adiponectin. In a preferred embodiment of the invention, the
pharmaceutical compositions of the invention comprise human
adiponectin. The cDNA sequence of human adiponectin is shown in SEQ
ID NO:6. The amino acid sequence of human adiponectin is shown in
SEQ ID NO:7.
[0238] In another aspect of the present invention, biologically
active adiponectin fragments or variants together with
undercarboxylated/uncarboxylated osteocalcin or biologically active
undercarboxylated/uncarboxylated osteocalcin fragments in a
pharmaceutical composition can be used in the treatment methods of
the present invention. Adiponectin fragments or variants encompass
the kinds of changes in the native sequence of adiponectin that are
described above for osteocalcin and can be produced by the same
methods that are described above for producing osteocalcin
fragments and variants.
[0239] Compositions Comprising Inhibitors of Gamma-Carboxylase
and/or OST-PTP
[0240] In another embodiment of the invention, the pharmaceutical
compositions of the invention comprise an inhibitor that reduces
the expression or activity of gamma-carboxylase or OST-PTP.
Preferably, the biological activity of gamma-carboxylase or OST-PTP
(as previously described) is inhibited. The inhibitors may be
antibodies (monoclonal or polyclonal) or fragments of antibodies,
small molecules, polypeptides or proteins, or nucleic acids (e.g.,
antisense DNA or RNA, siRNA).
[0241] In certain embodiments, the inhibitors reduce the activity
of OST-PTP having the amino acid sequene of SEQ ID NO:19. In other
embodiments, the inhibitors reduce the activity of an OST-PTP
having an amino acid sequence that is substantially homologous or
identical, as previouly described to the amino acid sequence of SEQ
ID NO:19.
[0242] In certain embodiments, the inhibitors reduce the activity
of gamma-carboxylase having the amino acid sequence of SEQ ID
NO:11. In other embodiments, the inhibitors reduce the activity of
an gamma-carboxylase having an amino acid sequence that is
substantially homologous or identical, as previouly described to
the amino acid sequence.
Small Molecule Inhibitors of OST-PTP and Gamma-Carboxylase
[0243] In certain embodiments, the agent is a small molecule. By
"small molecule" is meant organic compounds of molecular weight of
more than 100 and less than about 2,500 daltons, and preferably
less than 500 daltons. Such small molecules inhibit the biological
activity of OST-PTP or gamma-carboxylase.
[0244] The inhibitors may comprise agents that act as inhibitors of
vitamin K, beta blockers, statins, and/or thiol-specific inhibitors
that function to increase serum adiponectin, serum insulin and/or
serum osteocalcin levels, preferably
undercarboxylated/uncarboxylated osteocalcin. The agents may also
increase glucose tolerance, increase insulin sensitivity, increase
beta-cell proliferation, and/or cause other effects of biologically
active agents as previously described.
[0245] Warfarin and other vitamin K inhibitors, including coumadin
and other derivatives, beta-blockers, statins, and fragments and
modifications thereof, may be administered to patients who would
benefit from inhibition of gamma-carboxylase. In a specific
embodiment of the invention, the small molecule warfarin may be
used to inhibit the activity of gamma-carboxylase. Warfarin
derivatives are exemplified by acenocoumarol, phenprocoumon and
phenindione. Warfarin and other coumadin derivatives block vitamin
K-dependent gamma-carboxylation, thus increasing the level of
undercarboxylated/uncarboxylated osteocalcin.
[0246] Beta blockers are used to treat high blood pressure
(hypertension), congestive heart failure (CHF), abnormal heart
rhythms (arrhythmias), and chest pain (angina). Beta blockers are
sometimes used in heart attack patients to prevent future heart
attacks. There are 2 main beta receptors: beta 1 and beta 2. Some
beta blockers are selective, which means that they block beta 1
receptors more than they block beta 2 receptors. Beta 1 receptors
are responsible for heart rate and the strength of the heartbeat.
Nonselective beta blockers block both beta 1 and beta 2 receptors.
Beta 2 receptors are responsible for the function of smooth
muscles; they are also the only beta receptors expressed by
osteoblasts. Non-limiting examples of beta blockers include
sotalol, timolol, esmolol, carteolol, carvedilol, nadolol,
propranolol, betaxolol, penbutolol, metoprolol, labetalol,
acebutolol, atenolol, metoprolol, labetalol, pindolol, and
bisoprolol.
[0247] Statins are further exemplified by atorvastatin,
cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,
pravastatin, rosuvastatin, and simvastatin. Non-limiting examples
of beta-blockers include sotalol, carvedilol, metroprolol. Other
small molecules can be identified using the screens and assays
disclosed herein.
[0248] Other inhibitors include thiol specific inhibitors of
gamma-carboxylase. Cys and His residues of gamma carboxylase are
implicated in the carboxylase mechanism of gamma carboxylase, and
it is observed that the enzyme is inhibited by thiol-specific
inhibitors, such as N-ethylmaleimide (NEM) and mercurials such as
p-hydroxymurcuribenzoate (pHMB). Additional non-limiting examples
of these inhibitors include 5,5-dithiobis-(2-nitrobenzoic acid)
(DTNB), 2-nitro-5-thiocyanobenzoic acid (NTCB), iodoacetamide (IA),
N-phenylmaleimide (PheM), N-(1-pyrenyl) maleimide (PyrM),
naphthalene-1,5-dimaleimide (NDM), N,N'-(1,2-phenylene) dimaleimide
(oPDM), N,N'-1,4-phenylene dimaleimide (pPDM), N,N'-1,3-phenylene
dimaleimide (mPDM), 1,1-(methylenedi-4,1-phenylene)bismaleimide
(BM), 4-(N-maleimido)phenyltrimethylammonium (MPTM),
N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine (BMP),
N-succinimidyl 3-(2-pyridyldithio)propionate, diethyl
pyrocarbonate, p-chloromercuribenzene sulphonic acid and
thiosulfinates. These inhibitors may also be provided as conjugate
or derivative, such as with, e.g., BSA or aminodextran.
Antibody Inhibitors of OST-PTP and Gamma-Carboxylase
[0249] The present invention also provides compositions comprising
an antibody or antibodies, as well as biologically active fragments
or variants thereof, that are capable of binding to an epitope of
OST-PTP or gamma-carboxylase polypeptides. An antibody against
OST-PTP that decreases its activity can be used therapeutically. In
certain embodiments, the antibody against OST-PTP binds to the
extracellular domain of OST-PTP.
[0250] In certain embodiments, the antibody against OST-PTP binds
to an epitope in the mouse OST-PTP of SEQ ID NO:19 or an OST-PTP
having an amino acid sequence that is substantially homologous or
identical to SEQ ID NO:19. In other embodiments, the antibody
against OST-PTP binds to an epitope in an OST-PTP having an amino
acid sequence that is at least 70% homologous or identical to SEQ
ID NO:19.
[0251] Human OST-PTP can be obtained by isolating the human
ortholog of mouse OST-PTP (SEQ ID NO:18) (or rat OST-PTP; SEQ ID
NO:24) by methods known in the art. For example, one could prepare
a cDNA library from human osteoblasts and identify human OST-PTP
cDNA by hybridizing the cDNA clones from the library to a mouse
probe. The mouse probe could be based on a portion of mouse OST-PTP
(SEQ ID NO:18). Alternatively, PCR, using primers based on the
mouse sequence, can be used to obtain the human OST-PTP gene.
[0252] Gamma-carboxylase is an intracellular protein, so antibodies
or fragments of antibodies against it are preferably used
therapeutically when combined with technologies for delivering the
antibodies, fragments or variants into the interior of target cells
expressing gamma-carboxylase, e.g., osteoblasts. Antibodies,
fragments or variants against gamma-carboxylase can also be used
diagnostically or in drug screening assays. Antibodies or antibody
fragments or variants against osteocalcin and adiponectin similarly
can be used with technologies for delivering the antibodies or
fragments into the interior of target cells and can also be used in
diagnostics and drug screening assays.
[0253] In a particular embodiment, the present invention provides
antibodies, fragments or variants of antibodies that recognize an
epitope in OST-PTP that includes the amino acid at position 1316 of
mouse OST-PTP or the corresponding position of human OST-PTP. In
certain embodiments, these antibodies, fragments or variants of
antibodies block or inhibit the ability of OST-PTP to activate
gamma-carboxylase. In certain embodiments, use of these antibodies
or fragments results in OST-PTP losing 50%, 60%, 70%, 80%, 90%,
95%, or essentially all of its ability to activate
gamma-carboxylase.
[0254] The term "epitope" refers to an antigenic determinant on an
antigen to which an antibody binds. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains, and typically have specific
three-dimensional structural characteristics, as well as specific
charge characteristics. Epitopes generally have at least five
contiguous amino acids. The terms "antibody" and "antibodies"
include polyclonal antibodies, monoclonal antibodies, humanized or
chimeric antibodies, single chain Fv antibody fragments, Fab
fragments, and F(ab').sub.2 fragments. Polyclonal antibodies are
heterogeneous populations of antibody molecules that are specific
for a particular antigen, while monoclonal antibodies are
homogeneous populations of antibodies to a particular epitope
contained within an antigen. Monoclonal antibodies are particularly
useful in the present invention.
[0255] Antibody fragments that have specific binding affinity for
the polypeptide of interest (e.g., OST-PTP or gamma-carboxylase)
can be generated by known techniques. Such antibody fragments
include, but are not limited to, F(ab').sub.2 fragments that can be
produced by pepsin digestion of an antibody molecule, and Fab
fragments that can be generated by reducing the disulfide bridges
of F(ab').sub.2 fragments. Alternatively, Fab expression libraries
can be constructed. See, for example, Huse et al. (1989) Science
246:1275-1281. Single chain Fv antibody fragments are formed by
linking the heavy and light chain fragments of the Fv region via an
amino acid bridge (e.g., 15 to 18 amino acids), resulting in a
single chain polypeptide. Single chain Fv antibody fragments can be
produced through standard techniques, such as those disclosed in
U.S. Pat. No. 4,946,778.
[0256] Once produced, antibodies or fragments thereof can be tested
for recognition of the target polypeptide by standard immunoassay
methods including, for example, enzyme-linked immunosorbent assay
(ELISA) or radioimmunoassay assay (RIA). See, Short Protocols in
Molecular Biology eds. Ausubel et al., Green Publishing Associates
and John Wiley & Sons (1992).
[0257] The immunoassays, immunohistochemistry, RIA, IRMAs used
herein are based on the generation of various antibodies, including
those that specifically bind to osteocalcin, OST-PTP,
gamma-carboxylase, adiponectin, vitamin K, or their fragments or
variants. Antibodies and methods of using antibodies to quantitate
the amount of osteocalcin, in particular, in a sample are also
described in Hosoda et al. (U.S. Pat. No. 5,681,707). Hosoda et al.
disclose antibodies that bind to the N-terminal 20 amino acids, or
the C-terminal 14 amino acids of osteocalcin. Anti-OST-PTP
antibodies are commercially available.
[0258] In one embodiment, antibodies against OST-PTP or
gamma-carboxylase that reduce its activity are useful in the
treatment of a patient having a disorder related to the OST-PTP
pathway. Such disorders include metabolic syndrome, glucose
intolerance, diabetes types 1 and 2, atherosclerosis and obesity.
Such disorders are characterized by decreased insulin production,
decreased insulin sensitivity, decreased glucose tolerance and/or
increased fat mass.
Nucleic Acid Inhibitors of OST-PTP and Gamma-Carboxylase
[0259] Other embodiments of the present invention are directed to
the use of antisense nucleic acids or small interfering RNA (siRNA)
to reduce or inhibit expression and hence the biological acitivity
of proteins or peptides, particularly OST-PTP and
gamma-carboxylase. The cDNA sequences encoding OST-PTP and
gamma-carboxylase are set forth below. Based on these known
sequences, antisense DNA or RNA that hybridize sufficiently to the
respective gene or mRNA encoding OST-PTP and gamma-carboxylase to
turn off or reduce expression can be readily designed and
engineered, using methods known in the art.
[0260] In a specific embodiment of the invention, antisense or
siRNA molecules for use in the present invention are those that
bind under stringent conditions to the human gamma-carboxylase
nucleic sequence of SEQ ID NO:10. In yet another embodiment of the
invention, the antisense or siRNA molecules are those that that
bind under stringent conditions to the OST-PTP nucleic acid
sequence of SEQ ID NO:18, or sequences that are substantially
homologous to SEQ ID NO:18. In other embodiments, the antisense or
siRNA molecules bind under stringent conditions to sequences that
are substantially homologous or identical to SEQ ID NO:18.
[0261] Antisense-RNA and anti-sense DNA have been used
therapeutically in mammals to treat various diseases. See for
example Agrawal, S. and Zhao, Q. (1998) Curr. Opin. Chemical Biol.
Vol. 2, 519-528; Agrawal, S and Zhang, R. (1997) CIBA Found. Symp.
Vol. 209, 60-78; and Zhao, Q, et al., (1998), Antisense Nucleic
Acid Drug Dev. Vol 8, 451-458; the entire contents of which are
hereby incorporated by reference as if fully set forth herein.
Antisense oligodeoxyribonucleotides (antisense-DNA),
oligoribonucleotides (antisense-RNA), and other polymeric antisense
compounds (e.g., oligonucleotides composed of naturally-occurring
nucleobases, sugars and covalent internucleoside linkages and
non-naturally-occurring portions which function similarly) can base
pair with a gene or its transcript. An antisense
PS-oligodeoxyribonucleotide for treatment of cytomegalovirus
retinitis in AIDS patients is the first antisense
oligodeoxyribonucleotide approved for human use in the US.
Anderson, K. O., et al., (1996) Antimicrobiol. Agents Chemother.
Vol. 40, 2004-2011, and U.S. Pat. No. 6,828,151 by Borchers, et
al., entitled "Antisense modulation of hematopoietic cell protein
tyrosine kinase expression," describe methods for making and using
antisense nucleic acids and their formulation, the entire contents
of which are hereby incorporated by reference as if fully set forth
herein.
[0262] Methods of making antisense nucleic acids are well known in
the art. Further provided are methods of modulating the expression
of OST-PTP and gamma-carboxylase genes and mRNA in cells or tissues
by contacting the cells or tissues with one or more of the
antisense compounds or compositions of the invention. As used
herein, the terms "target nucleic acid" encompass DNA encoding
OST-PTP or gamma-carboxylase and RNA (including pre-mRNA and mRNA)
transcribed from such DNA. The specific hybridization of a nucleic
acid oligomeric compound with its target nucleic acid interferes
with the normal function of the target nucleic acid. This
modulation of function of a target nucleic acid by compounds which
specifically hybridize to it is generally referred to as
"antisense." The functions of DNA to be interfered with include
replication and transcription. The functions of RNA to be
interfered with include all vital functions such as, for example,
translocation of the RNA to the site of protein translation,
translation of protein from the RNA, and catalytic activity which
may be engaged in or facilitated by the RNA. The overall effect of
such interference with target nucleic acid function is modulation
of the expression of the protein encoded by the DNA or RNA. In the
context of the present invention, "modulation" means reducing or
inhibiting in the expression of the gene or mRNA for OST-PTP and/or
gamma-carboxylase. DNA is the preferred antisense nucleic acid.
[0263] The targeting process includes determination of a site or
sites within the target DNA or RNA encoding the OST-PTP and/or
gamma-carboxylase for the antisense interaction to occur such that
the desired inhibitory effect is achieved. Within the context of
the present invention, a preferred intragenic site is the region
encompassing the translation initiation or termination codon of the
open reading frame (ORF) of the mRNA for OST-PTP or
gamma-carboxylase, preferably human OST-PTP or gamma-carboxylase.
Since, as is known in the art, the translation initiation codon is
typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the
corresponding DNA molecule), the translation initiation codon is
also referred to as the "AUG codon," the "start codon" or the "AUG
start codon." A minority of genes have a translation initiation
codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA,
5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the
terms "translation initiation codon" and "start codon" can
encompass many codon sequences, even though the initiator amino
acid in each instance is typically methionine in eukaryotes. It is
also known in the art that eukaryotic genes may have two or more
alternative start codons, any one of which may be preferentially
utilized for translation initiation in a particular cell type or
tissue, or under a particular set of conditions. In the context of
the invention, "start codon" and "translation initiation codon"
refer to the codon or codons that are used in vivo to initiate
translation of an mRNA molecule transcribed from a gene. Routine
experimentation will determine the optimal sequence of the
antisense or siRNA.
[0264] It is also known in the art that a translation termination
codon (or "stop codon") of a gene may have one of three sequences,
i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences
are 5'-TAA, 5'-TAG and 5'-TGA, respectively).
[0265] The terms "start codon region" and "translation initiation
codon region" refer to a portion of such an mRNA or gene that
encompasses from about 25 to about 50 contiguous nucleotides in
either direction (i.e., 5' or 3') from a translation initiation
codon. Similarly, the terms "stop codon region" and "translation
termination codon region" refer to a portion of such an mRNA or
gene that encompasses from about 25 to about 50 contiguous
nucleotides in either direction (i.e., 5' or 3') from a translation
termination codon.
[0266] The open reading frame (ORF) or "coding region," which is
known in the art to refer to the region between the translation
initiation codon and the translation termination codon, is also a
region which may be targeted effectively. Other target regions
include the 5' untranslated region (5'UTR), known in the art to
refer to the portion of an mRNA in the 5' direction from the
translation initiation codon, and thus including nucleotides
between the 5' cap site and the translation initiation codon of an
mRNA or corresponding nucleotides on the gene, and the 3'
untranslated region (3'UTR), known in the art to refer to the
portion of an mRNA in the 3' direction from the translation
termination codon, and thus including nucleotides between the
translation termination codon and 3' end of an mRNA or
corresponding nucleotides on the gene.
[0267] It is also known in the art that variants can be produced
through the use of alternative signals to start or stop
transcription and that pre-mRNAs and mRNAs can possess more that
one start codon or stop codon. Variants that originate from a
pre-mRNA or mRNA that use alternative start codons are known as
"alternative start variants" of that pre-mRNA or mRNA. Those
transcripts that use an alternative stop codon are known as
"alternative stop variants" of that pre-mRNA or mRNA. One specific
type of alternative stop variant is the "polyA variant" in which
the multiple transcripts produced result from the alternative
selection of one of the "polyA stop signals" by the transcription
machinery, thereby producing transcripts that terminate at unique
polyA sites.
[0268] Once one or more target sites have been identified, nucleic
acids are chosen which are sufficiently complementary to the
target, i.e., hybridize sufficiently well and with sufficient
specificity, to give the desired effect of inhibiting gene
expression and transcription or mRNA translation.
[0269] In the context of this invention, "hybridization" means
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases. For example, adenine and thymine are
complementary nucleobases which pair through the formation of
hydrogen bonds. "Complementary," as used herein, refers to the
capacity for precise pairing between two nucleotides. For example,
if a nucleotide at a certain position of a nucleic acid is capable
of hydrogen bonding with a nucleotide at the same position of a DNA
or RNA molecule, then the nucleic acid and the DNA or RNA are
considered to be complementary to each other at that position. The
nucleic acid and the DNA or RNA are complementary to each other
when a sufficient number of corresponding positions in each
molecule are occupied by nucleotides which can hydrogen bond with
each other. Thus, "specifically hybridizable" and "complementary"
are terms which are used to indicate a sufficient degree of
complementarity or precise pairing such that stable and specific
binding occurs between the nucleic acid and the DNA or RNA target.
It is understood in the art that the sequence of an antisense
compound need not be 100% complementary to that of its target
nucleic acid to be specifically hybridizable. An antisense compound
is specifically hybridizable when binding of the compound to the
target DNA or RNA molecule interferes with the normal function of
the target DNA or RNA to cause a loss of function, and there is a
sufficient degree of complementarity to avoid non-specific binding
of the antisense compound to non-target sequences under conditions
in which specific binding is desired, i.e., under physiological
conditions in the case of in vivo assays or therapeutic treatment,
and in the case of in vitro assays, under conditions in which the
assays are performed.
[0270] Antisense nucleic acids have been employed as therapeutic
moieties in the treatment of disease states in animals and man.
Antisense nucleic acid drugs, including ribozymes, have been safely
and effectively administered to humans and numerous clinical trials
are presently underway. It is thus established that nucleic acids
can be useful therapeutic modalities that can be configured to be
useful in treatment regimes for treatment of cells, tissues and
animals, especially humans, for example to regulate expression of
OST-PTP and gamma-carboxylase.
[0271] Nucleic acids in the context of this invention includes
"oligonucleotides," which refers to an oligomer or polymer of
ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics
thereof. This term includes oligonucleotides composed of
naturally-occurring nucleobases, sugars and covalent
internucleoside (backbone) linkages as well as oligonucleotides
having non-naturally-occurring portions which function similarly.
Such modified or substituted oligonucleotides are often preferred
over native forms because of desirable properties such as, for
example, enhanced cellular uptake, enhanced affinity for nucleic
acid target and increased stability in the presence of
nucleases.
[0272] While antisense nucleic acids are a preferred form of
antisense compound, the present invention comprehends other
oligomeric antisense compounds, including but not limited to
oligonucleotide mimetics. The antisense compounds in accordance
with this invention preferably comprise from about 8 to about 50
nucleobases (i.e., from about 8 to about 50 linked nucleosides).
Particularly preferred antisense compounds are antisense nucleic
acids comprising from about 12 to about 30 nucleobases. Antisense
compounds include ribozymes, external guide sequence (EGS) nucleic
acids (oligozymes), and other short catalytic RNAs or catalytic
nucleic acids which hybridize to the target nucleic acid and
modulate its expression.
[0273] The antisense compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
nucleic acids such as the phosphorothioates and alkylated
derivatives.
[0274] The antisense compounds of the present invention can be
utilized for diagnostics, therapeutics, and prophylaxis and as
research reagents and kits. For therapeutics, an animal, preferably
a human, suspected of having a disease or disorder such as
metabolic syndrome, glucose intolerance, diabetes, atherosclerosis,
and/or obesity, which can be treated by modulating the expression
of gamma-carboxylase or OST-PTP, is treated by administering
antisense compounds in accordance with this invention. The
compounds of the invention can be utilized in pharmaceutical
compositions by adding an effective amount of an antisense compound
to a suitable pharmaceutically acceptable diluent or carrier. The
antisense compounds and methods of the invention are useful
prophylactically, e.g., to prevent or delay the appearance of
metabolic syndrome, glucose intolerance, diabetes, atherosclerosis
or obesity. The antisense compounds and methods of the invention
are also useful to retard the progression of metabolic syndrome,
glucose intolerance, diabetes, atherosclerosis or obesity.
[0275] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
of the invention that are administered to return the level of serum
insulin in diabetic patients (for example) to normal.
[0276] US Patent Application 2004/0023390 (the entire contents of
which are hereby incorporated by reference as if fully set forth
herein) teaches that double-stranded RNA (dsRNA) can induce
sequence-specific posttranscriptional gene silencing in many
organisms by a process known as RNA interference (RNAi). However,
in mammalian cells, dsRNA that is 30 base pairs or longer can
induce sequence-nonspecific responses that trigger a shut-down of
protein synthesis and even cell death through apoptosis. Recent
work shows that RNA fragments are the sequence-specific mediators
of RNAi (Elbashir et al., 2001). Interference of gene expression by
these small interfering RNA (siRNA) is now recognized as a
naturally occurring strategy for silencing genes in C. elegans,
Drosophila, plants, and in mouse embryonic stem cells, oocytes and
early embryos (Cogoni et al., 1994; Baulcombe, 1996; Kennerdell,
1998; Timmons, 1998; Waterhouse et al., 1998; Wianny and
Zernicka-Goetz, 2000; Yang et al., 2001; Svoboda et al., 2000).
[0277] In mammalian cell culture, a siRNA-mediated reduction in
gene expression has been accomplished by transfecting cells with
synthetic RNA nucleic acids (Caplan et al., 2001; Elbashir et al.,
2001). The 2004/0023390 application, the entire contents of which
are hereby incorporated by reference as if fully set forth herein,
provides exemplary methods using a viral vector containing an
expression cassette containing a pol II promoter operably-linked to
a nucleic acid sequence encoding a small interfering RNA molecule
(siRNA) targeted against a gene of interest.
[0278] As used herein RNAi is the process of RNA interference. A
typical mRNA produces approximately 5,000 copies of a protein. RNAi
is a process that interferes with or significantly reduces the
number of protein copies made by an mRNA, preferably encoding
OST-PTP or gamma-carboxylase. For example, a double-stranded short
interfering RNA (siRNA) molecule is engineered to complement and
match the protein-encoding nucleotide sequence of the target mRNA
to be interfered with. Following intracellular delivery, the siRNA
molecule associates with an RNA-induced silencing complex (RISC).
The siRNA-associated RISC binds the target mRNA (such as mRNA
encoding gamma-carboxylase and OST-PTP) through a base-pairing
interaction and degrades it. The RISC remains capable of degrading
additional copies of the targeted mRNA. Other forms of RNA can be
used such as short hairpin RNA and longer RNA molecules. Longer
molecules cause cell death, for example by instigating apoptosis
and inducing an interferon response. Cell death was the major
hurdle to achieving RNAi in mammals because dsRNAs longer than 30
nucleotides activated defense mechanisms that resulted in
non-specific degradation of RNA transcripts and a general shutdown
of the host cell. Using from about 20 to about 29 nucleotide siRNAs
to mediate gene-specific suppression in mammalian cells has
apparently overcome this obstacle. These siRNAs are long enough to
cause gene suppression but not of a length that induces an
interferon response. In a specific embodiment of the invention, the
targets of gene suppression are the OST-PTP gene and the gene for
gamma-carboxylase. siRNA molecules useful in the present invention
include those sequences that bind under stringent conditions to the
human gamma-carboxylase gene of SEQ ID:10 or the OST-PTP gene of
SEQ ID NO:18. siRNA molecules useful in the present invention also
include those sequences that bind under stringent conditions to
nucleic acids that are 80%, 85%, 90%, or 95% homologous to SEQ ID
NO:18.
Co-Administration of the Therapeutic Agents of the Present
Invention and Other Drugs
[0279] The undercarboxylated/uncarboxylated osteocalcin and
inhibitors of OST-PTP and gamma-carboxylase described herein may be
co-administered to a patient with other drugs such as
anti-coagulants, vasodilators, drugs used to treat atherosclerosis,
drugs used to treat glucose intolerance, drugs used to treat
diabetes, vitamin K inhibitors, statins, beta blockers, and other
drugs used to treat diseases associated with disorders related to
the OST-PTP signaling pathway, including, but not limited to
metabolic syndrome, glucose intolerance, type 1 or type 2 diabetes,
atherosclerosis, and obesity in amounts effective to provide
therapeutic benefit of the drug in the combination therapy. The
combination may provide increased, additive, or synergistic effect.
The co-administration of the undercarboxylated/uncarboxylated
osteocalcin, inhibitors of OST-PTP, inhibitors of gamma-carboxylase
and the other drugs may be done by administration of separate
pharmaceutical compositions or the the
undercarboxylated/uncarboxylated osteocalcin, inhibitors of
OST-PTP, inhibitors of gamma-carboxylase and the other drugs may be
present in a single pharmaceutical composition.
[0280] Anticoagulants useful in the invention are exemplified by
vitamin K antagonists, heparin and derivatives of heparin, and
direct thrombin inhibitors. Vitamin K antagonists are exemplified
by warfarin (also known under the brand names COUMADIN.RTM.,
JANTOVEN.RTM., MAREVAN.RTM., and WARAN.RTM.), warfarin derivatives,
acenocoumarol, phenprocoumon as well as phenindione. Heparin and
derivatives of heparin are exemplified by low molecular weight
heparin and fondaparinux. Direct thrombin inhibitors are
exemplified by argatroban, lepirudin, bivalirudin and
ximelagatran.
[0281] Vasodilators are useful in the present invention.
Vasodilators are exemplified by adenosine, amyl nitrite and other
nitrites, L-arginine, atrial natriuretic peptide (ANP), bradykinin,
ethanol, endothelium-derived hyperpolarizing factor (EDHF),
histamine, complement proteins C3a, C4a and C5a, niacin (nicotinic
acid), nitric oxide, glyceryl trinitrate (commonly known as
nitroglycerin), isosorbide mononitrate & isosorbide dinitrate,
pentaerythritol tetranitrate (PETN), sodium nitroprusside, PDE5
inhibitors, sildenafil, tadalafil, vardenafil, platelet activating
factor (PAF), prostacyclin (PGI.sub.2) as well as other
prostaglandins, tetrahydrocannabinol (THC), theobromine, and
papaverine.
[0282] Drugs used to treat atherosclerosis are useful in the
present invention. Drugs used to treat atherosclerosis are
exemplified by statins, scilostazol, benzothiazepines,
phenylalkylamines, dihydropyridines, epoprostenol, vitamin B3, and
aspirin. Statins are further exemplified by atorvastatin,
cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,
pravastatin, rosuvastatin, and simvastatin. Benzothiazepines are
exemplified by diltiazem. Phenylalkylamines are exemplified by
verapamil. Dihydropyridines are exemplified by amlodipine,
felodipine, isradipine, lacidipine, lercanidipine, nicardipine,
nifedipine, nimodipine, nisoldipine, and nitrendipine.
[0283] Drugs useful in the treatment of diabetes include, but are
not limited to, sulfonylureas, meglitinides, D-Phenylalanine
Derivatives (nateglinides), biguanides, thiazolidinediones,
alpha-glucose inhibitors, Dipeptidyl peptidase 4 (DPP4) inhibitors,
insulins (preferably human recombinant insulin) and incretins.
[0284] Sulfonylureas are exemplified by glimepiride, glyburide,
chlorpropamide, acetohexamide, glipizide, tolbutamide, and
tolazamide. Meglitinides are exemplified by Repaglinide.
D-Phenylalanine Derivatives are exemplified by nateglinide.
Biguanides are exemplified by metformin and metformin
hydrochloride. Thiazolidinediones are exemplified by pioglitazone
and rosiglitazone. Alpha-glucose inhibitors are exemplified by
miglitol and acarbose. Dipeptidyl peptidase 4 (DPP4) inhibitors are
exemplified by vildagliptin, sitagliptin and saxagliptin.
[0285] Generally, there are six categories of insulins:
rapid-acting, short-acting, intermediate acting, long acting, very
long acting, and premixed. Incretins are a type of gastrointestinal
hormone that cause an increase in the amount of insulin released
from the beta-cells of the islets of Langerhans after eating, even
before blood glucose levels become elevated. Incretins are
exemplified by glucagon-like peptide-1 (GLP-1) and Gastric
inhibitory peptide (aka glucose-dependent Insulinotropic peptide or
GIP).
[0286] Beta blockers are used to treat high blood pressure
(hypertension), congestive heart failure (CHF), abnormal heart
rhythms (arrhythmias), and chest pain (angina). Beta blockers are
sometimes used in heart attack patients to prevent future heart
attacks. Beta blockers work by blocking the effects of the hormone
epinephrine, also known as adrenaline. As a result, the heart beats
more slowly and with less force, thereby reducing blood pressure.
Beta blockers also help blood vessels relax and open up to improve
blood flow. Beta blockers also block the impulses that can cause an
arrhythmia. There are 2 main beta receptors: beta 1 and beta 2.
Some beta blockers are selective, which means that they block beta
1 receptors more than they block beta 2 receptors. Beta 1 receptors
are responsible for heart rate and the strength of the heartbeat.
Nonselective beta blockers block both beta 1 and beta 2 receptors.
Beta 2 receptors are responsible for the function of smooth
muscles; they are also the only beta receptors expressed by
osteoblasts.
[0287] Brand Names and generic claims of beta blockers commonly
used in the United States are: Betapace (sotalol), Blocadren
(timolol), Brevibloc (esmolol), Cartrol (carteolol), Coreg
(carvedilol), Corgard (nadolol), Inderal (propranolol), Inderal-LA
(propranolol), Kerlone (betaxolol), Levatol (penbutolol), Lopressor
(metoprolol), Normodyne (labetalol), Sectral (acebutolol), Tenormin
(atenolol), Toprol-XL (metoprolol), Trandate (labetalol), Visken
(pindolol), Zebeta (bisoprolol). Commonly Used Brand Names in
Canada are: Apo-Atenolol (atenolol), Apo-Metoprolol (metoprolol),
Apo-Propranolol (propranolol), Apo-Timol (timolol), Betaloc
(metoprolol), Blocadren (timolol), Corgard (nadolol), Inderal
(propranolol), Lopressor (metoprolol), Monitan (acebutolol),
Novo-Atenol (atenolol), Novometoprol (metoprolol), Novo-Pindol
(pindolol), Novo-Timol (timolol), Sectral (acebutolol), Sotacor
(sotalol), Tenormin (atenolol), Trandate (labetalol), Trasicor
(oxprenolol), Visken (pindolol).
Pharmaceutical Compositions and Administration
[0288] The present invention encompasses the use of the
polypeptides, nucleic acids, antibodies, small molecules and other
therapeutic agents described herein formulated in pharmaceutical
compositions to administer to a subject. The therapeutic agents
(also referred to as "active compounds") can be incorporated into
pharmaceutical compositions suitable for administration to a
subject, e.g., a human. Such compositions typically comprise the
polypeptides, nucleic acids, antibodies, small molecules and a
pharmaceutically acceptable carrier. Preferably, such compositions
are non-pyrogenic when administered to humans.
[0289] The pharmaceutical compositions of the invention are
adminstered in an amount sufficient to modulate the the OST-PTP
signaling pathway involving gamma-carboxylase, osteocalcin, insulin
and adiponectin.
[0290] As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, binders,
diluents, disintegrants, lubricants, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. As
long as any conventional media or agent is compatible with the
active compound, such media can be used in the compositions of the
invention. Supplementary active compounds or therapeutic agents can
also be incorporated into the compositions. A pharmaceutical
composition of the invention is formulated to be compatible with
its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
intranasal, subcutaneous, oral, inhalation, transdermal (topical),
transmucosal, and rectal administration.
[0291] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo as by transcription or translation of
polynucleotides that have been exogenously introduced into a
subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[0292] Solutions or suspensions used for parenteral, intradermal,
or subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylene diamine tetra acetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampules, disposable syringes or multiple dose vials made of glass
or plastic.
[0293] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where the therapeutic agents are
water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersion. For intravenous administration, suitable carriers
include physiological saline, bacteriostatic water, Cremophor
EL.RTM. (BASF, Parsippany, N.J.) or phosphate buffered saline
(PBS). In all cases, the composition must be sterile and should be
fluid to the extent that easy syringability exists. It should be
stable under the conditions of manufacture and storage and should
be preserved against the contaminating action of microorganisms
such as bacteria and fungi. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms can be achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, polyalcohols such as mannitol, sorbitol, sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0294] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g.,
undercarboxylated/uncarboxylated osteocalcin protein or
anti-OST-PTP antibody) in the required amount in an appropriate
solvent with one or a combination of the ingredients enumerated
above, as required, followed by filter sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0295] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. Depending on the specific conditions being
treated, pharmaceutical compositions of the present invention for
treatment of atherosclerosis or the other elements of metabolic
syndrome can be formulated and administered systemically or
locally. Techniques for formulation and administration can be found
in "Remington: The Science and Practice of Pharmacy" (20.sup.th
edition, Gennaro (ed.) and Gennaro, Lippincott, Williams &
Wilkins, 2000). For oral administration, the agent can be contained
in enteric forms to survive the stomach or further coated or mixed
to be released in a particular region of the GI tract by known
methods. For the purpose of oral therapeutic administration, the
active compound can be incorporated with excipients and used in the
form of tablets, troches, or capsules. Oral compositions can also
be prepared using a fluid carrier for use as a mouthwash, wherein
the compound in the fluid carrier is applied orally and swished and
expectorated or swallowed. Pharmaceutically compatible binding
agents, and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, PRIMOGEL.RTM., or corn
starch; a lubricant such as magnesium stearate or STEROTES.RTM.; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0296] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser, which contains a suitable propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
[0297] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0298] If appropriate, the compounds can also be prepared in the
form of suppositories (e.g., with conventional suppository bases
such as cocoa butter and other glycerides) or retention enemas for
rectal delivery.
[0299] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to particular cells with, e.g., monoclonal antibodies) can
also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811.
[0300] It is especially advantageous to formulate oral or
parenteral compositions in unit dosage form for ease of
administration and uniformity of dosage. "Unit dosage form" as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the unit dosage forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0301] As previously noted, the agent may be administered
continuously by pump or frequently during the day for extended
periods of time. In certain embodiments, the agent may be
administered at a rate of from about 0.3-100 ng/hour, preferably
about 1-75 ng/hour, more preferably about 5-50 ng/hour, and even
more preferably about 10-30 ng/hour. The agent may be administered
at a rate of from about 0.1-100 pg/hr, preferably about 1-75
.mu.g/hr, more preferably about 5-50 .mu.g/hr, and even more
preferably about 10-30 .mu.g/hr It will also be appreciated that
the effective dosage of antibody, protein, or polypeptide used for
treatment may increase or decrease over the course of a particular
treatment. Changes in dosage may result and become apparent from
monitoring the level of undercarboxylated/uncarboxylated
osteocalcin and/or adiponectin and/or insulin and/or monitoring
glycemia control in a biological sample, preferably blood or
serum.
[0302] In an embodiment of the invention, the agent can be
delivered by subcutaneous, long-term, automated drug delivery using
an osmotic pump to infuse a desired dose of the agent for a desired
time. Insulin pumps are widely available and are used by diabetics
to automatically deliver insulin over extended periods of time.
Such insulin pumps can be adapted to deliver the agent. The
delivery rate of the agent to control glucose intolerance, diabetes
types 1 or 2 can be readily adjusted through a large range to
accommodate changing insulin requirements of an individual (e.g.,
basal rates and bolus doses). New pumps permit a periodic dosing
manner, i.e., liquid is delivered in periodic discrete doses of a
small fixed volume rather than in a continuous flow manner. The
overall liquid delivery rate for the device is controlled and
adjusted by controlling and adjusting the dosing period. The pump
can be coupled with a continuous blood glucose monitoring device
and remote unit, such as a system described in U.S. Pat. No.
6,560,471, entitled "Analyte Monitoring Device and Methods of Use."
In such an arrangement, the hand-held remote unit that controls the
continuous blood glucose monitoring device could wirelessly
communicate with and control both the blood glucose monitoring unit
and the fluid delivery device delivering therapeutic agents of the
present invention.
[0303] A "therapeutically effective amount" of a protein or
polypeptide, small moleclue or nucleic acid is an amount that
achieves the desired therapeutic result. For example, if a
therapeutic agent is administered to treat or prevent
atherosclerosis, a therapeutically effective amount is an amount
that ameliorates one or more symptoms of the disease, or produces
at least one effect selected from the group consisting of a
reduction of oxidized phospholipids, a regression of
atherosclerotic plaques, a decrease in inflammatory protein
biosynthesis, a reduction in plasma cholesterol, a reduction in
vascular smooth muscle cell (VSMC) proliferation and number, a
decrease in the thickness of arterial plaque, a reduction in
clinical events such as heart attack, angina, or stroke, a decrease
in hypertension, and the like. If a therapeutic agent is used for
treating or preventing metabolic syndrome in an animal (including
mammals, including humans and laboratory animals) a therapeutically
effective amount is an amount that produces at least one effect
selected from the group consisting of increasing pancreatic
beta-cell proliferation, increasing insulin secretion, increasing
insulin sensitivity, increasing glucose tolerance, decreasing
weight gain, weight loss, decreasing fat mass, increasing serum
adiponectin, and an a decrease in or improved control of
atherosclerosis.
[0304] A therapeutically effective amount of protein or
polypeptide, small molecule or nucleic acid for use in the present
invention typically varies and can be an amount sufficient to
achieve serum therapeutic agent levels typically of between about 1
nanogram per milliliter and about 10 micrograms per milliliter in
the subject, or an amount sufficient to achieve serum therapeutic
agent levels of between about 1 nanogram per milliliter and about 7
micrograms per milliliter in the subject. Other preferred serum
therapeutic agent levels include about 0.1 nanogram per milliliter
to about 3 micrograms per milliliter, about 0.5 nanograms per
milliliter to about 1 microgram per milliliter, about 1 nanogram
per milliliter to about 750 nanograms per milliliter, about 5
nanograms per milliliter to about 500 nanograms per milliliter, and
about 5 nanograms per milliliter to about 100 nanograms per
milliliter.
[0305] Expressed as a daily dose, this amount can be between about
0.1 nanograms per kilogram body weight per day and about 20
milligrams per kilogram body weight per day, and between about 1
nanogram per kilogram body weight per day and about 10 milligrams
per kilogram body weight per day. Other preferred daily dosages
include about 1 nanogram per kilogram body weight per day to about
20 milligrams per kilogram body weight per day, about 5 nanograms
per kilogram body weight per day to about 5 milligrams per kilogram
body weight per day, about 20 nanograms per kilogram body weight
per day to about 500 micrograms per kilogram body weight per day,
and about 500 nanograms per kilogram body weight per day to about
100 micrograms per kilogram body weight per day. However, the
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the condition, previous treatments,
the general health and/or age of the subject, and other disorders
or diseases present.
[0306] In certain embodiments, the pharmaceutical compositions of
the present invention comprise about 0.1 mg to 5 g, about 0.5 mg to
about 1 g, about 1 mg to about 750 mg, about 5 mg to about 500 mg,
or about 10 mg to about 100 mg of therapeutic agent.
[0307] Moreover, treatment of a subject with a therapeutically
effective amount of a protein, polypeptide, nucleotide or antibody
can include a single treatment or, preferably, can include a series
of treatments.
[0308] In certain embodiments, treatment of a subject with
undercarboxylated/uncarboxylated osteocalcin leads to
undercarboxylated/uncarboxylated osteocalcin being about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, or about 50% of the total osteocalcin in the blood of the
patient.
[0309] It is understood that the appropriate dose of a small
molecule agent depends upon a number of factors within the ken of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, and the effect which the
practitioner desires the small molecule to have. It is furthermore
understood that appropriate doses of a small molecule depend upon
the potency of the small molecule with respect to the expression or
activity to be modulated. Such appropriate doses may be determined
using the assays described herein. When one or more of these small
molecules is to be administered to an animal (e.g., a human) in
order to modulate expression or activity of OST-PTP or
gamma-carboxylase, a relatively low dose may be prescribed at
first, with the dose subsequently increased until an appropriate
response is obtained. In addition, it is understood that the
specific dose level for any particular subject will depend upon a
variety of factors including the activity of the specific compound
employed, the age, body weight, general health, gender, and diet of
the subject, the time of administration, the route of
administration, the rate of excretion, any drug combination, and
the degree of expression or activity to be modulated.
[0310] For atherosclerosis prevention or treatment, a suitable
subject can be an individual who is suspected of having, has been
diagnosed as having, or is at risk of developing atherosclerosis,
coronary artery disease, stroke, restenosis, vascular fibromuscular
dysplasia, polyarteritis nodosa, Takayasu's arteritis, and like
conditions as can be determined by one knowledgeable in the art.
Another example of a suitable subject is an individual who is to
undergo vascular surgery, including but not limited to vascular
bypass surgery, atherectomy, endatherectomy, laser ablation,
angioplasty, balloon angioplasty, cardiac allograft (cardiac
transplant), insertion of a prosthesis, insertion of a graft,
insertion of a stent, catheterization, or arterial blockage
evaluation. Suitable routes of administration can include oral,
intestinal, parenteral, transmucosal, transdermal, intramuscular,
subcutaneous, transdermal, rectal, intramedullary, intrathecal,
intravenous, intraventricular, intraatrial, intraaortal,
intraarterial, or intraperitoneal administration. The
pharmaceutical compositions of the present invention can be
administered to the subject by a medical device, such as, but not
limited to, catheters, balloons, implantable devices, biodegradable
implants, prostheses, grafts, sutures, patches, shunts, or stents.
For atherosclerosis, pharmaceutical compositions of the present
invention can contain a therapeutic agent in an amount sufficient
to prevent/slow down the development of atherosclerotic lesions. In
one preferred embodiment, the therapeutic agent (e.g.,
undercarboxylated/uncarboxylated osteocalcin) can be coated on a
stent for localized administration to the target area. In this
situation a slow release preparation of
undercarboxylated/uncarboxylated osteocalcin, for example, is
preferred.
[0311] The compounds of the invention may also be admixed,
encapsulated, conjugated or otherwise associated with other
molecules, molecule structures or mixtures of compounds, as for
example, liposomes, receptor targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption assisting formulations include, but are not
limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.
[0312] In yet another aspect of the invention,
undercarboxylated/uncarboxylated osteocalcin is administered as a
pharmaceutical composition with a pharmaceutically acceptable
excipient. Exemplary pharmaceutical compositions for
undercarboxylated/uncarboxylated osteocalcin include injections as
solutions or injections as injectable self-setting or self-gelling
mineral polymer hybrids. Undercarboxylated/uncarboxylated
osteocalcin may be administered using a porous crystalline
biomimetic bioactive composition of calcium phosphate. See U.S.
Pat. Nos. 5,830,682; 6,514,514; 6,511,958 and U.S. Pat. Pub. No.:
2006/0063699; 2006/0052327; 2003/199615; 2003/0158302;
2004/0157864; 2006/0292670; 2007/0099831 and 2006/0257492, all of
which are incorporated herein in their entirety by reference.
Methods of Treatment
[0313] The present invention provides methods for modulating energy
metabolism through the OST-PTP signaling pathway for treating or
preventing a variety of different disorders relating to the OST-PTP
signaling pathway involving gamma-carboxylase, osteocalcin, insulin
and adiponectin. In particular, the methods are used to inhibit
OST-PTP phosphorylase activity, reduce gamma-carboxylase activity,
and/or increase undercarboxylated/uncarboxylated osteocalcin.
According to the invention, the methods provide an amount of an
agent effective to treat or prevent a disorder associated with the
OST-PTP signaling pathway. The agent may be selected from the group
consisting of small molecules, antibodies and nucleic acids. Such
disorders include, but are not limited to, metabolic syndrome,
glucose intolerance, diabetes type 1, diabetes type 2,
atherosclerosis, and/or obesity.
[0314] In certain embodiments, the methods comprise identifying a
patient in need of treatment or prevention of metabolic syndrome,
glucose intolerance, diabetes type 1, diabetes type 2, vascular
disorders (such as atherosclerosis), and/or obesity and then
applying the methods disclosed herein to the patient.
[0315] Vascular disorders include atherosclerosis, arteriosclerosis
and arteriolosclerosis. Arteriosclerosis, which means hardening
(sclerosis) of the arteries (arterio-), is a general term for
several diseases in which the wall of an artery becomes thicker and
less elastic. There are three types: atherosclerosis,
arteriolosclerosis, and Monckeberg's arteriosclerosis.
Atherosclerosis, the most common type, means hardening related to
atheromas, which are deposits of fatty materials. It affects
medium-sized and large arteries of the brain, heart, kidneys, other
vital organs, and legs. It is the most important and most common
type of arteriosclerosis, a general term for several diseases in
which the wall of an artery becomes thicker and less elastic.
[0316] Type 1 diabetes is usually diagnosed in children and young
adults, and was previously known as juvenile diabetes. In type 1
diabetes, the body does not produce insulin. Insulin is a hormone
that is needed to convert sugar (glucose), starches and other food
into energy needed for daily life. Conditions associated with type
1 diabetes include hyperglycemia, hypoglycemia, ketoacidosis and
celiac disease.
[0317] Type 2 diabetes is the most common form of diabetes. In type
2 diabetes, either the body does not produce enough insulin or the
cells ignore the insulin. Conditions associated with type 2
diabetes include hyperglycemia and hypoglycemia.
