U.S. patent application number 10/889121 was filed with the patent office on 2004-12-16 for enos mutations useful for gene therapy and therapeutic screening.
This patent application is currently assigned to Yale University. Invention is credited to Sessa, William C..
Application Number | 20040253685 10/889121 |
Document ID | / |
Family ID | 22440541 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253685 |
Kind Code |
A1 |
Sessa, William C. |
December 16, 2004 |
eNOS mutations useful for gene therapy and therapeutic
screening
Abstract
The present invention relates to new NOS variants or mutants
which contain structural alterations in the site of Akt dependent
phosphorylation. The altered NOS proteins or peptides, especially
the human eNOS proteins or peptides, Akt proteins or polypeptides
and their encoding nucleic acid molecules are useful as gene
therapy agents for the treatment of diseases including post
angioplasty restenosis, hypertension, atherosclerosis, heart
failure, diabetes and diseases with defective angiogenesis. NOS
proteins and peptides are also useful in methods of screening for
agents which modulate NOS activity.
Inventors: |
Sessa, William C.; (Madison,
CT) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP
1701 MARKET STREET
PHILADELPHIA
PA
19103-2921
US
|
Assignee: |
Yale University
|
Family ID: |
22440541 |
Appl. No.: |
10/889121 |
Filed: |
July 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10889121 |
Jul 12, 2004 |
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09956699 |
Sep 20, 2001 |
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09956699 |
Sep 20, 2001 |
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PCT/US00/09913 |
Apr 14, 2000 |
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60129550 |
Apr 16, 1999 |
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Current U.S.
Class: |
435/69.1 ;
435/191; 435/320.1; 435/325; 514/1.9; 514/13.3; 514/15.7; 514/16.4;
514/6.9; 530/350; 536/23.5 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61K 48/00 20130101; A61P 9/04 20180101; A61P 9/00 20180101; A61P
3/10 20180101; A61P 15/10 20180101; A61K 38/00 20130101; C12N
9/0075 20130101; A61P 9/12 20180101; A61P 9/10 20180101 |
Class at
Publication: |
435/069.1 ;
514/012; 530/350; 536/023.5; 435/320.1; 435/325; 435/191 |
International
Class: |
C07K 014/47; C12N
009/06; C07H 021/04 |
Goverment Interests
[0002] The present invention arose in part from research funded by
the following federal grant monies: HL 57665 and HL 61371.
Claims
1-43 (Canceled)
44. An isolated nucleic acid molecule encoding a nitric oxide
synthetase (NOS) polypeptide selected from the group consisting of
a constitutively active NOS polypeptide having an increased
activity of NO production, a NOS polypeptide having an increased
rate of NO production and a NOS polypeptide having an increased
reductase activity when compared with the wild-type NOS
polypeptide, wherein amino acid S/T of the consensus sequence motif
RXRXXS/T is mutated.
45. An isolated nucleic acid molecule comprising the sequence as
set forth in SEQ ID NO: 8 or a variant thereof, wherein said
nucleic acid molecule encodes a nitric oxide synthetase (NOS)
polypeptide selected from the group consisting of a constitutively
active NOS polypeptide having an increased activity of NO
production, a NOS polypeptide having an increased rate of NO
production and a NOS polypeptide having an increased reductase
activity when compared with the wild-type NOS polypeptide, wherein
amino acid S/T of the consensus sequence motif RXRXXS/T is
mutated.
46. The isolated nucleic acid molecule of claim 45, wherein the NOS
polypeptide comprises a substituted amino acid residue
corresponding to residue 1179 of bovine eNOS, residue 1177 of human
eNOS, residue 1412 of rat nNOS, or residue 1415 of human nNOS.
47. The isolated nucleic acid molecule of claim 46, wherein the
substituted amino acid residue corresponding to residue 1179 of
bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat nNOS,
or residue 1415 of human nNOS, mimics a phosphoserine.
48. The isolated nucleic acid molecule of claim 46, wherein a
serine corresponding to residue 1179 of bovine eNOS, residue 1177
of human eNOS, residue 1412 of rat nNOS, or residue 1415 of human
nNOS, has been substituted with an aspartic acid residue or a
glutamic acid residue.
49. The isolated nucleic acid molecule of claim 46, wherein the
substituted amino acid comprises an R group that mimics a phosphate
group.
50. The isolated nucleic acid molecule of claim 46, wherein the
substituted amino acid comprises a negatively charged R group.
51. An isolated nucleic acid molecule encoding a human eNOS
polypeptide as set forth in SEQ ID NO. 3, wherein the eNOS
polypeptide is selected from the group consisting of a
constitutively active NOS polypeptide having an increased activity
of NO production, a NOS polypeptide having an increased rate of NO
production and a NOS polypeptide having an increased reductase
activity when compared with the wild-type NOS polypeptide, wherein
amino acid S/T of the consensus sequence motif RXRXXS/T is
mutated.
52. An isolated nucleic acid molecule encoding a human eNOS
polypeptide which comprises a substituted amino acid residue
corresponding to residue 1177 of human eNOS as set forth in SEQ ID
NO. 5.
53. The isolated nucleic acid molecule of claim 52, wherein the
substituted amino acid is an aspartic acid residue as set forth in
SEQ ID NO. 6 or a glutamic acid residue as set forth in SEQ ID NO.
7.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/129,550, filed Apr. 16, 1999, which is herein
incorporated by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates to new NOS variants or mutants
which contain structural alterations in the site of Akt dependent
phosphorylation. The altered NOS proteins or peptides and their
encoding nucleic acid molecules are useful as gene therapy agents
for the treatment of diseases including post angioplasty
restenosis, hypertension, atherosclerosis, heart failure, diabetes
and diseases with defective angiogenesis.
BACKGROUND OF THE INVENTION
[0004] Atherosclerosis and vascular thrombosis are a major cause of
morbidity and mortality, leading to coronary artery disease,
myocardial infarction, and stroke. Atherosclerosis begins with an
alteration in the endothelium which lines the blood vessels. An
endothelial alteration may eventually result in the development of
an endothelial lesion caused, in part, by the uptake of oxidized
low-density lipoprotein (LDL) cholesterol. Rupture of this lesion
can lead to thrombosis and occlusion of the blood vessel. In the
case of a coronary artery, rupture of a complex lesion may
precipitate a myocardial infarction, whereas in the case of a
carotid artery, stroke may ensue.
[0005] In atherosclerotic coronary heart disease, endothelial
dysfunction may diminish production of vasodilatory substances,
such as nitric oxide. Myocardial ischemia results when
autoregulatory vasodilation is prevented, whether by flow-limiting
coronary arterial stenosis or by endothelial dysfunction. In both
cases, arterial blood flow can no longer increase proportional to
rising oxygen demands. In other situations, myocardial ischemia may
occur when oxygen demands are constant but there is a primary
decrease in coronary blood flow mediated via coronary artery spasm,
rapid evolution of the underlying atherosclerotic plaque leading to
a reduced coronary arterial lumen caliber, and/or intermittent
microvascular plugging by platelet aggregates.
[0006] Balloon angioplasty is commonly used to reopen a blood
vessel which is narrowed by plaque. Although balloon angioplasty is
successful in a high percentage of the cases in opening the vessel,
it often denudes the endothelium and injures the vessel in the
process. This damage causes the migration and proliferation of
vascular smooth muscle cells of the blood vessel into the area of
injury to form a lesion, known as myointimal hyperplasia or
restenosis. This new lesion leads to a recurrence of symptoms
within three to six months after the angioplasty in a significant
proportion of patients.