[0318] Disorders associated with energy metabolism include
diabetes, glucose intolerance, decreased insulin sensitivity,
decreased pancreatic beta-cell proliferation, decreased insulin
secretion, weight gain, increased fat mass and decreased serum
adiponectin
[0319] The methods of the invention increase serum osteocalcin
levels (preferably undercarboxylated/uncarboxylated osteocalcin),
serum adiponectin levels and/or serum insulin levels. The
pharmaceutical compositions may also increase glucose tolerance,
increase insulin sensitivity, and/or increase pancreatic beta-cell
proliferation.
[0320] As used herein, the terms "animal," "patient," or "subject"
include mammals, e.g., humans, dogs, cows, horses, kangaroos, pigs,
sheep, goats, cats, mice, rabbits, rats, and transgenic non-human
animals. The preferred animal, patient, or subject is a human.
[0321] In certain embodiments, the methods comprise administering
undercarboxylated/uncarboxylated osteocalcin to a patient,
preferably a human. In a specific embodiment of the invention, the
daily dose of undercarboxylated/uncarboxylated osteocalcin is
between about 0.1 nanograms per kilogram body weight per day and
about 20 milligrams per kilogram body weight per day, or between
about 1 nanogram per kilogram body weight per day and about 10
milligrams per kilogram body weight per day. Other preferred daily
dosages include about 1 nanogram per kilogram body weight per day
to about 20 milligrams per kilogram body weight per day, about 5
nanograms per kilogram body weight per day to about 5 milligrams
per kilogram body weight per day, about 20 nanograms per kilogram
body weight per day to about 500 micrograms per kilogram body
weight per day, and about 500 nanograms per kilogram body weight
per day to about 100 micrograms per kilogram body weight per
day.
[0322] In some embodiments, the daily dosage is about 0.1
milligrams per kilogram body weight per day, about 0.25 milligrams
per kilogram body weight per day, about 0.5 milligrams per kilogram
body weight per day, about 0.75 milligrams per kilogram body weight
per day, about 1 milligram per kilogram body weight per day, about
2 milligrams per kilogram body weight per day, about 5 milligrams
per kilogram body weight per day, about 10 milligrams per kilogram
body weight per day, or about 20 milligrams per kilogram body
weight per day.
[0323] In one embodiment of the invention, the method of treatment
comprises administering to a patient in need thereof a
therapeutically effective amount of
undercarboxylated/uncarboxylated osteocalcin sufficient to raise
the patient's blood level of undercarboxylated/uncarboxylated
osteocalcin compared to the pretreatment patient level. Preferably,
the patient is a human. In another embodiment, the method of
treatment comprises administering to a patient in need thereof a
therapeutically effective amount of
undercarboxylated/uncarboxylated osteocalcin sufficient to raise
the ratio of undercarboxylated/uncarboxylated osteocalcin to total
osteocalcin in the patient's blood compared to the pretreatment
patient ratio.
[0324] In another aspect of the invention, a method is provided for
treating or preventing metabolic syndrome in an animal comprising
administering to an animal in need thereof
undercarboxylated/uncarboxylated osteocalcin in a therapeutically
effective amount that produces at least one effect selected from
the group consisting of increasing pancreatic beta-cell
proliferation, increasing insulin secretion, increasing insulin
sensitivity, increasing glucose tolerance, decreasing weight gain,
decreasing fat mass, weight loss and increasing serum adiponectin
compared to pretreatment levels. Alternatively, the
undercarboxylated/uncarboxylated osteocalcin is administered in an
amount that produces at least one effect selected from the group
consisting of a reduction of oxidized phospholipids, a regression
of atherosclerotic plaques, a decrease in inflammatory protein
biosynthesis, a reduction in plasma cholesterol, a reduction in
vascular smooth muscle cell (VSMC) proliferation and number, a
decrease in the thickness of arterial plaque, a reduction in
clinical events such as heart attack, angina, or stroke, and a
decrease in hypertension as compared to pretreatment levels.
Preferably, the patient is a human.
[0325] Insulin sensitivity can be measured by the insulin tolerance
test or euglycemic hyperinsulinemic clamp. Glucose tolerance can be
measured by glucose tolerance tests. Insulin secretion can be
measured by the glucose stimulated insulin secretion test. The most
common test for glycemic control in diabetic patients is a blood
glucose test done typically by using test strips and one drop of
blood. To better monitor the level of glycemic control over time,
one can measure hemoglobin A1c (glycosylated hemoglobin).
[0326] In a specific embodiment of the invention, a method is
provided for treating or preventing type 1 or type 2 diabetes or
glucose intolerance in an animal comprising administering to an
animal in need thereof undercarboxylated/uncarboxylated osteocalcin
in a therapeutically effective amount that produces at least one
effect selected from the group comprising increasing pancreatic
beta-cell proliferation, increasing insulin secretion, increasing
insulin sensitivity, increasing glucose tolerance, decreasing
weight gain, decreasing fat mass, weight loss, and increasing serum
adiponectin compared to pretreatment levels. Preferably, the
patient is a human. In an embodiment of the invention, said method
further comprises co-administration of anti-diabetic drugs such as
insulin (preferably recombinant human insulin), incretins,
sulfonylureas, meglitinides, D-Phenylalanine Derivatives
(nateglinides), biguanides, thiazolidinediones, alpha-glucose
inhibitors,GLP-1, GLP-1 analogues such as liraglutide, exendin-4
LY5448806 and CJC-1131, as well as dipeptidyl peptidase IV
inhibitors.
[0327] Sulfonylureas are exemplified by glimepiride, glyburide,
chlorpropamide, acetohexamide, glipizide, tolbutamide, and
tolazamide. Meglitinides are exemplified by Repaglinide. Biguanides
are exemplified by metformin and metformin hydrochloride.
Thiazolidinediones are exemplified by pioglitazone and
rosiglitazone. Alpha-glucose inhibitors are exemplified by miglitol
and acarbose. Dipeptidyl peptidase 4 (DPP4) inhibitors are
exemplified by vildagliptin, sitagliptin and saxagliptin.
[0328] Generally, there are six categories of insulins:
rapid-acting, short-acting, intermediate acting, long acting, very
long acting, and premixed. Incretins are a type of gastrointestinal
hormone that cause an increase in the amount of insulin released
from the beta-cells of the islets of Langerhans after eating, even
before blood glucose levels become elevated. Incretins are
exemplified by glucagon-like peptide-1 (GLP-1) and Gastric
inhibitory peptide (aka glucose-dependent Insulinotropic peptide or
GIP).
[0329] The present invention further provides (i) methods for
treating or preventing obesity in an animal comprising
administering to an animal in need thereof
undercarboxylated/uncarboxylated osteocalcin in a therapeutically
effective amount that decreases weight gain, decreases fat mass or
results in loss of weight; (ii) methods for increasing insulin
sensitivity in an animal comprising administering to an animal in
need thereof undercarboxylated/uncarboxylated osteocalcin in a
therapeutically effective amount that increases insulin
sensitivity; (iii) methods for increasing glucose tolerance in an
animal comprising administering to an animal in need thereof
undercarboxylated/uncarboxylated osteocalcin in a therapeutically
effective amount that increases glucose tolerance; (iv) methods for
increasing insulin secretion in an animal comprising administering
to an animal in need thereof undercarboxylated/uncarboxylated
osteocalcin in a therapeutically effective amount that increases
insulin secretion; and (v) methods for increasing beta-cell
proliferation in an animal comprising administering to an animal in
need thereof undercarboxylated/uncarboxylated osteocalcin in a
therapeutically effective amount that increases beta-cell
proliferation. Preferably, the animal is a human.
[0330] In another aspect of the invention, a method is provided for
treating or preventing atherosclerosis in an animal comprising
administering to an animal in need thereof
undercarboxylated/uncarboxylated osteocalcin in a therapeutically
effective amount that produces at least one effect selected from
the group consisting of a reduction of oxidized phospholipids, a
regression of atherosclerotic plaques, a decrease in inflammatory
protein biosynthesis, a reduction in plasma cholesterol, a
reduction in vascular smooth muscle cell (VSMC) proliferation and
number, a decrease in the thickness of arterial plaque, a reduction
in clinical events such as heart attack, angina, or stroke, and a
decrease in hypertension. Preferably, the animal is a human. In an
embodiment of the invention, said method further comprises
co-administration of compounds used to treat atherosclerosis such
as low density lipoprotein peroxidation inhibitors,
antihyperlipidemic agents, anticoagulants, vasodilators, and other
drugs useful in the treatment of atherosclerosis.
[0331] Anticoagulants are exemplified by vitamin K antagonists,
heparin and derivatives of heparin, and direct thrombin inhibitors.
Vitamin K antagonists are exemplified by warfarin (also known under
the brand names COUMADIN.RTM., JANTOVEN.RTM., MAREVAN.RTM., and
WARAN.RTM.); acenocoumarol, phenprocoumon as well as phenindione.
Heparin and derivatives of heparin are exemplified by low molecular
weight heparin and fondaparinux. Direct thrombin inhibitors are
exemplified by argatroban, lepirudin, bivalirudin and
ximelagatran.
[0332] Vasodilators are exemplified by adenosine, amyl nitrite and
other nitrites, 1-arginine, atrial natriuretic peptide (ANP),
bradykinin, ethanol, endothelium-derived hyperpolarizing factor
(EDHF), histamine, complement proteins C3a, C4a and C5a, niacin
(nicotinic acid), nitric oxide, glyceryl trinitrate (commonly known
as nitroglycerin), isosorbide mononitrate & isosorbide
dinitrate, pentaerythritol tetranitrate (PETN),
sodium_nitroprusside, PDE5 inhibitors, sildenafil, tadalafil,
vardenafil, platelet activating factor (PAF), prostacyclin
(PGI.sub.2) as well as other prostaglandins, tetrahydrocannabinol
(THC), theobromine, and papaverine.
[0333] Other drugs useful in the treatment of atherosclerosis are
exemplified by statins, scilostazol, benzothiazepines,
phenylalkylamines, dihydropyridines, epoprostenol, vitamin B3, and
aspirin. Statins are further exemplified by atorvastatin,
cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,
pravastatin, rosuvastatin, and simvastatin. Benzothiazepines are
exemplified by diltiazem. Phenylalkylamines are exemplified by
verapamil. Dihydropyridines are exemplified by amlodipine,
felodipine, isradipine, lacidipine, lercanidipine, nicardipine,
nifedipine, nimodipine, nisoldipine, and nitrendipine.
[0334] In yet another embodiment of the invention, when
administering undercarboxylated/uncarboxylated osteocalcin, it may
be desirable to co-administered drugs normally administered to
treat osteoporosis. Such drugs include, for example, raloxifene,
calcitonin and alendronate.
[0335] In methods wherein adiponectin is co-administered with
undercarboxylated/uncarboxylated osteocalcin, the adiponectin and
undercarboxylated/uncarboxylated osteocalcin may be administered in
a single pharmaceutical composition. Alternatively, the
undercarboxylated/uncarboxylated osteocalcin and adiponectin may be
administered in separate pharmaceutical compositions. In another
embodiment of the invention, adiponectin and
undercarboxylated/uncarboxylated osteocalcin are administered on
the same day. In yet another embodiment, the adiponectin and
undercarboxylated/uncarboxylated osteocalcin are administered on
different days.
[0336] In an embodiment of the invention, a method is provided for
treating or preventing a disease associated with low levels of
serum osteocalcin in a patient comprising administering to a
patient in need thereof an amount of a beta blocker or a vitamin K
blocker or a combination thereof that causes an increase in the
level of serum osteocalcin compared to pretreatment levels.
Preferably, the patient is a human and the serum level of
undercarboxylated/uncarboxylated osteocalcin is increased.
[0337] The present invention further provides a method of treating
or preventing a disease including, but not limited to, metabolic
syndrome, glucose intolerance, diabetes types 1 and 2,
atherosclerosis and obesity in a patient comprising administering
to a patient in need thereof a therapeutically effective amount of
an agent that reduces OST-PTP expression or activity in osteoblasts
sufficient to raise the patient's level of
undercarboxylated/uncarboxylated osteocalcin. Preferably, the
patient is a human.
[0338] The invention further provides a method of treating or
preventing a disease selected from the group consisting of
metabolic syndrome, glucose intolerance, diabetes types 1 and 2,
atherosclerosis and obesity comprising administering to a patient
in need thereof a therapeutically effective amount of an agent that
reduces gamma-carboxylase expression or activity in osteoblasts
sufficient to raise the patient level of
undercarboxylated/uncarboxylated osteocalcin. Preferably, the
patient is a human. Preferably, the patient is a human.
[0339] In an embodiment of the invention, a method is provided for
treating or preventing metabolic syndrome in a patient comprising
administering to a patient in need thereof a therapeutically
effective amount of an agent that reduces OST-PTP expression or
activity in osteoblasts sufficient to produce at least one effect
selected from the group consisting of increasing pancreatic
beta-cell proliferation, increasing insulin secretion, increasing
insulin sensitivity, increasing glucose tolerance, decreasing
weight gain, decreasing fat mass, weight loss, and increasing serum
adiponectin, or which decreases or controls atherosclerosis
compared to pretreatment levels. Preferably, the patient is a
human.
[0340] In another aspect of the invention, a method is provided for
treating or preventing type 1 or type 2 diabetes or glucose
,intolerance, in an animal comprising administering to an animal in
need thereof adiponectin in a therapeutically effective amount so
as to reduce OST-PTP expression or activity in osteoblasts
sufficient to produce at least one effect selected from the group
consisting of increasing pancreatic beta-cell proliferation,
increasing insulin secretion, increasing insulin sensitivity,
increasing glucose tolerance, decreasing weight gain, and
increasing serum adiponectin compared to pretreatment levels.
Preferably, the animal is a human.
[0341] A method is provided for treating or preventing
atherosclerosis in an animal comprising administering to an animal
in need thereof in a therapeutically effective amount an agent that
reduces OST-PTP expression or activity in osteoblasts sufficient to
produce at least one effect selected from the group consisting of a
reduction of oxidized phospholipids, a regression of
atherosclerotic plaques, a decrease in inflammatory protein
biosynthesis, a reduction in plasma cholesterol, a reduction in
vascular smooth muscle cell (VSMC) proliferation and number, a
decrease in the thickness of arterial plaque, a reduction in
clinical events such as heart attack, angina, or stroke, and a
decrease in hypertension compared to pretreatment levels.
Preferably, the animal is a human.
[0342] In different embodiments of the invention, methods are
provided (i) for treating or preventing obesity in an animal
comprising administering to an animal in need thereof in a
therapeutically effective amount an agent that reduces OST-PTP
expression or activity in osteoblasts sufficient to decrease weight
gain, decrease fat mass or result in weight loss compared to
pretreatment levels; (ii) for treating or preventing glucose
intolerance in an animal comprising administering to an animal in
need thereof in a therapeutically effective amount an agent that
reduces OST-PTP expression or activity in osteoblasts sufficient to
increase glucose tolerance compared to pretreatment levels; (iii)
for increasing insulin sensitivity in an animal comprising
administering to an animal in need thereof in a therapeutically
effective amount an agent that reduces OST-PTP expression or
activity in osteoblasts sufficient to increase insulin sensitivity
compared to pretreatment levels. Preferably, the animal is a
human.
[0343] In another aspect of the invention, a method is provided for
treating or preventing metabolic syndrome in an animal comprising
administering to an animal in need thereof in a therapeutically
effective amount an agent that reduces gamma-carboxylase expression
or activity in osteoblasts sufficient to (1) produce at least one
effect selected from the group consisting of increasing pancreatic
beta-cell proliferation, increasing insulin secretion, increasing
insulin sensitivity, increasing glucose tolerance, decreasing
weight gain, decreasing fat mass, weight loss and increasing serum
adiponectin compared to pretreatment levels, or (2) to produce at
least one effect selected from the group consisting of a reduction
of oxidized phospholipids, a regression of atherosclerotic plaques,
a decrease in inflammatory protein biosynthesis, a reduction in
plasma cholesterol, a reduction in vascular smooth muscle cell
(VSMC) proliferation and number, a decrease in the thickness of
arterial plaque, a reduction in clinical events such as heart
attack, angina, or stroke, and a decrease in hypertension compared
to pretreatment levels. Preferably, the animal is a human.
[0344] In yet another embodiment of the invention, method for is
provided for treating or preventing type 1 or type 2 diabetes or
glucose intolerance in an animal comprising administering to an
animal in need thereof in a therapeutically effective amount an
agent that reduces gamma-carboxylase expression or activity in
osteoblasts sufficient to produce at least one effect selected from
the group consisting of increasing pancreatic beta-cell
proliferation, increasing insulin secretion, increasing insulin
sensitivity, increasing glucose tolerance, decreasing weight gain,
decreasing fat mass, weight loss and increasing serum adiponectin
compared to pretreatment levels. Preferably, the animal is a
human.
[0345] A method is also provided for treating or preventing
atherosclerosis in an animal comprising administering to an animal
in need thereof in a therapeutically effective amount an agent that
reduces gamma-carboxylase expression or activity in osteoblasts
sufficient to produce at least one effect selected from the group
consisting of a reduction of oxidized phospholipids, a regression
of atherosclerotic plaques, a decrease in inflammatory protein
biosynthesis, a reduction in plasma cholesterol, a reduction in
vascular smooth muscle cell (VSMC) proliferation and number, a
decrease in the thickness of arterial plaque, a reduction in
clinical events such as heart attack, angina, or stroke, and a
decrease in hypertension compared to pretreatment levels.
Preferably, the animal is a human.
[0346] The present invention is directed to methods (i) for
treating or preventing obesity in an animal comprising
administering to an animal in need thereof in a therapeutically
effective amount an agent that reduces gamma-carboxylase expression
or activity in osteoblasts sufficient to cause a decrease in weight
gain, a decrease in fat mass, or weight loss compared to
pretreatment levels (ii) for treating or preventing glucose
intolerance in an animal comprising administering to an animal in
need thereof in a therapeutically effective amount an agent that
reduces gamma-carboxylase expression or activity in osteoblasts
sufficient to increase glucose tolerance compared to pretreatment
levels or (iii) for increasing insulin sensitivity in an animal
comprising administering to an animal in need thereof in a
therapeutically effective amount an agent that reduces
gamma-carboxylase expression or activity in osteoblasts sufficient
to increase insulin sensitivity compared to pretreatment levels.
Preferably, the animal is a human. In an embodiment of the
invention, the agent is an isolated nucleic acid that is selected
from the group consisting of cDNA, antisense DNA, antisense RNA,
and small interfering RNA, which nucleic acid is sufficiently
complementary to the gene or mRNA encoding gamma-carboxylase to
permit specific hybridization to the gene or mRNA, and wherein the
hybridization prevents or reduces expression of gamma-carboxylase
in osteoblasts. In another embodiment of the invention, the nucleic
acid is conjugated to a phosphate group or other targeting ligand
to facilitate uptake by osteoblasts.
[0347] In the methods described herein, it will be understood that
"treating" a disease encompasses not only improving the disease or
its symptoms but also retarding the progression of the disease or
ameliorating the disease.
[0348] The present invention also encompasses the use of gene
therapy for treatment of metabolic syndrome, including obesity,
type 2 diabetes, glucose intolerance atherosclerosis, and type 1
diabetes. This can be accomplished by introducing a gene encoding
osteocalcin or a biologically active fragment or variant thereof
into a vector, and transfecting or infecting cells from a patient
afflicted with the disease or at a high risk of developing the
disease with the vector, according to various methods known in the
art. The cells may be transfected or infected by ex vivo or by in
vivo methods.
[0349] Adeno-associated virus (AAV) is one of the most promising
vectors for gene therapy and may be used in the methods of the
present invention. Conventional methods of gene transfer and gene
therapy are described in, e.g., Gene Therapy: Principles and
Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene
Therapy Protocols (Methods in Molecular Medicine), ed. P. D.
Robbins, Humana Press, 1997; and Retro-vectors for Human Gene
Therapy, ed. C. P. Hodgson, Springer Verlag, 1996. AAV is an
attractive vector system for human gene therapy because it is
non-pathogenic for humans, it has a high frequency of integration,
and it can infect non-dividing cells, thus making it useful for
delivery of genes into mammalian cells both in tissue culture and
in whole animals. Muzyczka, Curr. Top. Microbiol. Immunol.,
158:97-129, 1992. Recent studies have demonstrated AAV to be a
potentially useful vector for gene delivery. LaFace et al.,
Viology, 162:483-486, 1998; Zhou et al., Exp. Hematol. (NY),
21:928-933, 1993; Flotte et al., PNAS 90:10613-10617, 1993; and
Walsh et al., Blood 84:1492-1500, 1994. Recombinant AAV vectors
have been used successfully for in vitro and in vivo transduction
of marker genes (Kaplitt et al., Nature Genetics, 8:148-154, 1994;
Lebkowski et al., Mol. Cell. Biol. 8:3988-3996, 1988; Samulski et
al., J. Virol., 63:3822-3828, 1989; Shelling, A. N., and Smith, M.
G., Gene Therapy, 1:165-169, 1994; Yoder et al., Blood, 82:suppl.
1:347A, 1994; Zhou et al., J. Exp. Med., 179:1867-1875, 1994;
Hermonat, P. L. and Muzyczka, N., Proc. Natl. Acad. Sci. USA.,
81:6466-6470, 1984; Tratschin et al., Mol. Cell. Biol.,
4:2072-2081, 1984; McLaughlin et al., J. Virol., 62:1963-1973,
1988) as well as genes involved in human diseases (Flotte et al.,
Am. J. Respir. Cell Mol. Biol., 7:349-356, 1992; Luo et al., Blood,
82:suppl. 1,303A, 1994; Ohi et al., Gene, 89L:27914 282, 1990;
Walsh et al., PNAS 89:7257-7261, 1992; Wei et al., Gene Therapy,
1:261-268, 1994).
[0350] In certain other embodiments, the gene of interest (e.g.,
osteocalcin) can be transferred into a target cell using a
retroviral vector. Retroviruses refer to viruses that belong to the
Retroviridae family, and include oncoviruses, foamy viruses
(Russell, D. W. and Miller, A. D., J. Virol. 1996, 70:217-222; Wu,
M. et al., J. Virol. 1999, 73:4498-4501), and lentiviruses (for
example, HIV-1 (Naldini, L. et al., Science 1996, 272:263-267;
Poeschla, E. et al., Proc. Natl. Acad. Sci. USA 1996,
93:11395-11399; Srinivasakumar, N. et al., J. Virol. 1997,
71:5841-5848; Zufferey, R., et al. Nat. Biotechnol. 1997,
15:871-875; Kim, V. N., et al., J. Virol. 1998,72:811-816) and
feline immunodeficiency virus (Johnston, J. C. et al., J. Virol.
1999, 73:4991-5000; Johnston, J. and Power, C., J. Virol. 1999,
73:2491-2498; Poeschla, E. M. et al., Nat. Med. 1998, 4:354-357)).
Numerous gene therapy methods that take advantage of retroviral
vectors for treating a wide variety of diseases are well-known in
the art (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764;
Friedmann, 1989, Science, 244:1275-1281; Mulligan, 1993, Science,
260:926-932, R. Crystal, 1995, Science 270:404-410, and U.S. Pat.
No. 6,899,871, Kasahara, et al. each of which are incorporated
herein by reference in their entirety). An increasing number of
these methods are currently being applied in human clinical trials
(Morgan, R., 1993, BioPharm, 6(1):32-35; see also The Development
of Human Gene Therapy, Theodore Friedmann, Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1999. ISBN
0-87969-528-5, which is incorporated herein by reference in its
entirety).
[0351] Efficacy of the methods of treatment described herein can be
monitored by determining whether the methods ameliorate any of the
symptoms of the disease being treated. Alternatively, one can
monitor the level of serum undercarboxylated/uncarboxylated
osteocalcin (either in absolute terms or as a ratio of
undercarboxylated/uncarboxylated osteocalcin/total osteocalcin),
and/or serum adiponectin, and/or serum insulin, which levels should
increase in response to therapy. Alternatively efficacy can be
measured by monitoring glycemia in the subject being treated.
Diagnostics
[0352] The present invention provides methods and compositions for
diagnosing disorders such as those associated with decreased levels
of undercarboxylated/uncarboxylated osteocalcin. Such disorders
include, but are not limited to, metabolic syndrome, glucose
intolerance, diabetes types 1 and 2, atherosclerosis and
obesity.
[0353] In a specific embodiment of the invention, a method is
provided for diagnosing a patient at risk of developing glucose
intolerance or diabetes comprising: (i) determining a patient level
of undercarboxylated/uncarboxylated osteocalcin in a biological
sample taken from the patient and a control level of
undercarboxylated/uncarboxylated osteocalcin in a biological sample
taken from a subject that does not have glucose intolerance or
diabetes, (ii) comparing the patient and control levels, and (iii)
concluding that the patient is at risk of developing glucose
intolerance or diabetes if the patient level is lower than the test
level. In an embodiment of the invention, the diabetes is Type 1 or
Type 2.
[0354] "Biological samples" include solid and body fluid samples.
The biological samples of the present invention may include tissue,
organs, cells, protein or membrane extracts of cells, blood or
biological fluids such as blood, serum, ascites fluid or brain
fluid (e.g., cerebrospinal fluid).
[0355] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing glucose intolerance
or diabetes comprising: (i) determining a patient level of
undercarboxylated/uncarboxylated osteocalcin in a biological sample
taken from the patient; and (ii) comparing the patient level to a
standard level; where, if the patient level is lower than the
standard level, the patient is at risk of developing diabetes. In
instances where the method is practiced on humans, the standard
level can be a level of undercarboxylated/uncarboxylated
osteocalcin that has been previously determined to be the normal
range for people who are not at risk of developing diabetes. In
preferred embodiments, the biological sample is blood, serum,
plasma, cerebrospinal fluid, urine, a cell sample, or a tissue
sample. In an embodiment of the invention, the diabetes is Type 1
or Type 2.
[0356] A "standard level" of undercarboxylated/uncarboxylated
osteocalcin in humans can include values of 0.1 ng/ml to 10 ng/ml,
preferably 0.2 ng/ml to 7.5 ng/ml, more preferably 0.5 ng/ml to 5
ng/ml, and even more preferably 1 ng/ml to 5 ng/ml. A standard
level of undercarboxylated/uncarboxylated osteocalcin in humans can
also include about 0.1 ng/ml, about 0.5 ng/ml, about 1 ng/ml, about
2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6
ng/ml, about 7 ng/ml, or about 10 ng/ml.
[0357] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing glucose intolerance
or diabetes comprising: (i) determining the ratio of
undercarboxylated/uncarboxylated osteocalcin to total osteocalcin
in a biological sample taken from the patient; and (ii) comparing
the ratio to a standard ratio; where, if the patient ratio is lower
than the standard ratio, the patient is at risk of developing
glucose intolerance or diabetes. In certain embodiments, the
standard ratio is 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, or
30%-35%. In certain embodiments, the standard ratio is about 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
33%, 34%, or 35%. Preferably, the patient is a human. In preferred
embodiments, the biological sample is blood, serum, plasma,
cerebrospinal fluid, urine, a cell sample, or a tissue sample. In
an embodiment of the invention, the diabetes is Type 1 or Type
2.
[0358] The present invention further provides a method for
diagnosing a patient at risk of developing atherosclerosis
comprising: (i) determining a patient level of
undercarboxylated/uncarboxylated osteocalcin in a biological sample
taken from the patient and a control level of
undercarboxylated/uncarboxylated osteocalcin in a biological sample
taken from a subject that does not have atherosclerosis, (ii)
comparing the patient and control levels, and (iii) concluding that
the patient is at risk of developing atherosclerosis if the patient
level is lower than the test level.
[0359] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing atherosclerosis
comprising: (i) determining a patient level of
undercarboxylated/uncarboxylated osteocalcin in a biological sample
taken from the patient; and (ii) comparing the patient level to a
standard level; where, if the patient level is lower than the
standard level, the patient is at risk of developing
atherosclerosis. In instances where the method is practiced on
humans, the standard level can be a level of
undercarboxylated/uncarboxylated osteocalcin that has been
previously determined to be the normal range for people who are not
at risk of developing atherosclerosis. In preferred embodiments,
the biological sample is blood, serum, plasma, cerebrospinal fluid,
urine, a cell sample, or a tissue sample. Preferably, the patient
is a human.
[0360] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing atherosclerosis
comprising: (i) determining the ratio of
undercarboxylated/uncarboxylated osteocalcin to total osteocalcin
in a biological sample taken from the patient; and (ii) comparing
the ratio to a standard ratio; where, if the patient ratio is lower
than the standard ratio, the patient is at risk of developing
atherosclerosis. In certain embodiments, the standard ratio is
5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, or 30%-35%. In certain
embodiments, the standard ratio is 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,
25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%.
Preferably, the patient is a human. In preferred embodiments, the
biological sample is blood, serum, plasma, cerebrospinal fluid,
urine, a cell sample or a tissue sample.
[0361] In yet another embodiment of the invention, a method is
provided for diagnosing a patient at risk of developing metabolic
syndrome comprising: (i) determining a patient level of
undercarboxylated/uncarboxylated osteocalcin in a biological sample
taken from the patient and a control level of
undercarboxylated/uncarboxylated osteocalcin in a biological sample
taken from a subject that does not have metabolic syndrome, (ii)
comparing the patient and control levels, and (iii) concluding that
the patient is at risk of developing metabolic syndrome if the
patient level is lower than the test level.
[0362] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing metabolic syndrome
comprising: (i) determining a patient level of
undercarboxylated/uncarboxylated osteocalcin in a biological sample
taken from the patient; and (ii) comparing the patient level to a
standard level; where, if the patient level is lower than the
standard level, the patient is at risk of developing metabolic
syndrome. In instances where the method is practiced on humans, the
standard level can be a level of undercarboxylated/uncarboxylated
osteocalcin that has been previously determined to be the normal
range for people who are not at risk of developing metabolic
syndrome. In preferred embodiments, the biological sample is blood,
serum, plasma, cerebrospinal fluid, urine, a cell sample or a
tissue sample. Preferably, the patient is a human.
[0363] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing metabolic syndrome
comprising: (i) determining the ratio of
undercarboxylated/uncarboxylated osteocalcin to total osteocalcin
in a biological sample taken from the patient; and (ii) comparing
the ratio to a standard ratio; where, if the patient ratio is lower
than the standard ratio, the patient is at risk of developing
metabolic syndrome. In certain embodiments, the standard ratio is
5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, or 30%-35%. In certain
embodiments, the standard ratio is 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,
25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%.
Preferably, the patient is a human. In preferred embodiments, the
biological sample is blood, serum, plasma, cerebrospinal fluid,
urine, a cell sample or a tissue sample.
[0364] In another aspect of the invention a method for diagnosing a
patient at risk of developing obesity is provided comprising: (i)
determining a patient level of undercarboxylated/uncarboxylated
osteocalcin in a biological sample taken from the patient and a
control level of undercarboxylated/uncarboxylated osteocalcin in a
biological sample taken from a subject that does not have obesity,
(ii) comparing the patient and control levels, and (iii) concluding
that the patient is at risk of developing obesity if the patient
level is lower than the test level.
[0365] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing obesity comprising:
(i) determining a patient level of undercarboxylated/uncarboxylated
osteocalcin in a biological sample taken from the patient; and (ii)
comparing the patient level to a standard level; where, if the
patient level is lower than the standard level, the patient is at
risk of developing obesity. In instances where the method is
practiced on humans, the standard level can be a level of
undercarboxylated/uncarboxylated osteocalcin that has been
previously determined to be the normal range for people who are not
at risk of developing obesity. In preferred embodiments, the
biological sample is blood, serum, plasma, cerebrospinal fluid,
urine, a cell sample or a tissue sample. Preferably, the patient is
a human.
[0366] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing obesity comprising:
(i) determining the ratio of undercarboxylated/uncarboxylated
osteocalcin to total osteocalcin in a biological sample taken from
the patient; and (ii) comparing the ratio to a standard ratio;
where, if the patient ratio is lower than the standard ratio, the
patient is at risk of developing obesity. In certain embodiments,
the standard ratio is 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%,
or 30%-35%. In certain embodiments, the standard ratio is 5%, 6%,
7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,
21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,
34%, or 35%. Preferably, the patient is a human. In preferred
embodiments, the biological sample is blood, serum, plasma,
cerebrospinal fluid, urine, a cell sample or a tissue sample.
[0367] In yet another embodiment of the invention, a method is
provided for diagnosing a patient at risk of developing a disease
selected from the group consisting of glucose intolerance, impaired
pancreatic beta-cell proliferation, impaired insulin secretion, and
impaired insulin sensitivity comprising: (i) determining a patient
level of undercarboxylated/uncarboxylated osteocalcin in a
biological sample taken from the patient and a control level of
undercarboxylated/uncarboxylated osteocalcin in a biological sample
taken from a subject that does not have the disease, (ii) comparing
the patient and control levels, and (iii) concluding that the
patient is at risk of developing the disease if the patient level
is lower than the test level.
[0368] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing a disease selected
from the group consisting of glucose intolerance, impaired
pancreatic beta-cell proliferation, impaired insulin secretion, and
impaired insulin sensitivity comprising: (i) determining a patient
level of undercarboxylated/uncarboxylated osteocalcin in a
biological sample taken from the patient; and (ii) comparing the
patient level to a standard level; where, if the patient level is
lower than the standard level, the patient is at risk of developing
a disease selected from the group consisting of glucose
intolerance, impaired pancreatic beta-cell proliferation, impaired
insulin secretion, and impaired insulin sensitivity. In instances
where the method is practiced on humans, the standard level can be
a level of undercarboxylated/uncarboxylated osteocalcin that has
been previously determined to be the normal range for people who
are not at risk of developing a disease selected from the group
consisting of glucose intolerance, impaired pancreatic beta-cell
proliferation, impaired insulin secretion, and impaired insulin
sensitivity. In preferred embodiments, the biological sample is
blood, serum, plasma, cerebrospinal fluid, urine, a cell sample or
a tissue sample. Preferably, the patient is a human.
[0369] In another embodiment of the invention, a method is provided
for diagnosing a patient at risk of developing a disease selected
from the group consisting of glucose intolerance, impaired
pancreatic beta-cell proliferation, impaired insulin secretion, and
impaired insulin sensitivity comprising: (i) determining the ratio
of undercarboxylated/uncarboxylated osteocalcin to total
osteocalcin in a biological sample taken from the patient; and (ii)
comparing the ratio to a standard ratio; where, if the patient
ratio is lower than the standard ratio, the patient is at risk of
developing a disease selected from the group consisting of glucose
intolerance, impaired pancreatic beta-cell proliferation, impaired
insulin secretion, and impaired insulin sensitivity. In certain
embodiments, the standard ratio is 5%-10%, 10%-15%, 15%-20%,
20%-25%, 25%-30%, or 30%-35%. In certain embodiments, the standard
ratio is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,
17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
30%, 31%, 32%, 33%, 34%, or 35%. Preferably, the patient is a
human. In preferred embodiments, the biological sample is blood,
serum, plasma, cerebrospinal fluid, urine, a cell sample or a
tissue sample.
[0370] In addition to assaying for levels of
undercarboxylated/uncarboxylated osteocalcin, the present invention
also provides methods and compositions for diagnosing disorders
associated with decreased levels of adiponectin. Such disorders
include, but are not limited to, metabolic syndrome, glucose
intolerance, diabetes types 1 and 2, atherosclerosis and obesity.
In a specific embodiment of the invention, a method is provided for
diagnosing a patient at risk of developing diabetes comprising: (i)
determining a patient level of adiponectin in a biological sample
taken from the patient and a control level of adiponectin in a
biological sample taken from a subject that does not have diabetes,
(ii) comparing the patient and control levels, and (iii) concluding
that the patient is at risk of developing diabetes if the patient
level is lower than the test level.
[0371] In certain embodiments, the serum level of adiponectin and
insulin are both measured and, if the serum levels of adiponectin
and insulin in the patient are both lower than the levels in a
subject without the disease, then the patient is at risk of
developing the disease. In another embodiment, serum adiponectin
and the glycemic index for the patient are measured and, if the
serum level of adiponectin in the patient is lower than the level
in a subject without the disease and the patient also has high
glycemia, then the patient is at risk of developing the disease.
Alternatively, serum adiponectin and uncarboxylated osteocalcin can
be measured, or serum adiponectin, uncarboxylated osteocalcin and
insulin can be measured and compared to controls for diagnosis of
metabolic syndrome, its components, or type 1 diabetes.
[0372] In practicing the diagnostic methods of the invention, as
set forth above, the biological sample is selected from the group
consisting of blood, serum, plasma, cerebral spinal fluid, a cell
sample or a tissue sample. In another embodiment, the sample is
derived from a human.
[0373] Assays for detecting the levels of protein expression are
well known to those of skill in the art. Such assays include, for
example, antibody-based immunoassays. Methods for using antibodies
as disclosed herein are particularly applicable to the cells,
tissues and disorders that differentially express osteocalcin,
OST-PTP, or gamma-carboxylase or that are involved in conditions as
otherwise discussed herein. The methods use antibodies that
selectively bind to the protein of interest and its fragments or
variants. For therapeutic applications, antibodies that recognize
OST-PTP and reduce its ability to bind to or dephosphorylate
gamma-carboxylase are preferred. For diagnostic use, antibodies
against undercarboxylated/uncarboxylated osteocalcin,
gamma-carboxylase, adiponectin and vitamin K are preferred. An
antibody is considered to selectively bind even if it also binds to
other proteins that are not substantially homologous with the
protein of interest. These other proteins share homology with a
fragment or domain of the protein of interest. This conservation in
specific regions gives rise to antibodies that bind to both
proteins by virtue of the homologous sequence. In this case, it
would be understood that antibody binding to the protein of
interest is still selective. In certain embodiments, however, the
antibodies do not substantially bind to proteins other than the
protein of interest.
[0374] The amount of an antigen (e.g., osteocalcin or other protein
of interest) in a biological sample may be determined by an assay
such as a radioimmunoassay, an immunoradiometric assay, and/or an
enzyme immunoassay. A "radioimmunoassay" is a technique for
detecting and measuring the concentration of an antigen using a
labeled (e.g., radioactively labeled) form of the antigen. Examples
of radioactive labels for antigens include .sup.3H, .sup.14C, and
.sup.125I. The concentration of antigen (e.g., osteocalcin) in a
sample (e.g., biological sample) is measured by having the antigen
in the sample compete with a labeled (e.g., radioactively) antigen
for binding to an antibody to the antigen. To ensure competitive
binding between the labeled antigen and the unlabeled antigen, the
labeled antigen is present in a concentration sufficient to
saturate the binding sites of the antibody. The higher the
concentration of antigen in the sample, the lower the concentration
of labeled antigen that will bind to the antibody.
[0375] In a radioimmunoassay, to determine the concentration of
labeled antigen bound to antibody, the antigen-antibody complex
must be separated from the free antigen. One method for separating
the antigen-antibody complex from the free antigen is by
precipitating the antigen-antibody complex with an anti-isotype
antiserum. Another method for separating the antigen-antibody
complex from the free antigen is by precipitating the
antigen-antibody complex with formalin-killed S. aureus. Yet
another method for separating the antigen-antibody complex from the
free antigen is by performing a "solid-phase radioimmunoassay"
where the antibbdy is linked (e.g., covalently) to Sepharose beads,
polystyrene wells, polyvinylchloride wells, or microtiter wells. By
comparing the concentration of labeled antigen bound to antibody to
a standard curve based on samples having a known concentration of
antigen, the concentration of antigen in the biological sample can
be determined.
[0376] An "Immunoradiometric Assay" (IRMA) is an immunoassay in
which the antibody reagent is radioactively labeled. An IRMA
requires the production of a multivalent antigen conjugate, by
techniques such as conjugation to a protein, e.g., rabbit serum
albumin (RSA). The multivalent antigen conjugate must have at least
2 antigen residues per molecule and the antigen residues must be of
sufficient distance apart to allow binding by at least two
antibodies to the antigen. For example, in an IRMA the multivalent
antigen conjugate can be attached to a solid surface such as a
plastic sphere. Unlabeled "sample" antigen and antibody to antigen
which is radioactively labeled are added to a test tube containing
the multivalent antigen conjugate coated sphere. The antigen in the
sample competes with the multivalent antigen conjugate for antigen
antibody binding sites. After an appropriate incubation period, the
unbound reactants are removed by washing and the amount of
radioactivity on the solid phase is determined. The amount of bound
radioactive antibody is inversely proportional to the concentration
of antigen in the sample.
[0377] The most common enzyme immunoassay is the "Enzyme-Linked
Immunosorbent Assay (ELISA)." The "Enzyme-Linked Immunosorbent
Assay (ELISA)" is a technique for detecting and measuring the
concentration of an antigen using a labeled (e.g., enzyme linked)
form of the antibody. In a "sandwich ELISA", an antibody (e.g., to
osteocalcin) is linked to a solid phase (e.g., a microtiter plate)
and exposed to a biological sample containing antigen (e.g.,
osteocalcin). The solid phase is then washed to remove unbound
antigen. A labeled (e.g., enzyme linked) antibody is then bound to
the bound-antigen (if present) forming an antibody-antigen-antibody
sandwich. Examples of enzymes that can be linked to the antibody
are alkaline phosphatase, horseradish peroxidase, luciferase,
urease, and .beta.-galactosidase. The enzyme linked antibody reacts
with a substrate to generate a colored reaction product that can be
assayed.
[0378] In a "competitive ELISA," antibody is incubated with a
sample containing antigen (e.g., osteocalcin). The antigen-antibody
mixture is then contacted with an antigen-coated solid phase (e.g.,
a microtiter plate). The more antigen present in the sample, the
less free antibody that will be available to bind to the solid
phase. A labeled (e.g., enzyme linked) secondary antibody is then
added to the solid phase to determine the amount of primary
antibody bound to the solid phase.
[0379] In an "immunohistochemistry assay," a section of tissue is
tested for specific proteins by exposing the tissue to antibodies
that are specific for the protein that is being assayed. The
antibodies are then visualized by any of a number of methods to
determine the presence and amount of the protein present. Examples
of methods used to visualize antibodies are, for example, through
enzymes linked to the antibodies (e.g., luciferase, alkaline
phosphatase, horseradish peroxidase, or .beta.-galactosidase), or
chemical methods (e.g., DAB/Substrate chromagen).
[0380] In addition to detecting levels of protein expression, the
diagnostic assays of the invention may employ methods designed to
detect the level of RNA expression. Levels of RNA expression may be
determined using methods well known to those of skill in the art,
including, for example, the use of northern blots, RT-PCR or in
situ hybridizations.
[0381] Carboxylation of osteocalcin confers a greater affinity for
hydroxyapatite. Typically, total osteocalcin is measured by
immunoassay followed by incubation with hydroxyapatite and
centrifugation. The supernatant, which contains osteocalcin that
has not adsorbed to hyudroxyapatite is then measured using the same
immunoassay. The results of this procedure can be expressed either
as absolute concentrations or as a ratio of undercarbocylated to
carboxylated osteocalcin.
[0382] Another procedure uses monoclonal antibodies that
distinguish the carboxylation state of all or some of the Glu/Gla
residues of osteocalcin. For example, GluOC4-5 (TaKaRa catalog no.
M171) reacts with human osteocalcin with glutamic acid residues
(decarboxylated) at positions 21 and 24, and does not react with
react with Gla-type osteocalcin.
[0383] For a review of osteocalcin measurement methods, see Lee et
al., 2000, Ann.
[0384] Clin. Biochem. 37, 432-446.
Drug Screening and Assays
[0385] Cell-based and non-cell based methods of drug screening are
provided to identify candidate agents that reduce OST-PTP or
gamma-carboxylase activity or expression, or increase the level of
undercarboxylated/uncarboxylated osteocalcin activity or
expression. Such agents find use in treating or preventing a
disorder related to energy metabolism and the OST-PTP signaling
pathway. Such disorders include, metabolic syndrome, glucose
intolerance, type 1 or type 2 diabetes, atherosclerosis, or
obesity. Such agent may be used to treat disorders characterized by
decreased insulin production, deceased insulin sensitivity, and
decreased glucose tolerance or increased fat mass. Such assays may
also be used to assay for the effectiveness of an agent in treating
or preventing a disorder related to the OST-PTP pathway.
[0386] A non-cell based screening method is provided to identify
compounds that bind to OST-PTP, gamma-carboxylase or osteocalcin
and thereby modulate the activity of said proteins.
[0387] A screening method is provided to identify, or assay for, an
agent that binds to OST-PTP, the method comprising the steps of:
(i) providing a mixture comprising OST-PTP or a fragment or variant
thereof, (ii) contacting the mixture with an agent, (iii)
determining whether the agent binds to the OST-PTP, and (iv)
identifying the agent if it binds to the OST-PTP or a fragment or
variant thereof. The method may further comprise the step of
determining whether the agent reduces the ability of OST-PTP to
dephosphorylate gamma-carboxylase.
[0388] A screening method is provided to identify, or assay for, an
agent that binds to the phosphatase 1 domain of OST-PTP, the method
comprising the steps of (i) providing a mixture comprising the
phosphatase 1 domain of OST-PTP or a fragment or variant thereof,
(ii) contacting the mixture with an agent, (iii) determining
whether the agent binds to the phosphatase 1 domain of OST-PTP, and
(iv) identifying the agent if it binds to the phosphatase 1 domain
of OST-PTP or a fragment or variant thereof. The method may further
comprise the step of determining whether the agent reduces the
ability of OST-PTP to dephosphorylate gamma-carboxylase.
[0389] A screening method is provided to identify, or assay for, an
agent that binds to gamma-carboxylase, the method comprising the
steps of: (i) providing a mixture comprising the gamma-carboxylase
or a fragment or variant thereof, (ii) contacting the mixture with
an agent, (iii) determining whether the agent binds to the
gamma-carboxylase, and (iv) identifying the agent if it binds to
the gamma-carboxylase or a fragment or variant thereof The method
may further comprise the step of determining whether the agent
reduces gamma-carboxylase activity.
[0390] A screening method is provided to identify, or assay for, an
agent that binds to osteocalcin, the method comprising, the steps
of: (i) providing a mixture comprising osteocalcin or a fragment or
variant thereof, (ii) contacting the mixture with an agent, (iii)
determining whether the agent binds to the osteocalcin, and (iv)
identifying the agent if it binds to the osteocalcin or a fragment
or variant thereof. The method may further comprise the step of
determining whether the agent increases osteocalcin activity.
[0391] The binding of the agent may be determined through the use
of competitive binding assays. The competitor is a binding moiety
known to bind to the target molecule (i.e. one of the various
proteins), such as an antibody, peptide, binding partner, ligand,
etc. Under certain circumstances, there may be competitive binding
as between the agent and the binding moiety, with the binding
moiety displacing the agent.
[0392] The agent may be labeled. Either the agent, or the
competitor, or both, is added first to the protein for a time
sufficient to allow binding, if present. Incubations may be
performed at any temperature which facilitates optimal activity,
typically between 4 degrees Centigrade. and 40 degrees Centigrade.
Incubation periods are selected for optimum activity, but may also
optimized to facilitate rapid high through put screening. Typically
between 0.1 and 1 hour will be sufficient. Excess reagent is
generally removed or washed away. The second component is then
added, and the presence or absence of the labeled component is
followed, to indicate binding.
[0393] Using such assays, the competitor may be added first,
followed by the agent. Displacement of the competitor is an
indication that the agent is binding to one of the various proteins
and thus is capable of binding to, and potentially modulating, its
activity. In this embodiment, either component can be labeled.
Thus, for example, if the competitor is labeled, the presence of
label in the wash solution indicates displacement by the agent.
Alternatively, if the agent is labeled, the presence of the label
on the support indicates displacement.
[0394] In another example, the agent is added first, with
incubation and washing, followed by the competitor. The absence of
binding by the competitor may indicate that the agent is bound to
one of the various proteins with a higher affinity. Thus, if the
agent is labeled, the presence of the label on the support, coupled
with a lack of competitor binding, may indicate that the agent is
capable of binding to one of the various proteins.