[0007] In atherosclerosis, thrombosis and restenosis there is also
a loss of normal vascular function, such that vessels tend to
constrict, rather than dilate. The excessive vasoconstriction of
the vessel causes further narrowing of the vessel lumen, limiting
blood flow. This can cause symptoms such as angina (if a heart
artery is involved), or transient cerebral ischemia (i.e. a "small
stroke", if a brain vessel is involved). This abnormal vascular
function (excessive vasoconstriction or inadequate vasodilation)
occurs in other disease states as well. Hypertension (high blood
pressure) is caused by excessive vasoconstriction, as well as
thickening, of the vessel wall, particularly in the smaller vessels
of the circulation. This process may affect the lung vessels as
well causing pulmonary (lung) hypertension. Other disorders known
to be associated with excessive vasoconstriction, or inadequate
vasodilation include transplant atherosclerosis, congestive heart
failure, toxemia of pregnancy, Raynaud's phenomenon, Prinzmetal's
angina (coronary vasospasm), cerebral vasospasm, hemolytic-uremia
and impotence.
[0008] A substance released by the endothelium, initially referred
to as "endothelium derived relaxing factor" (EDRF), plays an
important role in inhibiting these pathologic processes. EDRF is
now known to be nitric oxide (NO). NO plays many roles in human
physiology, including the relaxation of vascular smooth muscle, the
inhibition of platelet aggregation, the inhibition of mitogenesis,
the proliferation of vascular smooth muscle, and leukocyte
adherence. Because NO is the most potent endogenous vasodilator,
and because it is largely responsible for exercise-induced
vasodilation in the conduit arteries, enhancement of NO synthesis
could also improve exercise capacity in normal individuals and
those with vascular disease.
[0009] Endothelial nitric oxide synthase (eNOS) is the nitric oxide
synthase (NOS) isoform responsible for the maintenance of systemic
blood pressure, vascular remodeling and angiogenesis (Shesely et
al., 1996; Huang et al., 1995; Rudic et al., 1998; Murohara et al.,
1998). As deficient endothelial production of NO is an early,
persistent feature of atherosclerosis and vascular injury, eNOS has
proven to be an attractive target for vascular gene therapy. While
the regulation of eNOS activation remains largely undefined, it is
known that eNOS is phosphorylated in response to various forms of
cellular stimulation (Michel et al., 1993; Garcia-Cardena et al.,
1996; Corson et al., 1996), however, the role of phosphorylation in
the regulation of nitric oxide (NO) production and the kinase(s)
responsible has not been previously elucidated.
SUMMARY OF THE INVENTION
[0010] The present inventions result, in part, from the new
discovery that the serine/threonine protein kinase, Akt (protein
kinase B), can directly phosphorylate eNOS on a serine residue
corresponding to residue 1179 in bovine eNOS or residue 1177 in
human eNOS and activate the enzyme leading to NO production. Mutant
eNOS (S1179A or S1177A) is resistant to Akt phosphorylation and
activation while mutant eNOS (S1179D and S1177D) or (S1179E and
S1177E) is constitutively active. Moreover, using adenoviral
mediated gene transfer activated Akt increases basal NO release
from endothelial cells and activation deficient Akt attenuates VEGF
stimulated NO production. Thus, eNOS is a newly described Akt
substrate linking signal transduction via Akt to the release of the
gaseous second messenger, NO. The present inventions are also based
in part on the findings that mutant eNOS (S1179D) exhibits an
increase in the rate of NO production and an increase in reductase
activity.
[0011] The present invention includes NOS, polypeptides or proteins
and their encoding isolated nucleic acid molecules, wherein the NOS
polypeptide or protein contains a substituted amino acid residue
corresponding to residue 1179 of bovine eNOS, residue 1177 of human
eNOS, residue 1412 of rat nNOS, or residue 1415 at human nNOS.
Preferred substitutions include amino acids with negatively charged
R groups, including aspartic acid and glutamic acid.
[0012] The present invention also includes NOS polypeptides or
proteins and their encoding isolated nucleic acid molecules,
wherein the NOS polypeptide or protein contains a substituted amino
acid residue corresponding to residue 1179 of bovine eNOS, residue
1177 of human eNOS, residue 1412 of rat nNOS, or residue 1415 at
human nNOS. Preferred substitutions include amino acids with
non-negatively charged R groups, such as alanine.