[0395] The method may comprise differential screening to identify
agents that are capable of modulating the activity of one of the
various proteins. In such an instance, the methods comprise
combining a protein and a competitor in a first sample. A second
sample comprises an agent, a protein and a competitor. Addition of
the agent is performed under conditions which allow the modulation
of one of the various proteins. The binding of the competitor is
determined for both samples, and a change, or difference in binding
between the two samples indicates the presence of an agent capable
of binding to one of the various proteins and potentially
modulating its activity. That is, if the binding of the competitor
is different in the second sample relative to the first sample, the
agent is capable of binding to one of the various proteins.
[0396] Positive controls and negative controls may be used in the
assays. Preferably all control and test samples are performed in at
least triplicate to obtain statistically significant results.
Incubation of all samples is for a time sufficient for the binding
of the agent to the protein. Following incubation, all samples are
washed free of non-specifically bound material and the amount of
bound, generally labeled agent determined. For example, where a
radiolabel is employed, the samples may be counted in a
scintillation counter to determine the amount of bound
compound.
[0397] A variety of other reagents may be included in the screening
assays. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc which may be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., may be used. The mixture of components
may be added in any order that provides for the requisite
binding.
[0398] Non-cell based screening for agents that modulate the
activity of one of the various proteins may also be done. Methods
for screening for an agent capable of modulating the activity of
one of the various proteins comprise the steps of adding an agent
to a sample of one of the various proteins, as above, and
determining an alteration in the biological activity of one of the
various proteins. "Modulating the activity of one of the various
proteins" includes an increase in activity, a decrease in activity,
or a change in the type or kind of activity present. Thus, the
agent should both bind to the protein (although this may not be
necessary), and alter its biological or biochemical activity as
defined herein.
[0399] Thus, in one example, the methods comprise combining a
protein sample and an agent, and evaluating the effect on OST-PTP,
gamma-carboxylase or osteocalcin. By enzyme activity, specifically
OST-PTP or gamma-carboxylase activity or grammatical equivalents
herein is meant one or more of the biological activities associated
with the enzyme. For OST-PTP this activity is preferably the
dephosphorylation of gamma-carboxylase or of the insulin receptor;
for gamma-carboxylase it is the carboxylation of osteocalcin. The
screening assay are designed to find agents that reduce OST-PTP or
gamma-carboxylase activity, or increase levels of
undercarboxylated/uncarboxylated osteocalcin and adiponectin in a
biological sample taken from the transformed animal or cell.
[0400] Specifically, a screening method is provided to identify, or
assay for, an agent that reduces OST-PTP activity, the method
comprising the steps of: (a) providing a control and a test mixture
comprising OST-PTP or a fragment or variant thereof, (b) contacting
the mixture with an agent, (c) determining the level of activity of
OST-PTP in the test mixture and in the control, and(d) selecting
the bioactive agent if the level of OST-PTP activity in the test
mixture is lower than the level in the control.
[0401] A screening method is provided to identify, or assay for, an
agent that reduces gamma-carboxylase activity, the method
comprising the steps of: (a) providing a control and a test mixture
comprising or a fragment or variant thereof, (b) adding to the test
mixture a bioactive agent under conditions that permit the
bioactive agent to bind to the gamma-carboxylase or a fragment or
variant thereof, (c) determining the level of activity of
gamma-carboxylase in the test mixture and in the control, and (d)
selecting the bioactive agent if the level of gamma-carboxylase
activity in the test mixture is lower than the level in the
control.
[0402] Cell-based screening methods are provided for identifying,
or assaying for, agents which decrease the level of expression of
the Esp gene encoding OST-PTP or the gene encoding
gamma-carboxylase. Alternatively, the drug screening assays may be
used to identify, or assay for, agents which increase the level of
osteocalcin gene expression.
[0403] The present invention also provides a screening method to
identify an agent that decarboxylates osteocalcin, the method
comprising the steps of: (a) providing a control and a test mixture
comprising carboxylated osteocalcin, (b) adding to the test mixture
an agent, (c) determining the level of carboxylated osteocalcin in
the test mixture and in the control, and (d) selecting an agent if
the level of carboxylated osteocalcin in the test mixture is lower
than the activity in the control.
[0404] A cell-based method is provided for identifying, or assaying
for, an agent that reduces OST-PTP gene expression, the method
comprising steps: (a) determining a first expression level of
OST-PTP in a cell, (b) determining a second expression level of
OST-PTP after contact with a test agent; and (c) comparing the
first expression level with the second expression level, wherein an
agent that is capable of reducing OST-PTP expression is identified
if the level of expression in the first is higher than the second
expression level. The level of OST-PTP gene expression may be
determined by measuring the amount of the OST-PTP mRNA made or the
amount of the OST-PTP protein made.
[0405] A cell-based method is provided for identifying, or assaying
for, an agent that reduces gamma-carboxylase gene expression, the
method comprising steps: (a) determining a first expression level
of gamma-carboxylase in a cell, (b) determining a second expression
level of gamma-carboxylase after contact with a test agent; and (c)
comparing the first expression level with the second expression
level, wherein an agent that is capable of reducing
gamma-carboxylase expression is identified if the level of
expression in the first is higher than the second expression level.
The level of gamma-carboxylase gene expression may be determined by
measuring the amount of the gamma-carboxylase mRNA made or the
amount of gamma-carboxylase protein made.
[0406] A cell-based method is provided for identifying, or assaying
for, an agent that increases osteocalcin gene expression, the
method comprising steps: (a) determining a first expression level
of osteocalcin in a cell, (b) determining a second expression level
of osteocalcin expression after contact with a test agent; and (c)
comparing the first expression level with the second expression
level, wherein an agent that is capable of increasing osteocalcin
expression is identified if the level of expression in the first is
lower than the second expression level. The level osteocalcin gene
expression may be determined by measuring the amount of the
osteocalcin mRNA made or the amount of osteocalcin protein
made.
[0407] A reporter gene may be utilized to screen for agent capable
of modulating gene expression. In such assays, cells are generated
that contain a gene construct wherein expression of a reporter gene
is placed under the control of native gene expression regulatory
elements of the native gene of interest, i.e., the OST-PTP,
gamma-carboxylase or osteocalcin gene. Reporter genes include, but
are not limited to CAT, LacZ, lucierase or GFP.
[0408] A cell-based method is provided for screening, or assaying,
for an agent that reduces OST-PTP gene expression, the method
comprising steps: (a) determining a first expression level of a
reporter gene in a cell wherein expression of the reporter gene is
under the control of native OST-PTP gene expression regulatory
elements, (b) determining a second expression level of reporter
gene exprssion after contact with a test agent; and (c) comparing
the first expression level with the second expression level,
wherein an agent that is capable of reducing reporter gene
expression is identified if the level of expression in the first is
higher than the second expression level.
[0409] A cell-based method is provided for screening, or assaying,
for an agent that reduces gamma-carboxylase gene expression, the
method comprising steps: (a) determining a first expression level
of a reporter gene in a cell wherein expression of the reporter
gene is under the control of native gamma-carboxylase gene
expression regulatory elements, (b) determining a second expression
level of reporter gene expression after contact with a test agent;
and (c) comparing the first expression level with the second
expression level, wherein an agent that is capable of reducing
gamma-carboxylase gene expression is identified if the level of
expression in the first is higher than the second expression
level.
[0410] A cell-based method is provided for screening, or assaying,
for an agent that increases osteocalcin gene expression, the method
comprising steps: (a) determining a first expression level of a
reporter gene in a cell wherein expression of the reporter gene is
under the control of native osteocalcin gene expression regulatory
elements, (b) determining a second expression level of reporter
gene expression after contact with a test agent; and (c) comparing
the first expression level with the second expression level,
wherein an agent that is capable of increasing osteocalcin gene
expression is identified if the level of expression in the first is
lower than the second expression level.
[0411] Cell-based screening assays are provided for identifying
agents that reduce OST-PTP or gamma-carboxylase activity.
[0412] Specifically, a cell-based method for screening for an agent
that reduces OST-PTP activity is provided, the method comprising
steps: (a) determining a first activity level in a first cell that
expresses the phosphatase 1 domain of OST-PTP, (b) contacting a
second cell that expresses the phosphatase 1 domain of OST-PTP with
an agent, (c) determining a second activity level in the second
cell that expresses the phosphatase 1 domain of OST-PTP; and (d)
comparing the first activity level with the second activity level,
wherein the agent reduces OST-PTP activity if the first activity
level is higher than the second activity level. The level of
OST-PTP activity may be determined by measuring the level of
gamma-carboxylase activity. The level of OST-PTP activity may be
determined by measuring the level of osteocalcin carboxylation.
[0413] A cell-based method for screening, or assaying, for an agent
that reduces gamma-carboxylase activity is provided, the method
comprising the steps: (a) determining a first activity level in a
first cell that expresses gamma-carboxylase, (b) contacting a
second cell that expresses gamma-carboxylase with an agent (c)
determining a second activity level in the second cell that
expresses gamma-carboxylase; and (d) comparing the first activity
level with the second activity level, wherein the agent reduces
gamma-carboxylase activity if the first activity level is higher
than the second activity level. Assays for measuring gamma
carboxylase activity are know to those of skill in the art (See,
for example, Hubbard et al., (1989) Proc. Natl. Acad. Sci. USA
86:6893-6897; Rehemtulla et al., (1993) Proc. Natl. acad. Sci USA
90:4611-4615).
[0414] Gamma carboxylase catalyzes the posttranslational
modification of specific glutamic acid residues within osteocalcin
to form .gamma.-carboxyglutamic acid residues. In an embodiment of
the invention, the level of gamma carboxylase activity or
decarboxylase activity is determined by measuring the level of
osteocalcin carboxylation.
[0415] A cell based method is provided for screening for an agent
that decarboxylates osteocalcin, the method comprising the steps
of: (a) determining a first level of carboxylated osteocalcin in a
first cell that expresses osteocalcin, (b) contacting a second cell
that expresses carboxylated osteocalcin with an agent, (c)
determining a second level of carboxylated, osteocalcin, and (d)
comparing the first level of carboxylated osteocalcin with the
second level of carboxylated osteocalcin, wherein the agent
decarboxylates osteocalcin if the first level of carboxylated
osteocalcin is higher than the second level.
[0416] Cells to be used in the screening, or assaying, methods
include cells that naturally express OST-PTP, gamma-carboxylase, or
osteocalcin, cells that have been genetically engineered to express
(or overexpress) OST-PTP, the phosphatase 1 domain of OST-PTP,
gamma-carboxylase, or osteocalcin, as well as cells derived from
the transgenic animals of the present invention. Such cells include
transformed osteoblast that overexpresses OST-PTP or
gamma-carboxylase.
[0417] A method is provided for testing an agent's effectiveness in
increasing adiponectin expression in adipocytes, comprising: (a)
co-culturing osteoblasts and adipocytes, (b) contacting the
osteoblasts with a candidate agent,(c) determining whether the
candidate agent increases the level of expression or secretion of
adiponectin or a fragment or variant thereof above a control level
measured in a control co-culture in which osteoblasts are not
contacted with the candidate agent, and (d) if the candidate agent
increases the level of adiponectin expression or secretion above
the control level, then selecting the candidate agent as an agent
that increases adiponectin expression or secretion in
adipocytes.
[0418] A method is provided for testing an agent's effectiveness in
increasing insulin expression or secretion in pancreatic beta
cells, comprising: (a) co-culturing the osteoblasts and pancreatic
beta cells, (b) contacting the osteoblasts with a candidate agent,
(c) determining whether the candidate agent increases the level of
insulin expression or secretion above a control level of insulin
expression measured in a control co-culture in which osteoblasts
are not contacted with the candidate agent, and (d) if the
candidate agent increases the level of insulin expression or
secretion above the control level, then selecting the candidate
agent as an agent that increases insulin expression or secretion in
pancreatic beta cells.
[0419] A method is provided for determining the ability of a
candidate agent to treat or prevent in an animal metabolic syndrome
or a phenotype associated with metabolic syndrome is provided that
is selected from the group comprising predisposition to type 1 or 2
diabetes, glucose intolerance, decreased insulin production,
decreased insulin sensitivity, decreased glucose tolerance,
atherosclerosis and increased fat mass, comprising: (a) providing a
test animal and a control animal, (b) administering the candidate
agent to the test animal, (c) comparing the level of
undercarboxylated/uncarboxylated osteocalcin in the test animal to
the level of undercarboxylated/uncarboxylated osteocalcin in the
control animal, and (d) selecting the candidate agent if the level
of undercarboxylated/uncarboxylated osteocalcin is higher in the
test animal than in the control animal. In a specific embodiment of
the invention the level of undercarboxylated/uncarboxylated
osteocalcin is measured in osteoblasts.
[0420] In one example, the level of
undercarboxylated/uncarboxylated osteocalcin is measured in
osteoblasts. The candidate agent may be bound to a phosphate group
that facilitates its uptake by osteoblasts.
[0421] A method is provided for screening a candidate agent for the
ability to treat or prevent metabolic syndrome in an animal or a
phenotype associated with metabolic syndrome including
predisposition to type 2 diabetes, glucose intolerance, decreased
insulin production, decreased insulin sensitivity, decreased
glucose tolerance, atherosclerosis and increased fat mass,
comprising: (a) providing a first and a second animal, (b)
administering to said first animal a candidate agent, and (c)
comparing the level of OST-PTP expression or activity in the first
animal of step (b) that was given the candidate agent to the level
of OST-PTP in the second animal of step (a) that was not
administered said candidate agent; wherein a candidate agent that
reduces the level of OST-PTP expression or activity is selected as
an agent that has effectiveness in treating metabolic syndrome or a
phenotype associated therewith.
[0422] The level of OST-PTP expression or activity may be measured
in osteoblasts. Further, the candidate agent may be bound to a
phosphate group that facilitates its uptake by osteoblasts.
[0423] A method is provided for screening a candidate agent for the
ability to treat or prevent metabolic syndrome in an animal, or a
phenotype associated with metabolic syndrome including
predisposition to type 1 and 2 diabetes, glucose intolerance,
decreased insulin production, decreased insulin sensitivity,
atherosclerosis, decreased glucose tolerance and increased fat
mass, comprising: (a) providing a first and a second animal, (b)
administering to said first animal a candidate agent, and (c)
comparing the level of expression or activity or secretion of
osteocalcin in the first animal of step (b) that was given the
candidate agent to the level of expression or activity of
osteocalcin in the second animal of step (a) that was not
administered said candidate agent; wherein a candidate agent that
in increases expression or activity or secretion of osteocalcin or
a fragment or variant thereof is selected as an agent that has
effectiveness in treating metabolic syndrome or a phenotype
associated therewith.
[0424] A method is provided for screening a candidate agent for the
ability to treat or prevent metabolic syndrome in an animal or a
phenotype associated with metabolic syndrome including
predisposition to type 1 or 2 diabetes, glucose intolerance,
decreased insulin production, decreased insulin sensitivity,
atherosclerosis, decreased glucose tolerance and increased fat
mass, comprising: (a) providing a first and a second animal, (b)
administering to said first animal a candidate agent, and (c)
comparing the level of expression or secretion of adiponectin or a
fragment or variant thereof in the first animal of step (b) that
was given the candidate agent to the level of adiponectin
expression or secretion in the second animal of step (a) that was
not administered said candidate agent; wherein a candidate agent
that increases the level of expression or secretion of adiponectin
or a fragment or variant thereof is selected as an agent that has
effectiveness in treating metabolic syndrome or a phenotype
associated therewith. In such a method, the level of adiponectin
expression or secretion is measured in adipocytes or in serum.
[0425] A method is provided for screening a candidate agent for the
ability to treat or prevent metabolic syndrome in a
osteocalcin-deficient mouse, wherein the osteocalcin-deficient
mouse exhibits a phenotype relative to a wild type mice, which
phenotype is selected from the group comprising reduced osteocalcin
expression, type 1 or 2 diabetes predisposition, decreased insulin
secretion, atherosclerosis, decreased insulin sensitivity,
decreased expression or secretion of adiponectin or a fragment or
variant thereof, decreased glucose tolerance, and increased fat
mass,comprising: (a) providing a first and a second
osteocalcin-deficient mouse that are both from the same strain as
the osteocalcin-deficient mouse; (b) administering to said first
osteocalcin-deficient mouse a candidate agent, and (c) comparing
the phenotype of the first osteocalcin-deficient mouse of step (b)
that was given the candidate agent to the phenotype of said second
osteocalcin-deficient mouse of step (a) that was not administered
said candidate agent; wherein a candidate agent that reduces or
ameliorates the phenotype is selected as an agent that has
effectiveness in treating metabolic syndrome.
[0426] A method is also provided for screening a candidate agent
for the ability to treat metabolic syndrome in an
adiponectin-deficient mouse, wherein the adiponectin-deficient
mouse exhibits a phenotype selected from the group comprising type
1 or 2 diabetes predisposition; decreased insulin secretion;
decreased insulin sensitivity; atherosclerosis, decreased glucose
tolerance, and increased fat mass, comprising: (a) providing a
first and a second adiponectin-deficient mouse that are both from
the same strain, (b) administering to said first
adiponectin-deficient mouse a candidate agent, and c) comparing the
phenotype of the first adiponectin-deficient mouse of step (b) that
was given the candidate agent to the phenotype of said second
adiponectin-deficient mouse of step (a) not administered said
candidate agent; wherein a candidate agent that reduces or
ameliorates the phenotype is selected as an agent that has
effectiveness in treating metabolic syndrome.
[0427] A method is provided for screening for an agent suspected to
reduce OST-PTP activity or expression in osteoblasts for use as a
therapeutic agent for treating or preventing a disease that is a
member of the group comprising metabolic syndrome, type I or II
diabetes, decreased insulin secretion, decreased insulin
sensitivity, decreased glucose tolerance, increased fat mass and
atherosclerosis, comprising: a) obtaining a control transgenic
mouse overexpressing OST-PTP selectively in osteoblasts, and a
second transgenic mouse from the same strain as the control, b)
subjecting the first mouse to a placebo and the second mouse to a
therapeutic agent, c) assaying for the level of OST-PTP activity in
a sample of osteoblasts from the first and second mice, d)
comparing the level of OST-PTP activity assayed in the first mouse
to that in the second mouse,and e) concluding that the agent is
useful as a therapeutic compound to treat or prevent the disease if
the level in the first mouse is higher than the level in the second
mouse.
[0428] A method is provided for screening a agent suspected to
reduce gamma-carboxylase activity or expression in osteoblasts for
use as a therapeutic compound for treating or preventing a disease
that is a member of the group comprising metabolic syndrome, type I
or II diabetes, decreased insulin secretion, decreased insulin
sensitivity, decreased glucose tolerance, increased fat mass and
atherosclerosis,comprising: a) obtaining a control transgenic mouse
overexpressing gamma-carboxylase selectively in osteoblasts, and a
second transgenic mouse from the same strain as the control, b)
subjecting the first mouse to a placebo and the second mouse to the
therapeutic compound under the same conditions that allow the
therapeutic compound to have an effect, c) assaying for the level
of gamma-carboxylase activity in a sample of osteoblasts from the
first and second mice, d) comparing the level of gamma-carboxylase
activity assayed in the first mouse to that in the second mouse, e)
concluding that bioactive agent is useful as a therapeutic compound
for use in reducing gamma-carboxylase activity or expression in
osteoblasts if the level in the first mouse is higher than the
level in the second mouse. The bioactive agent may be an enzyme
inhibitor.
[0429] A method is provided for screening a agent suspected of
having a therapeutic use to treat or prevent a disease that is a
member of the group comprising metabolic syndrome, type I or II
diabetes, decreased insulin secretion, decreased insulin
sensitivity, decreased glucose tolerance, increased fat mass and
atherosclerosis, comprising: (a) providing an animal that has the
disease, (b) determining the amount of
undercarboxylated/uncarboxylated osteocalcin in a pretreatment
biological sample taken from the animal, (c) administering the
bioactive agent to the test animal under conditions that permit the
agent to have an effect, (d) determining the amount of
undercarboxylated/uncarboxylated osteocalcin in a post-treatment
biological sample taken from the animal, and (e) if the bioactive
agent increases the amount of undercarboxylated/uncarboxylated
osteocalcin in the post-treatment biological sample compared to the
pre-treatment sample, concluding that the agent has the therapeutic
use.
[0430] A method is provided for screening a bioactive agent
suspected of having a therapeutic use to treat or prevent a disease
that is a member of the group comprising metabolic syndrome, type I
or II diabetes, decreased insulin secretion, decreased insulin
sensitivity, decreased glucose tolerance, increased fat mass and
atherosclerosis, comprising: (a) providing an animal that has the
disease, (b) determining the amount of adiponectin in a
pretreatment biological sample taken from the animal, (c)
administering the bioactive agent to the test animal under
conditions that permit the agent to have an effect, (d) determining
the amount of adiponectin in a post-treatment biological sample
taken from the animal, and (e) if the bioactive agent increases the
amount of adiponectin in the post-treatment biological sample
compared to the pre-treatment sample, concluding that the agent has
the therapeutic use.
[0431] The term "agent" or "exogeneous compound" as used herein
includes any molecule, e.g., protein, oligopeptide, small organic
molecule, polysaccharide, polynucleotide, lipid, etc., or mixtures
thereof, with the capability of directly or indirectly altering the
bioactivity of one of the various proteins (OST-PTP,
gamma-carboxylase, osteocalcin,). Some of the agents can be used
therapeutically. Generally a plurality of assay mixtures is run in
parallel with different agent concentrations to obtain a
differential response to the various concentrations. Typically, one
of these concentrations serves as a negative control, i.e., at zero
concentration or below the level of detection.
[0432] Agents for use in screening encompass numerous chemical
classes, though typically they are organic molecules, preferably
small organic compounds having a molecular weight of more than 100
and less than about 2,500 daltons, preferably less than about 500
daltons. Agents comprise functional groups necessary for structural
interaction with proteins, particularly hydrogen bonding, and
typically include at least an amine, carbonyl, hydroxyl or carboxyl
group, preferably at least two of the functional chemical groups.
The agent often comprise cyclical carbon or heterocyclic structures
and/or aromatic or polyaromatic structures substituted with one or
more of the above functional groups. Agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Particularly preferred are peptides.
[0433] Libraries of high-purity small organic ligands and peptide
agonists that have well-documented pharmacological activities are
available from numerous sources. One example is an NCI diversity
set which contains 1,866 drug-like compounds (small, intermediate
hydrophobicity). Another is an Institute of Chemistry and Cell
Biology (ICCB; maintained by Harvard Medical School) set of known
bioactives (467 compounds) which includes many extended, flexible
compounds. Some other examples of the ICCB libraries are: Chem
Bridge DiverSet E (16,320 compounds); Bionet 1 (4,800 compounds);
CEREP (4,800 compounds); Maybridge 1 (8,800 compounds); Maybridge 2
(704 compounds); Maybridge HitFinder (14,379 compounds); Peakdale 1
(2,816 compounds); Peakdale 2 (352 compounds); ChemDiv Combilab and
International (28,864 compounds); Mixed Commercial Plate 1 (352
compounds); Mixed Commercial Plate 2 (320 compounds); Mixed
Commercial Plate 3 (251 compounds); Mixed Commercial Plate 4 (331
compounds); ChemBridge Microformat (50,000 compounds); Commercial
Diversity Set1 (5,056 compounds). Other NCI Collections are:
Structural Diversity Set, version 2 (1,900 compounds); Mechanistic
Diversity Set (879 compounds); Open Collection 1 (90,000
compounds); Open Collection 2 (10,240 compounds); Known Bioactives
Collections: NINDS Custom Collection (1,040 compounds); ICCB
Bioactives 1 (489 compounds); SpecPlus Collection (960 compounds);
ICCB Discretes Collections. The following ICCB compounds were
collected individually from chemists at the ICCB, Harvard, and
other collaborating institutions: ICCB1 (190 compounds); ICCB2 (352
compounds); ICCB3 (352 compounds); ICCB4 (352 compounds). Natural
Product Extracts: NCI Marine Extracts (352 wells); Organic
fractions--NCI Plant and Fungal Extracts (1,408 wells); Philippines
Plant Extracts 1 (200 wells); ICCB-ICG Diversity Oriented Synthesis
(DOS) Collections; DDS1 (DOS Diversity Set) (9600 wells). Compound
libraries are also available from a commercial suppliers, such as
ActiMol, Albany Molecular, Bachem, Sigma-Aldrich, TimTec, and
others.
[0434] Known and novel pharmacological agents identified in screens
may be further subjected to directed or random chemical
modifications, such as acylation, alkylation, esterification,
amidification to produce structural analogs.
[0435] When screeing, designing or modifying compounds, other
factors to consider include the Lipinski rule-of-five (not more
than 5 hydrogen bond donors (OH and NH groups); not more than 10
hydrogen bond acceptors (notably N and O); molecular weight under
500 g/mol; partition coefficient log P less than 5), and Veber
criteria, which are recognized in the pharmaceutical art and relate
to properties and structural features that make molecules more or
less drug-like.
[0436] The agent may be a protein. By "protein" in this context is
meant at least two covalently attached amino acids, which includes
proteins, polypeptides, oligopeptides and peptides. The protein may
be made up of naturally occurring amino acids and peptide bounds,
or synthetic peptidomimetic structures. Thus "amino acid", or
"peptide residue", as used herein means both naturally occurring
and synthetic amino acids. For example, homo-phenylalanine,
citrulline and noreleucine are considered amino acids for the
purposes of the invention. "Amino acids" also includes imino acid
residues such as proline and hydroxyproline. The side chains may be
in either the (R) or the (S) configuration. In the preferred
embodiment, the amino acids are in the (S) or L-configuration. If
non-naturally occurring side chains are used, non-amino acid
substituents may be used, for example to prevent or retard in vivo
degradations.
[0437] The agent may be a naturally occurring protein or fragment
or variant of a naturally occurring protein. Thus, for example,
cellular extracts containing proteins, or random or directed
digests of proteinaceous cellular extracts, may be used. In this
way, libraries of prokaryotic and eukaryotic proteins may be made
for screening against one of the various proteins. Libraries of
bacterial, fungal, viral, and mammalian proteins, with the latter
being preferred, and human proteins being especially preferred may
be used.
[0438] Agents may be peptides of from about 5 to about 30 amino
acids, with from about 5 to about 20 amino acids being preferred,
and from about 7 to about 15 being particularly preferred. The
peptides may be digests of naturally occurring proteins as is
outlined above, random peptides, or "biased" random peptides. By
"randomized" or grammatical equivalents herein is meant that each
nucleic acid and peptide consists of essentially random nucleotides
and amino acids, respectively. Since generally these random
peptides (or nucleic acids, discussed below) are chemically
synthesized, they may incorporate any nucleotide or amino acid at
any position. The synthetic process can be designed to generate
randomized proteins or nucleic acids, to allow the formation of all
or most of the possible combinations over the length of the
sequence, thus forming a library of randomized agent bioactive
proteinaceous agents.
[0439] The library may be fully randomized, with no sequence
preferences or constants at any position. The library may be
biased. That is, some positions within the sequence are either held
constant, or are selected from a limited number of possibilities.
For example, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, etc., or to purines, etc.
[0440] The agent may be an isolated nucleic acid, preferably
antisense, siRNA, or cDNA that binds to either the gene encoding
the protein of interest, or its mRNA to block gene expression or
mRNA translation, respectively. By "nucleic acid" or
"oligonucleotide" or grammatical equivalents herein means at least
two nucleotides covalently linked together. Such nucleic acids will
generally contain phosphodiester bonds, although in some cases, as
outlined below, nucleic acid analogs are included that may have
alternate backbones, comprising, for example, phosphoramide
(Beaucage et al., Tetrahedron 49)10):1925 (1993) and references
therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al.,
Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.
14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et
al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica
Scripta 26:141 91986)), pohsphorothioate (Mag et al., Nucleic Acids
Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048),
phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989),
O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides
and Analogues: A Practical Approach, Oxford University Press), and
peptide nucleic acid backbones and linkages (see Egholm, J. Am.
Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.
31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,
Nature 380:207 (1996), all of which are incorporated by
reference).
[0441] Other analog nucleic acids include those with positive
backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097
(1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684,
5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem.
Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.
Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleoside
13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic &
Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular
NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose
backbones, including those described in U.S. Pat. Nos. 5,235,033
and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
"Carbohydrate Modifications in antisense Research", Ed. Y. S.
Sanghui and P. Can Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp
169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments. In addition, mixtures of naturally occurring acids
and analogs can be made. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made. The nucleic acids may be single
stranded or double stranded, as specified, or contain portions of
both double stranded or single stranded sequence. The nucleic acid
may be DNA, both genomic and cDNA, RNA or a hybrid, where the
nucleic acid contains any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xanthine hypoxathine,
isocytosine, isoguanine, etc.
[0442] As described above generally for proteins, nucleic acid
agents may be naturally occurring nucleic acids, random nucleic
acids, or "biased" random nucleic acids. For example, digests of
prokaryotic or eukaryotic genomes may be used as is outlined above
for proteins.
[0443] The agents may be obtained from combinatorial chemical
libraries, a wide variety of which are available in the literature.
By "combinatorial chemical library" herein is meant a collection of
diverse chemical compounds generated in a defined or random manner,
generally by chemical synthesis. Millions of chemical compounds can
be synthesized through combinatorial mixing.
[0444] The determination of the binding of the agent to one of the
various proteins may be done in a number of ways. In a preferred
embodiment, the agent is labeled, and binding determined directly.
For example, this may be done by attaching all or a portion of one
of the various proteins to a solid support, adding a labeled agent
(for example an agent comprising a fluorescent label), washing off
excess reagent, and determining whether the label is present on the
solid support. Various blocking and washing steps may be utilized
as is known in the art.
[0445] By "labeled" herein is meant that the agent is either
directly or indirectly labeled with a label which provides a
detectable signal, e.g. a radioisotope (such as .sup.3H, .sup.14C,
.sup.32P, .sup.33P, .sup.35S, or .sup.125I), a fluorescent or
chemiluminescent compound (such as fluorescein isothiocyanate,
rhodamine, or luciferin), an enzyme (such as alkaline phosphatase,
beta-galactosidase or horseradish peroxidase), antibodies,
particles such as magnetic particles, or specific binding
molecules, etc. Specific binding molecules include pairs, such as
biotin and streptavidin, digoxin and antidigoxin etc. For the
specific binding members, the complementary member would normally
be labeled with a molecule which provides for detection, in
accordance with known procedures, as outlined above. The label can
directly or indirectly provide a detectable signal. Only one of the
components may be labeled. Alternatively, more than one component
may be labeled with different labels.
Sequence Listings
[0446] Full nucleic acid and amino acid sequence listings relevant
to this application are listed below. Transgenic mice and isolated
cells from them (especially osteoblasts and adipocytes) that over
or under express any of the listed nucleic acids (cDNA for Esp,
osteocalcin, adiponectin, gamma-carboxylase, apolipoprotein E) can
be made using routine methods known in the art and described
herein, including knock in and knock out mice. In certain
instances, nucleic acids are inserted into the genome of the host
organism operably connected to and under the control of a promoter
and regulatory elements (endogenous or heterogeneous) that will
cause the organism to over express the nucleic acid gene or mRNA.
One example of an exogenous/heterogeneous promoter included in the
transfecting vector carrying the gene to be amplified is alpha 1(I)
collagen. Many such promoters are known in the art. Human
osteoblasts can be transfected with vectors carrying the cDNA for
human Esp or human osteocalcin (or fragments or variants thereof)
operably linked to known promoters and regulatory elements that
cause the transfected human osteoblast to overexpress osteocalcin
(or fragments or variants thereof). Disclosed herein are transgenic
mice and mouse cells, and transfected human cells over expressing
osteocalcin (or fragments or variants thereof), OST-PTP or
gamma-carboxylase. Also disclosed herein are double mutant mice
that have deletions of one or both alleles for osteocalcin, Esp,
gamma-carboxylase and adiponectin, and various combinations of
double mutants. Also disclosed herein are vectors carrying the cDNA
or mRNA encoding the proteins for insertion into the genome of a
target animal or cell. Such vectors can optionally include
promoters and regulatory elements operably linked to the cDNA or
mRNA. By "operably linked" is meant that promoters and regulatory
elements are connected to the cDNA or mRNA in such a way as to
permit expression of the cDNA or mRNA under the control of the
promoters and regulatory elements.
[0447] Antisense and small interfering RNAs for use in reducing
expression of OST-PTP and gamma-carboxylase thereby treating or
preventing metabolic syndrome or a component thereof in an animal
or type 1 diabetes, can be made that specifically hybridize to the
gene and mRNA encoding OST-PTP or gamma-carboxylase, respectively.
The sequence for mouse (OST-PTP, Ptprv) cDNA is set forth in SEQ ID
NO:18. The amino acid sequence for OST-PTP, Ptprv) protein is set
forth in SEQ ID NO:19. This cDNA will hybridize with mRNA for
OST-PTP and thereby interfere with its translation. Reducing
OST-PTP expression will increase undercarboxylate/uncarboxylated
osteocalcin. The cDNA for mouse gamma-carboxylase is identified by
SEQ ID NO:12, and its amino acid sequence is SEQ ID NO:13. This
cDNA will hybridize with mRNA for gamma-carboxylase and thereby
interfere with its translation and is a preferred embodiment. The
cDNA for human gamma-carboxylase is identified by SEQ ID NO:10, and
the amino acid sequence is SEQ ID NO:11. Human gamma-carboxylase
cDNA can be used therapeutically to reduce gamma-carboxylase
expression to treat or prevent metabolic syndrome and its
components and type 1 or type 2 diabetes.
Examples
[0448] The invention is illustrated herein by the experiments
described above and by the following examples, which should not be
construed as limiting. The contents of all references, pending
patent applications and published patents, cited throughout this
application are hereby expressly incorporated by reference. Those
skilled in the art will understand that this invention may be
embodied in many different forms and should not be construed as
limited to the embodiments set forth herein. Rather, these
embodiments are provided so that this disclosure will fully convey
the invention to those skilled in the art. Many modifications and
other embodiments of the invention will come to mind in one skilled
in the art to which this invention pertains having the benefit of
the teachings presented in the foregoing description. Although
specific terms are employed, they are used as in the art unless
otherwise indicated.
Materials and Methods
[0449] Esp-nLacZ mice refers to an Esp-deficient mouse model in
which one (+/-) or both alleles (-/-) for OST-PTP have been
inactivated in all of the cells in the animal. The nLacZ (or LacZ)
mouse is made by homologous recombination of a targeted OST-PTP
allele with a transgene having a sequence encoding a
nuclear-localized LacZ cassette, which is homologously recombined
into exon 6 of the OST-PTP allele, such that the transgene is in
frame with the OST-PTP gene, and expression of the transgene is
operably linked to the native gene expression regulatory sequences
of the OST-PTP allele. Esp KI (Knock In)=Esp nLacZ (-/-) mice.
[0450] Esp-nLacZ mice were generated using a targeting vector
designed to insert a nuclear-localized LacZ (nLacZ) cassette into
exon 6 such that LacZ was in frame with OST-PTP sequence (Dacquin
et al., 2004; Ducy et al., 1996). Genomic clones spanning the
entire mouse Ptprv gene were isolated from a mouse genomic library
(129ola strain) by using fragments of the mouse cDNA (Lee et al.,
[1996]). A targeting vector was constructed that contains an HPRT
hypoxanthine guanine phosphoribosyl transferase minigene selection
cassette, an internal ribosomal entry site (Mountford et al.,
1994), and a reporter containing SV40 nuclear localization
sequences fused to the LacZ gene (nLacZ). Into this were cloned 4.4
kb of homology from the 5' end of the gene and 1.9 kb of homology
from the 3' end of the gene. Gene targeting was conducted by using
standard techniques (Joyner, 1999) in E14Tg2A feeder-independent
embryonic stem (ES) cells (Hooper et al., 1987). Targeted ES cells
were selected in HAT (10 microM hypoxanthine, 9 microM aminopterin,
20 microM thymidine) selection medium as previously described
(Thompson et al., 1989). Tissue culture medium was GMEM (Glasgow
Modified Eagles Media; Gibco) supplemented with 10% fetal calf
serum (FCS), 0.1 mM 2 mercaptoethanol, 1 mM sodium pyruvate, and
approximately 10.sup.3 U/ml of leukemia inhibitory factor. A total
of 5.times.10.sup.6 cells were electroporated in 800 microliters of
phosphate buffered saline (PBS) with 20 micrograms of NotI
linearized vector DNA at 800 V and 3 microFD by using a Gene Pulser
(Bio-Rad) and plated onto gelatin coated 10-cm tissue culture
plates. After 48 hr, the cells were transferred to HAT selection
medium. Targeted ES cell clones were identified by Southern
hybridization using radiolabeled cDNA fragments complementary to
regions outside the homology of the targeting vector both 5' and 3'
of the integration site and by using a LacZ probe to check for
single copy integration. Targeted ES cells were injected in C57BL/6
blastocysts, which were subsequently transferred into foster
mothers. Chimeric males were mated with MF1 strain females, and
Southern blot analysis or polymerase chain reaction (PCR) of tail
tip DNA from grey offspring was used to identify heterozygous
animals. The mutation was crossed to the MF1 strain for five
generations to provide heterozygous mice for subsequent analysis.
This mutation resulted in deletion of most of OST-PTP extracellular
domain, its transmembrane and intracellular domains (1). This type
of mutant allele is referred to as the Esp nLacZ mutant allele or
as the Esp KI (Knock In) mutant allele. In the Esp nLacZ mutant
mice, one (+/-) or both alleles (-/-) for OST-PTP have been
inactivated in all of the cells in the animal thereby interfering
with OST-PTP expression.
[0451] "Esp osb mutant mice" are Esp-deficient mouse models in
which one (+/-) or both alleles (-/-) for OST-PTP have been deleted
or knocked out from osteoblasts only in the animal, thereby
blocking synthesis of OST-PTP selectively in osteoblasts. This is
not to be confused with the ob mutant which is lacking one or both
alleles of leptin. An Esp osb mouse carries a disruption in one or
both endogenous OST-PTP alleles in which exons 24 to 35 encoding
the phosphatase domain of the OST-PTP allele have been deleted and
replaced by a neomycin resistance gene floxed by loxP sites in one
(+/-) or both alleles (-/-) for OST-PTP.
[0452] A targeting vector harboring LoxP sites within introns 23
and 35 as well as a foxed neomycin resistance cassette was
electroporated into ES cells. Targeted ES cells were injected in
129Sv/EV blastocysts to generate chimeric mice harboring the floxed
allele (Esp.sub.flox). Esp.sub.flox/+ mice were crossed with
.alpha.1(I)collagen-Cre mice to generate Esp.sub.ob-/+ mice and
their progeny was intercrossed to obtain Esp.sub.ob-/- mice. The
mice harboring floxed Esp alleles can be crossed with transgenic
mice expressing the recombinase under the control of any promoter
of interest to specifically inactivate the Esp gene in the cells
where this promoter is active. In the Esp.sub.ob, one (+/-) or both
alleles (-/-) for OST-PTP have been inactivated in osteoblasts only
thereby interfering with OST-PTP expression only in these cells.
Molecular analysis showed that recombination occurred at high
frequency at the Esp locus in osteoblasts but not in any other
tissues or cell types including testis, adipocytes or pancreas
beta-cells (FIGS. 1C and 1D). Northern blot analysis verified that
it was a null allele while Souther blot hybridization was used to
demonstrate the efficiency of Esp excision in osteoblasts (FIG.
1C). Quantitative RT-PCR and Western analysis failed to detect Esp
mRNA or OST-PTP protein, respectively, in bone of Esp.sub.ob-/-
mice while both Esp mRNA and OST-PTP protein were present in the
testis of Esp.sub.osb-/- mice (FIG. 1D). These data indicate that
an osteoblast-specific inactivation of Esp was achieved.
[0453] As used herein, "Esp-deficient mice" means either of two
strains of transgenic mice in which both alleles for
osteotesticular protein tyrosine phosphatase OST-PTP (encoded by
the Esp gene) have been deleted (knocked out) as in the Esp osb -/-
mouse, or have been disrupted or (knocked in) as in the Esp-nLacz
-/- mouse.
[0454] FIG. 22 shows certain details of the method for generating
Esp.sub.osb-/- mice and normal bone formation in Esp-/- animals.
FIG. 23 compares 1 month-old WT and Esp-/- mice with respect to
various metabolic and physiologic parameters: serum levels of
C-peptide (A), serum Glucagon level (left) and glucagons content in
pancreas (right) (B) and serum levels of IGF-1 (C), PYY (D), and
amylin (E) in Esp-/- mice; (F) Ratio of muscle mass over body mass
calculated by proton magnetic resonance spectroscopy (.sup.1H-MRS)
in 10 week-old WT and Esp-/- mice; (G) Representative images of
proton .sup.1H-MRS of 10 week-old WT and Esp-/- mice; (H) Food
intake per day in 1 month- and 3 month-old Esp-/- and WT mice; (I
and J) Comparison of expression level by real time PCR (I) and of
serum levels (J) of TNF-.alpha. (left) and IL-6 (right) in 1
month-old Esp-/- and WT mice; and (K) Serum leptin (left) and
resistin (right) levels in 1 month-old Esp-/- and WT mice. In all
panels data represent the means .+-.SD of experiments. *, P<0.01
(t-test).
[0455] FIG. 24 shows the anatomy of the destruction of VMH nuclei
by GTG. FIG. 25 shows that there is no cell transdifferentiation
during the co-culture assays. FIG. 26 shows that Osteocalcin
expression is bone specific. In situ hybridization analysis of
osteocalcin and Esp expression in pancreas of 18.5 dpc embryos show
that neither gene is expressed in pancreas. Insulin expression was
used as a positive control. Hematoxylin-eosin staining of adjacent
sections was used to assess tissues integrity. Real time PCR
analysis of osteocalcin expression in osteoblasts, adipocytes, and
pancreatic islets collected from 1 month-old WT mice showed that
osteocalcin is not expressed in adipocytes or islets.
[0456] Generation of Collagen alpha 1(I)-PTP and Collagen alpha
1(I)-PTP.sub.ED transgenic mice. Transgenic mice over expressing
either the full length Esp cDNA (alpha1(I) collagen--OST-PTP) or a
truncated version of this cDNA encoding only the OST-PTP
extracellular (also herein referred to as the soluble domain)
domain (alpha 1(I) collagen--OST-PTP.sub.EC mice) were generated.
The extracellular domain is also herein referred to as the soluble
domain (SD). These cDNA genes were under the control of the
osteoblast-specific regulatory elements of the alpha 1(I) collagen
to make mice that over express ESP (OST-PTP) or the OST-PTP
extracellular domain in osteoblast in vivo.
[0457] At 1 month of age, the alpha 1(I) collagen--Esp transgenic
mice displayed an increase in serum glucose both after fasting and
after feeding, a decrease in insulin serum level after feeding, and
a decrease in energy expenditure. Accordingly, glucose tolerance
tests (GTT) showed that alpha1(I) collagen--Esp mice were
glucose-intolerant while insulin tolerance tests (ITT) established
that they were insulin-resistant (FIG. 4). Altogether the phenotype
of the transgenic mice is the mirror image (the opposite) of the
one observed in Esp-deficient mice. Moreover, this Esp cDNA full
transcript transgene corrected all the metabolic abnormalities in
Esp-deficient, diabetes-resistant mice. Transgenic mice
over-expressing either the full-length Esp cDNA (alpha1(I)
collagen--Esp) or a truncated version of this cDNA encoding only
the OST-PTP extracellular domain are herein referred to as the
soluble domain) domain (alpha 1(I) collagen--Esp.sub.EC mice.
[0458] Generation of ApoE-PTP, ApoE-PTP.sub.SD (also named
ApoE-PTP.sub.ED) transgenic mice. The full-length mouse Esp cDNA or
fragment of the Esp mouse cDNA encoding amino acids 1 to 1111 of
the extracellular domain (ED) was cloned into a vector directing
liver-specific expression using the promoter of the ApoE gene. In
contrast to expression of the full cDNA transcript for OST-PTP,
Apolipoprotein E--OST-PTP.sub.EC transgenic mice that express a
truncated version of this cDNA encoding only OST-PTP extracellular
domain were indistinguishable from wild type mice. These
experiments further prove that OST-PTP regulates energy metabolism
through its intracellular phosphatase domain.
[0459] Generation of Osteocalcin-deficient (also named Ocn-/- or
Bgp-/-) mice. "Osteocalcin-deficient mice" means a strain of mice
in which both osteocalcin alleles were deleted. In the osteocalcin
deficient transgenic mice described herein, Exon 4 of osteocalcin
gene 1 (OG1) coding for the mature protein, and the entire
osteocalcin gene 2 (OG2) sequence were deleted, while
osteocalcin-related gene (ORG) was left in place. Correct targeting
resulted in the replacement of the entire mature osteocalcin
protein-coding sequences by the pGKNeo selection cassette.
[0460] Generation of Osteocalcin-/- mice was previously reported
(Ducy et al., 1996). Exon 4 of osteocalcin gene 1 (OG1) coding for
the mature protein, and the entire osteocalcin gene 2 (OG2)
sequence were deleted, while osteocalcin-related gene (ORG) was
left in place. Correct targeting resulted in the replacement of the
entire mature osteocalcin protein-coding sequences by the pGKNeo
selection cassette. Analysis of these mice is reported FIGS. 5-7
and Table 1.
[0461] Generation of Adiponectin-deficient Mice and Ocn+/-;
Adiponectin +/- mice. Adiponectin-deficient mice were generated
according to a previously described strategy (Maeda et al., 2002)
where Exons 2 and 3 of either one (+/-) or both (-/-) alleles of
the adiponectin gene were deleted. Adiponectin +/- or -/- were then
crossed with Ocn-/- or +/- mice to generate Adiponectin+/-;Ocn+/-
mice. Analysis of these mice is reported FIG. 6.
[0462] Generation of SAP-Adiponectin transgenic Mice. Transgenic
mice may be generated which overexpress adiponectin. Such a
transgenic mouse's genome carries heterogeneous cDNA encoding
adiponectin under the control of the regulatory elements of the
mouse serum amyloid protein (SAP) gene, that produces an effect,
relative to a wild-type effect, that is selected from the group
consisting of an increase in adiponectin production, secretion and
activity. In some cases, the cDNA is defined by SEQ ID NO:8.
Constructs for use in generating such a mouse include one
comprising the cDNA for adiponectin under the control of the serum
amyloid protein promoter, which construct is designated pSAP-Adipo.
Cells, including adipocytes, may be isolated from such transgenic
animals.
[0463] To generate mice that over express adiponectin, the mouse
cDNA for adiponectin was subcloned upstream of a cassette
containing the human SAP promoter and rabbit .beta.-globin
non-coding exon/intron (FIG. 28). Fat pad weight was measured in WT
and adiponectin transgenic pups and mice of each sex at 3 months of
age (FIG. 28D). Food intake and energy expenditure were assessed in
WT and Sap-Adiponectin transgenic mice to ascertain if the increase
in energy expenditure observed in Esp-deficient mice is due solely
to their increase in adiponectin serum levels. It was also verified
that increasing serum adiponectin level will not affect appetite.
To that end, metabolic cages and equipment were used. Serum glucose
level was measured at birth, 2, 4, 8, and 16 weeks of age in WT and
Sap-Adiponectin transgenic mice. In adult mice, this was done both
after fasting and after feeding. In the same samples, serum insulin
and adiponectin levels were measured (FIGS. 28C and 28E). Serum
leptin levels were measured in serum of adult mice. Insulin
sensitivity was assessed by insulin tolerance test (FIG. 28F): mice
were fasted for six hours, injected IP with insulin (0.2 U/kg BW)
and glucose levels were measured at indicated times as described
(Mauvais-Jarvis et al., 2002). ITT data are presented as percentage
of initial blood glucose concentration. Insulin secretion was
assayed both by a glucose tolerance test performed following
glucose intraperitoneal injection and by a glucose stimulated
insulin secretion. Blood samples were obtained at 0, 2, 5, 15, and
30 minutes for GSIS or at 0, 15, 30, 60, and 120 minutes for GTT
after intraperitoneal injection of 2 g/kg dextrose. Whole blood
glucose values were determined using an automatic glucose monitor.
Histological analysis. We have observed that in Esp-deficient mice
there are fewer adipocytes than in WT mice yet they are larger,
suggesting that they cannot release fat. The same analysis may be
performed in 1 and 2 month old WT and Sap-Adiponectin transgenic
mice. To ascertain that the large size of the adipocytes betrays
their inability to release fat, WT, Esp-deficient and
Sap-Adiponectin 1 month-old mice may be fasted for 16 or 24 hours
and measured for free fatty acid (FFA) serum levels. It is expected
that FFA serum levels will not increase in Esp-deficient and
Sap-Adiponectin as it will in WT mice.
[0464] Generation of Sap-Insulin transgenic mice. A transgenic
mouse is disclosed herein whose genome carries cDNA encoding full
length mouse insulin under the control of the promoter and
regulatory elements of the mouse serum amyloid protein (SAP) gene,
that produces an effect, relative to a wild-type, comprising
increased insulin expression and secretion.
[0465] To generate mice that over express insulin, the mouse cDNA
for insulin was subcloned upstream of a cassette containing the
human SAP promoter and rabbit .beta.-globin non-coding exon/intron.
These transgenic mice were analyzed using the same batteries of
metabolic/molecular tests, including than the ones used for
studying the Sap-Adiponectin transgenic mice. These studies are
presented in FIG. 29.
[0466] Substrate Trapping. Plasmids for substrate trapping
experiments were made as follows: Rat OST-PTP sequences encoding
the first phosphatase domain (a.a 1116-a.a 1412) were cloned into
the BamHI site of pGEX 4T3 (Amersham) encoding GST (Glutathione
S-Transferase). This construct (GST-PTP) was used to generate
Asp1316Ala GST-PTP DA, which is a catalytic mutant form which leads
to the stabilization of the enzyme-substrate interaction, by site
directed mutagenesis. The mutation was made in the phosphatase 1
domain that is known to mediate the dephosphorylation function of
this class of phosphatases. The GST-PTP.sup.D1316A mutant has
reduced phosphatase activity but increased substrate binding
ability compared to wild type OST-PTP. It can thus retain, i.e.
"Trap," the substrate better than the wild type protein. Cells
expressing the mutant OST-PTP.sup.D1316A will trap any substrate
that is the usual target of OST-PTP, but the mutant enzyme cannot
dephosphorylate the substrate. It therefore holds onto the
substrate without releasing it. Protein complexes for each
experimental condition were then pulled down by centrifugation,
washed 4 times and analyzed by western blot.
[0467] For the substrate-trapping experiments, cells were lysed in
lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1%
Triton, 0.1% CHAPS, 5 mM iodoacetic acid, 10 mM sodium phosphate,
10 mM NaF). Cell lysates were incubated with either GST,
GST-PTP.sup.WT (a fusion of GST with the phosphatase domain I of
OST-PTP), or with GST-PTP.sup.D1316A (a trapping mutant of the Asp
of the phosphatase domain I). Recombinant proteins bound to
Sepharose beads for 1 hour at 4.degree. C. (insulin receptor
trapping) or for 2 h at 4.degree. C. (gamma-carboxylase substrate
trapping). Precipitates were collected, washed four times with
lysis buffer and resolved on SDS-PAGE, followed by western
blotting. Insulin receptor (InsR) was detected using rabbit
anti-insulin receptor antibodies (Santa-Cruz, C-19) and GST was
detected by mouse anti-GST antibodies (Santa-Cruz).
Gamma-carboxylase was detected using rabbit anti-gamma-carboxylase
antibody.
[0468] OST-PTP substrates are insulin receptor and
gamma-carboxylase. To determine if OST-PTP acts through
gamma-carboxylase, we conducted substrate-trapping experiments in
primary osteoblasts. Differentiated primary osteoblasts (d10) were
cultured for 10 days in alpha MEM/10% fetal bovine serum (FBS)
supplemented with ascorbic acid (100 micrograms/ml) and
Beta-glycerophosphate (5 mM). They were then starved for 24
hours.mu. in the same medium supplemented with 1% FBS only and
treated with pervanadate (100 .mu.M), an irreversible
protein-tyrosine phosphatase inhibitor, and 20% FBS for 30 minutes.
Cell lysates were incubated for 2 h at 4.degree. C. with either
GST, GST-PTP.sup.WT or GST-PTP.sup.D1316A. Different amounts of the
total cell extract were also loaded as control.
[0469] Transformed cells that over express full-length or truncated
OST-PTP. Eukaryotic expression vectors that express flag-tagged
full-length OST-PTP or flag-tagged truncated OST-PTP containing
only its extracellular domain (OST-PTP.sub.EC) were used to perform
DNA permanent transfection experiments in ROS (rat osteoblast
cells) 17/2.8 osteoblastic cells transfected with these flag-tagged
vectors, and as a negative control in COS 7 cells. Following
selection, and isolation of clones of cells that have incorporated
each of the two genes in chromosomes (flag-tagged full-length
OST-PTP or flag-tagged truncated OST-PTP containing only its
extracellular domain) it was verified that the genes were
transcribed and that the proteins were made using RT-PCR and
Western blot analysis of cell lysates respectively. The cells were
then cultured in a serum-free medium overnight. The supernatant of
cells transfected with the empty vector, or vector encoding the
full length or the truncated Esp cDNA was isolated and a Western
analysis was performed using a commercially available anti-Flag
antibody.
[0470] Bacterial expression vectors for osteocalcin production. We
have generated prokaryotic expression vectors for GST-tagged mouse
osteocalcin, GST-tagged human osteocalcin, GST-tagged mutants of
mouse and human osteocalcin and GST-tagged truncation mutants of
mouse and human osteocalcin.
[0471] Diet and GTG induced obesity and type 2 diabetes. For
diet-induced obesity, male and female six-week old WT and
osteocalcin-deficient mice (n=10 per group) were fed for 4, 6, 8,
or 12 weeks with either a normal or a "western" diet" of 45% fat,
35% carbohydrate, and 20% protein. For GTG-induced obesity, male
and female 4 week-old WT and osteocalcin-deficient mice (n=10 per
group) were injected with 0.5 mg/kg of GTG and sacrificed at 12
weeks of age. In both type of experiments, WT and mutant mice were
analyzed as follows. Physical inspection: Whole body weight of each
mouse at the start of the experiment and every week thereafter
until sacrifice was measured. Food intake: This parameter was
assessed to ascertain in particular that GTG lesions induce an
increase in food intake. To that end, metabolic cages and equipment
were used. Metabolic studies: Serum glucose and insulin levels were
measured after fasting overnight and after feeding. Serum
adiponectin and leptin levels were also measured in each mouse.
Insulin secretion was assayed both by a glucose tolerance test
(GTT) performed following glucose intraperitoneal injection and or
glucose stimulated insulin secretion test (GSIS). Blood samples
were obtained at 0, 2, 5, 15 and 30 minutes or at 0, 15, 30, 60 and
120 minutes following intraperitoneal injection of 2 g/kg dextrose
for GTT. Whole blood glucose values were determined using an
automatic glucose monitor. Molecular analysis: Expression of
multiple markers of insulin sensitivity in hepatocytes, adipocytes
and myoblasts at the end of each experiment was measured.
[0472] Co-culture of osteoblasts and adipocytes to study regulation
of adiponectin expression/secretion by osteocalcin. A co-culture
assay was developed between osteoblasts and adipocytes to analyze
modifications in adiponectin expression. We used in this assay
osteoblast from WT, Esp-deficient or osteocalcin-deficient mice
along with primary adipocytes taken from any of these same mice. As
a negative control, we co-cultured mouse embryonic fibroblasts of
each genotype with adipocytes. Osteoblasts and fibroblasts were
prepared according standard protocols that have been routinely used
in the laboratory for the last twelve years (Ducy and Karsenty
1995), incorporated by reference as if set forth fully herein.
Osteoblasts or fibroblasts were plated at 70% confluence in alpha
MEM, 10% fetal bovine serum (FBS) 36 h prior to the beginning of
the experiment. Prior to adding adipocytes, culture medium was
changed to decrease the FBS concentration to 1%. Adipocytes were
added for 0, 2, 4, 8, or 12 hours the following morning. At the end
of the experiment, adipocytes that were present as non adherent
cells were collected by centrifuging the culture medium. Adipocytes
were used to extract RNA and to measure by real time the PCR
expression of adiponectin and possibly other adipocyte-derived
hormones including leptin. Culture medium was used to measure
osteocalcin, adiponectin, leptin and other adipokine levels.
[0473] Co-culture of osteoblasts and beta-cells to study regulation
of insulin expression/secretion by osteocalcin. A co-culture assay
between osteoblasts and pancreatic beta-cells was developed to
analyze modifications in insulin expression. Osteoblasts from WT,
Esp-deficient or osteocalcin-deficient mice were used along with
pancreatic beta-cells taken from any of these same mice. As a
negative control, mouse embryonic fibroblasts of each genotype were
co-cultured with adipocytes. Osteoblasts and fibroblasts were
prepared according standard protocols that have been routinely used
in the laboratory for the last twelve years. (Duey and Karsenty
1995, incorporated by reference as if set forth fully herein).
Osteoblasts or fibroblasts were plated at 70% confluence in alpha
MEM 10% fetal bovine serum (FBS) 36 h prior to the beginning of the
experiment. Prior to adding beta-cells, culture medium was changed
to decrease the FBS concentration to 1%. Beta-cells were added for
0, 2, 4, 8, or 12 hours the following morning. At the end of the
experiment, beta-cells that were present as non adherent cells were
collected by centrifuging the culture medium. Beta-cells were used
to extract RNA and to measure by real time the PCR expression of
insulin and other beta-cell-derived hormones as well as the
expression of molecules known to regulate insulin expression and
cell proliferation. Culture medium was used to measure osteocalcin,
adiponectin, insulin and other cytokine levels.
[0474] Metabolic studies. For glucose tolerance test (GTT), glucose
(2 g/kg body weight (BW)) was injected intraperitoneally (IP) after
an overnight fast and blood glucose was monitored using blood
glucose strips and the Accu-Check glucometer (Roche) at indicated
times. For glucose stimulated insulin secretion test (GSIS),
glucose (3 g/kg BW) was injected IP after an overnight fast; sera
were collected from tails and insulin measured as described
(Mauvais-Jarvis et al., 2000). For insulin tolerance test (ITT),
mice were fasted for six hours, injected IP with insulin (0.2 U/kg
BW) and blood glucose levels were measured at indicated times as
described (Mauvais-Jarvis et al., 2002). ITT data are presented as
percentage of initial blood glucose concentration. Gold thioglucose
(600 mg/kg BW, USP) was injected IP after an overnight fast, mice
were sacrificed 3 months later for analysis. Streptozotocin (150
mg/ml single injection, Sigma) was injected IP and blood glucose
measured as described above every 2 days thereafter. After 8 days,
pancreases were isolated to measure insulin content as previously
described (Mauvais-Jarvis et al., 2000). Food intake was measured
using metabolic cages as the daily change of food weight. Energy
expenditure was measured using metabolic cages connected to a
calorimeter (Columbus Instrument). Heat values (Kcal/Hr) were
recorded over 2 days and reported to each mouse BW.
[0475] Laboratory measurements. Blood was collected by heart
puncture of isoflurane anesthetized mice in the fed and fasted
states. Colorimetric assays were used to measure serum levels of
free fatty acids (Wako Chemicals) and of triglycerides (Sigma).
Serum levels of insulin (Crystal Chem Inc. kit), adiponectin (Linco
kit), leptin (Crystal Chem Inc. kit) and resistin (Linco kit) were
quantified by ELISA, osteocalcin levels by IRMA (Immunotopics kit).
There is no IRA, IRMA, or ELISA designed to differentiate
carboxylated from undercarboxylated osteocalcin in mice. The
existing kits measure total osteocalcin, but cannot specifically
recognize undercarboxylated osteocalcin. Therefore, hydroxyapatite
(HA) resin was used to separate the two forms. The carboxylated
form is the only one that binds to the HA.
[0476] Mouse islets and adipocytes isolation. Islets were isolated
using a Histopaque gradient (1077, Sigma). In brief, after clamping
the common bile duct at its entrance to the duodenum, lmg/ml
collagenase P (Sigma) in M199 medium (GIBCO) was injected into the
duct. The swollen pancreas was surgically removed and incubated at
37.degree. C. for 17 min. Digested pancreata were dispersed by
pipetting and rinsed twice with the same medium. After filtering
the tissue suspension through a Spectra-mesh (400 .mu.m), the
digested tissue was resuspended in Histopaque and overlaid with
M199 medium. The sample was then centrifuged at 1,700 g for 20 min,
and the islets were collected from the interface. The recovered
material was washed twice with cold M199 medium, resuspended in
M199/1% NCS or aMEM/1% FBS (GIBCO) medium and cultured at
37.degree. C. in 5% CO.sub.2.
[0477] Primary adipocytes were isolated from epididymal fat pads by
collagenase digestion. Briefly, minced adipose tissue was digested
by lmg/ml collagenase P in KRP Buffer (20 mM HEPES, 120 mM NaCl, 6
mM KCl, 1.2 mM MgSO.sub.4, 1 mM CaCl.sub.2, 0.6 mM
Na.sub.2HPO.sub.4, 0.4 mM NaH.sub.2PO.sub.4, 2.5 mM D-glucose, 2%
BSA, pH 7.4) for 1 h at 37.degree. C. The isolated cells were
washed twice with KRP Buffer before being cultured in .alpha.MEM/1%
FBS at 37.degree. C. in 5% CO.sub.2.
[0478] Cell culture experiments. Primary osteoblasts were prepared
from calvaria of 5 day-old pups as previously described (Ducy et
al., 2000a) and were cultured in .alpha.MEM/10% FBS in the,
presence of 100 .mu.g/ml ascorbic acid and 5 mM
.beta.-glycerophosphate for 5 days. Skin fibroblasts were isolated
by collagenase digestion (0.5 mg/ml) and were cultured in
.quadrature.MEM/10% FBS. Twenty-four hours before addition of
primary islets (or adipocytes), osteoblasts (or fibroblasts) were
placed in .alpha.MEM/1% FBS. For warfarin treatment, ROS17/2.8
osteoblastic cells were maintained in DMEM/F12/10% FBS until being
supplemented with 50 .mu.M warfarin or vehicle in DMEM/F12/1% FBS
for 48 h prior to co-culture with adipocytes. After 4 h of
co-culture, either in the presence or absence of (1 .mu.m) culture
inserts (Falcon) islets (or adipocytes) were collected for RNA
isolation using TRIZOL (Invitrogen).
[0479] Gene expression analyses. All gene expression analyses were
performed using real time PCR. DNAse I-treated total RNA was
converted to cDNA with the SuperScript III kit (Invitrogen).
Real-time PCR were performed using the Taq SYBR Green Supermix with
ROX (Biorad) on an MX3000 instrument (Stratagene); beta-actin
amplification was used as an internal reference for each sample.
All primers were from SuperArray.
[0480] Osteocalcin/hydroxyapatite (HA) binding assay. Sera from 1
month-old mice, obese patients or supernatant from warfarin-treated
osteoblast cultures were added to HA slurry to achieve a final
concentration of 25 mg slurry/ml. After 15 min (mouse sera,
supernatant) or 30 min (human sera), HA beads were pelleted by
centrifugation and HA-bound osteocalcin was eluted with 0.5M sodium
phosphate buffer, pH 8.0. Osteocalcin present in eluates and
initial samples was measured by IRMA. Values represent percentage
of HA-bound osteocalcin over initial osteocalcin content. Hauschka,
P. V., et al., Physiol Review 69, 990-1047 (1989).
[0481] Statistical analyses. Results are given as means.+-.standard
deviations except in FIGS. 2B and 5F where means.+-.standard errors
of the mean are shown. Statistical analyses were performed using
unpaired, two-tailed Student's t or ANOVA tests followed by post
hoc tests. A p value <0.05 was considered significant and is
indicated by a star in all figures unless otherwise indicated.
[0482] Recombinant Osteocalcin. Recombinant osteocalcin was
bacterially produced and purified on glutathione beads according to
standard procedures. Osteocalcin was then cleaved from the GST
subunit using thrombin digestion. Thrombin contamination was
removed using an affinity column. The purity of the product was
qualitatively assessed by SDS-PAGE. Bacteria do not have a
gamma-carboxylase gene. Therefore, recombinant osteocalcin produced
in bacteria is always completely undercarboxylated at all three
sites. Osteocalcin can be made in many ways known in the art,
including being chemically synthesized, since it can be made
without gamma-carboxylation when chemically synthesized.
[0483] Human studies. This study enrolled a group of obese and
non-obese Caucasian women participating in a Clinical investigation
performed at the Center of Research on Human Nutrition, Hotel-Dieu
Hospital, Paris, France (PHRC protocol N.degree. A0R076). This
study was approved by the Ethics Committees of Hotel-Dieu (Paris).
All subjects gave their informed consent. Subjects were weight
stable for at least 3 months before the investigation day. Clinical
and biochemical parameters were assessed in the morning (8:00 am)
at the fasting state.
[0484] Histology. Frozen sections of livers were cryoembedded,
sectioned at 5 .mu.m and stained with Oil red O. Fat and pancreatic
tissues were fixed overnight in 10% neutral formalin, embedded in
paraffin, and sectioned at 5 .mu.m. Histology sections were stained
with hematoxylin and eosin (H&E). Immunohistochemistry was
performed using rabbit anti-insulin (SantaCruz, 1:100) and mouse
anti-Ki67 (Vector, 1:100) antibodies and ABC Elite kits (Vector).
Hypothalamic histology was performed as described (Takeda et al.,
2002). To evaluate cell sizes or numbers, 5 to 10 sections (each 50
micrometers apart) were analyzed using a 40.times. objective on a
Leica microscope outfitted with a CCD camera (SONY). Images were
processed using the Osteomeasure software. Beta-cell area
represents the surface positive for insulin immunostaining divided
by the total pancreatic surface. Beta-cell mass was calculated as
beta-cell area multiplied by pancreatic weight. At least 3 mice
were analyzed per condition. Tibia anterior muscles were fixed in
4% PFA/2% glutaraldehyde/0.1 M sodium cacodylate ph 7.3, post-fixed
in 1% osmium tetraoxide and embedded in epoxy resin (Epon).
Ultrathin sections were stained in 4% aqueous Uranyl Acetate and 2
min in Reynolds' Lead Citrate and examined with a JEOL 2000FX. Ten
electron micrographs per mouse were digitized and the area of each
clearly distinguishable mitochondrion was analyzed using ImageJ
software. Fifteen to 25 individual mitochondria were measured in 4
mice of each genotype.
[0485] Results
[0486] Generation and perinatal lethality of Esp-/- mouse models.
To study OST-PTP, Esp was disrupted in a classical way (Esp-nLacZ)
(Dacquin et al., 2004) and in an osteoblast-specific manner
(Esp.sub.ob-/-) by deleting exons 24 to 35 that encode the
phosphatase domain using the LoxP/Cre recombinase technology (FIG.
22A). Mice harboring Esp floxed alleles were crossed with
.alpha.1(1) collagen-Cre mice (Dacquin et al., 2002) to generate
osteoblast-specific Esp-deficient mice (Esp.sub.ob-/-) (FIG. 22B).
Southern blot analysis showed that recombination occurred at high
frequency at the Esp locus in osteoblasts (FIG. 1C). Accordingly,
Esp expression was reduced nearly 90% in Esp.sub.ob-/- osteoblasts
and was unaffected in testis, the other site of Esp expression
(FIG. 1D). Esp expression could not be detected in adipocytes or
pancreatic beta-cells (data not shown). These data established that
an osteoblast-specific inactivation of Esp was achieved. For the
sake of clarity, reference to Esp-/- mice will be made when both
Esp-nLacZ and Esp.sub.ob-/- mice were studied.
[0487] When analyzed at weaning, intercrosses of Esp-/- mice in
either a 129Sv/EV or a C57BL/6 genetic background yielded only
about 25% of Esp-/- mice (FIG. 1F). To determine if this early
post-natal lethality was due to a delay in skeletal development,
skeletal preparations of newborn wild-type (WT) and Esp-/- pups
were stained. No abnormality of bone formation was detected that
could explain this lethality (FIGS. 22D-22F). Experiments were
conducted to determine whether Esp-/- pup lethality could be due to
a maternal effect, possibly a humoral abnormality. If it were the
case, mutant pups born from homozygous mutant mothers should die at
a higher frequency than those born from heterozygous mothers. That
is precisely what was observed. While lethality of Esp-/- pups born
from Esp+/- mothers never reached 15%, up to 35% of Esp-/- pups
born from Esp-/- mothers died before weaning (FIG. 1F). These data
indicate that the lethality of Esp-/- pups was caused in part by a
maternal effect.
[0488] Increased beta-cell proliferation and insulin secretion in
Esp-/- mice. To determine if the maternal effect responsible of
Esp-/- mice perinatal lethality was caused by a humoral
abnormality, metabolic parameters were measured in newborn pups
prior to milk ingestion. Esp-/- pups, regardless of genetic
background, sex, and type of deletion performed, showed only one
abnormality: a 3-fold reduction of blood glucose levels (FIG. 1G).
In some mutant pups blood glucose levels were even too low to be
detected. Albeit less severe, a significant decrease in blood
glucose level was also observed in 1 and 3 month-old Esp-/- mice
after feeding (FIG. 1G). This hypoglycemia was explained by a
significant hyperinsulinemia in newborn, 1 and 3 month-old fed
Esp-/- mice (FIG. 1H). On the other hand, expression of Glucagon, a
hormone secreted by pancreatic beta-cells, was normal (FIG. 23B),
thus indicating that Esp mutation affects beta-cells
specifically.
[0489] To establish more firmly that there was an increase in
insulin secretion in the Esp-/- mice, intraperitoneal (IP) glucose
stimulated insulin secretion tests (GSIS) were performed at 1 and 3
months of age. These assays showed that insulin secretion was
enhanced by the absence of OST-PTP (FIGS. 1H and 1L). To assess how
this increase in insulin secretion affects the ability to dispose
of a glucose load, glucose tolerance tests were performed following
IP injection of glucose (2 g/kg of body weight) after an overnight
fast (GTT). These tests revealed that 1 and 3 month-old Esp-/- mice
had a significantly higher tolerance to glucose than WT mice (FIG.
1J).
[0490] Histological and immunochemical analyses showed an increase
in pancreas insulin content, the number of islets, islet size and
overall of beta-cell mass in the Esp-/- pancreas (FIGS. 1K and 1L).
A TUNEL assay failed to detect any abnormal apoptosis, and Ki67
immunostaining performed in 5 day-old pups (P5) and 1 month-old
mice showed that beta-cell proliferation was increased 60 to 300%
in Esp-/- mice (FIG. 1M). These data demonstrate that OST-PTP
expressed in osteoblasts influences a pathway regulating beta-cell
proliferation.
[0491] Increased insulin sensitivity in Esp-/- mice. To determine
whether the enhanced ability of Esp-/- mice to dispose of a glucose
load was secondary to an increase in insulin sensitivity, insulin
tolerance tests (ITT) were performed. Insulin sensitivity, defined
by the drop in blood glucose level following IP insulin injection,
was significantly increased in 1 and 3 month-old Esp-/- compared to
WT mice (FIG. 2A). Accordingly, expression of molecular markers of
insulin sensitivity in fat (PPAR.alpha., PPAR.gamma.), liver
(Foxa2, PPAR.alpha.) and skeletal muscle (Pgc-1.alpha., Nrf-1,
Mcad) were also markedly increased in Esp-/- compared to WT mice.
Pepck expression was decreased in Esp-/- liver indicating that
gluconeogenesis was inhibited in this organ (FIG. 2E). It was
speculated that as a result of these molecular events energy
expenditure was increased in Esp-/- mice (FIG. 2G). In all
analyses, heterozygous Esp+/- mice behaved as their WT
littermates.
[0492] The experimental data show that Esp (OST-PTP) inactivation
causes hypoglycemia, potentially lethal in newborn pups, that is
associated with an increase in insulin secretion and sensitivity.
That these abnormalities were observed to the same extent in both
Esp-nLacZ-/- and in Esp.sub.ob-/- mice established that it is the
Esp gene expressed in osteoblasts, and not in any other cells or
tissues, that is responsible for the metabolic phenotype.
[0493] One and 3 month-old Esp-/- mice displayed another phenotypic
abnormality; their fat pads were significantly lighter than those
of their WT littermates (FIG. 2F). Serum triglyceride levels were
also lower in Esp-/- than in WT mice (FIG. 2H). Since Esp is not
expressed in fat and food intake is normal in Esp-/- mice (FIG.
23H), this decrease in fat mass is secondary to the increase in
insulin sensitivity. Although there were fewer adipocytes in Esp-/-
than in WT mice (WT, 93.2.+-.10.7.times.10.sup.3 adipocytes/fat pad
(n=5); Esp-/-, 37.+-.5.1.times.10.sup.3 adipocytes/fat pad (n=3))
they were larger (FIG. 21). To understand this phenotype the
expression of multiple molecular markers was studied. C/EBP.alpha.,
Srebp1c, Fatty acid synthase (FAS) and Lipoprotein lipase (LPL)
were similarly expressed in Esp-/- and WT adipocytes, showing that
adipogenesis, lipogenesis and fat uptake were not overtly affected
by the mutation (FIG. 2J). In contrast, expression of molecular
markers of insulin sensitivity (PPAR.gamma. and the regulator of
fat oxidation PPAR.alpha.) was increased, thus explaining enhanced
insulin sensitivity without fat accumulation. Furthermore,
expression of Perilipin and Triglyceride lipase (TGL), two
anti-lipolytic proteins, was markedly decreased in Esp-/- compared
to WT adipocytes (FIG. 2J) indicating that lipolysis is inhibited
in Esp-/- mice. Accordingly, the serum level of free fatty acid did
not increase following an overnight fast in Esp-/- mice as it did
in WT littermates (FIG. 2K). The combination of increased insulin
sensitivity and fat oxidation with inhibition of fat release from
adipocytes synergized to produce the observed phenotype of low
adiposity with large adipocytes in Esp-/- mice. These results are
consistent with the increase in insulin secretion in Esp-deficient
mice because insulin is a potent inhibitor of lipolysis.
[0494] Increased adiponectin expression in Esp-/- mice. Experiments
were conducted to determine whether there was a humoral basis for
the increase in insulin sensitivity observed in Esp-/- mice.
Expression and serum levels of Resistin, an adipokine mediating
insulin resistance, were virtually unaffected by Esp deletion. The
same was true for leptin, an insulin-sensitizing hormone (Friedman
and Halaas, 1998; Steppan et al., 2001) (FIGS. 2L and 23K). This
latter observation is in agreement with the fact that food intake
was normal in Esp-/- mice (FIG. 23H). By contrast, expression and
serum levels of adiponectin, an adipokine able to enhance
sensitivity to insulin (Yamauchi et al., 2001), were respectively
increased three and two-fold in Esp-/- mice at birth, 1 and 3
months of age regardless of their sex and genetic background (FIGS.
2L and 23M). Accordingly, it was observed that expression of
adiponectin target genes such as Acyl-CoA Oxidase, PPAR.alpha. and
Ucp2 was increased in Esp-/- mice (FIG. 2N) (Kadowaki and Yamauchi,
2005). This increase in adiponectin expression and serum levels
provides one mechanism to explain the increase in insulin
sensitivity observed in Esp-/- mice.
[0495] In summary, Esp inactivation caused hypoglycemia as a result
of increased pancreatic beta-cell proliferation, enhanced insulin
secretion and improved insulin sensitivity in peripheral tissues
with decreased adiposity. That these abnormalities were observed in
both Esp-nLacZ-/- and Esp.sub.ob-/- mice demonstrated that the
skeleton via osteoblasts is involved in regulating glucose
homeostasis.
[0496] Esp-/- mice are protected from obesity and glucose
intolerance. The increase in insulin secretion and sensitivity
characterizing Esp-/- mice raised the prospect that these mutant
mice could be protected from obesity and diabetes. Esp-nLacZ-/- and
Esp.sub.ob-/- showed identical metabolic and molecular
abnormalities. In some experiments only one or the other model were
tested so for the sake of clarity we will refer to Esp-/- in this
case.
[0497] First, gold thioglucose (GTG) was injected in 1 month-old
mice to induce specific lesions in the ventromedial hypothalamus
(Brecher et al., 1965). As expected, GTG induced ventromedial
hypothalamic lesions (FIG. 24) and hyperphagia (FIG. 3A) in both WT
and Esp-/- mice. When analyzed 3 months after injection,
GTG-treated WT mice were obese and their fat pad mass and serum
triglyceride levels were significantly increased. GTT and ITT
analyses showed that glucose intolerance and insulin resistance
also increased (FIGS. 3E-3F). By contrast, GTG-treated Esp-/- mice
were not obese, had fat pad mass and serum triglyceride levels
similar to those of PBS-treated WT mice, and they displayed no
evidence of glucose intolerance or of insulin insensitivity (FIGS.
3E-3F).
[0498] Next, 1 month-old WT and Esp-deficient mice were fed with a
high fat diet (HFD) (58% fat kcal) for six weeks. It was discovered
that body weights were significantly lower in Esp-nLacZ-/- mice
than in WT mice at the end of this six week period (FIGS. 3G-3I).
Glucose tolerance testing (GTT) demonstrated that after being fed a
HFD for six weeks Esp-nLacZ-/- mice kept a normal tolerance to
glucose, and insulin sensitivity determined by ITT remained normal.
By contrast these parameters were altered in WT mice fed a high fat
diet (HFD).
[0499] Whether the increase in insulin sensitivity could protect
Esp-/- mice from pancreatic beta-cell failure was determined. To
that end, mice were injected with streptozotocin (STZ) to provoke
oxidative stress in beta-cells and cell death as are seen in type 2
diabetes (Le May et al., 2006). STZ treatment markedly decreased
pancreas insulin content and insulin serum level in both genotypes
(FIGS. 3J and 3K). Eight days after STZ injection, 3 of the 7
STZ-treated WT mice had died and all the surviving ones had serum
glucose levels above 500 mg/dl (FIGS. 3L and 3M). On the other
hand, only one STZ-treated Esp-/- mouse died during this period and
the blood glucose level of the surviving ones did not exceed 250
mg/dl. Unlike STZ-treated WT mice, glucose could not be detected in
urine of STZ-injected Esp-/- mice (FIG. 3N). Since both STZ-treated
WT and Esp-/- mice had a major decrease in islet insulin content,
the absence of an overt diabetic phenotype in STZ-treated Esp-/-
mice showed that their increase in insulin sensitivity occurred
independently of their increase in insulin secretion. These results
establish that Esp function (OST-PTP) is required for the
development of obesity and glucose intolerance in mice.
[0500] Esp influences the biological activity of an
osteoblast-secreted molecule. The next question was how Esp,
through its expression in osteoblasts could regulate insulin
secretion and sensitivity. Cell-based assays failed to provide
evidence that the OST-PTP extracellular domain could be either
cleaved and secreted or expressed independently of the phosphatase
domain. Therefore, COS cells that do not normally express Esp, were
transfected with vectors expressing either a full-length flag
tagged OST-PTP or its flag-tagged extracellular domain only. The
cells were transfected using the standard calcium phosphate method
well known in the art. At the end of the experiment, supernatant
was collected, cells were lysed and both supernatant and cell
lysate assayed for the presence of OST-PTP. Western blot analysis
using either cell lysates or cell supernatants was then performed.
Recombinant full-length or truncated proteins were detected in cell
lysates but never in the supernatants, showing that OST-PTP
extracellular domain is not normally secreted by cells. An antibody
was made against the OST-PTP extracellular domain to be able to
perform these experiments; certain embodiments of this invention
are directed to this antibody and to other antibodies that bind to
the OST-PTP extracellular domain. The OST-PTP extracellular domain
is accessible to antibodies because it is not sequestered inside
the cell membrane. An antibody against the transmembrane domain of
OST also exists. Both of these antibodies are polyclonal and could
be administered to an animal to inhibit OST-PTP, thereby increasing
osteocalcin activity, which in turn increases adiponectin
production and secretion from adipocytes, which in turn increases
insulin production and sensitivity. Of course, monoclonal
antibodies can be used as well.
[0501] To further study OST-PTP function, transgenic mice
expressing either full-length OST-PTP or its extracellular domain
only in osteoblasts were generated and analysed. Transgenic mice
overexpressing full-length Esp cDNA selectively in osteoblasts
(alpha1(I)-OST-PTP mice) were made that displayed decreased
beta-cell proliferation, lower beta-cell mass, hypoinsulinemia in
the fed state and impaired insulin secretion in response to glucose
(FIGS. 4A-C). They also showed lower adiponectin serum
concentrations (FIG. 4B). As a result, alpha1(I)-OST-PTP mice
developed hyperglycemia on regular chow, glucose intolerance and
insulin resistance (FIGS. 4B, 4D and 4E). The fact that this
phenotype, which is the minor image of the one observed in Esp-/-
mice, is only observed in transgenic mice over expressing
full-length OST-PTP shows that the phosphatase activity of OST-PTP
is required to affect glucose homeostasis. Furthermore, the fact
that these mice over expressed Esp in osteoblasts further supports
the conclusion that OST-PTP regulates the bioactivity of an
osteoblast-derived secreted molecule that in turn regulates glucose
homeostasis. By contrast, alpha.sub.1(I) collagen-Esp.sub.sd mice,
which express only OST-PTP extracellular domain, had no energy
metabolism abnormalities of any kind. These results taken with the
well-described fact that OST-PTP phosphatase domain is an active
one, show that it is through its phosphatase domain and not through
its extracellular domain that OST-PTP regulates insulin secretion
and adiponectin expression and further confirm that OST-PTP act on
the regulation of energy metabolism via its expression in
osteoblasts.
[0502] Apolipoprotein E--OST-PTP.sub.EC transgenic mice were also
generated that express the OST-PTP extracellular domain and release
it into the general circulation. The apolipoprotein E promoter was
used to direct Esp expression in liver cells thereby causing
release of the OST-PTP extracellular domain into the general
circulation. These transgenic mice were indistinguishable from wild
type mice, further proving that OST-PTP regulates energy metabolism
through its intracellular phosphatase domain and its expression in
osteoblasts.
[0503] To further prove that osteoblasts secrete a factor that acts
on pancreatic beta-cells and adipocytes, osteoblasts, which are
adherent cells, were co-cultured with either pancreatic islets or
adipocytes, which are non-adherent cells. Co-culture of
differentiated WT osteoblasts with islets isolated from WT mice
increased insulin expression in islets 40% (FIG. 4F). In full
agreement with the increase in insulin secretion observed in Esp-/-
mice, Esp-/- osteoblasts further enhanced insulin expression (FIG.
4F). Osteoblasts or fibroblasts were also co-cultured with
adipocytes. WT osteoblasts, but not fibroblasts, increased
expression of adiponectin and Esp-/- osteoblasts were twice as
potent as WT osteoblasts in enhancing adiponectin expression (FIG.
4G). In this assay, adiponectin was the only adipokine whose
expression was affected (FIG. 4G). Control experiments using WT
osteoblasts co-cultured with Esp-/- islets or adipocytes showed the
same increase in insulin and adiponectin expression as seen when
using WT islets or adipocytes (FIG. 4H).
[0504] To establish that osteoblasts influence insulin and
adiponectin expression via the release of secreted molecule(s),
additional experiments were performed. First, osteoblasts were
co-cultured with either islets or adipocytes using a filter
preventing cell-cell contact. Second, islets and adipocytes were
co-cultured in the presence of supernatant of primary osteoblast
cultures. In both cases, a significant increase in insulin and
adiponectin expression was observed (FIGS. 41 and 4J). Taken
together, these data indicate that Esp expressed in osteoblasts
regulates the expression or activity of a secreted molecule that
affects insulin and adiponectin expression in beta-cells and
adipocytes.
[0505] Osteocalcin is the osteoblast-derived secreted molecule that
increases proliferation, insulin secretion and insulin sensitivity.
To identify the molecule(s) secreted by osteoblasts that regulate
glucose homeostasis, energy metabolism parameters were analysed in
mutant mouse strains lacking osteocalcin, an osteoblast-specific
secreted molecule present in serum. In earlier studies, it was
observed that, upon their generation, oc-/- mice were abnormally
fat. Ducy et al Nature 1996, herein incorporated by reference. At
the time there was no explanation for why these animals were so fat
and therefore the obesity aspect of these mice was observed but not
published. Both homozygous (Oc-/-) and heterozygous strains (Oc+/-)
were made.
[0506] Osteocalcin is one of the major non-collagenous proteins
made by osteoblasts and is also an osteoblast-specific molecule.
Like many secreted proteins, including peptide hormones,
osteocalcin is generated as pre-pro-osteocalcin and undergoes
cleavage and post-translational modifications in the cytoplasm
before being secreted. In addition, osteocalcin belongs to the
family of gla proteins in which some glutamic acid residues are
carboxylated by a gamma-carboxylase to form gla residues. Hence the
other name of osteocalcin: bone gla protein (BGP). Gla residues
confer on gla proteins a high affinity for mineral ions.
[0507] Osteocalcin-/- mice had higher blood glucose and lower
insulin serum levels than WT mice (FIGS. 5A and 5B). Insulin
secretion and sensitivity as well as glucose tolerance analyzed by
GSIS, GTT and ITT were all decreased in Osteocalcin-/- mice, as was
energy expenditure (FIGS. 5C-5E and 5G). Accordingly, the
expression of genes involved in insulin action was decreased in
skeletal muscle and liver while Pepck expression was increased
(FIG. 5H). Islet size and number, beta-cell mass, pancreas insulin
content and insulin immunoreactivity were all markedly decreased in
Osteocalcin-/- mice (FIG. 5I). Beta-cell proliferation measured by
Ki67 immunostaining was decreased two fold in Osteocalcin-/-
pancreas in P5 pups and at 3 months of age (FIG. 5I). Accompanying
this marked decrease in beta-cell proliferation, insulin secretion
and sensitivity, was an increase in fat pad mass, adipocyte number
(WT, 93.2.+-.10.7.times.10.sup.3 adipocytes/fat pad (n=5);
Osteocalcin-/-, 125.6.+-.10.6.times.10.sup.3 adipocytes/fat pad
(n=3)) and serum triglyceride levels (FIGS. 5J and 5K). Adiponectin
expression and serum levels were significantly lower in
Osteocalcin-/- than in WT mice, especially considering their
increased fat pad mass, while expression of other adipokines was
not affected (FIGS. 5L and 5M). Expression of molecular targets of
adiponectin action was decreased in Osteocalcin-/- mice (FIG. 5N).
However, Osteocalcin+/- mice were undistinguishable from WT
littermates (data not shown). The cDNA sequence for mouse
adiponectin is SEQ ID NO:8; and it identified also by amino acid
SEQ ID NO:9. The cDNA sequence for human adiponectin is SEQ ID
NO:6; and it identified also by amino acid SEQ ID NO:7.
[0508] To demonstrate that osteocalcin is the molecule secreted by
osteoblasts that affects insulin and adiponectin expression,
further co-culture experiments were performed. Unlike WT
osteoblasts, Osteocalcin-/- osteoblasts failed to enhance
expression of insulin and adiponectin in islets and adipocytes,
respectively (FIGS. 5O and 5P). In a converse experiment, forced
expression of osteocalcin in COS cells allowed these cells to
increase insulin expression in islets and adiponectin expression in
adipocytes (FIG. 5Q). WT immature osteoblasts, that do not express
osteocalcin (Ducy et al., 2000b) were co-cultured with either
islets or adipocytes. These cells failed to induce either insulin
or adiponectin expression (FIG. 5R). Taken together, these data
provide genetic and cellular evidence indicating that osteocalcin
is the molecule secreted by differentiated osteoblasts that
regulates insulin and adiponectin expression.
[0509] Osteocalcin regulates insulin sensitivity through
adiponectin. To determine whether insulin and adiponectin both
contribute, independently of each other, to the metabolic phenotype
of the Osteocalcin-/- mice, two related questions were asked.
First, does osteocalcin regulate adiponectin expression
independently of its action on insulin secretion, and if so, does
the decrease in adiponectin expression noted in the Osteocalcin-/-
mice explain the decrease in insulin sensitivity? If both
hypotheses are correct, then compound heterozygote Osteocalcin+/-;
Adiponectin+/- mice should have lower expression of adiponectin
than WT littermates and should show a decrease in insulin
sensitivity similar to the one observed in the Osteocalcin-/- or in
the Adiponectin-/- mice (Maeda et al., 2002). Certain embodiments
are directed to these heterozygous transgenic strains.
[0510] As shown in FIGS. 6A-D, insulin sensitivity was markedly
decreased in Osteocalcin+/-; Adiponectin+/- mice while blood
glucose levels, insulin serum levels and insulin secretion as
determined by GSIS test remained within the normal range.
Adiponectin serum levels were also significantly decreased in
Osteocalcin+/-; Adiponectin+/- compared to WT or single
heterozygote mice (FIG. 6E). These observations are consistent with
the notion that osteocalcin regulates insulin sensitivity at least
in part through its regulation of adiponectin expression and
secretion.
[0511] To show that the increase in insulin sensitivity and
decrease in fat weight observed in the Esp-deficient mice was
secondary to the increase in adiponectin expression,
Sap-Adiponectin transgenic mice harboring a two-fold increase in
serum adiponectin level similar to the one observed in
Esp-deficient mice were generated. The Sap-Adiponectin transgenic
mice also showed the phenotype of low fat pad weight, high energy
expenditure and metabolic and molecular evidence of increased
insulin sensitivity similar to those observed in Esp-deficient mice
(FIG. 22). This result shows that the increase in adiponectin
expression was the main identifiable cause of the increase in
insulin production and sensitivity in Esp-deficient mice. Certain
embodiments of the invention are thus directed to human cells
transfected with the gene for adiponectin under the control of a
promoter that causes the cell to over express adiponectin.
[0512] OST-PTP regulates osteocalcin bioactivity by influencing
indirectly its carboxylation. The metabolic phenotype of
Osteocalcin-/- mice is the mirror image of the one observed in
Esp-/- mice suggesting that in the latter there is a gain of
osteocalcin activity. To further prove that Esp-deficient mice
(OST-PTP-/-) are a model of a gain of activity of osteocalcin,
double mutants were made by introducing additional mutations into
Esp-deficient transgenic mice, specifically by making them
osteocalcin+/-.
[0513] It was hypothesized that the metabolic abnormalities of
Esp-/- mice would be reversible by reducing osteocalcin expression.
This is exactly what was observed: Esp-/- mice lacking one allele
of osteocalcin showed a remarkable reversal of all their metabolic
abnormalities such as blood glucose, insulin and adiponectin serum
levels, glucose tolerance, insulin secretion and sensitivity (FIGS.
7A-F). Ki67 staining showed that beta-cell proliferation was also
reduced in these mutant mice (FIG. 7G).
[0514] Indeed, Esp-/-; Ocn+/- mice display a decrease in insulin
synthesis and sensitivity compared to Esp-/- without any
osteocalcin deletion, showing a fully corrected/normalization of
all metabolic abnormalities of the Esp-/- mice compared to wt mice.
This experiment established genetically that OST-PTP and
osteocalcin are in the same signaling cascade, and that the
Esp.sub.ob-/- mouse phenotype is a model of a gain of activity of
osteocalcin. In other words, the metabolic phenotype seen in
Esp.sub.ob-/- mice is due to an increase in osteocalcin
activity.
[0515] Because osteocalcin expression and serum levels were normal
in Esp-/- mice, OST-PTP regulation of osteocalcin expression was
ruled out (FIG. 20) In contrast, Esp-/- mice showed a decreased
ratio of serum carboxylated osteocalcin to total osteocalcin (FIG.
7H). Carboxylated osteocalcin has a higher affinity for
hydroxyapatite (HA) than undercarboxylated osteocalcin (Hauschka et
al., 1989; Price, 1989). An assay was used where carboxylated
osteocalcin is measured as the % of total osteocalcin able to bind
to hydroxyapatite (HA). This assay showed that this value is
decreased by 20% in Esp-/- mice compared to wt mice. In the
presence of an equal amount of total osteocalcin, this means that
undercarboxylated osteocalcin is increased 20% in Esp-/- mice
compared to WT.
[0516] This experiment suggested that OST-PTP influences
osteocalcin function by regulating its degree of
gamma-carboxylation and that it was the undercarboxylated form of
osteocalcin that regulated glucose homeostasis. To determine
whether the was the case, two additional experiments were
performed. WT primary osteoblasts were treated with warfarin, an
inhibitor of gamma-carboxylation (Bergner, 2005) prior to and
during co-culture assays. This treatment resulted in a marked
decrease in the percentage of osteocalcin bound to HA, indicating
that, as expected, these osteoblasts secrete less carboxylated
osteocalcin (FIG. 7I). Nevertheless, and despite secreting less
osteocalcin than WT osteoblasts (+vehicle, 10 ng/ml; +warfarin, 2
ng/ml) (Hauschka et al., 1989), warfarin-treated osteoblasts
induced adiponectin expression to a significantly higher extent
than vehicle-treated osteoblasts (FIG. 7J). Second, carboxylated
osteocalcin and bacterially produced mouse osteocalcin, which is
not carboxylated, were used in cell-based assays. While
carboxylated osteocalcin failed to induce adiponectin expression,
bacterially produced osteocalcin did (FIG. 7K). Likewise,
undercarboxylated osteocalcin induced insulin expression as well as
expression of Cyclin D1, a molecular marker of beta-cell
proliferation (Kushner et al., 2005) (FIG. 7L). Lastly, we studied
human obese patients that are hyperinsulinemic but not diabetic
(FIG. 7M). The amount of uncarboxylated osteocalcin was
significantly increased in these patients while osteocalcin serum
levels were not affected (FIGS. 7M-O). Taken together these data
indicate that OST-PTP influences osteocalcin bioactivity by
enhancing its degree of carboxylation.
[0517] OST-PTP affects enzymes involved in the carboxylation
process. A mandatory event in every function of any cell type is
the ability of intracellular proteins to be phosphorylated by
protein kinases and/or dephosphorylated by protein phosphatase. In
particular phosphorylation of tyrosine residues accounts for 0.1%
of the total cellular phosphoamino acid content; as a result
protein tyrosine phosphatases (PTPs) are critically important
intracellular proteins (23).
[0518] Protein tyrosine phosphatases can be schematically grouped
into four classes: the classical receptor-like PTPs that have an
extracellular domain that sometimes is cleaved (RPTPs); OST-PTP is
a receptor-like PTP. Other classes include the classical
non-receptor PTPs, the dual specificity PTPs and the low molecular
weight PTPs (24). There are approximately 20 RPTPs in the human
genome. RPTPs that are predominantly localized in the plasma
membrane can be involved in cell to cell functions, cell-cell
adhesion and in hormone signaling. However, two questions remain
often unanswered regarding their biology. One is to determine the
identity of substrate(s) for their phosphatase activity and the
second one is to identify their ligands.
[0519] Results suggested that OST-PTP could dephosphorylate a
specific substrate present in osteoblasts, thereby increasing the
substrate's expression and/or activity. This substrate would then
be released by osteoblasts and signal to pancreatic beta-cells and
adipocytes, thereby affecting insulin secretion and sensitivity.
While osteocalcin was a logical target candidate for OST-PTP
physiologically speaking, osteocalcin is not phosphorylated. It was
thus eliminated as a direct target.
[0520] To decipher how OST-PTP might influence osteocalcin activity
we asked whether it was regulating gamma-carboxylation of
osteocalcin, which is the main post-translational modification
known for this molecule (Hauschka et al., 1989). This
post-translational modification occurs both in rodent and in human;
Poser et al analyzed the primary structure of human osteocalcin,
and reported that human osteocalcin is a mixture of Glu.sup.7
osteocalcin (herein "Oc-glu") with the 17-position being glutamic
acid, and Gla.sup.7 osteocalcin with the 17-position being
gamma-carboxyglutamic acid (herein "BGP", also bone Gla-protein)
[Poser, J. W. et al., Proc. Natl. Acad. Sci. U.S., 255, 8685-8691
(1980)]. Gla residues usually confer on proteins a high affinity
for mineral ions. However, loss and gain of function experiments
failed to identify a function for osteocalcin in extracellular
matrix mineralization in vivo (Ducy et al., 1996; Murshed et al.,
2004).
[0521] OST-PTP Substrates Include Insulin Receptor and
Gamma-Carboxylase. A computer search revealed that
gamma-carboxylase, also known as vitamin K-dependent gamma-glutamyl
carboxylase, has PTP consensus sites. This enzyme catalyzes the
conversion of glutamic acid to gamma-carboxyglutamic acid in
substrate proteins like osteocalcin. To determine if OST-PTP acts
on gamma-carboxylase, substrate-trapping experiments were conducted
in COS cells, Ros17/2.8 osteoblast cells and differentiated primary
osteoblasts. d10 bone-derived cells were cultured for 10 days in
alpha MEM/10% fetal bovine serum (FBS) supplemented with ascorbic
acid (100 micrograms/ml) and beta-glycerophosphate (5 mM)). These
cells were then starved for 24 hours in alpha MEM culture medium
supplemented with 1% FBS only and treated with pervanadate (100
mM), an irreversible protein-tyrosine phosphatase inhibitor, and
20% FBS for 30 minutes. Cell lysates were incubated for 2 h at
4.degree. C. with either GST, GST-PTP.sup.WT or GST-PTP.sup.D1316A.
Different amounts of the total cell extract were also loaded as
control.
[0522] The results in FIG. 9 showed that the mutant enzyme
GST-PTP.sup.D1316A trapped gamma-carboxylase, thereby demonstrating
that gamma-carboxylase is a substrate of OST-PTP. This, however,
does not mean that gamma-carboxylase is the only substrate of
OST-PTP. There was no binding in the GST lane because there is no
PTP transfected. It is a control to show that if there is trapping,
it is not due to the GST part of any GST fusion protein. There was
also no trapping with GST-PTP.sup.WT because this form
dephosphorylated the substrate gamma-carboxylase, which is then
released. A band is clearly seen in the lane having the mutant
GST-PTP.sup.D1316A because the mutation engineered a defect in
OST-PTP phosphatase activity that allowed the substrate to
irreversibly bind to and be retained by the enzyme.
[0523] These results show that gamma-carboxylase is a substrate for
OST-PTP in osteoblasts. This enabled elucidation of part of the
biochemical pathway through which OST-PTP regulates osteocalcin
bioactivity: OST-PTP dephosphorylates gamma-carboxylase, thereby
activating it. Activated gamma-carboxylase in turn causes an
increase in carboxylated osteocalcin. There is less
dephosphorylated active gamma-carboxylase in OST-PTP-deficient
mice, which leads to secretion of more undercarboxylated
osteocalcin. This explains why the OST-PTP-deficient mice have
elevated levels of undercarboxylated osteocalcin, which itself
causes resistance to metabolic syndrome and diabetes.
[0524] Using the same substrate trapping assay, it was also
discovered that the insulin receptor which is expressed in
osteoblasts is a substrate for OST-PTP (FIG. 8). The results of the
substrate-trapping experiments show that mutated OST-PTP
(GST-PTP.sup.DA) interacts with the insulin receptor (InsR)
expressed in COS cells (left upper panel) and ROS17/2.8 osteoblasts
cells (right upper panel) (third lane). By contrast, WT OST-PTP
(GST-PTP.sup.WT) did not interact with the insulin receptor (second
lane). The same amount of GST fusion proteins were used for
substrate trapping.
[0525] Human Patient Data. FIG. 7O shows that human obese patients
that are hyperinsulinemic but not diabetic, have significantly
elevated levels of undercarboxylated osteocalcin (about 30% higher)
compared to normal patients, even though osteocalcin serum levels
(7M) are about the same. This shows that in mice and in humans the
level of carboxylation of osteocalcin influences its bioactivity.
FIG. 7O further shows that obese non-diabetic patients have an
increase in undercarboxylated osteocalcin compared to patients who
are obese and diabetic. The ratio of carboxylated osteocalcin
compared to total osteocalcin was measured in serum from
non-medicated normal, obese non-diabetic and obese-diabetic
patients.
[0526] An in vivo experiment was conducted in which the effect of
undercarboxylated osteocalcin on glycemia was monitored. Wild type
mice were infused with 3 different amounts of mouse recombinant
undercarboxylated osteocalcin or placebo (PBS) subcutaneously for
28 days (0.3, 1 and 3 ng/hour). Compared to the control animal
infused with placebo, all three doses of undercarboxylated
osteocalcin decreased glycemia in vivo over the 28 day period (FIG.
10).
[0527] In another in vivo experiment, the effect of uncarboxylated
osteocalcin on glucose tolerance was investigated. Wild type mice
were infused subcutaneously with either 0.3 or 3 ng/hour doses of
recombinant uncarboxylated osteocalcin or PBS for 14 days before
receiving a single injection of glucose. Blood glucose was measured
thereafter at the indicated times. The results show that both doses
of uncarboxylated osteocalcin increased glucose tolerance above
control levels over the 120 minute time period following the
glucose injection (FIG. 11).
[0528] The effect of uncarboxylated osteocalcin on insulin
sensitivity was also examined. Wild type mice were infused
subcutaneously with 0.3 or 3 ng/hour doses of recombinant
osteocalcin or PBS for 18 days before receiving a single injection
of insulin. Blood glucose was measured thereafter at the indicated
times from 0-120 minutes after injection. The results show that
insulin sensitivity was increased by both doses of uncarboxylated
osteocalcin (FIG. 12).
[0529] In another in vivo experiment, the effect of uncarboxylated
osteocalcin on body weight and fat pad mass was monitored (FIG.
13). Wild type mice were infused subcutaneously for 28 days with
PBS or uncarboxylated osteocalcin at 0.3, 1 or 3 ng/hour. The
results show that body weight was slightly reduced by
uncarboxylated osteocalcin with the highest dose being the most
effective. (FIG. 13) Gonadal fat pad mass, measured after 28 days,
decreased by about %18 with 3 ng/hour uncarboxylated osteocalcin
treatment. The other doses did not significantly decrease fat pad
mass in that period.
[0530] The effect of uncarboxylated osteocalcin on GTG-induced
obesity was investigated (FIG. 14). Wild type mice were injected
with gold thioglucose (GTG) to induce hyperphagia and obesity or
vehicle. Two weeks later they were implanted with subcutaneous
osmotic pumps infusing lng/hr of recombinant uncarboxylated
osteocalcin or PBS for 28 days. Body weight gain was significantly
reduced with both doses of uncarboxylated osteocalcin by the first
time point checked, 7 days, and remained lower than controls for
the entire 28 day period. At 28 days, body weight was reduced by
about 15% with uncarboxylated osteocalcin treatment.
[0531] A fragment of uncarboxylated osteocalcin is biologically
active. Experiments were performed to test whether truncated
osteocalcin was as effective as full-length uncarboxylated
osteocalcin in stimulating adiponectin secretion from mouse
adipocytes in vitro. Wild type adipocytes were treated for 4 hours
with recombinant full-length osteocalcin (1-46) or a truncated form
(1-36) (having a deletion of the first ten amino acids from the
C-terminal end) or vehicle. Adiponectin expression was then
quantified by real time PCR. The results show that full-length
uncarboxylated osteocalcin produced about a 1.5 fold increase, and
the 1-36 fragment of uncarboxylated osteocalcin produced about a
1.8 fold increase (FIG. 15). Thus, the full length molecule is not
needed for biological activity; at least up to 10 amino acids can
be deleted from the C-terminal end of the mouse osteocalcin
molecule to achieve the same biological effects on adipocytes and
beta-cells. Certain embodiments of the invention are directed to
osteocalcin from which the first ten amino acids from the
C-terminal end have been deleted, preferably human osteocalcin,
preferably undercarboxylated osteocalcin.
[0532] The primary sequence of osteocalcin is highly conserved
among species and it is one of the ten most abundant proteins in
the human body (FIG. 21), suggesting that its function is preserved
throughout evolution. Conserved features importantly include 3 Gla
residues at positions 17, 21, and 24, a disulfide bridge between
Cys23 and Cys29, and most species contain a hydroxyproline at
position 9. The N-terminus of osteocalcin shows highest sequence
variation in comparison to other parts of the molecule. The high
degree of conservation of human and mouse osteocalcin underscores
the relevance of the mouse as an animal model for the human, in
both healthy and diseased states, and validates our claims to the
therapeutic and diagnostic use of osteocalcin to treat or prevent
metabolic syndrome or any of its components and type 1
diabetes.
[0533] Vitamin K and Statins Increase Osteocalcin. Vitamin K is
required for gamma-carboxylation. Warfarin and other COUMADIN.RTM.
derivatives block vitamin K-dependent gamma-carboxylation, thus
increasing the level of active, undercarboxylated osteocalcin. This
is in agreement with data showing that warfarin-treated osteoblasts
produce elevated levels of undercarboxylated osteocalcin compared
to vehicle-treated osteoblasts (FIG. 7I). Others have shown that
four weeks of treatment of osteoporotic patients with Vitamin K
caused a dramatic percentage mean decrease in undercarboxylated
osteocalcin of 85% compared to controls without Vitamin K
treatment. Vitamin D had no significant effect alone or when
administered together with Vitamin K. Takahashi, et al, Clinical
Endocrinology (2001) 54, 291-224. See also Sugiyama, T., J Bone
Miner Metabolism (2001) 19,146-159. This observation suggests that
warfarin or another COUMADIN.RTM. derivative could be used to block
vitamin K-dependent gamma-carboxylation and increase the level of
undercarboxylated osteocalcin in patients with the goal to
prevent/treat metabolic disorders.
[0534] Warfarin, sold as the brand name COUMADIN.RTM., is used as
an oral anticoagulant that inhibits the synthesis of clotting
factors, thus preventing blood clot formation. However,
COUMADIN.RTM. can cause bleeding and necrosis (gangrene) of the
skin. Many drugs, both prescription and nonprescription (OTC), can
affect the anticoagulant action of COUMADIN.RTM.. Some medications
can enhance the action of COUMADIN.RTM. and cause excessive blood
thinning and life-threatening bleeding. A few examples of such
medications include Aspirin, TYLENOL.RTM., alcohol, ibuprofen
(MOTRIN.RTM.), cimetidine (TAGAMET.RTM.), oxandrolone
(OXANDRIN.RTM.), certain vitamins, and antibiotics.
[0535] Others have shown that the statin ZOCOR.RTM. (simvastatin at
20 mg/day in humans) significantly increased serum levels of
osteocalcin (p value less than 0.05) after four weeks of treatment,
although undercarboxylated osteocalcin could not be distinguished
from intact osteocalcin. Chan, M. H., et al., J Clin Endocrinology
and Metabolism (2001) Vol 86(9), 4556-59. Even though there is no
experimental proof that the level of undercarboxylated osteocalcin
was increased by statins, a significant increase in the overall
expression of osteocalcin, could cause a saturation of the
gamma-carboxylase activity and an inability to carboxylate all of
the osteocalcin produced. As a result statins could indirectly
increase the amount of undercarboxylated osteocalcin released in
the blood. Moreover, administering statins together with drugs that
block gamma-carboxylation, such as warfarin, which blocks vitamin
K, or inhibitors of OST-PTP and gamma-carboxylase, could work
together to elevate serum undercarboxylated osteocalcin and have
therapeutic use. Statins and vitamin K inhibitors could be
administered in a single preparation or in separate
preparations.
[0536] Therefore certain aspects of the present invention are
directed to the use of vitamin K inhibitors and statins to increase
undercarboxylated osteocalcin levels in serum, and to their
therapeutic use in treating metabolic syndrome and its various
components.
[0537] The sympathetic nervous system positively regulates OST-PTP
expression. It was discovered that sympathetic nervous system (SNS)
activity positively regulates Esp expression in osteoblasts.
Indeed, FIG. 16 shows that stimulation of SNS signaling with
isoproterenol, a beta adrenergic receptor agonist, increased Esp
expression by about 80% by 4 hours, and that this increase remains
steady even at 8 hours. However, increased SNS activity did not
increase expression of gamma-carboxylase (ggcx), vkor (an enzyme
involved in recycling of vitamin K that is necessary for ggcx
activity) or osteocalcin. This experiment shows that SNS signaling
positively regulates Esp expression in osteoblasts. Thus,
decreasing sympathetic activity should lead to a decrease in Esp
expression and thereby to an increase in the undercarboxylated,
active form of osteocalcin.
[0538] In vivo experiments using ob/ob mice, which have low
sympathetic activity, confirmed this hypothesis, and showed that
there is a genetic link between leptin (the product of the ob gene)
and osteocalcin. It has been shown that leptin signals to
osteoblasts via the SNS. Thus, ob/ob mice are a model of decreased
SNS signaling on osteoblasts. It has been shown that ob/ob mice
display an increase in insulin before they develop any other
metabolic abnormalities. This increase could be due to decreased
SNS activity on osteoblasts, which as a result would express less
OST-PTP and secrete more bioactive undercarboxylated osteocalcin,
leading to increased insulin expression. The level of serum insulin
was measured in one week old mice having various genotypes: WT
mice, ob -/+ mice (hemizygous for obesity), ob/ob mice, Bgp -/+
(hemizygous for osteocalcin), BGP-/- mice, and ob/ob mice that are
also Bgp-/- (Ocn deficient). One week old mice were chosen because
ob/ob mice at one week are not yet obese and they are relatively
normal metabolically, except for having high serum insulin levels.
The results show that ob/ob mice indeed have increased serum
insulin levels but if both alleles of the Bgp gene (encoding
osteocalcin) are deleted in ob/ob mice their serum insulin levels
returns to normal. FIG. 17. This experiment demonstrates that the
increase in insulin observed in ob/ob mice is dependent on
osteocalcin.
[0539] Taken together the results presented above mean that
administering beta blockers, which decrease SNS activity, will
likewise decrease Esp expression and thereby increase the level of
undercarboxylated osteocalcin. As such they could be used to
prevent/treat metabolic disorders via an increase in osteocalcin
activity. Beta blockers have been used clinically for a long time,
therefore the amounts that are safe for human use are established.
Routine experimentation will determine the optimum amount of a
particular beta blocker to administer to achieve increased levels
of serum undercarboxylated osteocalcin. New beta-blockers,
targeting more preferentially the cells of the skeleton could also
be developed to more specifically increase osteocalcin activity and
reduce the risk of side effects.
TABLE-US-00004 TABLE 2 Wild-type Esp-/- Ocn-/- (n = 4) (n = 3) (n =
4) Basal hepatic glucose 13.2 .+-. 1.9 16.6 .+-. 1.3 15.5 .+-. 1.8
production (mg/kg/min) Clamp hepatic glucose 10.3 .+-. 1.9 -5.5
.+-. 1.8* 16.1 .+-. 1.9* production (mg/kg/min) Glucose turnover
41.8 .+-. 1.3 50.6 .+-. 2.6* 38.2 .+-. 3.7 (mg/kg/min) Glycogen
synthesis 19.2 .+-. 2.7 19.4 .+-. 3.2 11.5 .+-. 1.3* (mg/kg/min)
Muscle glucose uptake 229 .+-. 29 358 .+-. 65* 188 .+-. 15
(nmol/g/min) White adipose 16.7 .+-. 2.7 31.7 .+-. 8.2* 7.5 .+-.
0.9* glucose uptake (nmol/g/min) Brown adipose 2022 .+-. 205 3330
.+-. 263* ND glucose uptake (nmol/g/min) *p .ltoreq. 0.05, Student
t-test, ND, not done
Analysis of 3 month-old Esp-/- and Ocn-/- mice by
hyperinsulinemic-euglycemic clamps.
TABLE-US-00005 TABLE 3 SEQ ID NO: Amino GenBank cDNA Acid Accession
No: Human Osteocalcin cDNA 1 2 NM_199173 Mouse osteocalcin gene 1 3
5 NM_007541 Mouse osteocalcin gene 2 4 5 NM_001032298 Human
Adiponectin 6 7 NM_004797 Mouse Adiponectin 8 9 NM_009605 Human
Gamma-glutamyl 10 11 NM_000821 carboxylase Mouse Gamma-glutamyl 12
13 NM_019802 carboxylase Human ApoE 14 15 NM_000041 Mouse ApoE 16
17 NM_009696 Mouse Esp (OST-PTP, Ptprv) 18 19 NM_007955 E. coli
.beta.-galactosidase 20 21 Rat (OST-PTP, Ptprv) 24 25 NM_033099
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Sequence CWU 1
1
251498DNAHomo sapiensCDS(19)..(318) 1cgcagccacc gagacacc atg aga
gcc ctc aca ctc ctc gcc cta ttg gcc 51 Met Arg Ala Leu Thr Leu Leu
Ala Leu Leu Ala 1 5 10ctg gcc gca ctt tgc atc gct ggc cag gca ggt
gcg aag ccc agc ggt 99Leu Ala Ala Leu Cys Ile Ala Gly Gln Ala Gly
Ala Lys Pro Ser Gly 15 20 25gca gag tcc agc aaa ggt gca gcc ttt gtg
tcc aag cag gag ggc agc 147Ala Glu Ser Ser Lys Gly Ala Ala Phe Val
Ser Lys Gln Glu Gly Ser 30 35 40gag gta gtg aag aga ccc agg cgc tac
ctg tat caa tgg ctg gga gcc 195Glu Val Val Lys Arg Pro Arg Arg Tyr
Leu Tyr Gln Trp Leu Gly Ala 45 50 55cca gtc ccc tac ccg gat ccc ctg
gag ccc agg agg gag gtg tgt gag 243Pro Val Pro Tyr Pro Asp Pro Leu
Glu Pro Arg Arg Glu Val Cys Glu60 65 70 75ctc aat ccg gac tgt gac
gag ttg gct gac cac atc ggc ttt cag gag 291Leu Asn Pro Asp Cys Asp
Glu Leu Ala Asp His Ile Gly Phe Gln Glu 80 85 90gcc tat cgg cgc ttc
tac ggc ccg gtc tagggtgtcg ctctgctggc 338Ala Tyr Arg Arg Phe Tyr
Gly Pro Val 95 100ctggccggca accccagttc tgctcctctc caggcaccct
tctttcctct tccccttgcc 398cttgccctga cctcccagcc ctatggatgt
ggggtcccca tcatcccagc tgctcccaaa 458taaactccag aagaggaatc
tgaaaaaaaa aaaaaaaaaa 4982100PRTHomo sapiens 2Met Arg Ala Leu Thr
Leu Leu Ala Leu Leu Ala Leu Ala Ala Leu Cys1 5 10 15Ile Ala Gly Gln
Ala Gly Ala Lys Pro Ser Gly Ala Glu Ser Ser Lys 20 25 30Gly Ala Ala
Phe Val Ser Lys Gln Glu Gly Ser Glu Val Val Lys Arg 35 40 45Pro Arg
Arg Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro 50 55 60Asp
Pro Leu Glu Pro Arg Arg Glu Val Cys Glu Leu Asn Pro Asp Cys65 70 75
80Asp Glu Leu Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe
85 90 95Tyr Gly Pro Val 1003494DNAmus musculus 3agaacagaca
agtcccacac agcagcttgg cccagaccta gcagacacca tgaggaccat 60ctttctgctc
actctgctga ccctggctgc gctctgtctc tctgacctca cagatgccaa
120gcccagcggc cctgagtctg acaaagcctt catgtccaag caggagggca
ataaggtagt 180gaacagactc cggcgctacc ttggagcctc agtccccagc
ccagatcccc tggagcccac 240ccgggagcag tgtgagctta accctgcttg
tgacgagcta tcagaccagt atggcttgaa 300gaccgcctac aaacgcatct
atggtatcac tatttaggac ctgtgctgcc ctaaagccaa 360actctggcag
ctcggctttg gctgctctcc gggacttgat cctccctgtc ctctctctct
420gccctgcaag tatggatgtc acagcagctc caaaataaag ttcagatgag
gaagtgcaaa 480aaaaaaaaaa aaaa 4944470DNAmus musculusCDS(49)..(333)
4gaacagacaa gtcccacaca gcagcttggt gcacacctag cagacacc atg agg acc
57 Met Arg Thr 1ctc tct ctg ctc act ctg ctg gcc ctg gct gcg ctc tgt
ctc tct gac 105Leu Ser Leu Leu Thr Leu Leu Ala Leu Ala Ala Leu Cys
Leu Ser Asp 5 10 15ctc aca gat ccc aag ccc agc ggc cct gag tct gac
aaa gcc ttc atg 153Leu Thr Asp Pro Lys Pro Ser Gly Pro Glu Ser Asp
Lys Ala Phe Met20 25 30 35tcc aag cag gag ggc aat aag gta gtg aac
aga ctc cgg cgc tac ctt 201Ser Lys Gln Glu Gly Asn Lys Val Val Asn
Arg Leu Arg Arg Tyr Leu 40 45 50gga gcc tca gtc ccc agc cca gat ccc
ctg gag ccc acc cgg gag cag 249Gly Ala Ser Val Pro Ser Pro Asp Pro
Leu Glu Pro Thr Arg Glu Gln 55 60 65tgt gag ctt aac cct gct tgt gac
gag cta tca gac cag tat ggc ttg 297Cys Glu Leu Asn Pro Ala Cys Asp
Glu Leu Ser Asp Gln Tyr Gly Leu 70 75 80aag acc gcc tac aaa cgc atc
tac ggt atc act att taggacctgt 343Lys Thr Ala Tyr Lys Arg Ile Tyr
Gly Ile Thr Ile 85 90 95gctgccctaa agccaaactc tggcagctcg gctttggctg
ctctccggga cttgatcctc 403cctgtcctct ctctctgccc tgcaagtatg
gatgtcacag cagctccaaa ataaagttca 463gatgagg 470595PRTmus musculus
5Met Arg Thr Leu Ser Leu Leu Thr Leu Leu Ala Leu Ala Ala Leu Cys1 5
10 15Leu Ser Asp Leu Thr Asp Pro Lys Pro Ser Gly Pro Glu Ser Asp
Lys 20 25 30Ala Phe Met Ser Lys Gln Glu Gly Asn Lys Val Val Asn Arg
Leu Arg 35 40 45Arg Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu
Glu Pro Thr 50 55 60Arg Glu Gln Cys Glu Leu Asn Pro Ala Cys Asp Glu
Leu Ser Asp Gln65 70 75 80Tyr Gly Leu Lys Thr Ala Tyr Lys Arg Ile
Tyr Gly Ile Thr Ile 85 90 9564592DNAhomo sapiensCDS(85)..(816)
6aggctgttga ggctgggcca tctcctcctc acttccattc tgactgcagt ctgtggttct
60gattccatac cagaggggct cagg atg ctg ttg ctg gga gct gtt cta ctg
111 Met Leu Leu Leu Gly Ala Val Leu Leu 1 5cta tta gct ctg ccc ggt
cat gac cag gaa acc acg act caa ggg ccc 159Leu Leu Ala Leu Pro Gly
His Asp Gln Glu Thr Thr Thr Gln Gly Pro10 15 20 25gga gtc ctg ctt
ccc ctg ccc aag ggg gcc tgc aca ggt tgg atg gcg 207Gly Val Leu Leu
Pro Leu Pro Lys Gly Ala Cys Thr Gly Trp Met Ala 30 35 40ggc atc cca
ggg cat ccg ggc cat aat ggg gcc cca ggc cgt gat ggc 255Gly Ile Pro
Gly His Pro Gly His Asn Gly Ala Pro Gly Arg Asp Gly 45 50 55aga gat
ggc acc cct ggt gag aag ggt gag aaa gga gat cca ggt ctt 303Arg Asp
Gly Thr Pro Gly Glu Lys Gly Glu Lys Gly Asp Pro Gly Leu 60 65 70att
ggt cct aag gga gac atc ggt gaa acc gga gta ccc ggg gct gaa 351Ile
Gly Pro Lys Gly Asp Ile Gly Glu Thr Gly Val Pro Gly Ala Glu 75 80
85ggt ccc cga ggc ttt ccg gga atc caa ggc agg aaa gga gaa cct gga
399Gly Pro Arg Gly Phe Pro Gly Ile Gln Gly Arg Lys Gly Glu Pro
Gly90 95 100 105gaa ggt gcc tat gta tac cgc tca gca ttc agt gtg gga
ttg gag act 447Glu Gly Ala Tyr Val Tyr Arg Ser Ala Phe Ser Val Gly
Leu Glu Thr 110 115 120tac gtt act atc ccc aac atg ccc att cgc ttt
acc aag atc ttc tac 495Tyr Val Thr Ile Pro Asn Met Pro Ile Arg Phe
Thr Lys Ile Phe Tyr 125 130 135aat cag caa aac cac tat gat ggc tcc
act ggt aaa ttc cac tgc aac 543Asn Gln Gln Asn His Tyr Asp Gly Ser
Thr Gly Lys Phe His Cys Asn 140 145 150att cct ggg ctg tac tac ttt
gcc tac cac atc aca gtc tat atg aag 591Ile Pro Gly Leu Tyr Tyr Phe
Ala Tyr His Ile Thr Val Tyr Met Lys 155 160 165gat gtg aag gtc agc
ctc ttc aag aag gac aag gct atg ctc ttc acc 639Asp Val Lys Val Ser
Leu Phe Lys Lys Asp Lys Ala Met Leu Phe Thr170 175 180 185tat gat
cag tac cag gaa aat aat gtg gac cag gcc tcc ggc tct gtg 687Tyr Asp
Gln Tyr Gln Glu Asn Asn Val Asp Gln Ala Ser Gly Ser Val 190 195
200ctc ctg cat ctg gag gtg ggc gac caa gtc tgg ctc cag gtg tat ggg
735Leu Leu His Leu Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly
205 210 215gaa gga gag cgt aat gga ctc tat gct gat aat gac aat gac
tcc acc 783Glu Gly Glu Arg Asn Gly Leu Tyr Ala Asp Asn Asp Asn Asp
Ser Thr 220 225 230ttc aca ggc ttt ctt ctc tac cat gac acc aac
tgatcaccac taactcagag 836Phe Thr Gly Phe Leu Leu Tyr His Asp Thr
Asn 235 240cctcctccag gccaaacagc cccaaagtca attaaaggct ttcagtacgg
ttaggaagtt 896gattattatt tagttggagg cctttagata ttattcattc
atttactcat tcatttattc 956attcattcat caagtaactt taaaaaaatc
atatgctatg ttcccagtcc tggggagctt 1016cacaaacatg accagataac
tgactagaaa gaagtagttg acagtgctat tttgtgccca 1076ctgtctctcc
tgatgctcat atcaatccta taaggcacag ggaacaagca ttctcctgtt
1136tttacagatt gtatcctgag gctgagagag ttaagtgaat gtctaaggtc
acacagtatt 1196aagtgacagt gctagaaatc aaacccagag ctgtggactt
tgttcactag actgtgccct 1256tttatagagg tacatgttct ctttggagtg
ttggtaggtg tctgtttccc acctcacctg 1316agagccattg aatttgcctt
cctcatgaat taaaacctcc cccaagcaga gcttcctcag 1376agaaagtggt
tctatgatga agtcctgtct tggaaggact actactcaat ggcccctgca
1436ctactctact tcctcttacc tatgtccctt ctcatgcctt tccctccaac
ggggaaagcc 1496aactccatct ctaagtgctg aactcatccc tgttcctcaa
ggccacctgg ccaggagctt 1556ctctgatgtg atatccactt tttttttttt
tgagatggag tctcactctg tcacccaggc 1616tggagtacag tgacacgacc
tcggctcact gcagcctcct tctcctgggt ccaagcaatt 1676attgtgcctc
agcctcccga gtagctgaga cttcaggtgc attccaccac acatggctaa
1736tttttgtatt tttagtagaa atggggtttc gtcatgttgg ccaggctggt
ctcgaactcc 1796tggcctaggt gatccacccg cctcgacctc ccaaagtgct
gggattacag gcatgagcca 1856ccatgcccag tcgatatctc actttttatt
ttgccatgga tgagagtcct gggtgtgagg 1916aacacctccc accaggctag
aggcaactgc ccaggaagga ctgtgcttcc gtcacctcta 1976aatcccttgc
agatccttga taaatgcctc atgaagacca atctcttgaa tcccatatct
2036acccagaatt aactccattc cagtctctgc atgtaatcag ttttatccac
agaaacattt 2096tcattttagg aaatccctgg ttttaagtat caatccttgt
tcagctggac aatatgaatc 2156ttttccactg aagttaggga tgactgtgat
tttcagaaca cgtccagaat ttttcatcaa 2216gaaggtagct tgagcctgaa
atgcaaaacc catggaggaa ttctgaagcc attgtctcct 2276tgagtaccaa
cagggtcagg gaagactggg cctcctgaat ttattattgt tctttaagaa
2336ttacaggttg aggtagttga tggtggtaaa cattctctca ggagacaata
actccagtga 2396tgttcttcaa agattttagc aaaaacagag taaatagcat
tctctatcaa tatataaatt 2456taaaaaacta tctttttgct tacagtttta
aattctgaac aattctctct tatatgtgta 2516ttgctaatca ttaaggtatt
attttttcca catataaagc tttgtctttt tgttgttgtt 2576gttgttttta
agatggagtt tccctctgtt gccaggctag agtgcagtgg catgatctcg
2636gcttactgca acctttgcct cccaggttca agcgattctt ctgcctcagc
ctcccgagta 2696gctgggacca caggtgccta ccaccatgcc aggctaattt
ttgtattttt agtaaagaca 2756gggtttcacc atattggcca ggctggtctc
gaactcctga ccttgtgatc tgcccgcctc 2816catttttgtt gttatttttt
gagaaagata gatatgaggt ttagagaggg atgaagaggt 2876gagagtaagc
cttgtgttag tcagaactct gtgttgtgaa tgtcattcac aacagaaaac
2936ccaaaatatt atgcaaacta ctgtaagcaa gaaaaataaa ggaaaaatgg
aaacatttat 2996tcctttgcat aatagaaatt accagagttg ttctgtcttt
agataaggtt tgaaccaaag 3056ctcaaaacaa tcaagaccct tttctgtatg
tccttctgtt ctgccttccg cagtgtaggc 3116tttaccctca ggtgctacac
agtatagttc tagggtttcc ctcccgatat caaaaagact 3176gtggcctgcc
cagctctcgt atccccaagc cacaccatct ggctaaatgg acatcatgtt
3236ttctggtgat gcccaaagag gagagaggaa gctctctttc ccagatgccc
cagcaagtgt 3296aaccttgcat ctcattgctc tggctgagtt gtgtgcctgt
ttctgaccaa tcactgagtc 3356aggaggatga aatattcata ttgacttaat
tgcagcttaa gttaggggta tgtagaggta 3416ttttccctaa agcaaaattg
ggacactgtt atcagaaata ggagagtgga tgatagatgc 3476aaaataatac
ctgtccacaa caaactctta atgctgtgtt tgagctttca tgagtttccc
3536agagagacat agctggaaaa ttcctattga ttttctctaa aatttcaaca
agtagctaaa 3596gtctggctat gctcacagtc tcacatctgg ttggggtggg
ctccttacag aacacgcttt 3656cacagttacc ctaaactctc tggggcaggg
ttattccttt gtggaaccag aggcacagag 3716agagtcaact gaggccaaaa
gaggcctgag agaaactgag gtcaagattt caggattaat 3776ggtcctgtga
tgctttgaag tacaattgtg gatttgtcca attctcttta gttctgtcag
3836cttttgcttc atatatttta gcgctctatt attagatata tacatgttta
gtattatgtc 3896ttattggtgc atttactctc ttatcattat gtaatgtcct
tctttatctg tgataatttt 3956ctgtgttctg aagtctactt tgtctaaaaa
taacatacgc actcaacttc cttttctttc 4016ttccttcctt tctttcttcc
ttcctttctt tctctctctc tctctttcct tccttccttc 4076ctccttttct
ttctctctct ctctctctct ctttttttga cagactctcg ttctgtggcc
4136ctggctggag ttcagtggtg tgatcttggc tcactgctac ctctaccatg
agcaattctc 4196ctgcctcagc ctcccaagta gctggaacta caggctcatg
ccactgcgcc cagctaattt 4256ttgtattttt cgtagagacg gggtttcacc
acattcgtca ggttggtttc aaactcctga 4316ctttgtgatc cacccgcctc
ggcctcccaa agtgctggga ttacaggcat gagccatcac 4376acctggtcaa
ctttcttttg attagtgttt ttgtggtata tctttttcca tcatgttact
4436ttaaatatat ctatattatt gtatttaaaa tgtgtttctt acagactgca
tgtagttggg 4496tataattttt atccagtcta aaaatatctg tcttttaatt
ggtgtttaga caatttatat 4556ttaataaaat tgttgaattt aaaaaaaaaa aaaaaa
45927244PRThomo sapiens 7Met Leu Leu Leu Gly Ala Val Leu Leu Leu
Leu Ala Leu Pro Gly His1 5 10 15Asp Gln Glu Thr Thr Thr Gln Gly Pro
Gly Val Leu Leu Pro Leu Pro 20 25 30Lys Gly Ala Cys Thr Gly Trp Met
Ala Gly Ile Pro Gly His Pro Gly 35 40 45His Asn Gly Ala Pro Gly Arg
Asp Gly Arg Asp Gly Thr Pro Gly Glu 50 55 60Lys Gly Glu Lys Gly Asp
Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile65 70 75 80Gly Glu Thr Gly
Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly 85 90 95Ile Gln Gly
Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg 100 105 110Ser
Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met 115 120
125Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp
130 135 140Gly Ser Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr
Tyr Phe145 150 155 160Ala Tyr His Ile Thr Val Tyr Met Lys Asp Val
Lys Val Ser Leu Phe 165 170 175Lys Lys Asp Lys Ala Met Leu Phe Thr
Tyr Asp Gln Tyr Gln Glu Asn 180 185 190Asn Val Asp Gln Ala Ser Gly
Ser Val Leu Leu His Leu Glu Val Gly 195 200 205Asp Gln Val Trp Leu
Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu 210 215 220Tyr Ala Asp
Asn Asp Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr225 230 235
240His Asp Thr Asn81233DNAmus musculusCDS(115)..(855) 8taagctgggg
tctgcctgtc cccatgagta ccagactaat gagacctggc cactttctcc 60tcatttctgt
ctgtacgatt gtcagtggat ctgacgacac caaaagggct cagg atg 117 Met 1cta
ctg ttg caa gct ctc ctg ttc ctc tta atc ctg ccc agt cat gcc 165Leu
Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His Ala 5 10
15gaa gat gac gtt act aca act gaa gag cta gct cct gct ttg gtc cct
213Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val Pro
20 25 30cca ccc aag gga act tgt gca ggt tgg atg gca ggc atc cca gga
cat 261Pro Pro Lys Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly
His 35 40 45cct ggc cac aat ggc aca cca ggc cgt gat ggc aga gat ggc
act cct 309Pro Gly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly
Thr Pro50 55 60 65gga gag aag gga gag aaa gga gat gca ggt ctt ctt
ggt cct aag ggt 357Gly Glu Lys Gly Glu Lys Gly Asp Ala Gly Leu Leu
Gly Pro Lys Gly 70 75 80gag aca gga gat gtt gga atg aca gga gct gaa
ggg cca cgg ggc ttc 405Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu
Gly Pro Arg Gly Phe 85 90 95ccc gga acc cct ggc agg aaa gga gag cct
gga gaa gcc gct tat gtg 453Pro Gly Thr Pro Gly Arg Lys Gly Glu Pro
Gly Glu Ala Ala Tyr Val 100 105 110tat cgc tca gcg ttc agt gtg ggg
ctg gag acc cgc gtc act gtt ccc 501Tyr Arg Ser Ala Phe Ser Val Gly
Leu Glu Thr Arg Val Thr Val Pro 115 120 125aat gta ccc att cgc ttt
act aag atc ttc tac aac caa cag aat cat 549Asn Val Pro Ile Arg Phe
Thr Lys Ile Phe Tyr Asn Gln Gln Asn His130 135 140 145tat gac ggc
agc act ggc aag ttc tac tgc aac att ccg gga ctc tac 597Tyr Asp Gly
Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu Tyr 150 155 160tac
ttc tct tac cac atc acg gtg tac atg aaa gat gtg aag gtg agc 645Tyr
Phe Ser Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser 165 170
175ctc ttc aag aag gac aag gcc gtt ctc ttc acc tac gac cag tat cag
693Leu Phe Lys Lys Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr Gln
180 185 190gaa aag aat gtg gac cag gcc tct ggc tct gtg ctc ctc cat
ctg gag 741Glu Lys Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His
Leu Glu 195 200 205gtg gga gac caa gtc tgg ctc cag gtg tat ggg gat
ggg gac cac aat 789Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly Asp
Gly Asp His Asn210 215 220 225gga ctc tat gca gat aac gtc aac gac
tct aca ttt act ggc ttt ctt 837Gly Leu Tyr Ala Asp Asn Val Asn Asp
Ser Thr Phe Thr Gly Phe Leu 230 235 240ctc tac cat gat acc aac
tgactgcaac tacccatagc ccatacacca 885Leu Tyr His Asp Thr Asn
245ggagaatcat ggaacagtcg acacactttc agcttagttt gagagattga
ttttattgct 945tagtttgaga gtcctgagta ttatccacac gtgtactcac
ttgttcatta aacgacttta 1005taaaaaataa tttgtgttcc tagtccagaa
aaaaaggcac tccctggtct ccacgactct 1065tacatggtag caataacaga
atgaaaatca catttggtat gggggcttca caatattcgc 1125atgactgtct
ggaagtagac catgctattt ttctgctcac tgtacacaaa tattgttcac
1185ataaacccta taatgtaaat atgaaataca gtgattactc ttctcact
12339247PRTmus musculus 9Met Leu Leu Leu Gln Ala Leu Leu Phe Leu
Leu Ile Leu Pro Ser His1 5 10 15Ala Glu Asp Asp Val Thr Thr Thr Glu
Glu Leu Ala Pro Ala Leu Val 20 25 30Pro Pro Pro Lys Gly Thr Cys Ala
Gly Trp Met Ala Gly Ile Pro Gly 35 40 45His Pro Gly His Asn Gly Thr
Pro Gly Arg Asp Gly Arg Asp Gly Thr 50 55 60Pro Gly Glu Lys Gly Glu
Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys65 70 75 80Gly Glu Thr Gly
Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly 85 90 95Phe Pro Gly
Thr Pro Gly Arg Lys Gly Glu Pro Gly Glu Ala Ala Tyr 100 105 110Val
Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val 115 120
125Pro Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn
130 135 140His Tyr Asp Gly Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro
Gly Leu145 150 155 160Tyr Tyr Phe Ser Tyr His Ile Thr Val Tyr Met
Lys Asp Val Lys Val 165 170 175Ser Leu Phe Lys Lys Asp Lys Ala Val
Leu Phe Thr Tyr Asp Gln Tyr 180 185 190Gln Glu Lys Asn Val Asp Gln
Ala Ser Gly Ser Val Leu Leu His Leu 195 200 205Glu Val Gly Asp Gln
Val Trp Leu Gln Val Tyr Gly Asp Gly Asp His 210 215 220Asn Gly Leu
Tyr Ala Asp Asn Val Asn Asp Ser Thr Phe Thr Gly Phe225 230 235
240Leu Leu Tyr His Asp Thr Asn 245103236DNAhomo
sapiensCDS(29)..(2302) 10gtgacccacc tgcctcctcc gcagagca atg gcg gtg
tct gcc ggg tcc gcg 52 Met Ala Val Ser Ala Gly Ser Ala 1 5cgg acc
tcg ccc agc tca gat aaa gta cag aaa gac aag gct gaa ctg 100Arg Thr
Ser Pro Ser Ser Asp Lys Val Gln Lys Asp Lys Ala Glu Leu 10 15 20atc
tca ggg ccc agg cag gac agc cga ata ggg aaa ctc ttg ggt ttt 148Ile
Ser Gly Pro Arg Gln Asp Ser Arg Ile Gly Lys Leu Leu Gly Phe25 30 35
40gag tgg aca gat ttg tcc agt tgg cgg agg ctg gtg acc ctg ctg aat
196Glu Trp Thr Asp Leu Ser Ser Trp Arg Arg Leu Val Thr Leu Leu Asn
45 50 55cga cca acg gac cct gca agc tta gct gtc ttt cgt ttt ctt ttt
ggg 244Arg Pro Thr Asp Pro Ala Ser Leu Ala Val Phe Arg Phe Leu Phe
Gly 60 65 70ttc ttg atg gtg cta gac att ccc cag gag cgg ggg ctc agc
tct ctg 292Phe Leu Met Val Leu Asp Ile Pro Gln Glu Arg Gly Leu Ser
Ser Leu 75 80 85gac cgg aaa tac ctt gat ggg ctg gat gtg tgc cgc ttc
ccc ttg ctg 340Asp Arg Lys Tyr Leu Asp Gly Leu Asp Val Cys Arg Phe
Pro Leu Leu 90 95 100gat gcc cta cgc cca ctg cca ctt gac tgg atg
tat ctt gtc tac acc 388Asp Ala Leu Arg Pro Leu Pro Leu Asp Trp Met
Tyr Leu Val Tyr Thr105 110 115 120atc atg ttt ctg ggg gca ctg ggc
atg atg ctg ggc ctg tgc tac cgg 436Ile Met Phe Leu Gly Ala Leu Gly
Met Met Leu Gly Leu Cys Tyr Arg 125 130 135ata agc tgt gtg tta ttc
ctg ctg cca tac tgg tat gtg ttt ctc ctg 484Ile Ser Cys Val Leu Phe
Leu Leu Pro Tyr Trp Tyr Val Phe Leu Leu 140 145 150gac aag aca tca
tgg aac aac cac tcc tat ctg tat ggg ttg ttg gcc 532Asp Lys Thr Ser
Trp Asn Asn His Ser Tyr Leu Tyr Gly Leu Leu Ala 155 160 165ttt cag
cta aca ttc atg gat gca aac cac tac tgg tct gtg gac ggt 580Phe Gln
Leu Thr Phe Met Asp Ala Asn His Tyr Trp Ser Val Asp Gly 170 175
180ctg ctg aat gcc cat agg agg aat gcc cac gtg ccc ctt tgg aac tat
628Leu Leu Asn Ala His Arg Arg Asn Ala His Val Pro Leu Trp Asn
Tyr185 190 195 200gca gtg ctc cgt ggc cag atc ttc att gtg tac ttc
att gcg ggt gtg 676Ala Val Leu Arg Gly Gln Ile Phe Ile Val Tyr Phe
Ile Ala Gly Val 205 210 215aaa aag ctg gat gca gac tgg gtt gaa ggc
tat tcc atg gaa tat ttg 724Lys Lys Leu Asp Ala Asp Trp Val Glu Gly
Tyr Ser Met Glu Tyr Leu 220 225 230tcc cgg cac tgg ctc ttc agt ccc
ttc aaa ctg ctg ttg tct gag gag 772Ser Arg His Trp Leu Phe Ser Pro
Phe Lys Leu Leu Leu Ser Glu Glu 235 240 245ctg act agc ctg ctg gtc
gtg cac tgg ggt ggg ctg ctg ctt gac ctc 820Leu Thr Ser Leu Leu Val
Val His Trp Gly Gly Leu Leu Leu Asp Leu 250 255 260tca gct ggt ttc
ctg ctc ttt ttt gat gtc tca aga tcc att ggc ctg 868Ser Ala Gly Phe
Leu Leu Phe Phe Asp Val Ser Arg Ser Ile Gly Leu265 270 275 280ttc
ttt gtg tcc tac ttc cac tgc atg aat tcc cag ctt ttc agc att 916Phe
Phe Val Ser Tyr Phe His Cys Met Asn Ser Gln Leu Phe Ser Ile 285 290
295ggt atg ttc tcc tac gtc atg ctg gcc agc agc cct ctc ttc tgc tcc
964Gly Met Phe Ser Tyr Val Met Leu Ala Ser Ser Pro Leu Phe Cys Ser
300 305 310cct gag tgg cct cgg aag ctg gtg tcc tac tgc ccc cga agg
ttg caa 1012Pro Glu Trp Pro Arg Lys Leu Val Ser Tyr Cys Pro Arg Arg
Leu Gln 315 320 325caa ctg ttg ccc ctc aag gca gcc cct cag ccc agt
gtt tcc tgt gtg 1060Gln Leu Leu Pro Leu Lys Ala Ala Pro Gln Pro Ser
Val Ser Cys Val 330 335 340tat aag agg agc cgg ggc aaa agt ggc cag
aag cca ggg ctg cgc cat 1108Tyr Lys Arg Ser Arg Gly Lys Ser Gly Gln
Lys Pro Gly Leu Arg His345 350 355 360cag ctg gga gct gcc ttc acc
ctg ctc tac ctc ctg gag cag cta ttc 1156Gln Leu Gly Ala Ala Phe Thr
Leu Leu Tyr Leu Leu Glu Gln Leu Phe 365 370 375ctg ccc tat tct cat
ttt ctc acc cag ggc tat aac aac tgg aca aat 1204Leu Pro Tyr Ser His
Phe Leu Thr Gln Gly Tyr Asn Asn Trp Thr Asn 380 385 390ggg ctg tat
ggc tat tcc tgg gac atg atg gtg cac tcc cgc tcc cac 1252Gly Leu Tyr
Gly Tyr Ser Trp Asp Met Met Val His Ser Arg Ser His 395 400 405cag
cac gtg aag atc acc tac cgt gat ggc cgc act ggc gaa ctg ggc 1300Gln
His Val Lys Ile Thr Tyr Arg Asp Gly Arg Thr Gly Glu Leu Gly 410 415
420tac ctt aac cct ggg gta ttt aca cag agt cgg cga tgg aag gat cat
1348Tyr Leu Asn Pro Gly Val Phe Thr Gln Ser Arg Arg Trp Lys Asp
His425 430 435 440gca gac atg ctg aag caa tat gcc act tgc ctg agc
cgc ctg ctt ccc 1396Ala Asp Met Leu Lys Gln Tyr Ala Thr Cys Leu Ser
Arg Leu Leu Pro 445 450 455aag tat aat gtc act gag ccc cag atc tac
ttt gat att tgg gtc tcc 1444Lys Tyr Asn Val Thr Glu Pro Gln Ile Tyr
Phe Asp Ile Trp Val Ser 460 465 470atc aat gac cgc ttc cag cag agg
att ttt gac cct cgt gtg gac atc 1492Ile Asn Asp Arg Phe Gln Gln Arg
Ile Phe Asp Pro Arg Val Asp Ile 475 480 485gtg cag gcc gct tgg tca
ccc ttt cag cgc aca tcc tgg gtg caa cca 1540Val Gln Ala Ala Trp Ser
Pro Phe Gln Arg Thr Ser Trp Val Gln Pro 490 495 500ctc ttg atg gac
ctg tct ccc tgg agg gcc aag tta cag gaa atc aag 1588Leu Leu Met Asp
Leu Ser Pro Trp Arg Ala Lys Leu Gln Glu Ile Lys505 510 515 520agc
agc cta gac aac cac act gag gtg gtc ttc att gca gat ttc cct 1636Ser
Ser Leu Asp Asn His Thr Glu Val Val Phe Ile Ala Asp Phe Pro 525 530
535gga ctg cac ttg gag aat ttt gtg agt gaa gac ctg ggc aac act agc
1684Gly Leu His Leu Glu Asn Phe Val Ser Glu Asp Leu Gly Asn Thr Ser
540 545 550atc cag ctg ctg cag ggg gaa gtg act gtg gag ctt gtg gca
gaa cag 1732Ile Gln Leu Leu Gln Gly Glu Val Thr Val Glu Leu Val Ala
Glu Gln 555 560 565aag aac cag act ctt cga gag gga gaa aaa atg cag
ttg cct gct ggt 1780Lys Asn Gln Thr Leu Arg Glu Gly Glu Lys Met Gln
Leu Pro Ala Gly 570 575 580gag tac cat aag gtg tat acg aca tca cct
agc cct tct tgc tac atg 1828Glu Tyr His Lys Val Tyr Thr Thr Ser Pro
Ser Pro Ser Cys Tyr Met585 590 595 600tac gtc tat gtc aac act aca
gag ctt gca ctg gag caa gac ctg gca 1876Tyr Val Tyr Val Asn Thr Thr
Glu Leu Ala Leu Glu Gln Asp Leu Ala 605 610 615tat ctg caa gaa tta
aag gaa aag gtg gag aat gga agt gaa aca ggg 1924Tyr Leu Gln Glu Leu
Lys Glu Lys Val Glu Asn Gly Ser Glu Thr Gly 620 625 630cct cta ccc
cca gag ctg cag cct ctg ttg gaa ggg gaa gta aaa ggg 1972Pro Leu Pro
Pro Glu Leu Gln Pro Leu Leu Glu Gly Glu Val Lys Gly 635 640 645ggc
cct gag cca aca cct ctg gtt cag acc ttt ctt aga cgc caa caa 2020Gly
Pro Glu Pro Thr Pro Leu Val Gln Thr Phe Leu Arg Arg Gln Gln 650 655
660agg ctc cag gag att gaa cgc cgg cga aat act cct ttc cat gag cga
2068Arg Leu Gln Glu Ile Glu Arg Arg Arg Asn Thr Pro Phe His Glu
Arg665 670 675 680ttc ttc cgc ttc ttg ttg cga aag ctc tat gtc ttt
cgc cgc agc ttc 2116Phe Phe Arg Phe Leu Leu Arg Lys Leu Tyr Val Phe
Arg Arg Ser Phe 685 690 695ctg atg act tgt atc tca ctt cga aat ctg
ata tta ggc cgt cct tcc 2164Leu Met Thr Cys Ile Ser Leu Arg Asn Leu
Ile Leu Gly Arg Pro Ser 700 705 710ctg gag cag ctg gcc cag gag gtg
act tat gca aac ttg aga ccc ttt 2212Leu Glu Gln Leu Ala Gln Glu Val
Thr Tyr Ala Asn Leu Arg Pro Phe 715 720 725gag gca gtt gga gaa ctg
aat ccc tca aac acg gat tct tca cat tct 2260Glu Ala Val Gly Glu Leu
Asn Pro Ser Asn Thr Asp Ser Ser His Ser 730 735 740aat cct cct gag
tca aat cct gat cct gtc cac tca gag ttc 2302Asn Pro Pro Glu Ser Asn
Pro Asp Pro Val His Ser Glu Phe745 750 755tgaagggggc cagatgttgg
gtgcagatgt agaagcagcc agtcacagac ccattctatg 2362caatggacat
ttatttgaaa aaaattctca aaagtttttt tttttttttt gggggggcgg
2422ggttctaaag ctgtttttaa ctccgagatt acaacttaga ggaaccaagg
aaataaagca 2482aataagattt aacaacccaa gattaagagg ccaggaagag
gttagacgca atgtgaaact 2542gtcctcctag gataaggttt aaagtggctt
tttgggggct gggtgccgtg gctcacgcct 2602gtaatcccag cattttggga
ggctgaggtg ggcagatcac ttgaggccag gagttcgaga 2662ccagcctggc
caacatggca aaaccccttc tctactaaaa atacaaaaat tagccagacg
2722tggtggtggg tgcctgtaat ccaactaccc aggaggctga ggcatgagaa
tcgcttgggc 2782ccaggaggtg gaggttgcag tgagccgaga tcgagccact
gcactcctgg gcaacagagc 2842aagacttcgt ctcaaaataa ataaataaag
tggctcttgg ggaaaagcaa tttaatgtac 2902cacgatgaat agctaactgt
tcccaagtgt ttgctatgtg caacacaccg cgtgagcagt 2962gttacctgca
ttattacatt aggctgagag gtaaaataat ttgcccgaag acatacagct
3022agtgacgaat ggactgatgg tttgaactta acgtctattt gacttaaggt
cctgcaccct 3082gccacttgta attttcagaa tcactgataa tctgaaataa
tgcagcttaa aacatgtttt 3142cttaattaaa agtataaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3202aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaa 323611758PRThomo sapiens 11Met Ala Val Ser Ala Gly
Ser Ala Arg Thr Ser Pro Ser Ser Asp Lys1 5 10 15Val Gln Lys Asp Lys
Ala Glu Leu Ile Ser Gly Pro Arg Gln Asp Ser 20 25 30Arg Ile Gly Lys
Leu Leu Gly Phe Glu Trp Thr Asp Leu Ser Ser Trp 35 40 45Arg Arg Leu
Val Thr Leu Leu Asn Arg Pro Thr Asp Pro Ala Ser Leu 50 55 60Ala Val
Phe Arg Phe Leu Phe Gly Phe Leu Met Val Leu Asp Ile Pro65 70 75
80Gln Glu Arg Gly Leu Ser Ser Leu Asp Arg Lys Tyr Leu Asp Gly Leu
85 90 95Asp Val Cys Arg Phe Pro Leu Leu Asp Ala Leu Arg Pro Leu Pro
Leu 100 105 110Asp Trp Met Tyr Leu Val Tyr Thr Ile Met Phe Leu Gly
Ala Leu Gly 115 120 125Met Met Leu Gly Leu Cys Tyr Arg Ile Ser Cys
Val Leu Phe Leu Leu 130 135 140Pro Tyr Trp Tyr Val Phe Leu Leu Asp
Lys Thr Ser Trp Asn Asn His145 150 155 160Ser Tyr Leu Tyr Gly Leu
Leu Ala Phe Gln Leu Thr Phe Met Asp Ala 165 170 175Asn His Tyr Trp
Ser Val Asp Gly Leu Leu Asn Ala His Arg Arg Asn 180 185 190Ala His
Val Pro Leu Trp Asn Tyr Ala Val Leu Arg Gly Gln Ile Phe 195 200
205Ile Val Tyr Phe Ile Ala Gly Val Lys Lys Leu Asp Ala Asp Trp Val
210 215 220Glu Gly Tyr Ser Met Glu Tyr Leu Ser Arg His Trp Leu Phe
Ser Pro225 230 235 240Phe Lys Leu Leu Leu Ser Glu Glu Leu Thr Ser
Leu Leu Val Val His 245 250 255Trp Gly Gly Leu Leu Leu Asp Leu Ser
Ala Gly Phe Leu Leu Phe Phe 260 265 270Asp Val Ser Arg Ser Ile Gly
Leu Phe Phe Val Ser Tyr Phe His Cys 275 280 285Met Asn Ser Gln Leu
Phe Ser Ile Gly Met Phe Ser Tyr Val Met Leu 290 295 300Ala Ser Ser
Pro Leu Phe Cys Ser Pro Glu Trp Pro Arg Lys Leu Val305 310 315
320Ser Tyr Cys Pro Arg Arg Leu Gln Gln Leu Leu Pro Leu Lys Ala Ala
325 330 335Pro Gln Pro Ser Val Ser Cys Val Tyr Lys Arg Ser Arg Gly
Lys Ser 340 345 350Gly Gln Lys Pro Gly Leu Arg His Gln Leu Gly Ala
Ala Phe Thr Leu 355 360 365Leu Tyr Leu Leu Glu Gln Leu Phe Leu Pro
Tyr Ser His Phe Leu Thr 370 375 380Gln Gly Tyr Asn Asn Trp Thr Asn
Gly Leu Tyr Gly Tyr Ser Trp Asp385 390 395 400Met Met Val His Ser
Arg Ser His Gln His Val Lys Ile Thr Tyr Arg 405 410 415Asp Gly Arg
Thr Gly Glu Leu Gly Tyr Leu Asn Pro Gly Val Phe Thr 420 425 430Gln
Ser Arg Arg Trp Lys Asp His Ala Asp Met Leu Lys Gln Tyr Ala 435 440
445Thr Cys Leu Ser Arg Leu Leu Pro Lys Tyr Asn Val Thr Glu Pro Gln
450 455 460Ile Tyr Phe Asp Ile Trp Val Ser Ile Asn Asp Arg Phe Gln
Gln Arg465 470 475 480Ile Phe Asp Pro Arg Val Asp Ile Val Gln Ala
Ala Trp Ser Pro Phe 485 490 495Gln Arg Thr Ser Trp Val Gln Pro Leu
Leu Met Asp Leu Ser Pro Trp 500 505 510Arg Ala Lys Leu Gln Glu Ile
Lys Ser Ser Leu Asp Asn His Thr Glu 515 520 525Val Val Phe Ile Ala
Asp Phe Pro Gly Leu His Leu Glu Asn Phe Val 530 535 540Ser Glu Asp
Leu Gly Asn Thr Ser Ile Gln Leu Leu Gln Gly Glu Val545 550 555
560Thr Val Glu Leu Val Ala Glu Gln Lys Asn Gln Thr Leu Arg Glu Gly
565 570 575Glu Lys Met Gln Leu Pro Ala Gly Glu Tyr His Lys Val Tyr
Thr Thr 580 585 590Ser Pro Ser Pro Ser Cys Tyr Met Tyr Val Tyr Val
Asn Thr Thr Glu 595 600 605Leu Ala Leu Glu Gln Asp Leu Ala Tyr Leu
Gln Glu Leu Lys Glu Lys 610 615 620Val Glu Asn Gly Ser Glu Thr Gly
Pro Leu Pro Pro Glu Leu Gln Pro625 630 635 640Leu Leu Glu Gly Glu
Val Lys Gly Gly Pro Glu Pro Thr Pro Leu Val 645 650 655Gln Thr Phe
Leu Arg Arg Gln Gln Arg Leu Gln Glu Ile Glu Arg Arg 660 665 670Arg
Asn Thr Pro Phe His Glu Arg Phe Phe Arg Phe Leu Leu Arg Lys 675 680
685Leu Tyr Val Phe Arg Arg Ser Phe Leu Met Thr Cys Ile Ser Leu Arg
690 695 700Asn Leu Ile Leu Gly Arg Pro Ser Leu Glu Gln Leu Ala Gln
Glu Val705 710 715 720Thr Tyr Ala Asn Leu Arg Pro Phe Glu Ala Val
Gly Glu Leu Asn Pro 725 730 735Ser Asn Thr Asp Ser Ser His Ser Asn
Pro Pro Glu Ser Asn Pro Asp 740 745 750Pro Val His Ser Glu Phe
755122905DNAmus musculusCDS(75)..(2345) 12agacagcaag tctaagtctg
gaggttccac tgggtccgac ctggctgcag agaggctcac 60ctgtccctgc agtc atg
gct gtg cac cgc ggc tcc gca ctg gtt gct ccc 110 Met Ala Val His Arg
Gly Ser Ala Leu Val Ala Pro 1 5 10gcc tca gat aaa gta cag aaa aac
aag tct gca cag aca
tca gga ctg 158Ala Ser Asp Lys Val Gln Lys Asn Lys Ser Ala Gln Thr
Ser Gly Leu 15 20 25aaa cag ggc agc cga atg gag aaa att tta ggg ttt
gaa tgg aca gat 206Lys Gln Gly Ser Arg Met Glu Lys Ile Leu Gly Phe
Glu Trp Thr Asp 30 35 40tta tct agc tgg cag agt gtc gtg acc ctg ctt
aac aaa cca acg gac 254Leu Ser Ser Trp Gln Ser Val Val Thr Leu Leu
Asn Lys Pro Thr Asp45 50 55 60cct gca aac ctg gct gtc ttt cgt ttt
ctc ttt gct ttc ttg atg ctg 302Pro Ala Asn Leu Ala Val Phe Arg Phe
Leu Phe Ala Phe Leu Met Leu 65 70 75ctg gac att ccc cag gaa cgc ggc
ctt agc tcc ctg gac cga aaa tac 350Leu Asp Ile Pro Gln Glu Arg Gly
Leu Ser Ser Leu Asp Arg Lys Tyr 80 85 90ttg gat ggg ctg gat gtg tgc
cgt ttc ccc ttg ctg gat gcc ttg cgc 398Leu Asp Gly Leu Asp Val Cys
Arg Phe Pro Leu Leu Asp Ala Leu Arg 95 100 105cca ctg cca ctg gac
tgg atg tat ctt gtc tac acc atc atg ttt ctg 446Pro Leu Pro Leu Asp
Trp Met Tyr Leu Val Tyr Thr Ile Met Phe Leu 110 115 120ggg gca ctg
ggc atg atg ctg ggg cta tgc tac cgg cta agc tgt gtg 494Gly Ala Leu
Gly Met Met Leu Gly Leu Cys Tyr Arg Leu Ser Cys Val125 130 135
140tta ttc ctg cta ccg tac tgg tac gtg ttt ctc ctg gac aag act tcg
542Leu Phe Leu Leu Pro Tyr Trp Tyr Val Phe Leu Leu Asp Lys Thr Ser
145 150 155tgg aac aat cac tcc tat ctg tat ggt ttg ttg gcc ttt cag
ttg aca 590Trp Asn Asn His Ser Tyr Leu Tyr Gly Leu Leu Ala Phe Gln
Leu Thr 160 165 170ttc atg gat gca aac cac tac tgg tct gtg gat ggc
ttg ctg aat gcc 638Phe Met Asp Ala Asn His Tyr Trp Ser Val Asp Gly
Leu Leu Asn Ala 175 180 185cga aag aag aat gct cac gtg ccc ctt tgg
aac tac aca gtt ctg cgt 686Arg Lys Lys Asn Ala His Val Pro Leu Trp
Asn Tyr Thr Val Leu Arg 190 195 200ggc cag atc ttc atc gtg tac ttc
atc gcg ggt gtg aag aag ctc gat 734Gly Gln Ile Phe Ile Val Tyr Phe
Ile Ala Gly Val Lys Lys Leu Asp205 210 215 220gct gac tgg gtt ggg
ggc tac tcc atg gag cac ctg tcc cgg cac tgg 782Ala Asp Trp Val Gly
Gly Tyr Ser Met Glu His Leu Ser Arg His Trp 225 230 235ctc ttc agt
ccc ttc aag ctg gtg ttg tcg gag gag ctg aca agc ctg 830Leu Phe Ser
Pro Phe Lys Leu Val Leu Ser Glu Glu Leu Thr Ser Leu 240 245 250ctg
gta gta cac tgg tgt ggg ctt ctc ctt gac ctc tcg gct ggc ttc 878Leu
Val Val His Trp Cys Gly Leu Leu Leu Asp Leu Ser Ala Gly Phe 255 260
265ctg ctc ttc ttt gat gcc tcc aga ccc gtc ggc ctg ttc ttc gtg tcc
926Leu Leu Phe Phe Asp Ala Ser Arg Pro Val Gly Leu Phe Phe Val Ser
270 275 280tac ttt cac tgc atg aac tcg cag ctc ttc agc atc ggg atg
ttt ccc 974Tyr Phe His Cys Met Asn Ser Gln Leu Phe Ser Ile Gly Met
Phe Pro285 290 295 300tat gtc atg ctg gcc agc agc cct ctc ttc tgc
tca gct gaa tgg cct 1022Tyr Val Met Leu Ala Ser Ser Pro Leu Phe Cys
Ser Ala Glu Trp Pro 305 310 315cgg aag ttg gta gcc cga tgc ccg aaa
agg ctg caa gag ctg ctg ccc 1070Arg Lys Leu Val Ala Arg Cys Pro Lys
Arg Leu Gln Glu Leu Leu Pro 320 325 330acc aaa gcc gct cct cgg cct
agt gct tcc tgt gtg tat aag agg tcc 1118Thr Lys Ala Ala Pro Arg Pro
Ser Ala Ser Cys Val Tyr Lys Arg Ser 335 340 345cgg ggc aaa gct ggc
ccg aag ccc ggg ctg cgc cac cag ctg gga gcc 1166Arg Gly Lys Ala Gly
Pro Lys Pro Gly Leu Arg His Gln Leu Gly Ala 350 355 360atc ttc acc
ctg ctc tac ctc cta gag cag ctc ttc ctg ccc tat tcc 1214Ile Phe Thr
Leu Leu Tyr Leu Leu Glu Gln Leu Phe Leu Pro Tyr Ser365 370 375
380cac ttc ctg acc cag ggt tac aat aac tgg aca aat ggg ctg tat ggc
1262His Phe Leu Thr Gln Gly Tyr Asn Asn Trp Thr Asn Gly Leu Tyr Gly
385 390 395tat tcc tgg gac atg atg gtg cac tcc cgc tcc cac cag cac
gta aag 1310Tyr Ser Trp Asp Met Met Val His Ser Arg Ser His Gln His
Val Lys 400 405 410atc acc tac cgc gac ggc ctc acg ggc gag cta ggc
tac ctt aac cct 1358Ile Thr Tyr Arg Asp Gly Leu Thr Gly Glu Leu Gly
Tyr Leu Asn Pro 415 420 425ggg gta ttc aca cag agc cgg cga tgg aag
gat cat gca gac atg ctg 1406Gly Val Phe Thr Gln Ser Arg Arg Trp Lys
Asp His Ala Asp Met Leu 430 435 440aag caa tat gcc act tgc ctg agc
ctc ctg ctt ccc aag tac aat gtc 1454Lys Gln Tyr Ala Thr Cys Leu Ser
Leu Leu Leu Pro Lys Tyr Asn Val445 450 455 460act gag ccc cag atc
tac ttt gat att tgg gtc tcc atc aat gac cgc 1502Thr Glu Pro Gln Ile
Tyr Phe Asp Ile Trp Val Ser Ile Asn Asp Arg 465 470 475ttc cag cag
agg ctt ttt gac cct cgt gtg gac atc gtg cag gct gtc 1550Phe Gln Gln
Arg Leu Phe Asp Pro Arg Val Asp Ile Val Gln Ala Val 480 485 490tgg
tcc ccc ttc cag cgc aca cct tgg gtg cag cca ctc ttg atg gat 1598Trp
Ser Pro Phe Gln Arg Thr Pro Trp Val Gln Pro Leu Leu Met Asp 495 500
505tta tct ccc tgg agg acc aag tta cag gat att aag agc agt ctg gac
1646Leu Ser Pro Trp Arg Thr Lys Leu Gln Asp Ile Lys Ser Ser Leu Asp
510 515 520aac cac acc gag gtg gtc ttc att gca gat ttc cct ggg ctt
cac ttg 1694Asn His Thr Glu Val Val Phe Ile Ala Asp Phe Pro Gly Leu
His Leu525 530 535 540gag aat ttt gtg agt gaa gac ctg ggc aac act
agc atc cag ctg ctg 1742Glu Asn Phe Val Ser Glu Asp Leu Gly Asn Thr
Ser Ile Gln Leu Leu 545 550 555cag gga gaa gtc acc gtg gaa ttg gtg
gca gaa cag aaa aat cag act 1790Gln Gly Glu Val Thr Val Glu Leu Val
Ala Glu Gln Lys Asn Gln Thr 560 565 570ctt caa gaa gga gag aaa atg
cag ttg cct gct gga gag tac cat aaa 1838Leu Gln Glu Gly Glu Lys Met
Gln Leu Pro Ala Gly Glu Tyr His Lys 575 580 585gtc tat act gta tca
tct agt cct tcc tgc tac atg tac gtc tat gtc 1886Val Tyr Thr Val Ser
Ser Ser Pro Ser Cys Tyr Met Tyr Val Tyr Val 590 595 600aac act aca
gag gtc gca ctg gag caa gac ctg gca tat ctg caa gaa 1934Asn Thr Thr
Glu Val Ala Leu Glu Gln Asp Leu Ala Tyr Leu Gln Glu605 610 615
620tta aag gag aag gtg gag aac gga agt gaa aca ggg ccc ctg cct cca
1982Leu Lys Glu Lys Val Glu Asn Gly Ser Glu Thr Gly Pro Leu Pro Pro
625 630 635gaa ctt cag cct ctt ttg gaa ggg gaa gta aaa ggg ggc cct
gag cca 2030Glu Leu Gln Pro Leu Leu Glu Gly Glu Val Lys Gly Gly Pro
Glu Pro 640 645 650aca cct ctg gtc caa act ttt ctc aga cga cag agg
aag ctc caa gaa 2078Thr Pro Leu Val Gln Thr Phe Leu Arg Arg Gln Arg
Lys Leu Gln Glu 655 660 665att gaa cgc agg cga aat agc cct ttc cat
gag cga ttt ctc cgc ttc 2126Ile Glu Arg Arg Arg Asn Ser Pro Phe His
Glu Arg Phe Leu Arg Phe 670 675 680gtg ctg cga aag ctc tac gtc ttt
cga cgc agc ttc ctg atg act cga 2174Val Leu Arg Lys Leu Tyr Val Phe
Arg Arg Ser Phe Leu Met Thr Arg685 690 695 700att tca ctc cga aac
ctg cta tta ggc cgc cct tcc cta gag caa cta 2222Ile Ser Leu Arg Asn
Leu Leu Leu Gly Arg Pro Ser Leu Glu Gln Leu 705 710 715gcc caa gag
gtg aca tat gca aac ttg cga cca ttt gaa cca gtt gat 2270Ala Gln Glu
Val Thr Tyr Ala Asn Leu Arg Pro Phe Glu Pro Val Asp 720 725 730gag
tca agt gct tca aac aca gat tct tca aat cac ccg tca gag cca 2318Glu
Ser Ser Ala Ser Asn Thr Asp Ser Ser Asn His Pro Ser Glu Pro 735 740
745gat tct gag cat gtt cac tct gag ttc tgagggatgt acagatgctc
2365Asp Ser Glu His Val His Ser Glu Phe 750 755tgtgcagatg
tgggggcagc ctgttatagg cttattgtct acgcaaagaa catatttttg
2425gagaaaaatg atatgggaca ggctttcaca gtacagccca ggctggcctc
aaactcatgg 2485ttggtccctc tgcttcagcc tgttttgtaa ttacatagta
tcaccaaacc tagttgcttt 2545tccctttaca ttttttcccc ttataagttc
tttaaaatta tagcttacat tttttctttt 2605ttcttttttt tttttttgta
ttttttcttt gtcaagacag gtctctctct gtgtagcact 2665ggctgtcctg
gaactcactc tgtagtccag gctggcctcc aactcagaaa ttctcctgcc
2725tctgcctccc aagtgctggg attaaaggtg tgtgccacca cgccccactg
ggcttttagt 2785ttttatagac aagatttctc catgtagacc agaccagctc
tcctgagtgc tgaaattaaa 2845ggcacgggac atcactacct ggctttctta
ttaaacttgt tttagtggtc tcaacaaaaa 290513757PRTmus musculus 13Met Ala
Val His Arg Gly Ser Ala Leu Val Ala Pro Ala Ser Asp Lys1 5 10 15Val
Gln Lys Asn Lys Ser Ala Gln Thr Ser Gly Leu Lys Gln Gly Ser 20 25
30Arg Met Glu Lys Ile Leu Gly Phe Glu Trp Thr Asp Leu Ser Ser Trp
35 40 45Gln Ser Val Val Thr Leu Leu Asn Lys Pro Thr Asp Pro Ala Asn
Leu 50 55 60Ala Val Phe Arg Phe Leu Phe Ala Phe Leu Met Leu Leu Asp
Ile Pro65 70 75 80Gln Glu Arg Gly Leu Ser Ser Leu Asp Arg Lys Tyr
Leu Asp Gly Leu 85 90 95Asp Val Cys Arg Phe Pro Leu Leu Asp Ala Leu
Arg Pro Leu Pro Leu 100 105 110Asp Trp Met Tyr Leu Val Tyr Thr Ile
Met Phe Leu Gly Ala Leu Gly 115 120 125Met Met Leu Gly Leu Cys Tyr
Arg Leu Ser Cys Val Leu Phe Leu Leu 130 135 140Pro Tyr Trp Tyr Val
Phe Leu Leu Asp Lys Thr Ser Trp Asn Asn His145 150 155 160Ser Tyr
Leu Tyr Gly Leu Leu Ala Phe Gln Leu Thr Phe Met Asp Ala 165 170
175Asn His Tyr Trp Ser Val Asp Gly Leu Leu Asn Ala Arg Lys Lys Asn
180 185 190Ala His Val Pro Leu Trp Asn Tyr Thr Val Leu Arg Gly Gln
Ile Phe 195 200 205Ile Val Tyr Phe Ile Ala Gly Val Lys Lys Leu Asp
Ala Asp Trp Val 210 215 220Gly Gly Tyr Ser Met Glu His Leu Ser Arg
His Trp Leu Phe Ser Pro225 230 235 240Phe Lys Leu Val Leu Ser Glu
Glu Leu Thr Ser Leu Leu Val Val His 245 250 255Trp Cys Gly Leu Leu
Leu Asp Leu Ser Ala Gly Phe Leu Leu Phe Phe 260 265 270Asp Ala Ser
Arg Pro Val Gly Leu Phe Phe Val Ser Tyr Phe His Cys 275 280 285Met
Asn Ser Gln Leu Phe Ser Ile Gly Met Phe Pro Tyr Val Met Leu 290 295
300Ala Ser Ser Pro Leu Phe Cys Ser Ala Glu Trp Pro Arg Lys Leu
Val305 310 315 320Ala Arg Cys Pro Lys Arg Leu Gln Glu Leu Leu Pro
Thr Lys Ala Ala 325 330 335Pro Arg Pro Ser Ala Ser Cys Val Tyr Lys
Arg Ser Arg Gly Lys Ala 340 345 350Gly Pro Lys Pro Gly Leu Arg His
Gln Leu Gly Ala Ile Phe Thr Leu 355 360 365Leu Tyr Leu Leu Glu Gln
Leu Phe Leu Pro Tyr Ser His Phe Leu Thr 370 375 380Gln Gly Tyr Asn
Asn Trp Thr Asn Gly Leu Tyr Gly Tyr Ser Trp Asp385 390 395 400Met
Met Val His Ser Arg Ser His Gln His Val Lys Ile Thr Tyr Arg 405 410
415Asp Gly Leu Thr Gly Glu Leu Gly Tyr Leu Asn Pro Gly Val Phe Thr
420 425 430Gln Ser Arg Arg Trp Lys Asp His Ala Asp Met Leu Lys Gln
Tyr Ala 435 440 445Thr Cys Leu Ser Leu Leu Leu Pro Lys Tyr Asn Val
Thr Glu Pro Gln 450 455 460Ile Tyr Phe Asp Ile Trp Val Ser Ile Asn
Asp Arg Phe Gln Gln Arg465 470 475 480Leu Phe Asp Pro Arg Val Asp
Ile Val Gln Ala Val Trp Ser Pro Phe 485 490 495Gln Arg Thr Pro Trp
Val Gln Pro Leu Leu Met Asp Leu Ser Pro Trp 500 505 510Arg Thr Lys
Leu Gln Asp Ile Lys Ser Ser Leu Asp Asn His Thr Glu 515 520 525Val
Val Phe Ile Ala Asp Phe Pro Gly Leu His Leu Glu Asn Phe Val 530 535
540Ser Glu Asp Leu Gly Asn Thr Ser Ile Gln Leu Leu Gln Gly Glu
Val545 550 555 560Thr Val Glu Leu Val Ala Glu Gln Lys Asn Gln Thr
Leu Gln Glu Gly 565 570 575Glu Lys Met Gln Leu Pro Ala Gly Glu Tyr
His Lys Val Tyr Thr Val 580 585 590Ser Ser Ser Pro Ser Cys Tyr Met
Tyr Val Tyr Val Asn Thr Thr Glu 595 600 605Val Ala Leu Glu Gln Asp
Leu Ala Tyr Leu Gln Glu Leu Lys Glu Lys 610 615 620Val Glu Asn Gly
Ser Glu Thr Gly Pro Leu Pro Pro Glu Leu Gln Pro625 630 635 640Leu
Leu Glu Gly Glu Val Lys Gly Gly Pro Glu Pro Thr Pro Leu Val 645 650
655Gln Thr Phe Leu Arg Arg Gln Arg Lys Leu Gln Glu Ile Glu Arg Arg
660 665 670Arg Asn Ser Pro Phe His Glu Arg Phe Leu Arg Phe Val Leu
Arg Lys 675 680 685Leu Tyr Val Phe Arg Arg Ser Phe Leu Met Thr Arg
Ile Ser Leu Arg 690 695 700Asn Leu Leu Leu Gly Arg Pro Ser Leu Glu
Gln Leu Ala Gln Glu Val705 710 715 720Thr Tyr Ala Asn Leu Arg Pro
Phe Glu Pro Val Asp Glu Ser Ser Ala 725 730 735Ser Asn Thr Asp Ser
Ser Asn His Pro Ser Glu Pro Asp Ser Glu His 740 745 750Val His Ser
Glu Phe 755141223DNAhomo sapiensCDS(84)..(1034) 14gggatccttg
agtcctactc agccccagcg gaggtgaagg acgtccttcc ccaggagccg 60actggccaat
cacaggcagg aag atg aag gtt ctg tgg gct gcg ttg ctg gtc 113 Met Lys
Val Leu Trp Ala Ala Leu Leu Val 1 5 10aca ttc ctg gca gga tgc cag
gcc aag gtg gag caa gcg gtg gag aca 161Thr Phe Leu Ala Gly Cys Gln
Ala Lys Val Glu Gln Ala Val Glu Thr 15 20 25gag ccg gag ccc gag ctg
cgc cag cag acc gag tgg cag agc ggc cag 209Glu Pro Glu Pro Glu Leu
Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln 30 35 40cgc tgg gaa ctg gca
ctg ggt cgc ttt tgg gat tac ctg cgc tgg gtg 257Arg Trp Glu Leu Ala
Leu Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val 45 50 55cag aca ctg tct
gag cag gtg cag gag gag ctg ctc agc tcc cag gtc 305Gln Thr Leu Ser
Glu Gln Val Gln Glu Glu Leu Leu Ser Ser Gln Val 60 65 70acc cag gaa
ctg agg gcg ctg atg gac gag acc atg aag gag ttg aag 353Thr Gln Glu
Leu Arg Ala Leu Met Asp Glu Thr Met Lys Glu Leu Lys75 80 85 90gcc
tac aaa tcg gaa ctg gag gaa caa ctg acc ccg gtg gcg gag gag 401Ala
Tyr Lys Ser Glu Leu Glu Glu Gln Leu Thr Pro Val Ala Glu Glu 95 100
105acg cgg gca cgg ctg tcc aag gag ctg cag gcg gcg cag gcc cgg ctg
449Thr Arg Ala Arg Leu Ser Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu
110 115 120ggc gcg gac atg gag gac gtg tgc ggc cgc ctg gtg cag tac
cgc ggc 497Gly Ala Asp Met Glu Asp Val Cys Gly Arg Leu Val Gln Tyr
Arg Gly 125 130 135gag gtg cag gcc atg ctc ggc cag agc acc gag gag
ctg cgg gtg cgc 545Glu Val Gln Ala Met Leu Gly Gln Ser Thr Glu Glu
Leu Arg Val Arg 140 145 150ctc gcc tcc cac ctg cgc aag ctg cgt aag
cgg ctc ctc cgc gat gcc 593Leu Ala Ser His Leu Arg Lys Leu Arg Lys
Arg Leu Leu Arg Asp Ala155 160 165 170gat gac ctg cag aag cgc ctg
gca gtg tac cag gcc ggg gcc cgc gag 641Asp Asp Leu Gln Lys Arg Leu
Ala Val Tyr Gln Ala Gly Ala Arg Glu 175 180 185ggc gcc gag cgc ggc
ctc agc gcc atc cgc gag cgc ctg ggg ccc ctg 689Gly Ala Glu Arg Gly
Leu Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu 190 195 200gtg gaa cag
ggc cgc gtg cgg gcc gcc act gtg ggc tcc ctg gcc ggc 737Val Glu Gln
Gly Arg Val Arg Ala Ala Thr Val Gly Ser Leu Ala Gly 205 210 215cag
ccg cta cag gag cgg gcc cag gcc tgg ggc gag cgg ctg cgc gcg 785Gln
Pro Leu Gln Glu Arg Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala 220 225
230cgg atg gag gag atg ggc agc cgg acc cgc gac cgc ctg gac gag gtg
833Arg Met Glu Glu Met Gly Ser Arg Thr Arg Asp Arg Leu Asp Glu
Val235 240 245 250aag gag cag gtg gcg gag gtg cgc gcc aag
ctg gag gag cag gcc cag 881Lys Glu Gln Val Ala Glu Val Arg Ala Lys
Leu Glu Glu Gln Ala Gln 255 260 265cag ata cgc ctg cag gcc gag gcc
ttc cag gcc cgc ctc aag agc tgg 929Gln Ile Arg Leu Gln Ala Glu Ala
Phe Gln Ala Arg Leu Lys Ser Trp 270 275 280ttc gag ccc ctg gtg gaa
gac atg cag cgc cag tgg gcc ggg ctg gtg 977Phe Glu Pro Leu Val Glu
Asp Met Gln Arg Gln Trp Ala Gly Leu Val 285 290 295gag aag gtg cag
gct gcc gtg ggc acc agc gcc gcc cct gtg ccc agc 1025Glu Lys Val Gln
Ala Ala Val Gly Thr Ser Ala Ala Pro Val Pro Ser 300 305 310gac aat
cac tgaacgccga agcctgcagc catgcgaccc cacgccaccc 1074Asp Asn
His315cgtgcctcct gcctccgcgc agcctgcagc gggagaccct gtccccgccc
cagccgtcct 1134cctggggtgg accctagttt aataaagatt caccaagttt
cacgcaaaaa aaaaaaaaaa 1194aaaaaaaaaa aaaaaaaaaa aaaaaaaaa
122315317PRThomo sapiens 15Met Lys Val Leu Trp Ala Ala Leu Leu Val
Thr Phe Leu Ala Gly Cys1 5 10 15Gln Ala Lys Val Glu Gln Ala Val Glu
Thr Glu Pro Glu Pro Glu Leu 20 25 30Arg Gln Gln Thr Glu Trp Gln Ser
Gly Gln Arg Trp Glu Leu Ala Leu 35 40 45Gly Arg Phe Trp Asp Tyr Leu
Arg Trp Val Gln Thr Leu Ser Glu Gln 50 55 60Val Gln Glu Glu Leu Leu
Ser Ser Gln Val Thr Gln Glu Leu Arg Ala65 70 75 80Leu Met Asp Glu
Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu 85 90 95Glu Glu Gln
Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser 100 105 110Lys
Glu Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp 115 120
125Val Cys Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu
130 135 140Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His
Leu Arg145 150 155 160Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp
Asp Leu Gln Lys Arg 165 170 175Leu Ala Val Tyr Gln Ala Gly Ala Arg
Glu Gly Ala Glu Arg Gly Leu 180 185 190Ser Ala Ile Arg Glu Arg Leu
Gly Pro Leu Val Glu Gln Gly Arg Val 195 200 205Arg Ala Ala Thr Val
Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg 210 215 220Ala Gln Ala
Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly225 230 235
240Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu
245 250 255Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu
Gln Ala 260 265 270Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu
Pro Leu Val Glu 275 280 285Asp Met Gln Arg Gln Trp Ala Gly Leu Val
Glu Lys Val Gln Ala Ala 290 295 300Val Gly Thr Ser Ala Ala Pro Val
Pro Ser Asp Asn His305 310 315161104DNAmus musculusCDS(46)..(978)
16ggacttgttt cggaaggagc tgactggcca atcacaattg cgaag atg aag gct ctg
57 Met Lys Ala Leu 1tgg gcc gtg ctg ttg gtc aca ttg ctg aca gga tgc
cta gcc gag gga 105Trp Ala Val Leu Leu Val Thr Leu Leu Thr Gly Cys
Leu Ala Glu Gly5 10 15 20gag ccg gag gtg aca gat cag ctc gag tgg
caa agc aac caa ccc tgg 153Glu Pro Glu Val Thr Asp Gln Leu Glu Trp
Gln Ser Asn Gln Pro Trp 25 30 35gag cag gcc ctg aac cgc ttc tgg gat
tac ctg cgc tgg gtg cag acg 201Glu Gln Ala Leu Asn Arg Phe Trp Asp
Tyr Leu Arg Trp Val Gln Thr 40 45 50ctg tct gac cag gtc cag gaa gag
ctg cag agc tcc caa gtc aca caa 249Leu Ser Asp Gln Val Gln Glu Glu
Leu Gln Ser Ser Gln Val Thr Gln 55 60 65gaa ctg acg gca ctg atg gag
gac act atg acg gaa gta aag gct tac 297Glu Leu Thr Ala Leu Met Glu
Asp Thr Met Thr Glu Val Lys Ala Tyr 70 75 80aaa aag gag ctg gag gaa
cag ctg ggt cca gtg gcg gag gag aca cgg 345Lys Lys Glu Leu Glu Glu
Gln Leu Gly Pro Val Ala Glu Glu Thr Arg85 90 95 100gcc agg ctg ggc
aaa gag gtg cag gcg gca cag gcc cga ctc gga gcc 393Ala Arg Leu Gly
Lys Glu Val Gln Ala Ala Gln Ala Arg Leu Gly Ala 105 110 115gac atg
gag gat cta cgc aac cga ctc ggg cag tac cgc aac gag gtg 441Asp Met
Glu Asp Leu Arg Asn Arg Leu Gly Gln Tyr Arg Asn Glu Val 120 125
130cac acc atg ctg ggc cag agc aca gag gag ata cgg gcg cgg ctc tcc
489His Thr Met Leu Gly Gln Ser Thr Glu Glu Ile Arg Ala Arg Leu Ser
135 140 145aca cac ctg cgc aag atg cgc aag cgc ttg atg cgg gat gcc
gat gat 537Thr His Leu Arg Lys Met Arg Lys Arg Leu Met Arg Asp Ala
Asp Asp 150 155 160ctg cag aag cgc cta gct gtg tac aag gca ggg gca
cgc gag ggc gcc 585Leu Gln Lys Arg Leu Ala Val Tyr Lys Ala Gly Ala
Arg Glu Gly Ala165 170 175 180gag cgc ggt gtg agt gcc atc cgt gag
cgc ctg ggg cct ctg gtg gag 633Glu Arg Gly Val Ser Ala Ile Arg Glu
Arg Leu Gly Pro Leu Val Glu 185 190 195caa ggt cgc cag cgc act gcc
aac cta ggc gct ggg gcc gcc cag cct 681Gln Gly Arg Gln Arg Thr Ala
Asn Leu Gly Ala Gly Ala Ala Gln Pro 200 205 210ctg cgc gat cgc gcc
cag gct ttt ggt gac cgc atc cga ggg cgg ctg 729Leu Arg Asp Arg Ala
Gln Ala Phe Gly Asp Arg Ile Arg Gly Arg Leu 215 220 225gag gaa gtg
ggc aac cag gcc cgt gac cgc cta gag gag gtg cgt gag 777Glu Glu Val
Gly Asn Gln Ala Arg Asp Arg Leu Glu Glu Val Arg Glu 230 235 240cac
atg gag gag gtg cgc tcc aag atg gag gaa cag acc cag caa ata 825His
Met Glu Glu Val Arg Ser Lys Met Glu Glu Gln Thr Gln Gln Ile245 250
255 260cgc ctg cag gcg gag atc ttc cag gcc cgc ctc aag ggc tgg ttc
gag 873Arg Leu Gln Ala Glu Ile Phe Gln Ala Arg Leu Lys Gly Trp Phe
Glu 265 270 275cca ata gtg gaa gac atg cat cgc cag tgg gca aac ctg
atg gag aag 921Pro Ile Val Glu Asp Met His Arg Gln Trp Ala Asn Leu
Met Glu Lys 280 285 290ata cag gcc tct gtg gct acc aac ccc atc atc
acc cca gtg gcc cag 969Ile Gln Ala Ser Val Ala Thr Asn Pro Ile Ile
Thr Pro Val Ala Gln 295 300 305gag aat caa tgagtatcct tctcctgtcc
tgcaacaaca tccatatcca 1018Glu Asn Gln 310gccaggtggc cctgtctcaa
gcacctctct ggccctctgg tggcccttgc ttaataaaga 1078ttctccgagc
aaaaaaaaaa aaaaaa 110417311PRTmus musculus 17Met Lys Ala Leu Trp
Ala Val Leu Leu Val Thr Leu Leu Thr Gly Cys1 5 10 15Leu Ala Glu Gly
Glu Pro Glu Val Thr Asp Gln Leu Glu Trp Gln Ser 20 25 30Asn Gln Pro
Trp Glu Gln Ala Leu Asn Arg Phe Trp Asp Tyr Leu Arg 35 40 45Trp Val
Gln Thr Leu Ser Asp Gln Val Gln Glu Glu Leu Gln Ser Ser 50 55 60Gln
Val Thr Gln Glu Leu Thr Ala Leu Met Glu Asp Thr Met Thr Glu65 70 75
80Val Lys Ala Tyr Lys Lys Glu Leu Glu Glu Gln Leu Gly Pro Val Ala
85 90 95Glu Glu Thr Arg Ala Arg Leu Gly Lys Glu Val Gln Ala Ala Gln
Ala 100 105 110Arg Leu Gly Ala Asp Met Glu Asp Leu Arg Asn Arg Leu
Gly Gln Tyr 115 120 125Arg Asn Glu Val His Thr Met Leu Gly Gln Ser
Thr Glu Glu Ile Arg 130 135 140Ala Arg Leu Ser Thr His Leu Arg Lys
Met Arg Lys Arg Leu Met Arg145 150 155 160Asp Ala Asp Asp Leu Gln
Lys Arg Leu Ala Val Tyr Lys Ala Gly Ala 165 170 175Arg Glu Gly Ala
Glu Arg Gly Val Ser Ala Ile Arg Glu Arg Leu Gly 180 185 190Pro Leu
Val Glu Gln Gly Arg Gln Arg Thr Ala Asn Leu Gly Ala Gly 195 200
205Ala Ala Gln Pro Leu Arg Asp Arg Ala Gln Ala Phe Gly Asp Arg Ile
210 215 220Arg Gly Arg Leu Glu Glu Val Gly Asn Gln Ala Arg Asp Arg
Leu Glu225 230 235 240Glu Val Arg Glu His Met Glu Glu Val Arg Ser
Lys Met Glu Glu Gln 245 250 255Thr Gln Gln Ile Arg Leu Gln Ala Glu
Ile Phe Gln Ala Arg Leu Lys 260 265 270Gly Trp Phe Glu Pro Ile Val
Glu Asp Met His Arg Gln Trp Ala Asn 275 280 285Leu Met Glu Lys Ile
Gln Ala Ser Val Ala Thr Asn Pro Ile Ile Thr 290 295 300Pro Val Ala
Gln Glu Asn Gln305 310185555DNAmus musculusCDS(316)..(5430)
18ggctgtggga gagcagaaga ggagctggaa gagcagccta caacagctgt cgggagggac
60cagggctagt tcacacttgg aagctgggat gccaggaccg gccctcctgc ctctctcggt
120ctccatcggc ctcctggtca gctcactcca cactgagacg attctgaagt
aagatgctcc 180tggctcctca cagactctgc tacaagagac agagtgaagt
gtccccaggg ctcagagcct 240ttgactctgc tccttccctt cccacggctg
agttggcaca ggagcacctg ggtgagctgc 300accagactta agaag atg agg ccc
ctg att ctg tta gct gcc ctc ctc tgg 351 Met Arg Pro Leu Ile Leu Leu
Ala Ala Leu Leu Trp 1 5 10ctc cag gac tct ttg gcc cag gaa gat gta
tgc tca tcc ttg gat ggg 399Leu Gln Asp Ser Leu Ala Gln Glu Asp Val
Cys Ser Ser Leu Asp Gly 15 20 25agc cca gac agg cag ggt gga ggt cca
cct ctg agt gtg aac gtc agc 447Ser Pro Asp Arg Gln Gly Gly Gly Pro
Pro Leu Ser Val Asn Val Ser 30 35 40agc cgc gga aag cct acc agc ctg
ttt ctg agc tgg gta gct gca gag 495Ser Arg Gly Lys Pro Thr Ser Leu
Phe Leu Ser Trp Val Ala Ala Glu45 50 55 60cca ggt gga ttt gac tat
gcc ctc tgc ctc agg gct atg aac ttg tcg 543Pro Gly Gly Phe Asp Tyr
Ala Leu Cys Leu Arg Ala Met Asn Leu Ser 65 70 75ggt ttt cca gaa ggg
caa cag ctc caa gct cat acc aac gag tcc agc 591Gly Phe Pro Glu Gly
Gln Gln Leu Gln Ala His Thr Asn Glu Ser Ser 80 85 90ttt gag ttc cat
ggc ctg gtg cca ggg agt cgc tac cag ctg gaa ctg 639Phe Glu Phe His
Gly Leu Val Pro Gly Ser Arg Tyr Gln Leu Glu Leu 95 100 105act gtc
cta aga ccc tgt tgg cag aat gtc aca att acc ctc act gct 687Thr Val
Leu Arg Pro Cys Trp Gln Asn Val Thr Ile Thr Leu Thr Ala 110 115
120cga act gcc cct aca gtg gtc cgt gga ctg caa ctg cat agc act ggg
735Arg Thr Ala Pro Thr Val Val Arg Gly Leu Gln Leu His Ser Thr
Gly125 130 135 140agc cca gcc agc ctg gaa gcc tca tgg agc gat gcc
tct ggg gat caa 783Ser Pro Ala Ser Leu Glu Ala Ser Trp Ser Asp Ala
Ser Gly Asp Gln 145 150 155gac agc tat caa ctt ctc ctc tac cac ccg
gaa tcc cac act ctg gca 831Asp Ser Tyr Gln Leu Leu Leu Tyr His Pro
Glu Ser His Thr Leu Ala 160 165 170tgt aat gtc tct gtg tcc cct gac
acc ctg tct tac aat ttt ggt gac 879Cys Asn Val Ser Val Ser Pro Asp
Thr Leu Ser Tyr Asn Phe Gly Asp 175 180 185ctc ttg cca ggt agt cag
tat gtc ttg gag gtt atc acc tgg gct ggc 927Leu Leu Pro Gly Ser Gln
Tyr Val Leu Glu Val Ile Thr Trp Ala Gly 190 195 200agt ctc cat gcg
aag act agc atc ctc caa tgg aca gag cct gtc cct 975Ser Leu His Ala
Lys Thr Ser Ile Leu Gln Trp Thr Glu Pro Val Pro205 210 215 220cct
gat cac cta aca ctg cgt gcc ttg ggt acc agt agc ctg caa gcc 1023Pro
Asp His Leu Thr Leu Arg Ala Leu Gly Thr Ser Ser Leu Gln Ala 225 230
235ttc tgg aac agc tct gaa ggg gcc acc tgg ttt cac ctg ata ctt aca
1071Phe Trp Asn Ser Ser Glu Gly Ala Thr Trp Phe His Leu Ile Leu Thr
240 245 250gac ctc cta gag ggt acc aac ctg acc aaa gtg gtc aga caa
ggc atc 1119Asp Leu Leu Glu Gly Thr Asn Leu Thr Lys Val Val Arg Gln
Gly Ile 255 260 265tca acc cac acc ttc ctt cgc ctg tct ccg ggt aca
cct tac cag ctg 1167Ser Thr His Thr Phe Leu Arg Leu Ser Pro Gly Thr
Pro Tyr Gln Leu 270 275 280aag atc tgt gct gct gct ggg ccc cac cag
att tgg gga ccc aat gcc 1215Lys Ile Cys Ala Ala Ala Gly Pro His Gln
Ile Trp Gly Pro Asn Ala285 290 295 300act gag tgg acc tat ccc tct
tac cca tct gac ctg gtg ctg acc ccc 1263Thr Glu Trp Thr Tyr Pro Ser
Tyr Pro Ser Asp Leu Val Leu Thr Pro 305 310 315tta tgg aat gag ctc
tgg gca agc tgg aag gca ggg cag gga gcc cgg 1311Leu Trp Asn Glu Leu
Trp Ala Ser Trp Lys Ala Gly Gln Gly Ala Arg 320 325 330gat ggc tat
gta ctg aag tta agt ggg cca gtg gag aat aca act act 1359Asp Gly Tyr
Val Leu Lys Leu Ser Gly Pro Val Glu Asn Thr Thr Thr 335 340 345ctg
ggt cct gag gag tgc aac gct gtc ttc cca ggg ccc ctg cct cca 1407Leu
Gly Pro Glu Glu Cys Asn Ala Val Phe Pro Gly Pro Leu Pro Pro 350 355
360gga cac tac act ttg ggg ctg agg gtt cta gct gga cct tat gat gcc
1455Gly His Tyr Thr Leu Gly Leu Arg Val Leu Ala Gly Pro Tyr Asp
Ala365 370 375 380tgg gta gag ggc agt atc tgg ctg gct gaa tct gct
gct cgt ccc atg 1503Trp Val Glu Gly Ser Ile Trp Leu Ala Glu Ser Ala
Ala Arg Pro Met 385 390 395gag gtc cct ggt gcc aga ctg tgg cta gaa
gga ctg gaa gct act aag 1551Glu Val Pro Gly Ala Arg Leu Trp Leu Glu
Gly Leu Glu Ala Thr Lys 400 405 410caa cct ggg aga cgg gcg ctg ctc
tat tct gtt gat gcc cca ggc ctc 1599Gln Pro Gly Arg Arg Ala Leu Leu
Tyr Ser Val Asp Ala Pro Gly Leu 415 420 425cta ggg aac atc tct gtg
tct tct ggt gcc act cat gtc acc ttc tgt 1647Leu Gly Asn Ile Ser Val
Ser Ser Gly Ala Thr His Val Thr Phe Cys 430 435 440ggc ttg gta ccc
gga gcg cac tac agg gtg gac att gcc tca tcc atg 1695Gly Leu Val Pro
Gly Ala His Tyr Arg Val Asp Ile Ala Ser Ser Met445 450 455 460gga
gac atc act cag agc ctc aca ggc tac aca agt ccc ctg cca cca 1743Gly
Asp Ile Thr Gln Ser Leu Thr Gly Tyr Thr Ser Pro Leu Pro Pro 465 470
475cag tct ctg gag atc atc agc cgg aac agc cca tct gac ctg act atc
1791Gln Ser Leu Glu Ile Ile Ser Arg Asn Ser Pro Ser Asp Leu Thr Ile
480 485 490ggt tgg gct cca gca cca ggg cag atg gaa ggt tat aag gtc
acc tgg 1839Gly Trp Ala Pro Ala Pro Gly Gln Met Glu Gly Tyr Lys Val
Thr Trp 495 500 505cat cag gat ggc agc cag agg tca cct ggc gac ctt
gtt gac ttg ggc 1887His Gln Asp Gly Ser Gln Arg Ser Pro Gly Asp Leu
Val Asp Leu Gly 510 515 520cct gac att tcg agc ctg act ctg aaa tct
ctg gta cct ggt tcc tgc 1935Pro Asp Ile Ser Ser Leu Thr Leu Lys Ser
Leu Val Pro Gly Ser Cys525 530 535 540tac acc gtg tca gca tgg gcc
tgg tct ggg aac ctc agc tct gac tct 1983Tyr Thr Val Ser Ala Trp Ala
Trp Ser Gly Asn Leu Ser Ser Asp Ser 545 550 555cag aag att cac agt
tgc acc cgt ccc gct cct ccc acc aac ctg agc 2031Gln Lys Ile His Ser
Cys Thr Arg Pro Ala Pro Pro Thr Asn Leu Ser 560 565 570ctg ggc ttt
gcc cac cag cct gca aca ctg agg gct tcc tgg tgt cac 2079Leu Gly Phe
Ala His Gln Pro Ala Thr Leu Arg Ala Ser Trp Cys His 575 580 585cca
ccg ggt ggc agg gat gcc ttt cag tta cgg ctt tac agg ctg agg 2127Pro
Pro Gly Gly Arg Asp Ala Phe Gln Leu Arg Leu Tyr Arg Leu Arg 590 595
600ccc ctg aca ctg gaa agt gag aag atc cta tcc cag gag gcc cag aac
2175Pro Leu Thr Leu Glu Ser Glu Lys Ile Leu Ser Gln Glu Ala Gln
Asn605 610 615 620ttc tcc tgg gcc cag ctg cct gca ggc tat gaa ttc
cag gta cag ctg 2223Phe Ser Trp Ala Gln Leu Pro Ala Gly Tyr Glu Phe
Gln Val Gln Leu 625 630 635tct acc ttg tgg ggg tcg gag gag agc ggc
agt gcc aac acc aca ggc 2271Ser Thr Leu Trp Gly Ser Glu Glu Ser Gly
Ser Ala Asn Thr Thr Gly 640 645 650tgg aca ccc ccc tca gct cct aca
ttg gta aat gtg acc agt gaa gcc 2319Trp Thr Pro Pro Ser Ala Pro Thr
Leu Val Asn Val Thr Ser Glu Ala 655 660 665ccc acc cag ctc cac gta
tcc tgg gtc cac gct gct ggg gac cgg agc 2367Pro Thr Gln Leu His Val
Ser Trp Val His Ala Ala Gly Asp Arg Ser 670 675
680agc tac caa gtg acc cta tac cag gag agc act cgg aca gcc acc agc
2415Ser Tyr Gln Val Thr Leu Tyr Gln Glu Ser Thr Arg Thr Ala Thr
Ser685 690 695 700att gtg ggg ccc aag gca gac agc aca agc ttt tgg
ggt ttg act cct 2463Ile Val Gly Pro Lys Ala Asp Ser Thr Ser Phe Trp
Gly Leu Thr Pro 705 710 715ggc act aag tac aag gtg gaa gcc atc tcc
tgg gct ggg ccc ctt tac 2511Gly Thr Lys Tyr Lys Val Glu Ala Ile Ser
Trp Ala Gly Pro Leu Tyr 720 725 730act gca gca gcc aac gtt tct gct
tgg acc tac cca ctc aca ccc aat 2559Thr Ala Ala Ala Asn Val Ser Ala
Trp Thr Tyr Pro Leu Thr Pro Asn 735 740 745gag ctg ctc gcc tct atg
cag gca ggc agt gct gtg gtt aac ctg gcc 2607Glu Leu Leu Ala Ser Met
Gln Ala Gly Ser Ala Val Val Asn Leu Ala 750 755 760tgg ccc agt ggt
ccc ttg ggg caa ggg aca tgc cat gcc caa ctc tca 2655Trp Pro Ser Gly
Pro Leu Gly Gln Gly Thr Cys His Ala Gln Leu Ser765 770 775 780gat
gct gga cac ctt tca tgg gag caa ccg ctg tcg cta ggc caa gac 2703Asp
Ala Gly His Leu Ser Trp Glu Gln Pro Leu Ser Leu Gly Gln Asp 785 790
795ctc ctc atg cta agg aat ctt ata cca gga cat acg gtt tca ttg tct
2751Leu Leu Met Leu Arg Asn Leu Ile Pro Gly His Thr Val Ser Leu Ser
800 805 810gtg aag tgt cgg gca gga cca ctc cag gcc tcc act cac ccc
ctg gtg 2799Val Lys Cys Arg Ala Gly Pro Leu Gln Ala Ser Thr His Pro
Leu Val 815 820 825ctg tct gta gag cct ggc cct gtg gaa gat gtg ttc
tgt caa cct gag 2847Leu Ser Val Glu Pro Gly Pro Val Glu Asp Val Phe
Cys Gln Pro Glu 830 835 840gcc acc tac ctg tcc ctg aac tgg acg atg
cct act gga gat gtg gct 2895Ala Thr Tyr Leu Ser Leu Asn Trp Thr Met
Pro Thr Gly Asp Val Ala845 850 855 860gtc tgt ctg gtg gag gta gag
cag ctg gtg cca gga ggg agc gct cat 2943Val Cys Leu Val Glu Val Glu
Gln Leu Val Pro Gly Gly Ser Ala His 865 870 875ttt gtc ttc cag gtc
aac acc tcg gag gat gca ctt ctg ctg ccc aac 2991Phe Val Phe Gln Val
Asn Thr Ser Glu Asp Ala Leu Leu Leu Pro Asn 880 885 890ttg acg ccc
acc act tct tac cgc ctt agc ctc act gtg ctg ggt ggg 3039Leu Thr Pro
Thr Thr Ser Tyr Arg Leu Ser Leu Thr Val Leu Gly Gly 895 900 905aat
cgc cag tgg agc cgg gcg gtt acc ctg gtg tgc act act tct gct 3087Asn
Arg Gln Trp Ser Arg Ala Val Thr Leu Val Cys Thr Thr Ser Ala 910 915
920gag gtt tgg cac ccc cca gag cta gct gag gcc ccc cag gtg gag ctg
3135Glu Val Trp His Pro Pro Glu Leu Ala Glu Ala Pro Gln Val Glu
Leu925 930 935 940ggg aca ggg atg ggt gtg aca gtc aca cgt ggc atg
ttt ggt aaa gat 3183Gly Thr Gly Met Gly Val Thr Val Thr Arg Gly Met
Phe Gly Lys Asp 945 950 955gac ggg cag atc cag tgg tat ggc ata att
gcc acc atc aac atg aca 3231Asp Gly Gln Ile Gln Trp Tyr Gly Ile Ile
Ala Thr Ile Asn Met Thr 960 965 970ctg gcc cag cct tcc cag gaa gcc
atc aac cac aca tgg tat gac cac 3279Leu Ala Gln Pro Ser Gln Glu Ala
Ile Asn His Thr Trp Tyr Asp His 975 980 985tac tat aga gga cat gac
tcc tac ctg gct ctc ctg ttc cca aac ccc 3327Tyr Tyr Arg Gly His Asp
Ser Tyr Leu Ala Leu Leu Phe Pro Asn Pro 990 995 1000ttc tac cca gag
cct tgg gct gtg cca aga tcc tgg aca gta cct 3372Phe Tyr Pro Glu Pro
Trp Ala Val Pro Arg Ser Trp Thr Val Pro1005 1010 1015gtg ggt aca
gag gac tgt gac aac acc cag gag ata tgc aat ggg 3417Val Gly Thr Glu
Asp Cys Asp Asn Thr Gln Glu Ile Cys Asn Gly1020 1025 1030cat ctc
aag cca ggc ttc cag tat agg ttc agc att gca gcc ttt 3462His Leu Lys
Pro Gly Phe Gln Tyr Arg Phe Ser Ile Ala Ala Phe1035 1040 1045agt
agg ctc agc tct cca gag acc atc ctg gcc ttc tcc gcc ttc 3507Ser Arg
Leu Ser Ser Pro Glu Thr Ile Leu Ala Phe Ser Ala Phe1050 1055
1060tca gag cct cag gct agc atc tct ctg gtg gcc atg ccc ctg aca
3552Ser Glu Pro Gln Ala Ser Ile Ser Leu Val Ala Met Pro Leu Thr1065
1070 1075gtt atg atg ggg act gtg gtg ggc tgc atc atc att gtg tgt
gca 3597Val Met Met Gly Thr Val Val Gly Cys Ile Ile Ile Val Cys
Ala1080 1085 1090gtg cta tgc ttg ttg tgc cgg cgg cgc ctg aag gga
cca agg tca 3642Val Leu Cys Leu Leu Cys Arg Arg Arg Leu Lys Gly Pro
Arg Ser1095 1100 1105gag aag aat ggc ttt tcc cag gag ttg atg cct
tac aac ctg tgg 3687Glu Lys Asn Gly Phe Ser Gln Glu Leu Met Pro Tyr
Asn Leu Trp1110 1115 1120cgg acc cat cgg ccc atc ccc agc cat agc
ttc cgg cag agc tat 3732Arg Thr His Arg Pro Ile Pro Ser His Ser Phe
Arg Gln Ser Tyr1125 1130 1135gag gcc aag agt gca cgt gca cac cag
gcc ttc ttc cag gaa ttt 3777Glu Ala Lys Ser Ala Arg Ala His Gln Ala
Phe Phe Gln Glu Phe1140 1145 1150gag gag ctg aag gag gtg ggc aag
gac cag ccc aga cta gag gct 3822Glu Glu Leu Lys Glu Val Gly Lys Asp
Gln Pro Arg Leu Glu Ala1155 1160 1165gag cat cct gcc aac atc acc
aag aac cgg tac cca cac gtg cta 3867Glu His Pro Ala Asn Ile Thr Lys
Asn Arg Tyr Pro His Val Leu1170 1175 1180cct tat gac cac tcc agg
gtc agg ctg acc cag cta tca gga gag 3912Pro Tyr Asp His Ser Arg Val
Arg Leu Thr Gln Leu Ser Gly Glu1185 1190 1195cct cat tct gac tac
atc aat gcc aac ttc atc cca ggc tat agc 3957Pro His Ser Asp Tyr Ile
Asn Ala Asn Phe Ile Pro Gly Tyr Ser1200 1205 1210cac cca cag gag
atc att gcc acc cag ggg cct ctc aaa aag acg 4002His Pro Gln Glu Ile
Ile Ala Thr Gln Gly Pro Leu Lys Lys Thr1215 1220 1225gtc gag gac
ttc tgg cgg ttg gtg tgg gag cag caa gtc cac gtg 4047Val Glu Asp Phe
Trp Arg Leu Val Trp Glu Gln Gln Val His Val1230 1235 1240atc atc
atg cta act gtg ggc atg gag aat ggg cgg gta ctg tgt 4092Ile Ile Met
Leu Thr Val Gly Met Glu Asn Gly Arg Val Leu Cys1245 1250 1255gag
cac tac tgg cca gtc aac tcc acg cct gtc acc cac ggt cac 4137Glu His
Tyr Trp Pro Val Asn Ser Thr Pro Val Thr His Gly His1260 1265
1270atc acc acc cac ctc ctg gca gag gaa tct gag gac gag tgg acc
4182Ile Thr Thr His Leu Leu Ala Glu Glu Ser Glu Asp Glu Trp Thr1275
1280 1285agg agg gaa ttc cag ctg cag cac ggt gca gag caa aaa cag
agg 4227Arg Arg Glu Phe Gln Leu Gln His Gly Ala Glu Gln Lys Gln
Arg1290 1295 1300cgc gtg aag cag ctg cag ttc acg acc tgg cca gac
cac agt gtc 4272Arg Val Lys Gln Leu Gln Phe Thr Thr Trp Pro Asp His
Ser Val1305 1310 1315ccc gag gct ccc agc tct ctg ctc gct ttt gtg
gaa ctg gtg cag 4317Pro Glu Ala Pro Ser Ser Leu Leu Ala Phe Val Glu
Leu Val Gln1320 1325 1330gag gag gtg aag gca act cag ggc aag ggg
ccc atc ctg gtg cat 4362Glu Glu Val Lys Ala Thr Gln Gly Lys Gly Pro
Ile Leu Val His1335 1340 1345tgc agt gcg ggt gtg ggc agg aca ggc
acc ttt gtg gct ctc tta 4407Cys Ser Ala Gly Val Gly Arg Thr Gly Thr
Phe Val Ala Leu Leu1350 1355 1360ccg gct gtt cga caa cta gag gaa
gaa cag gtg gtc gat gtg ttc 4452Pro Ala Val Arg Gln Leu Glu Glu Glu
Gln Val Val Asp Val Phe1365 1370 1375aac act gtg tac ata ctc cgg
ctg cac cgg ccc ctc atg atc cag 4497Asn Thr Val Tyr Ile Leu Arg Leu
His Arg Pro Leu Met Ile Gln1380 1385 1390acc ttg agt caa tac atc
ttc ctg cac agc tgc ctg ctg aac aag 4542Thr Leu Ser Gln Tyr Ile Phe
Leu His Ser Cys Leu Leu Asn Lys1395 1400 1405att ctg gaa ggg ccc
tct gac gcc tca gac tcc ggc ccc atc cct 4587Ile Leu Glu Gly Pro Ser
Asp Ala Ser Asp Ser Gly Pro Ile Pro1410 1415 1420gtg atg aat ttt
gca caa gct tgt gcc aag agg gca gcc aat gcc 4632Val Met Asn Phe Ala
Gln Ala Cys Ala Lys Arg Ala Ala Asn Ala1425 1430 1435aat gcc ggt
ttc ttg aag gag tac agg ctc ctg aag cag gcc atc 4677Asn Ala Gly Phe
Leu Lys Glu Tyr Arg Leu Leu Lys Gln Ala Ile1440 1445 1450aag gat
gag act ggc tct ctg ctg ccc tct cct gac tat aat cag 4722Lys Asp Glu
Thr Gly Ser Leu Leu Pro Ser Pro Asp Tyr Asn Gln1455 1460 1465aac
agc atc gcc tcc tgt cat cat tct cag gag cag ttg gcc ctg 4767Asn Ser
Ile Ala Ser Cys His His Ser Gln Glu Gln Leu Ala Leu1470 1475
1480gtg gag gag agc cct gct gat aac atg ctg gca gcc tcg ctc ttc
4812Val Glu Glu Ser Pro Ala Asp Asn Met Leu Ala Ala Ser Leu Phe1485
1490 1495cct ggt ggg ccg tct ggt cgc gac cat gtg gtg ctg act ggc
tcg 4857Pro Gly Gly Pro Ser Gly Arg Asp His Val Val Leu Thr Gly
Ser1500 1505 1510gcc gga cca aag gaa ctc tgg gaa atg gtg tgg gaa
cat ggc gcc 4902Ala Gly Pro Lys Glu Leu Trp Glu Met Val Trp Glu His
Gly Ala1515 1520 1525tat gtg ctt gtc tcc ctg ggt ctg cct gat acc
aag gag aag cca 4947Tyr Val Leu Val Ser Leu Gly Leu Pro Asp Thr Lys
Glu Lys Pro1530 1535 1540caa gac atc tgg cca atg gag atg cag cct
att gtc aca gac atg 4992Gln Asp Ile Trp Pro Met Glu Met Gln Pro Ile
Val Thr Asp Met1545 1550 1555gtg aca gtg cac aga gtg gct gag agc
aac aca gct ggc tgg ccc 5037Val Thr Val His Arg Val Ala Glu Ser Asn
Thr Ala Gly Trp Pro1560 1565 1570agt acc ctc atc aga gtt ata cat
ggg gac agt ggg acg gaa agg 5082Ser Thr Leu Ile Arg Val Ile His Gly
Asp Ser Gly Thr Glu Arg1575 1580 1585cag gtt caa tgc ctg cag ttt
cca cac tgc gag act ggg agt gag 5127Gln Val Gln Cys Leu Gln Phe Pro
His Cys Glu Thr Gly Ser Glu1590 1595 1600ctc cca gct aac acc cta
ctg acc ttc ctt gat gct gtg ggc cag 5172Leu Pro Ala Asn Thr Leu Leu
Thr Phe Leu Asp Ala Val Gly Gln1605 1610 1615tgc tgc tcc cgg ggc
aat agc aag aag cca ggg acc ctg ctc agt 5217Cys Cys Ser Arg Gly Asn
Ser Lys Lys Pro Gly Thr Leu Leu Ser1620 1625 1630cac tcc agc aag
gtc aca aac cag ctg agc acc ttc ttg gct atg 5262His Ser Ser Lys Val
Thr Asn Gln Leu Ser Thr Phe Leu Ala Met1635 1640 1645gaa cag ctg
cta cag caa gca ggg acc gag cgc aca gtg gat gtc 5307Glu Gln Leu Leu
Gln Gln Ala Gly Thr Glu Arg Thr Val Asp Val1650 1655 1660ttc agt
gtg gcc ctg aag cag aca cag gcc tgt ggc ctt aag acc 5352Phe Ser Val
Ala Leu Lys Gln Thr Gln Ala Cys Gly Leu Lys Thr1665 1670 1675cca
acg ctg gag cag tat atc tac ctc tac aac tgt ctg aac agc 5397Pro Thr
Leu Glu Gln Tyr Ile Tyr Leu Tyr Asn Cys Leu Asn Ser1680 1685
1690gca ttg agg aac agg ctg ccc cga gct agg aag tgaccttgcc 5440Ala
Leu Arg Asn Arg Leu Pro Arg Ala Arg Lys1695 1700 1705ctgctaggca
tcacgttcca gcaatccacc caggcctggc ttccccagga gaacagatct
5500attcggcctc acgctgtcaa agggcagagt ctgggaataa agggtaaatc tcgag
5555191705PRTmus musculus 19Met Arg Pro Leu Ile Leu Leu Ala Ala Leu
Leu Trp Leu Gln Asp Ser1 5 10 15Leu Ala Gln Glu Asp Val Cys Ser Ser
Leu Asp Gly Ser Pro Asp Arg 20 25 30Gln Gly Gly Gly Pro Pro Leu Ser
Val Asn Val Ser Ser Arg Gly Lys 35 40 45Pro Thr Ser Leu Phe Leu Ser
Trp Val Ala Ala Glu Pro Gly Gly Phe 50 55 60Asp Tyr Ala Leu Cys Leu
Arg Ala Met Asn Leu Ser Gly Phe Pro Glu65 70 75 80Gly Gln Gln Leu
Gln Ala His Thr Asn Glu Ser Ser Phe Glu Phe His 85 90 95Gly Leu Val
Pro Gly Ser Arg Tyr Gln Leu Glu Leu Thr Val Leu Arg 100 105 110Pro
Cys Trp Gln Asn Val Thr Ile Thr Leu Thr Ala Arg Thr Ala Pro 115 120
125Thr Val Val Arg Gly Leu Gln Leu His Ser Thr Gly Ser Pro Ala Ser
130 135 140Leu Glu Ala Ser Trp Ser Asp Ala Ser Gly Asp Gln Asp Ser
Tyr Gln145 150 155 160Leu Leu Leu Tyr His Pro Glu Ser His Thr Leu
Ala Cys Asn Val Ser 165 170 175Val Ser Pro Asp Thr Leu Ser Tyr Asn
Phe Gly Asp Leu Leu Pro Gly 180 185 190Ser Gln Tyr Val Leu Glu Val
Ile Thr Trp Ala Gly Ser Leu His Ala 195 200 205Lys Thr Ser Ile Leu
Gln Trp Thr Glu Pro Val Pro Pro Asp His Leu 210 215 220Thr Leu Arg
Ala Leu Gly Thr Ser Ser Leu Gln Ala Phe Trp Asn Ser225 230 235
240Ser Glu Gly Ala Thr Trp Phe His Leu Ile Leu Thr Asp Leu Leu Glu
245 250 255Gly Thr Asn Leu Thr Lys Val Val Arg Gln Gly Ile Ser Thr
His Thr 260 265 270Phe Leu Arg Leu Ser Pro Gly Thr Pro Tyr Gln Leu
Lys Ile Cys Ala 275 280 285Ala Ala Gly Pro His Gln Ile Trp Gly Pro
Asn Ala Thr Glu Trp Thr 290 295 300Tyr Pro Ser Tyr Pro Ser Asp Leu
Val Leu Thr Pro Leu Trp Asn Glu305 310 315 320Leu Trp Ala Ser Trp
Lys Ala Gly Gln Gly Ala Arg Asp Gly Tyr Val 325 330 335Leu Lys Leu
Ser Gly Pro Val Glu Asn Thr Thr Thr Leu Gly Pro Glu 340 345 350Glu
Cys Asn Ala Val Phe Pro Gly Pro Leu Pro Pro Gly His Tyr Thr 355 360
365Leu Gly Leu Arg Val Leu Ala Gly Pro Tyr Asp Ala Trp Val Glu Gly
370 375 380Ser Ile Trp Leu Ala Glu Ser Ala Ala Arg Pro Met Glu Val
Pro Gly385 390 395 400Ala Arg Leu Trp Leu Glu Gly Leu Glu Ala Thr
Lys Gln Pro Gly Arg 405 410 415Arg Ala Leu Leu Tyr Ser Val Asp Ala
Pro Gly Leu Leu Gly Asn Ile 420 425 430Ser Val Ser Ser Gly Ala Thr
His Val Thr Phe Cys Gly Leu Val Pro 435 440 445Gly Ala His Tyr Arg
Val Asp Ile Ala Ser Ser Met Gly Asp Ile Thr 450 455 460Gln Ser Leu
Thr Gly Tyr Thr Ser Pro Leu Pro Pro Gln Ser Leu Glu465 470 475
480Ile Ile Ser Arg Asn Ser Pro Ser Asp Leu Thr Ile Gly Trp Ala Pro
485 490 495Ala Pro Gly Gln Met Glu Gly Tyr Lys Val Thr Trp His Gln
Asp Gly 500 505 510Ser Gln Arg Ser Pro Gly Asp Leu Val Asp Leu Gly
Pro Asp Ile Ser 515 520 525Ser Leu Thr Leu Lys Ser Leu Val Pro Gly
Ser Cys Tyr Thr Val Ser 530 535 540Ala Trp Ala Trp Ser Gly Asn Leu
Ser Ser Asp Ser Gln Lys Ile His545 550 555 560Ser Cys Thr Arg Pro
Ala Pro Pro Thr Asn Leu Ser Leu Gly Phe Ala 565 570 575His Gln Pro
Ala Thr Leu Arg Ala Ser Trp Cys His Pro Pro Gly Gly 580 585 590Arg
Asp Ala Phe Gln Leu Arg Leu Tyr Arg Leu Arg Pro Leu Thr Leu 595 600
605Glu Ser Glu Lys Ile Leu Ser Gln Glu Ala Gln Asn Phe Ser Trp Ala
610 615 620Gln Leu Pro Ala Gly Tyr Glu Phe Gln Val Gln Leu Ser Thr
Leu Trp625 630 635 640Gly Ser Glu Glu Ser Gly Ser Ala Asn Thr Thr
Gly Trp Thr Pro Pro 645 650 655Ser Ala Pro Thr Leu Val Asn Val Thr
Ser Glu Ala Pro Thr Gln Leu 660 665 670His Val Ser Trp Val His Ala
Ala Gly Asp Arg Ser Ser Tyr Gln Val 675 680 685Thr Leu Tyr Gln Glu
Ser Thr Arg Thr Ala Thr Ser Ile Val Gly Pro 690 695 700Lys Ala Asp
Ser Thr Ser Phe Trp Gly Leu Thr Pro Gly Thr Lys Tyr705 710 715
720Lys Val Glu Ala Ile Ser Trp Ala Gly Pro Leu Tyr Thr Ala Ala Ala
725 730 735Asn Val Ser Ala Trp Thr Tyr Pro Leu Thr Pro Asn Glu Leu
Leu Ala 740 745 750Ser Met Gln Ala Gly Ser Ala Val Val Asn Leu Ala
Trp Pro Ser Gly 755 760 765Pro Leu Gly Gln Gly Thr Cys His Ala Gln
Leu Ser Asp Ala Gly His 770 775
780Leu Ser Trp Glu Gln Pro Leu Ser Leu Gly Gln Asp Leu Leu Met
Leu785 790 795 800Arg Asn Leu Ile Pro Gly His Thr Val Ser Leu Ser
Val Lys Cys Arg 805 810 815Ala Gly Pro Leu Gln Ala Ser Thr His Pro
Leu Val Leu Ser Val Glu 820 825 830Pro Gly Pro Val Glu Asp Val Phe
Cys Gln Pro Glu Ala Thr Tyr Leu 835 840 845Ser Leu Asn Trp Thr Met
Pro Thr Gly Asp Val Ala Val Cys Leu Val 850 855 860Glu Val Glu Gln
Leu Val Pro Gly Gly Ser Ala His Phe Val Phe Gln865 870 875 880Val
Asn Thr Ser Glu Asp Ala Leu Leu Leu Pro Asn Leu Thr Pro Thr 885 890
895Thr Ser Tyr Arg Leu Ser Leu Thr Val Leu Gly Gly Asn Arg Gln Trp
900 905 910Ser Arg Ala Val Thr Leu Val Cys Thr Thr Ser Ala Glu Val
Trp His 915 920 925Pro Pro Glu Leu Ala Glu Ala Pro Gln Val Glu Leu
Gly Thr Gly Met 930 935 940Gly Val Thr Val Thr Arg Gly Met Phe Gly
Lys Asp Asp Gly Gln Ile945 950 955 960Gln Trp Tyr Gly Ile Ile Ala
Thr Ile Asn Met Thr Leu Ala Gln Pro 965 970 975Ser Gln Glu Ala Ile
Asn His Thr Trp Tyr Asp His Tyr Tyr Arg Gly 980 985 990His Asp Ser
Tyr Leu Ala Leu Leu Phe Pro Asn Pro Phe Tyr Pro Glu 995 1000
1005Pro Trp Ala Val Pro Arg Ser Trp Thr Val Pro Val Gly Thr Glu
1010 1015 1020Asp Cys Asp Asn Thr Gln Glu Ile Cys Asn Gly His Leu
Lys Pro 1025 1030 1035Gly Phe Gln Tyr Arg Phe Ser Ile Ala Ala Phe
Ser Arg Leu Ser 1040 1045 1050Ser Pro Glu Thr Ile Leu Ala Phe Ser
Ala Phe Ser Glu Pro Gln 1055 1060 1065Ala Ser Ile Ser Leu Val Ala
Met Pro Leu Thr Val Met Met Gly 1070 1075 1080Thr Val Val Gly Cys
Ile Ile Ile Val Cys Ala Val Leu Cys Leu 1085 1090 1095Leu Cys Arg
Arg Arg Leu Lys Gly Pro Arg Ser Glu Lys Asn Gly 1100 1105 1110Phe
Ser Gln Glu Leu Met Pro Tyr Asn Leu Trp Arg Thr His Arg 1115 1120
1125Pro Ile Pro Ser His Ser Phe Arg Gln Ser Tyr Glu Ala Lys Ser
1130 1135 1140Ala Arg Ala His Gln Ala Phe Phe Gln Glu Phe Glu Glu
Leu Lys 1145 1150 1155Glu Val Gly Lys Asp Gln Pro Arg Leu Glu Ala
Glu His Pro Ala 1160 1165 1170Asn Ile Thr Lys Asn Arg Tyr Pro His
Val Leu Pro Tyr Asp His 1175 1180 1185Ser Arg Val Arg Leu Thr Gln
Leu Ser Gly Glu Pro His Ser Asp 1190 1195 1200Tyr Ile Asn Ala Asn
Phe Ile Pro Gly Tyr Ser His Pro Gln Glu 1205 1210 1215Ile Ile Ala
Thr Gln Gly Pro Leu Lys Lys Thr Val Glu Asp Phe 1220 1225 1230Trp
Arg Leu Val Trp Glu Gln Gln Val His Val Ile Ile Met Leu 1235 1240
1245Thr Val Gly Met Glu Asn Gly Arg Val Leu Cys Glu His Tyr Trp
1250 1255 1260Pro Val Asn Ser Thr Pro Val Thr His Gly His Ile Thr
Thr His 1265 1270 1275Leu Leu Ala Glu Glu Ser Glu Asp Glu Trp Thr
Arg Arg Glu Phe 1280 1285 1290Gln Leu Gln His Gly Ala Glu Gln Lys
Gln Arg Arg Val Lys Gln 1295 1300 1305Leu Gln Phe Thr Thr Trp Pro
Asp His Ser Val Pro Glu Ala Pro 1310 1315 1320Ser Ser Leu Leu Ala
Phe Val Glu Leu Val Gln Glu Glu Val Lys 1325 1330 1335Ala Thr Gln
Gly Lys Gly Pro Ile Leu Val His Cys Ser Ala Gly 1340 1345 1350Val
Gly Arg Thr Gly Thr Phe Val Ala Leu Leu Pro Ala Val Arg 1355 1360
1365Gln Leu Glu Glu Glu Gln Val Val Asp Val Phe Asn Thr Val Tyr
1370 1375 1380Ile Leu Arg Leu His Arg Pro Leu Met Ile Gln Thr Leu
Ser Gln 1385 1390 1395Tyr Ile Phe Leu His Ser Cys Leu Leu Asn Lys
Ile Leu Glu Gly 1400 1405 1410Pro Ser Asp Ala Ser Asp Ser Gly Pro
Ile Pro Val Met Asn Phe 1415 1420 1425Ala Gln Ala Cys Ala Lys Arg
Ala Ala Asn Ala Asn Ala Gly Phe 1430 1435 1440Leu Lys Glu Tyr Arg
Leu Leu Lys Gln Ala Ile Lys Asp Glu Thr 1445 1450 1455Gly Ser Leu
Leu Pro Ser Pro Asp Tyr Asn Gln Asn Ser Ile Ala 1460 1465 1470Ser
Cys His His Ser Gln Glu Gln Leu Ala Leu Val Glu Glu Ser 1475 1480
1485Pro Ala Asp Asn Met Leu Ala Ala Ser Leu Phe Pro Gly Gly Pro
1490 1495 1500Ser Gly Arg Asp His Val Val Leu Thr Gly Ser Ala Gly
Pro Lys 1505 1510 1515Glu Leu Trp Glu Met Val Trp Glu His Gly Ala
Tyr Val Leu Val 1520 1525 1530Ser Leu Gly Leu Pro Asp Thr Lys Glu
Lys Pro Gln Asp Ile Trp 1535 1540 1545Pro Met Glu Met Gln Pro Ile
Val Thr Asp Met Val Thr Val His 1550 1555 1560Arg Val Ala Glu Ser
Asn Thr Ala Gly Trp Pro Ser Thr Leu Ile 1565 1570 1575Arg Val Ile
His Gly Asp Ser Gly Thr Glu Arg Gln Val Gln Cys 1580 1585 1590Leu
Gln Phe Pro His Cys Glu Thr Gly Ser Glu Leu Pro Ala Asn 1595 1600
1605Thr Leu Leu Thr Phe Leu Asp Ala Val Gly Gln Cys Cys Ser Arg
1610 1615 1620Gly Asn Ser Lys Lys Pro Gly Thr Leu Leu Ser His Ser
Ser Lys 1625 1630 1635Val Thr Asn Gln Leu Ser Thr Phe Leu Ala Met
Glu Gln Leu Leu 1640 1645 1650Gln Gln Ala Gly Thr Glu Arg Thr Val
Asp Val Phe Ser Val Ala 1655 1660 1665Leu Lys Gln Thr Gln Ala Cys
Gly Leu Lys Thr Pro Thr Leu Glu 1670 1675 1680Gln Tyr Ile Tyr Leu
Tyr Asn Cys Leu Asn Ser Ala Leu Arg Asn 1685 1690 1695Arg Leu Pro
Arg Ala Arg Lys 1700 1705203078DNAEscherichia coliCDS(1)..(3078)
20acc atg att acg gat tca ctg gcc gtc gtt tta caa cgt cgt gac tgg
48Thr Met Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp Trp1
5 10 15gaa aac cct ggc gtt acc caa ctt aat cgc ctt gca gca cat ccc
cct 96Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro
Pro 20 25 30ttc gcc agc tgg cgt aat agc gaa gag gcc cgc acc gat cgc
cct tcc 144Phe Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg
Pro Ser 35 40 45caa cag ttg cgc agc ctg aat ggc gaa tgg cgc ttt gcc
tgg ttt ccg 192Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg Phe Ala
Trp Phe Pro 50 55 60gca cca gaa gcg gtg ccg gaa agc tgg ctg gag tgc
gat ctt cct gag 240Ala Pro Glu Ala Val Pro Glu Ser Trp Leu Glu Cys
Asp Leu Pro Glu65 70 75 80gcc gat act gtc gtc gtc ccc tca aac tgg
cag atg cac ggt tac gat 288Ala Asp Thr Val Val Val Pro Ser Asn Trp
Gln Met His Gly Tyr Asp 85 90 95gcg ccc atc tac acc aac gta acc tat
ccc att acg gtc aat ccg ccg 336Ala Pro Ile Tyr Thr Asn Val Thr Tyr
Pro Ile Thr Val Asn Pro Pro 100 105 110ttt gtt ccc acg gag aat ccg
acg ggt tgt tac tcg ctc aca ttt aat 384Phe Val Pro Thr Glu Asn Pro
Thr Gly Cys Tyr Ser Leu Thr Phe Asn 115 120 125gtt gat gaa agc tgg
cta cag gaa ggc cag acg cga att att ttt gat 432Val Asp Glu Ser Trp
Leu Gln Glu Gly Gln Thr Arg Ile Ile Phe Asp 130 135 140ggc gtt aac
tcg gcg ttt cat ctg tgg tgc aac ggg cgc tgg gtc ggt 480Gly Val Asn
Ser Ala Phe His Leu Trp Cys Asn Gly Arg Trp Val Gly145 150 155
160tac ggc cag gac agt cgt ttg ccg tct gaa ttt gac ctg agc gca ttt
528Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe Asp Leu Ser Ala Phe
165 170 175tta cgc gcc gga gaa aac cgc ctc gcg gtg atg gtg ctg cgt
tgg agt 576Leu Arg Ala Gly Glu Asn Arg Leu Ala Val Met Val Leu Arg
Trp Ser 180 185 190gac ggc agt tat ctg gaa gat cag gat atg tgg cgg
atg agc ggc att 624Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met Trp Arg
Met Ser Gly Ile 195 200 205ttc cgt gac gtc tcg ttg ctg cat aaa ccg
act aca caa atc agc gat 672Phe Arg Asp Val Ser Leu Leu His Lys Pro
Thr Thr Gln Ile Ser Asp 210 215 220ttc cat gtt gcc act cgc ttt aat
gat gat ttc agc cgc gct gta ctg 720Phe His Val Ala Thr Arg Phe Asn
Asp Asp Phe Ser Arg Ala Val Leu225 230 235 240gag gct gaa gtt cag
atg tgc ggc gag ttg cgt gac tac cta cgg gta 768Glu Ala Glu Val Gln
Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg Val 245 250 255aca gtt tct
tta tgg cag ggt gaa acg cag gtc gcc agc ggc acc gcg 816Thr Val Ser
Leu Trp Gln Gly Glu Thr Gln Val Ala Ser Gly Thr Ala 260 265 270cct
ttc ggc ggt gaa att atc gat gag cgt ggt ggt tat gcc gat cgc 864Pro
Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly Gly Tyr Ala Asp Arg 275 280
285gtc aca cta cgt ctg aac gtc gaa aac ccg aaa ctg tgg agc gcc gaa
912Val Thr Leu Arg Leu Asn Val Glu Asn Pro Lys Leu Trp Ser Ala Glu
290 295 300atc ccg aat ctc tat cgt gcg gtg gtt gaa ctg cac acc gcc
gac ggc 960Ile Pro Asn Leu Tyr Arg Ala Val Val Glu Leu His Thr Ala
Asp Gly305 310 315 320acg ctg att gaa gca gaa gcc tgc gat gtc ggt
ttc cgc gag gtg cgg 1008Thr Leu Ile Glu Ala Glu Ala Cys Asp Val Gly
Phe Arg Glu Val Arg 325 330 335att gaa aat ggt ctg ctg ctg ctg aac
ggc aag ccg ttg ctg att cga 1056Ile Glu Asn Gly Leu Leu Leu Leu Asn
Gly Lys Pro Leu Leu Ile Arg 340 345 350ggc gtt aac cgt cac gag cat
cat cct ctg cat ggt cag gtc atg gat 1104Gly Val Asn Arg His Glu His
His Pro Leu His Gly Gln Val Met Asp 355 360 365gag cag acg atg gtg
cag gat atc ctg ctg atg aag cag aac aac ttt 1152Glu Gln Thr Met Val
Gln Asp Ile Leu Leu Met Lys Gln Asn Asn Phe 370 375 380aac gcc gtg
cgc tgt tcg cat tat ccg aac cat ccg ctg tgg tac acg 1200Asn Ala Val
Arg Cys Ser His Tyr Pro Asn His Pro Leu Trp Tyr Thr385 390 395
400ctg tgc gac cgc tac ggc ctg tat gtg gtg gat gaa gcc aat att gaa
1248Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp Glu Ala Asn Ile Glu
405 410 415acc cac ggc atg gtg cca atg aat cgt ctg acc gat gat ccg
cgc tgg 1296Thr His Gly Met Val Pro Met Asn Arg Leu Thr Asp Asp Pro
Arg Trp 420 425 430cta ccg gcg atg agc gaa cgc gta acg cga atg gtg
cag cgc gat cgt 1344Leu Pro Ala Met Ser Glu Arg Val Thr Arg Met Val
Gln Arg Asp Arg 435 440 445aat cac ccg agt gtg atc atc tgg tcg ctg
ggg aat gaa tca ggc cac 1392Asn His Pro Ser Val Ile Ile Trp Ser Leu
Gly Asn Glu Ser Gly His 450 455 460ggc gct aat cac gac gcg ctg tat
cgc tgg atc aaa tct gtc gat cct 1440Gly Ala Asn His Asp Ala Leu Tyr
Arg Trp Ile Lys Ser Val Asp Pro465 470 475 480tcc cgc ccg gtg cag
tat gaa ggc ggc gga gcc gac acc acg gcc acc 1488Ser Arg Pro Val Gln
Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala Thr 485 490 495gat att att
tgc ccg atg tac gcg cgc gtg gat gaa gac cag ccc ttc 1536Asp Ile Ile
Cys Pro Met Tyr Ala Arg Val Asp Glu Asp Gln Pro Phe 500 505 510ccg
gct gtg ccg aaa tgg tcc atc aaa aaa tgg ctt tcg cta cct gga 1584Pro
Ala Val Pro Lys Trp Ser Ile Lys Lys Trp Leu Ser Leu Pro Gly 515 520
525gag acg cgc ccg ctg atc ctt tgc gaa tac gcc cac gcg atg ggt aac
1632Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr Ala His Ala Met Gly Asn
530 535 540agt ctt ggc ggt ttc gct aaa tac tgg cag gcg ttt cgt cag
tat ccc 1680Ser Leu Gly Gly Phe Ala Lys Tyr Trp Gln Ala Phe Arg Gln
Tyr Pro545 550 555 560cgt tta cag ggc ggc ttc gtc tgg gac tgg gtg
gat cag tcg ctg att 1728Arg Leu Gln Gly Gly Phe Val Trp Asp Trp Val
Asp Gln Ser Leu Ile 565 570 575aaa tat gat gaa aac ggc aac ccg tgg
tcg gct tac ggc ggt gat ttt 1776Lys Tyr Asp Glu Asn Gly Asn Pro Trp
Ser Ala Tyr Gly Gly Asp Phe 580 585 590ggc gat acg ccg aac gat cgc
cag ttc tgt atg aac ggt ctg gtc ttt 1824Gly Asp Thr Pro Asn Asp Arg
Gln Phe Cys Met Asn Gly Leu Val Phe 595 600 605gcc gac cgc acg ccg
cat cca gcg ctg acg gaa gca aaa cac cag cag 1872Ala Asp Arg Thr Pro
His Pro Ala Leu Thr Glu Ala Lys His Gln Gln 610 615 620cag ttt ttc
cag ttc cgt tta tcc ggg caa acc atc gaa gtg acc agc 1920Gln Phe Phe
Gln Phe Arg Leu Ser Gly Gln Thr Ile Glu Val Thr Ser625 630 635
640gaa tac ctg ttc cgt cat agc gat aac gag ctc ctg cac tgg atg gtg
1968Glu Tyr Leu Phe Arg His Ser Asp Asn Glu Leu Leu His Trp Met Val
645 650 655gcg ctg gat ggt aag ccg ctg gca agc ggt gaa gtg cct ctg
gat gtc 2016Ala Leu Asp Gly Lys Pro Leu Ala Ser Gly Glu Val Pro Leu
Asp Val 660 665 670gct cca caa ggt aaa cag ttg att gaa ctg cct gaa
cta ccg cag ccg 2064Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu Pro Glu
Leu Pro Gln Pro 675 680 685gag agc gcc ggg caa ctc tgg ctc aca gta
cgc gta gtg caa ccg aac 2112Glu Ser Ala Gly Gln Leu Trp Leu Thr Val
Arg Val Val Gln Pro Asn 690 695 700gcg acc gca tgg tca gaa gcc ggg
cac atc agc gcc tgg cag cag tgg 2160Ala Thr Ala Trp Ser Glu Ala Gly
His Ile Ser Ala Trp Gln Gln Trp705 710 715 720cgt ctg gcg gaa aac
ctc agt gtg acg ctc ccc gcc gcg tcc cac gcc 2208Arg Leu Ala Glu Asn
Leu Ser Val Thr Leu Pro Ala Ala Ser His Ala 725 730 735atc ccg cat
ctg acc acc agc gaa atg gat ttt tgc atc gag ctg ggt 2256Ile Pro His
Leu Thr Thr Ser Glu Met Asp Phe Cys Ile Glu Leu Gly 740 745 750aat
aag cgt tgg caa ttt aac cgc cag tca ggc ttt ctt tca cag atg 2304Asn
Lys Arg Trp Gln Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln Met 755 760
765tgg att ggc gat aaa aaa caa ctg ctg acg ccg ctg cgc gat cag ttc
2352Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln Phe
770 775 780acc cgt gca ccg ctg gat aac gac att ggc gta agt gaa gcg
acc cgc 2400Thr Arg Ala Pro Leu Asp Asn Asp Ile Gly Val Ser Glu Ala
Thr Arg785 790 795 800att gac cct aac gcc tgg gtc gaa cgc tgg aag
gcg gcg ggc cat tac 2448Ile Asp Pro Asn Ala Trp Val Glu Arg Trp Lys
Ala Ala Gly His Tyr 805 810 815cag gcc gaa gca gcg ttg ttg cag tgc
acg gca gat aca ctt gct gat 2496Gln Ala Glu Ala Ala Leu Leu Gln Cys
Thr Ala Asp Thr Leu Ala Asp 820 825 830gcg gtg ctg att acg acc gct
cac gcg tgg cag cat cag ggg aaa acc 2544Ala Val Leu Ile Thr Thr Ala
His Ala Trp Gln His Gln Gly Lys Thr 835 840 845tta ttt atc agc cgg
aaa acc tac cgg att gat ggt agt ggt caa atg 2592Leu Phe Ile Ser Arg
Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln Met 850 855 860gcg att acc
gtt gat gtt gaa gtg gcg agc gat aca ccg cat ccg gcg 2640Ala Ile Thr
Val Asp Val Glu Val Ala Ser Asp Thr Pro His Pro Ala865 870 875
880cgg att ggc ctg aac tgc cag ctg gcg cag gta gca gag cgg gta aac
2688Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala Glu Arg Val Asn
885 890 895tgg ctc gga tta ggg ccg caa gaa aac tat ccc gac cgc ctt
act gcc 2736Trp Leu Gly Leu Gly Pro Gln Glu Asn Tyr Pro Asp Arg Leu
Thr Ala 900 905 910gcc tgt ttt gac cgc tgg gat ctg cca ttg tca gac
atg tat acc ccg 2784Ala Cys Phe Asp Arg Trp Asp Leu Pro Leu Ser Asp
Met Tyr Thr Pro 915 920 925tac gtc ttc ccg agc gaa aac ggt ctg cgc
tgc ggg acg cgc gaa ttg 2832Tyr Val Phe Pro Ser Glu Asn Gly Leu Arg
Cys Gly Thr Arg Glu Leu 930 935 940aat tat ggc cca cac cag tgg cgc
ggc gac ttc cag ttc aac atc agc 2880Asn Tyr Gly Pro His Gln Trp Arg
Gly Asp Phe Gln Phe Asn Ile Ser945
950 955 960cgc tac agt caa cag caa ctg atg gaa acc agc cat cgc cat
ctg ctg 2928Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His
Leu Leu 965 970 975cac gcg gaa gaa ggc aca tgg ctg aat atc gac ggt
ttc cat atg ggg 2976His Ala Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly
Phe His Met Gly 980 985 990att ggt ggc gac gac tcc tgg agc ccg tca
gta tcg gcg gaa ttc cag 3024Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser
Val Ser Ala Glu Phe Gln 995 1000 1005ctg agc gcc ggt cgc tac cat
tac cag ttg gtc tgg tgt caa aaa 3069Leu Ser Ala Gly Arg Tyr His Tyr
Gln Leu Val Trp Cys Gln Lys 1010 1015 1020taa taa taa
3078211023PRTEscherichia coli 21Thr Met Ile Thr Asp Ser Leu Ala Val
Val Leu Gln Arg Arg Asp Trp1 5 10 15Glu Asn Pro Gly Val Thr Gln Leu
Asn Arg Leu Ala Ala His Pro Pro 20 25 30Phe Ala Ser Trp Arg Asn Ser
Glu Glu Ala Arg Thr Asp Arg Pro Ser 35 40 45Gln Gln Leu Arg Ser Leu
Asn Gly Glu Trp Arg Phe Ala Trp Phe Pro 50 55 60Ala Pro Glu Ala Val
Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro Glu65 70 75 80Ala Asp Thr
Val Val Val Pro Ser Asn Trp Gln Met His Gly Tyr Asp 85 90 95Ala Pro
Ile Tyr Thr Asn Val Thr Tyr Pro Ile Thr Val Asn Pro Pro 100 105
110Phe Val Pro Thr Glu Asn Pro Thr Gly Cys Tyr Ser Leu Thr Phe Asn
115 120 125Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr Arg Ile Ile
Phe Asp 130 135 140Gly Val Asn Ser Ala Phe His Leu Trp Cys Asn Gly
Arg Trp Val Gly145 150 155 160Tyr Gly Gln Asp Ser Arg Leu Pro Ser
Glu Phe Asp Leu Ser Ala Phe 165 170 175Leu Arg Ala Gly Glu Asn Arg
Leu Ala Val Met Val Leu Arg Trp Ser 180 185 190Asp Gly Ser Tyr Leu
Glu Asp Gln Asp Met Trp Arg Met Ser Gly Ile 195 200 205Phe Arg Asp
Val Ser Leu Leu His Lys Pro Thr Thr Gln Ile Ser Asp 210 215 220Phe
His Val Ala Thr Arg Phe Asn Asp Asp Phe Ser Arg Ala Val Leu225 230
235 240Glu Ala Glu Val Gln Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg
Val 245 250 255Thr Val Ser Leu Trp Gln Gly Glu Thr Gln Val Ala Ser
Gly Thr Ala 260 265 270Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly
Gly Tyr Ala Asp Arg 275 280 285Val Thr Leu Arg Leu Asn Val Glu Asn
Pro Lys Leu Trp Ser Ala Glu 290 295 300Ile Pro Asn Leu Tyr Arg Ala
Val Val Glu Leu His Thr Ala Asp Gly305 310 315 320Thr Leu Ile Glu
Ala Glu Ala Cys Asp Val Gly Phe Arg Glu Val Arg 325 330 335Ile Glu
Asn Gly Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu Ile Arg 340 345
350Gly Val Asn Arg His Glu His His Pro Leu His Gly Gln Val Met Asp
355 360 365Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met Lys Gln Asn
Asn Phe 370 375 380Asn Ala Val Arg Cys Ser His Tyr Pro Asn His Pro
Leu Trp Tyr Thr385 390 395 400Leu Cys Asp Arg Tyr Gly Leu Tyr Val
Val Asp Glu Ala Asn Ile Glu 405 410 415Thr His Gly Met Val Pro Met
Asn Arg Leu Thr Asp Asp Pro Arg Trp 420 425 430Leu Pro Ala Met Ser
Glu Arg Val Thr Arg Met Val Gln Arg Asp Arg 435 440 445Asn His Pro
Ser Val Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly His 450 455 460Gly
Ala Asn His Asp Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp Pro465 470
475 480Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala
Thr 485 490 495Asp Ile Ile Cys Pro Met Tyr Ala Arg Val Asp Glu Asp
Gln Pro Phe 500 505 510Pro Ala Val Pro Lys Trp Ser Ile Lys Lys Trp
Leu Ser Leu Pro Gly 515 520 525Glu Thr Arg Pro Leu Ile Leu Cys Glu
Tyr Ala His Ala Met Gly Asn 530 535 540Ser Leu Gly Gly Phe Ala Lys
Tyr Trp Gln Ala Phe Arg Gln Tyr Pro545 550 555 560Arg Leu Gln Gly
Gly Phe Val Trp Asp Trp Val Asp Gln Ser Leu Ile 565 570 575Lys Tyr
Asp Glu Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp Phe 580 585
590Gly Asp Thr Pro Asn Asp Arg Gln Phe Cys Met Asn Gly Leu Val Phe
595 600 605Ala Asp Arg Thr Pro His Pro Ala Leu Thr Glu Ala Lys His
Gln Gln 610 615 620Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln Thr Ile
Glu Val Thr Ser625 630 635 640Glu Tyr Leu Phe Arg His Ser Asp Asn
Glu Leu Leu His Trp Met Val 645 650 655Ala Leu Asp Gly Lys Pro Leu
Ala Ser Gly Glu Val Pro Leu Asp Val 660 665 670Ala Pro Gln Gly Lys
Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro 675 680 685Glu Ser Ala
Gly Gln Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn 690 695 700Ala
Thr Ala Trp Ser Glu Ala Gly His Ile Ser Ala Trp Gln Gln Trp705 710
715 720Arg Leu Ala Glu Asn Leu Ser Val Thr Leu Pro Ala Ala Ser His
Ala 725 730 735Ile Pro His Leu Thr Thr Ser Glu Met Asp Phe Cys Ile
Glu Leu Gly 740 745 750Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser Gly
Phe Leu Ser Gln Met 755 760 765Trp Ile Gly Asp Lys Lys Gln Leu Leu
Thr Pro Leu Arg Asp Gln Phe 770 775 780Thr Arg Ala Pro Leu Asp Asn
Asp Ile Gly Val Ser Glu Ala Thr Arg785 790 795 800Ile Asp Pro Asn
Ala Trp Val Glu Arg Trp Lys Ala Ala Gly His Tyr 805 810 815Gln Ala
Glu Ala Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp 820 825
830Ala Val Leu Ile Thr Thr Ala His Ala Trp Gln His Gln Gly Lys Thr
835 840 845Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly
Gln Met 850 855 860Ala Ile Thr Val Asp Val Glu Val Ala Ser Asp Thr
Pro His Pro Ala865 870 875 880Arg Ile Gly Leu Asn Cys Gln Leu Ala
Gln Val Ala Glu Arg Val Asn 885 890 895Trp Leu Gly Leu Gly Pro Gln
Glu Asn Tyr Pro Asp Arg Leu Thr Ala 900 905 910Ala Cys Phe Asp Arg
Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr Pro 915 920 925Tyr Val Phe
Pro Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu 930 935 940Asn
Tyr Gly Pro His Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser945 950
955 960Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His Leu
Leu 965 970 975His Ala Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe
His Met Gly 980 985 990Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser Val
Ser Ala Glu Phe Gln 995 1000 1005Leu Ser Ala Gly Arg Tyr His Tyr
Gln Leu Val Trp Cys Gln Lys 1010 1015 10202249PRThomo sapiens 22Tyr
Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu1 5 10
15Glu Pro Arg Arg Glu Val Cys Glu Leu Asn Pro Asp Cys Asp Glu Leu
20 25 30Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr Gly
Pro 35 40 45Val2349PRThomo sapiensVARIANT(17)..(17)Xaa can be any
amino acid. 23Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro
Asp Pro Leu1 5 10 15Xaa Pro Arg Arg Xaa Val Cys Xaa Leu Asn Pro Asp
Cys Asp Glu Leu 20 25 30Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg
Arg Phe Tyr Gly Pro 35 40 45Val245136DNARattus
norvegicusCDS(1)..(5136) 24atg agg ccc ctg att ctg tta gct gcc ctc
ctc tgg ctc cag ggc ttt 48Met Arg Pro Leu Ile Leu Leu Ala Ala Leu
Leu Trp Leu Gln Gly Phe1 5 10 15ttg gcc gag gac gac gca tgc tca tcc
ttg gaa ggg agc cca gac agg 96Leu Ala Glu Asp Asp Ala Cys Ser Ser
Leu Glu Gly Ser Pro Asp Arg 20 25 30cag ggt gga ggt cca ctt ctg agt
gtg aac gtc agt agc cat gga aag 144Gln Gly Gly Gly Pro Leu Leu Ser
Val Asn Val Ser Ser His Gly Lys 35 40 45tct acc agc ctg ttt ctg agc
tgg gta gct gca gag ctg ggc gga ttt 192Ser Thr Ser Leu Phe Leu Ser
Trp Val Ala Ala Glu Leu Gly Gly Phe 50 55 60gac tat gcc ctc agc ctc
agg agt gtg aac tcc tca ggt tct cca gaa 240Asp Tyr Ala Leu Ser Leu
Arg Ser Val Asn Ser Ser Gly Ser Pro Glu65 70 75 80ggg caa cag ctc
cag gct cac aca aat gag tcc ggc ttt gag ttc cat 288Gly Gln Gln Leu
Gln Ala His Thr Asn Glu Ser Gly Phe Glu Phe His 85 90 95ggc ctg gtg
cca ggg agt cgc tac cag cta aaa ctg act gtc cta aga 336Gly Leu Val
Pro Gly Ser Arg Tyr Gln Leu Lys Leu Thr Val Leu Arg 100 105 110ccc
tgt tgg cag aat gtc aca att acc ctc act gcc cga act gcc ccg 384Pro
Cys Trp Gln Asn Val Thr Ile Thr Leu Thr Ala Arg Thr Ala Pro 115 120
125aca gtg gtc cgt gga ctg cag ctg cat agc gct ggg agc cca gcc agg
432Thr Val Val Arg Gly Leu Gln Leu His Ser Ala Gly Ser Pro Ala Arg
130 135 140ctg gaa gcc tcg tgg agt gat gcc cct gga gat caa gac agc
tac caa 480Leu Glu Ala Ser Trp Ser Asp Ala Pro Gly Asp Gln Asp Ser
Tyr Gln145 150 155 160ctt ctc ctc tac cac ctg gaa tcc caa act ctg
gca tgc aat gtc tct 528Leu Leu Leu Tyr His Leu Glu Ser Gln Thr Leu
Ala Cys Asn Val Ser 165 170 175gtg tcc cct gac acc ctg tct tac agt
ttt ggc gac ctt ttg cca ggt 576Val Ser Pro Asp Thr Leu Ser Tyr Ser
Phe Gly Asp Leu Leu Pro Gly 180 185 190act cag tat gtc ttg gag gtt
atc acc tgg gct ggc agt ctc cat gcg 624Thr Gln Tyr Val Leu Glu Val
Ile Thr Trp Ala Gly Ser Leu His Ala 195 200 205aag act agt atc ctc
cag tgg aca gag cct gtc cct cct gat cac cta 672Lys Thr Ser Ile Leu
Gln Trp Thr Glu Pro Val Pro Pro Asp His Leu 210 215 220gca cta cgt
gcc ttg ggt acc agt agc ctg caa gcc ttc tgg aac agc 720Ala Leu Arg
Ala Leu Gly Thr Ser Ser Leu Gln Ala Phe Trp Asn Ser225 230 235
240tct gaa ggg gcc acc tcg ttt cac ctg atg ctc aca gac ctc ctc ggg
768Ser Glu Gly Ala Thr Ser Phe His Leu Met Leu Thr Asp Leu Leu Gly
245 250 255ggc acc aac acg act gcg gtg atc aga caa ggg gtc tcg acc
cac acc 816Gly Thr Asn Thr Thr Ala Val Ile Arg Gln Gly Val Ser Thr
His Thr 260 265 270ttt ctt cac cta tct ccg ggt aca cct cat gag ctg
aag att tgt gct 864Phe Leu His Leu Ser Pro Gly Thr Pro His Glu Leu
Lys Ile Cys Ala 275 280 285tct gct ggg ccc cac cag atc tgg gga ccc
agt gcc acc gag tgg acc 912Ser Ala Gly Pro His Gln Ile Trp Gly Pro
Ser Ala Thr Glu Trp Thr 290 295 300tat ccc tct tac cca tct gac ctg
gtg ctg act ccc tta cgg aat gag 960Tyr Pro Ser Tyr Pro Ser Asp Leu
Val Leu Thr Pro Leu Arg Asn Glu305 310 315 320ctc tgg gcc agc tgg
aag gca ggg ctg gga gcc cgg gac ggc tat gta 1008Leu Trp Ala Ser Trp
Lys Ala Gly Leu Gly Ala Arg Asp Gly Tyr Val 325 330 335ctg aag tta
agt ggg cca atg gag agt acg tct acc ctg ggc ccg gaa 1056Leu Lys Leu
Ser Gly Pro Met Glu Ser Thr Ser Thr Leu Gly Pro Glu 340 345 350gag
tgc aat gca gtc ttc cca ggg ccc ctg cct ccg gga cac tac act 1104Glu
Cys Asn Ala Val Phe Pro Gly Pro Leu Pro Pro Gly His Tyr Thr 355 360
365ttg cag ctg aag gtt cta gct gga cct tat gat gcc tgg gtg gag ggc
1152Leu Gln Leu Lys Val Leu Ala Gly Pro Tyr Asp Ala Trp Val Glu Gly
370 375 380agt acc tgg ctg gct gaa tct gct gcc ctt ccc agg gag gtc
cct ggt 1200Ser Thr Trp Leu Ala Glu Ser Ala Ala Leu Pro Arg Glu Val
Pro Gly385 390 395 400gcc aga ctg tgg cta gat gga ctg gaa gct tcc
aag cag cct ggg aga 1248Ala Arg Leu Trp Leu Asp Gly Leu Glu Ala Ser
Lys Gln Pro Gly Arg 405 410 415cgg gcg cta ctc tat tct gac gat gcc
cca ggc tcc cta ggg aac atc 1296Arg Ala Leu Leu Tyr Ser Asp Asp Ala
Pro Gly Ser Leu Gly Asn Ile 420 425 430tct gtg ccc tct ggt gcc act
cac gtc att ttc tgt ggc ctg gta cct 1344Ser Val Pro Ser Gly Ala Thr
His Val Ile Phe Cys Gly Leu Val Pro 435 440 445gga gcc cac tat agg
gtg gac att gcc tca tcc acg ggg gac atc tct 1392Gly Ala His Tyr Arg
Val Asp Ile Ala Ser Ser Thr Gly Asp Ile Ser 450 455 460cag agc atc
tca ggc tat aca agt ccc ctg cca ccg cag tca ctg gag 1440Gln Ser Ile
Ser Gly Tyr Thr Ser Pro Leu Pro Pro Gln Ser Leu Glu465 470 475
480gtc atc agc agg agc agc cca tct gac ctg act att gct tgg ggt cca
1488Val Ile Ser Arg Ser Ser Pro Ser Asp Leu Thr Ile Ala Trp Gly Pro
485 490 495gca cca ggg cag ctg gaa ggt tat aag gtt acc tgg cat cag
gat ggc 1536Ala Pro Gly Gln Leu Glu Gly Tyr Lys Val Thr Trp His Gln
Asp Gly 500 505 510agc cag agg tct cct ggc gac ctt gtt gac ttg ggc
cct gac act ttg 1584Ser Gln Arg Ser Pro Gly Asp Leu Val Asp Leu Gly
Pro Asp Thr Leu 515 520 525agc ctg act ctg aaa tct ctg gta ccc ggc
tcc tgc tac acc gtg tca 1632Ser Leu Thr Leu Lys Ser Leu Val Pro Gly
Ser Cys Tyr Thr Val Ser 530 535 540gca tgg gcc tgg gcc ggg aac ctc
gac tct gac tct cag aag att cac 1680Ala Trp Ala Trp Ala Gly Asn Leu
Asp Ser Asp Ser Gln Lys Ile His545 550 555 560agc tgc acc cgc ccc
gct cct ccc acc aac ctg agt ctg ggc ttt gcc 1728Ser Cys Thr Arg Pro
Ala Pro Pro Thr Asn Leu Ser Leu Gly Phe Ala 565 570 575cac cag cct
gcg gca ctg aag gct tcc tgg tat cac cca ccg ggt ggc 1776His Gln Pro
Ala Ala Leu Lys Ala Ser Trp Tyr His Pro Pro Gly Gly 580 585 590agg
gat gcc ttt cac tta cgg ctt tac agg ctg agg cct ctg aca ctg 1824Arg
Asp Ala Phe His Leu Arg Leu Tyr Arg Leu Arg Pro Leu Thr Leu 595 600
605gaa agt gag aag gtc cta cct cgg gag gcc cag aac ttc tcc tgg gcc
1872Glu Ser Glu Lys Val Leu Pro Arg Glu Ala Gln Asn Phe Ser Trp Ala
610 615 620cag ctg act gca ggc tgt gag ttc cag gta cag ctg tct acc
ttg tgg 1920Gln Leu Thr Ala Gly Cys Glu Phe Gln Val Gln Leu Ser Thr
Leu Trp625 630 635 640ggg tct gag aga agc agc agt gcc aac gcc aca
ggc tgg aca ccc cct 1968Gly Ser Glu Arg Ser Ser Ser Ala Asn Ala Thr
Gly Trp Thr Pro Pro 645 650 655tca gct cct aca ctg gta aac gtg acc
agc gat gct cct acc cag ctc 2016Ser Ala Pro Thr Leu Val Asn Val Thr
Ser Asp Ala Pro Thr Gln Leu 660 665 670caa gta tcc tgg gcc cac gtt
cct ggg ggc cgg agc cgc tac caa gtg 2064Gln Val Ser Trp Ala His Val
Pro Gly Gly Arg Ser Arg Tyr Gln Val 675 680 685acc cta tac cag gag
agt acc cgg aca gcc acc agc atc atg ggg ccc 2112Thr Leu Tyr Gln Glu
Ser Thr Arg Thr Ala Thr Ser Ile Met Gly Pro 690 695 700aag gaa gat
ggc acg agc ttt ttg ggt ttg act cct ggc act aag tac 2160Lys Glu Asp
Gly Thr Ser Phe Leu Gly Leu Thr Pro Gly Thr Lys Tyr705 710 715
720aag gtg gaa gtc atc tcc tgg gct ggg ccc ctc tac act gca gca gcc
2208Lys Val Glu Val Ile Ser Trp Ala Gly Pro Leu Tyr Thr Ala Ala Ala
725 730 735aac gtt tct gcc tgg acc tac cca ctc ata ccc aat gag ctg
ctc gtg 2256Asn Val Ser Ala Trp Thr Tyr Pro Leu Ile Pro Asn Glu Leu
Leu Val 740 745 750tca atg cag gca ggc agt
gct gtg gtt aac ctg gcc tgg ccc agt ggt 2304Ser Met Gln Ala Gly Ser
Ala Val Val Asn Leu Ala Trp Pro Ser Gly 755 760 765ccc ctg ggg caa
ggg gca tgc cac gcc caa ctc tca gat gct gga cac 2352Pro Leu Gly Gln
Gly Ala Cys His Ala Gln Leu Ser Asp Ala Gly His 770 775 780ctc tca
tgg gag caa ccc ctg aaa cta ggc caa gag ctc ttc atg cta 2400Leu Ser
Trp Glu Gln Pro Leu Lys Leu Gly Gln Glu Leu Phe Met Leu785 790 795
800agg gat ctc aca cca gga cat acc atc tcg atg tca gtg agg tgt cgg
2448Arg Asp Leu Thr Pro Gly His Thr Ile Ser Met Ser Val Arg Cys Arg
805 810 815gca ggg ccg ctc cag gcc tct acg cac ctt gtg gtg ctg tct
gtg gag 2496Ala Gly Pro Leu Gln Ala Ser Thr His Leu Val Val Leu Ser
Val Glu 820 825 830cct ggc cct gtg gaa gat gtg ctc tgt cat cca gag
gcc acc tac ctg 2544Pro Gly Pro Val Glu Asp Val Leu Cys His Pro Glu
Ala Thr Tyr Leu 835 840 845gcc ctg aac tgg acg atg cct gct gga gac
gtg gat gtc tgt ctg gtg 2592Ala Leu Asn Trp Thr Met Pro Ala Gly Asp
Val Asp Val Cys Leu Val 850 855 860gtg gta gag cgg ctg gtg ccg gga
ggg ggc act cat ttt gtc ttc cag 2640Val Val Glu Arg Leu Val Pro Gly
Gly Gly Thr His Phe Val Phe Gln865 870 875 880gtc aac acc tca ggg
gat gct ctt ctg ttg ccc aac ttg atg ccc acc 2688Val Asn Thr Ser Gly
Asp Ala Leu Leu Leu Pro Asn Leu Met Pro Thr 885 890 895act tct tac
cgc ctt agc ctc acc gtt ctg ggc agg aat agt cgg tgg 2736Thr Ser Tyr
Arg Leu Ser Leu Thr Val Leu Gly Arg Asn Ser Arg Trp 900 905 910agc
cgg gcg gtt tcc ctg gtg tgc agt act tct gct gag gct tgg cac 2784Ser
Arg Ala Val Ser Leu Val Cys Ser Thr Ser Ala Glu Ala Trp His 915 920
925ccc cca gag cta gct gag ccc ccc cag gtg gag ctg ggg aca ggg atg
2832Pro Pro Glu Leu Ala Glu Pro Pro Gln Val Glu Leu Gly Thr Gly Met
930 935 940ggt gtg aca gtc atg cgt ggc atg ttt ggt aaa gat gac ggg
cag atc 2880Gly Val Thr Val Met Arg Gly Met Phe Gly Lys Asp Asp Gly
Gln Ile945 950 955 960cag tgg tat ggc ata att gcc acc atc aac atg
acg ctg gcc cag cct 2928Gln Trp Tyr Gly Ile Ile Ala Thr Ile Asn Met
Thr Leu Ala Gln Pro 965 970 975tcc cgg gaa gcc atc aat tac aca tgg
tat gac cac tac tat aga gga 2976Ser Arg Glu Ala Ile Asn Tyr Thr Trp
Tyr Asp His Tyr Tyr Arg Gly 980 985 990tgt gag tcc ttc ctg gct ctc
ctg ttc cca aac ccc ttc tac cca gag 3024Cys Glu Ser Phe Leu Ala Leu
Leu Phe Pro Asn Pro Phe Tyr Pro Glu 995 1000 1005cct tgg gct ggg
cca aga tcc tgg aca gta cct gtg ggt act gag 3069Pro Trp Ala Gly Pro
Arg Ser Trp Thr Val Pro Val Gly Thr Glu 1010 1015 1020gac tgt gac
aac acc caa gag ata tgc aat ggg cgt ctc aag tca 3114Asp Cys Asp Asn
Thr Gln Glu Ile Cys Asn Gly Arg Leu Lys Ser 1025 1030 1035ggc ttc
cag tat agg ttc agc gtt gtg gcc ttt agt agg ctc aac 3159Gly Phe Gln
Tyr Arg Phe Ser Val Val Ala Phe Ser Arg Leu Asn 1040 1045 1050act
cca gag acc atc ctc gcc ttc tcg gcc ttc tca gag ccc cgg 3204Thr Pro
Glu Thr Ile Leu Ala Phe Ser Ala Phe Ser Glu Pro Arg 1055 1060
1065gcc agc atc tct ctg gcg atc att ccc ctg aca gtt atg ctg ggg
3249Ala Ser Ile Ser Leu Ala Ile Ile Pro Leu Thr Val Met Leu Gly
1070 1075 1080gct gtg gtg ggc agc att gtc att gtg tgt gca gtg cta
tgc ttg 3294Ala Val Val Gly Ser Ile Val Ile Val Cys Ala Val Leu Cys
Leu 1085 1090 1095ctc cgc tgg cgg tgc ctg aag gga cca aga tca gag
aag gat ggc 3339Leu Arg Trp Arg Cys Leu Lys Gly Pro Arg Ser Glu Lys
Asp Gly 1100 1105 1110ttt tcc aag gag ctg atg cct tac aac ctg tgg
cgg acc cat cgg 3384Phe Ser Lys Glu Leu Met Pro Tyr Asn Leu Trp Arg
Thr His Arg 1115 1120 1125cct atc ccc atc cat agc ttc cgg cag agc
tat gag gcc aag agc 3429Pro Ile Pro Ile His Ser Phe Arg Gln Ser Tyr
Glu Ala Lys Ser 1130 1135 1140gca cat gca cac cag acc ttc ttc cag
gaa ttt gag gag ttg aag 3474Ala His Ala His Gln Thr Phe Phe Gln Glu
Phe Glu Glu Leu Lys 1145 1150 1155gag gta ggc aag gac cag ccc cga
cta gag gct gag cat ccg gac 3519Glu Val Gly Lys Asp Gln Pro Arg Leu
Glu Ala Glu His Pro Asp 1160 1165 1170aac atc atc aag aac cgg tac
cca cac gtg ctg ccc tat gac cac 3564Asn Ile Ile Lys Asn Arg Tyr Pro
His Val Leu Pro Tyr Asp His 1175 1180 1185tcc agg gtc agg ctg acc
cag cta cca gga gag cct cat tct gac 3609Ser Arg Val Arg Leu Thr Gln
Leu Pro Gly Glu Pro His Ser Asp 1190 1195 1200tac atc aat gcc aac
ttc atc cca ggc tat agc cac aca cag gag 3654Tyr Ile Asn Ala Asn Phe
Ile Pro Gly Tyr Ser His Thr Gln Glu 1205 1210 1215atc att gcc acc
cag ggg cct ctc aaa aag acg cta gag gac ttc 3699Ile Ile Ala Thr Gln
Gly Pro Leu Lys Lys Thr Leu Glu Asp Phe 1220 1225 1230tgg cgg ttg
gta tgg gag cag caa gtc cac gtg atc atc atg ctg 3744Trp Arg Leu Val
Trp Glu Gln Gln Val His Val Ile Ile Met Leu 1235 1240 1245act gtg
ggc atg gag aac ggg cgg gta ctg tgt gag cac tac tgg 3789Thr Val Gly
Met Glu Asn Gly Arg Val Leu Cys Glu His Tyr Trp 1250 1255 1260cca
gcc aac tcc acg cct gtt act cac ggt cac atc acc atc cac 3834Pro Ala
Asn Ser Thr Pro Val Thr His Gly His Ile Thr Ile His 1265 1270
1275ctc ctg gca gag gag cct gag gat gag tgg acc agg agg gaa ttc
3879Leu Leu Ala Glu Glu Pro Glu Asp Glu Trp Thr Arg Arg Glu Phe
1280 1285 1290cag ctg cag cac ggt acc gag caa aaa cag agg cga gtg
aag cag 3924Gln Leu Gln His Gly Thr Glu Gln Lys Gln Arg Arg Val Lys
Gln 1295 1300 1305ctg cag ttc act acc tgg cca gac cac agt gtc ccg
gag gct ccc 3969Leu Gln Phe Thr Thr Trp Pro Asp His Ser Val Pro Glu
Ala Pro 1310 1315 1320agc tct ctg ctc gct ttt gta gaa ctg gta cag
gag cag gtg cag 4014Ser Ser Leu Leu Ala Phe Val Glu Leu Val Gln Glu
Gln Val Gln 1325 1330 1335gcc act cag ggc aag gga ccc atc ctg gtg
cat tgc agt gct ggc 4059Ala Thr Gln Gly Lys Gly Pro Ile Leu Val His
Cys Ser Ala Gly 1340 1345 1350gtg ggg agg aca ggc acc ttt gtg gct
ctc ttg cgg cta ctg cga 4104Val Gly Arg Thr Gly Thr Phe Val Ala Leu
Leu Arg Leu Leu Arg 1355 1360 1365caa cta gag gaa gag aag gtg gcc
gat gtg ttc aac act gtg tac 4149Gln Leu Glu Glu Glu Lys Val Ala Asp
Val Phe Asn Thr Val Tyr 1370 1375 1380ata ctc cgg ttg cac cgg ccc
ctc atg atc cag acc ctg agt caa 4194Ile Leu Arg Leu His Arg Pro Leu
Met Ile Gln Thr Leu Ser Gln 1385 1390 1395tac atc ttc ctg cac agt
tgc ctg ctg aac aag att ctg gaa ggg 4239Tyr Ile Phe Leu His Ser Cys
Leu Leu Asn Lys Ile Leu Glu Gly 1400 1405 1410ccc cct gac agc tcc
gac tcc ggc ccc atc tct gtg atg gat ttt 4284Pro Pro Asp Ser Ser Asp
Ser Gly Pro Ile Ser Val Met Asp Phe 1415 1420 1425gca cag gct tgt
gcc aag agg gca gcc aac gcc aat gct ggt ttc 4329Ala Gln Ala Cys Ala
Lys Arg Ala Ala Asn Ala Asn Ala Gly Phe 1430 1435 1440ttg aag gag
tac aag ctc ctg aag cag gcc atc aag gat ggg act 4374Leu Lys Glu Tyr
Lys Leu Leu Lys Gln Ala Ile Lys Asp Gly Thr 1445 1450 1455ggc tct
ctg ctg ccc cct cct gac tac aat cag aac agc att gtc 4419Gly Ser Leu
Leu Pro Pro Pro Asp Tyr Asn Gln Asn Ser Ile Val 1460 1465 1470tcc
cgt cgt cat tct cag gag cag ttc gcc ctg gtg gag gag tgc 4464Ser Arg
Arg His Ser Gln Glu Gln Phe Ala Leu Val Glu Glu Cys 1475 1480
1485cct gag gat agc atg ctg gaa gcc tca ctc ttc cct ggt ggt ccg
4509Pro Glu Asp Ser Met Leu Glu Ala Ser Leu Phe Pro Gly Gly Pro
1490 1495 1500tct ggt tgt gat cat gtg gtg ctg act ggc tca gcc gga
cca aag 4554Ser Gly Cys Asp His Val Val Leu Thr Gly Ser Ala Gly Pro
Lys 1505 1510 1515gaa ctc tgg gaa atg gtg tgg gag cat gat gcc cat
gtg ctc gtc 4599Glu Leu Trp Glu Met Val Trp Glu His Asp Ala His Val
Leu Val 1520 1525 1530tcc ctg ggc ctg cct gat acc aag gag aag cca
cca gac atc tgg 4644Ser Leu Gly Leu Pro Asp Thr Lys Glu Lys Pro Pro
Asp Ile Trp 1535 1540 1545cca gtg gag atg cag cct att gtc aca gac
atg gtg aca gtg cac 4689Pro Val Glu Met Gln Pro Ile Val Thr Asp Met
Val Thr Val His 1550 1555 1560aga gtg tct gag agc aac aca aca act
ggc tgg ccc agc acc ctc 4734Arg Val Ser Glu Ser Asn Thr Thr Thr Gly
Trp Pro Ser Thr Leu 1565 1570 1575ttc aga gtc ata cac ggg gag agt
gga aag gaa agg cag gtt caa 4779Phe Arg Val Ile His Gly Glu Ser Gly
Lys Glu Arg Gln Val Gln 1580 1585 1590tgc ctg caa ttt cca tgc tct
gag tct ggg tgt gag ctc cca gct 4824Cys Leu Gln Phe Pro Cys Ser Glu
Ser Gly Cys Glu Leu Pro Ala 1595 1600 1605aac acc cta ctg acc ttc
ctt gat gct gtg ggc cag tgc tgc ttc 4869Asn Thr Leu Leu Thr Phe Leu
Asp Ala Val Gly Gln Cys Cys Phe 1610 1615 1620cgg ggc aag agc aag
aag cca ggg acc ctg ctc agc cac tcc agc 4914Arg Gly Lys Ser Lys Lys
Pro Gly Thr Leu Leu Ser His Ser Ser 1625 1630 1635aaa aac aca aac
cag ctg ggc acc ttc ttg gct atg gaa cag ctg 4959Lys Asn Thr Asn Gln
Leu Gly Thr Phe Leu Ala Met Glu Gln Leu 1640 1645 1650tta cag caa
gca ggg aca gag cgc aca gtg gac gtc ttc aat gtg 5004Leu Gln Gln Ala
Gly Thr Glu Arg Thr Val Asp Val Phe Asn Val 1655 1660 1665gcc ctg
aag cag tca cag gcc tgc ggc ctt atg acc cca aca ctg 5049Ala Leu Lys
Gln Ser Gln Ala Cys Gly Leu Met Thr Pro Thr Leu 1670 1675 1680gag
cag tat atc tac ctc tac aac tgt ctg aac agc gca ctg ctg 5094Glu Gln
Tyr Ile Tyr Leu Tyr Asn Cys Leu Asn Ser Ala Leu Leu 1685 1690
1695aac ggg ctg ccc aga gct ggg aag tgg cct gcg ccc tgc tag 5136Asn
Gly Leu Pro Arg Ala Gly Lys Trp Pro Ala Pro Cys 1700 1705
1710251711PRTRattus norvegicus 25Met Arg Pro Leu Ile Leu Leu Ala
Ala Leu Leu Trp Leu Gln Gly Phe1 5 10 15Leu Ala Glu Asp Asp Ala Cys
Ser Ser Leu Glu Gly Ser Pro Asp Arg 20 25 30Gln Gly Gly Gly Pro Leu
Leu Ser Val Asn Val Ser Ser His Gly Lys 35 40 45Ser Thr Ser Leu Phe
Leu Ser Trp Val Ala Ala Glu Leu Gly Gly Phe 50 55 60Asp Tyr Ala Leu
Ser Leu Arg Ser Val Asn Ser Ser Gly Ser Pro Glu65 70 75 80Gly Gln
Gln Leu Gln Ala His Thr Asn Glu Ser Gly Phe Glu Phe His 85 90 95Gly
Leu Val Pro Gly Ser Arg Tyr Gln Leu Lys Leu Thr Val Leu Arg 100 105
110Pro Cys Trp Gln Asn Val Thr Ile Thr Leu Thr Ala Arg Thr Ala Pro
115 120 125Thr Val Val Arg Gly Leu Gln Leu His Ser Ala Gly Ser Pro
Ala Arg 130 135 140Leu Glu Ala Ser Trp Ser Asp Ala Pro Gly Asp Gln
Asp Ser Tyr Gln145 150 155 160Leu Leu Leu Tyr His Leu Glu Ser Gln
Thr Leu Ala Cys Asn Val Ser 165 170 175Val Ser Pro Asp Thr Leu Ser
Tyr Ser Phe Gly Asp Leu Leu Pro Gly 180 185 190Thr Gln Tyr Val Leu
Glu Val Ile Thr Trp Ala Gly Ser Leu His Ala 195 200 205Lys Thr Ser
Ile Leu Gln Trp Thr Glu Pro Val Pro Pro Asp His Leu 210 215 220Ala
Leu Arg Ala Leu Gly Thr Ser Ser Leu Gln Ala Phe Trp Asn Ser225 230
235 240Ser Glu Gly Ala Thr Ser Phe His Leu Met Leu Thr Asp Leu Leu
Gly 245 250 255Gly Thr Asn Thr Thr Ala Val Ile Arg Gln Gly Val Ser
Thr His Thr 260 265 270Phe Leu His Leu Ser Pro Gly Thr Pro His Glu
Leu Lys Ile Cys Ala 275 280 285Ser Ala Gly Pro His Gln Ile Trp Gly
Pro Ser Ala Thr Glu Trp Thr 290 295 300Tyr Pro Ser Tyr Pro Ser Asp
Leu Val Leu Thr Pro Leu Arg Asn Glu305 310 315 320Leu Trp Ala Ser
Trp Lys Ala Gly Leu Gly Ala Arg Asp Gly Tyr Val 325 330 335Leu Lys
Leu Ser Gly Pro Met Glu Ser Thr Ser Thr Leu Gly Pro Glu 340 345
350Glu Cys Asn Ala Val Phe Pro Gly Pro Leu Pro Pro Gly His Tyr Thr
355 360 365Leu Gln Leu Lys Val Leu Ala Gly Pro Tyr Asp Ala Trp Val
Glu Gly 370 375 380Ser Thr Trp Leu Ala Glu Ser Ala Ala Leu Pro Arg
Glu Val Pro Gly385 390 395 400Ala Arg Leu Trp Leu Asp Gly Leu Glu
Ala Ser Lys Gln Pro Gly Arg 405 410 415Arg Ala Leu Leu Tyr Ser Asp
Asp Ala Pro Gly Ser Leu Gly Asn Ile 420 425 430Ser Val Pro Ser Gly
Ala Thr His Val Ile Phe Cys Gly Leu Val Pro 435 440 445Gly Ala His
Tyr Arg Val Asp Ile Ala Ser Ser Thr Gly Asp Ile Ser 450 455 460Gln
Ser Ile Ser Gly Tyr Thr Ser Pro Leu Pro Pro Gln Ser Leu Glu465 470
475 480Val Ile Ser Arg Ser Ser Pro Ser Asp Leu Thr Ile Ala Trp Gly
Pro 485 490 495Ala Pro Gly Gln Leu Glu Gly Tyr Lys Val Thr Trp His
Gln Asp Gly 500 505 510Ser Gln Arg Ser Pro Gly Asp Leu Val Asp Leu
Gly Pro Asp Thr Leu 515 520 525Ser Leu Thr Leu Lys Ser Leu Val Pro
Gly Ser Cys Tyr Thr Val Ser 530 535 540Ala Trp Ala Trp Ala Gly Asn
Leu Asp Ser Asp Ser Gln Lys Ile His545 550 555 560Ser Cys Thr Arg
Pro Ala Pro Pro Thr Asn Leu Ser Leu Gly Phe Ala 565 570 575His Gln
Pro Ala Ala Leu Lys Ala Ser Trp Tyr His Pro Pro Gly Gly 580 585
590Arg Asp Ala Phe His Leu Arg Leu Tyr Arg Leu Arg Pro Leu Thr Leu
595 600 605Glu Ser Glu Lys Val Leu Pro Arg Glu Ala Gln Asn Phe Ser
Trp Ala 610 615 620Gln Leu Thr Ala Gly Cys Glu Phe Gln Val Gln Leu
Ser Thr Leu Trp625 630 635 640Gly Ser Glu Arg Ser Ser Ser Ala Asn
Ala Thr Gly Trp Thr Pro Pro 645 650 655Ser Ala Pro Thr Leu Val Asn
Val Thr Ser Asp Ala Pro Thr Gln Leu 660 665 670Gln Val Ser Trp Ala
His Val Pro Gly Gly Arg Ser Arg Tyr Gln Val 675 680 685Thr Leu Tyr
Gln Glu Ser Thr Arg Thr Ala Thr Ser Ile Met Gly Pro 690 695 700Lys
Glu Asp Gly Thr Ser Phe Leu Gly Leu Thr Pro Gly Thr Lys Tyr705 710
715 720Lys Val Glu Val Ile Ser Trp Ala Gly Pro Leu Tyr Thr Ala Ala
Ala 725 730 735Asn Val Ser Ala Trp Thr Tyr Pro Leu Ile Pro Asn Glu
Leu Leu Val 740 745 750Ser Met Gln Ala Gly Ser Ala Val Val Asn Leu
Ala Trp Pro Ser Gly 755 760 765Pro Leu Gly Gln Gly Ala Cys His Ala
Gln Leu Ser Asp Ala Gly His 770 775 780Leu Ser Trp Glu Gln Pro Leu
Lys Leu Gly Gln Glu Leu Phe Met Leu785 790 795 800Arg Asp Leu Thr
Pro Gly His Thr Ile Ser Met Ser Val Arg Cys Arg 805 810 815Ala Gly
Pro Leu Gln Ala Ser Thr His Leu Val Val Leu Ser Val Glu 820 825
830Pro Gly Pro Val Glu Asp Val Leu Cys His Pro Glu Ala Thr Tyr Leu
835 840 845Ala Leu Asn Trp Thr Met Pro Ala Gly Asp Val Asp Val Cys
Leu Val 850 855 860Val Val Glu Arg Leu Val Pro Gly Gly Gly Thr His
Phe Val Phe Gln865 870 875 880Val Asn Thr Ser Gly Asp Ala Leu Leu
Leu
Pro Asn Leu Met Pro Thr 885 890 895Thr Ser Tyr Arg Leu Ser Leu Thr
Val Leu Gly Arg Asn Ser Arg Trp 900 905 910Ser Arg Ala Val Ser Leu
Val Cys Ser Thr Ser Ala Glu Ala Trp His 915 920 925Pro Pro Glu Leu
Ala Glu Pro Pro Gln Val Glu Leu Gly Thr Gly Met 930 935 940Gly Val
Thr Val Met Arg Gly Met Phe Gly Lys Asp Asp Gly Gln Ile945 950 955
960Gln Trp Tyr Gly Ile Ile Ala Thr Ile Asn Met Thr Leu Ala Gln Pro
965 970 975Ser Arg Glu Ala Ile Asn Tyr Thr Trp Tyr Asp His Tyr Tyr
Arg Gly 980 985 990Cys Glu Ser Phe Leu Ala Leu Leu Phe Pro Asn Pro
Phe Tyr Pro Glu 995 1000 1005Pro Trp Ala Gly Pro Arg Ser Trp Thr
Val Pro Val Gly Thr Glu 1010 1015 1020Asp Cys Asp Asn Thr Gln Glu
Ile Cys Asn Gly Arg Leu Lys Ser 1025 1030 1035Gly Phe Gln Tyr Arg
Phe Ser Val Val Ala Phe Ser Arg Leu Asn 1040 1045 1050Thr Pro Glu
Thr Ile Leu Ala Phe Ser Ala Phe Ser Glu Pro Arg 1055 1060 1065Ala
Ser Ile Ser Leu Ala Ile Ile Pro Leu Thr Val Met Leu Gly 1070 1075
1080Ala Val Val Gly Ser Ile Val Ile Val Cys Ala Val Leu Cys Leu
1085 1090 1095Leu Arg Trp Arg Cys Leu Lys Gly Pro Arg Ser Glu Lys
Asp Gly 1100 1105 1110Phe Ser Lys Glu Leu Met Pro Tyr Asn Leu Trp
Arg Thr His Arg 1115 1120 1125Pro Ile Pro Ile His Ser Phe Arg Gln
Ser Tyr Glu Ala Lys Ser 1130 1135 1140Ala His Ala His Gln Thr Phe
Phe Gln Glu Phe Glu Glu Leu Lys 1145 1150 1155Glu Val Gly Lys Asp
Gln Pro Arg Leu Glu Ala Glu His Pro Asp 1160 1165 1170Asn Ile Ile
Lys Asn Arg Tyr Pro His Val Leu Pro Tyr Asp His 1175 1180 1185Ser
Arg Val Arg Leu Thr Gln Leu Pro Gly Glu Pro His Ser Asp 1190 1195
1200Tyr Ile Asn Ala Asn Phe Ile Pro Gly Tyr Ser His Thr Gln Glu
1205 1210 1215Ile Ile Ala Thr Gln Gly Pro Leu Lys Lys Thr Leu Glu
Asp Phe 1220 1225 1230Trp Arg Leu Val Trp Glu Gln Gln Val His Val
Ile Ile Met Leu 1235 1240 1245Thr Val Gly Met Glu Asn Gly Arg Val
Leu Cys Glu His Tyr Trp 1250 1255 1260Pro Ala Asn Ser Thr Pro Val
Thr His Gly His Ile Thr Ile His 1265 1270 1275Leu Leu Ala Glu Glu
Pro Glu Asp Glu Trp Thr Arg Arg Glu Phe 1280 1285 1290Gln Leu Gln
His Gly Thr Glu Gln Lys Gln Arg Arg Val Lys Gln 1295 1300 1305Leu
Gln Phe Thr Thr Trp Pro Asp His Ser Val Pro Glu Ala Pro 1310 1315
1320Ser Ser Leu Leu Ala Phe Val Glu Leu Val Gln Glu Gln Val Gln
1325 1330 1335Ala Thr Gln Gly Lys Gly Pro Ile Leu Val His Cys Ser
Ala Gly 1340 1345 1350Val Gly Arg Thr Gly Thr Phe Val Ala Leu Leu
Arg Leu Leu Arg 1355 1360 1365Gln Leu Glu Glu Glu Lys Val Ala Asp
Val Phe Asn Thr Val Tyr 1370 1375 1380Ile Leu Arg Leu His Arg Pro
Leu Met Ile Gln Thr Leu Ser Gln 1385 1390 1395Tyr Ile Phe Leu His
Ser Cys Leu Leu Asn Lys Ile Leu Glu Gly 1400 1405 1410Pro Pro Asp
Ser Ser Asp Ser Gly Pro Ile Ser Val Met Asp Phe 1415 1420 1425Ala
Gln Ala Cys Ala Lys Arg Ala Ala Asn Ala Asn Ala Gly Phe 1430 1435
1440Leu Lys Glu Tyr Lys Leu Leu Lys Gln Ala Ile Lys Asp Gly Thr
1445 1450 1455Gly Ser Leu Leu Pro Pro Pro Asp Tyr Asn Gln Asn Ser
Ile Val 1460 1465 1470Ser Arg Arg His Ser Gln Glu Gln Phe Ala Leu
Val Glu Glu Cys 1475 1480 1485Pro Glu Asp Ser Met Leu Glu Ala Ser
Leu Phe Pro Gly Gly Pro 1490 1495 1500Ser Gly Cys Asp His Val Val
Leu Thr Gly Ser Ala Gly Pro Lys 1505 1510 1515Glu Leu Trp Glu Met
Val Trp Glu His Asp Ala His Val Leu Val 1520 1525 1530Ser Leu Gly
Leu Pro Asp Thr Lys Glu Lys Pro Pro Asp Ile Trp 1535 1540 1545Pro
Val Glu Met Gln Pro Ile Val Thr Asp Met Val Thr Val His 1550 1555
1560Arg Val Ser Glu Ser Asn Thr Thr Thr Gly Trp Pro Ser Thr Leu
1565 1570 1575Phe Arg Val Ile His Gly Glu Ser Gly Lys Glu Arg Gln
Val Gln 1580 1585 1590Cys Leu Gln Phe Pro Cys Ser Glu Ser Gly Cys
Glu Leu Pro Ala 1595 1600 1605Asn Thr Leu Leu Thr Phe Leu Asp Ala
Val Gly Gln Cys Cys Phe 1610 1615 1620Arg Gly Lys Ser Lys Lys Pro
Gly Thr Leu Leu Ser His Ser Ser 1625 1630 1635Lys Asn Thr Asn Gln
Leu Gly Thr Phe Leu Ala Met Glu Gln Leu 1640 1645 1650Leu Gln Gln
Ala Gly Thr Glu Arg Thr Val Asp Val Phe Asn Val 1655 1660 1665Ala
Leu Lys Gln Ser Gln Ala Cys Gly Leu Met Thr Pro Thr Leu 1670 1675
1680Glu Gln Tyr Ile Tyr Leu Tyr Asn Cys Leu Asn Ser Ala Leu Leu
1685 1690 1695Asn Gly Leu Pro Arg Ala Gly Lys Trp Pro Ala Pro Cys
1700 1705 1710
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