[0013] The present invention provides methods for stimulating
collateral vessel development in ischemic diseases with deficient
endogenous angiogenesis, specifically peripheral vascular disease
and/or myocardial ischemia in a patient comprising delivering a
transgene coding for an NOS polypeptide of the invention or an Akt
polypeptide.
[0014] The invention further includes a non-human transgenic animal
which express an NOS polypeptide of the invention.
[0015] Lastly, the invention includes methods of identifying an
agent which modulates the Akt regulated activity of NOS, comprising
the general steps of: (a) exposing purified NOS, preferably eNOS or
nNOS, or a cell that expresses NOS, preferably eNOS or nNOS, and
Akt to an agent; and (b) measuring the Akt regulated activity of
NOS, preferably eNOS or nNOS.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A-1B. Wild-type Akt, but not kinase inactive Akt
increases NO release from cells expressing membrane associated
eNOS. In FIG. 1A, COS cells were transfected with plasmids for
eNOS, in the absence or presence of Akt or kinase inactive Akt
(K179M) and the production of NO (assayed as NO.sub.2.sup.-)
determined by chemiluminescence. In FIG. 1B, COS cells were
transfected with various NOS plasmids as above. In both FIG. 1A and
FIG. 1B, values for NO.sub.2.sup.- production were subtracted from
levels obtained from cells transfected with the
.beta.-galactosidase cDNA only. The inset shows the expression of
proteins in total cell lysates. Data are mean.+-.SEM, n=3-7
experiments; * denotes p<0.05.
[0017] FIGS. 2A-2D. Phosphorylation of eNOS by active Akt in vitro
and in vivo. In FIG. 2A, COS cells were transfected with HA-Akt or
HA-Akt(K179M), lysates were immunoprecipitated and placed into an
in vitro kinase reaction with histone 2B (25 mg) or recombinant
eNOS (3 mg) as substrates. The top panel depicts the incorporation
of .sup.32P into the substrates and the bottom panel shows the
amount of substrate by Coomassie staining of the gel. In FIG. 2B,
.sup.32P labeled wild-type or the double serine mutant of eNOS
(eNOS S635/1179) was affinity purified from transfected COS cells
and subjected to radiography (upper panel) or Western blotting
(lower panel). The graphical data in FIG. 2B reflects the relative
amount of labeled protein to the amount of immunoreactive eNOS in
the gel. In FIG. 2C, labeled eNOS was digested with trypsin and
peptides separated by RP-HPLC. The upper chromatogram documents a
predominant labeled tryptic peptide that co-migrates with a
unlabeled synthetic phosphopeptide standard (bottom chromatogram).
The insets demonstrate by linear mode MS of labeled peptide (top)
and phosphopeptide standard identical mass ions. In FIG. 2D,
recombinant wild-type eNOS or eNOS S1179A were purified and equal
amounts (2.4 mg) placed into an in vitro kinase reaction with
recombinant Akt as described in Methods. The top panel in FIG. 2D
depicts the incorporation of .sup.32P into eNOS and the bottom
panel shows the amount of substrate by Coomassie staining of the
gel. The graphical data (n=3) reflects the relative amount of
labeled eNOS to the mass of eNOS (Coomassie) in the in vitro kinase
reaction.
[0018] FIG. 3. Evidence that serine 1179 is functionally important
for Akt stimulated NO release. COS cells were transfected with
plasmids for wild-type eNOS or eNOS mutants, in the absence or
presence of Akt and the expression of the proteins and production
of NO (assayed as NO.sub.2.sup.-) determined. Interestingly,
constructs with the S1179 mutation to A were not activated by Akt
and mutation of S1179 to D resulted in a gain of function. In A,
data are mean.+-.SEM of 4-7 experiments; * represent significant
differences (p<0.05).
[0019] FIGS. 4A-4C. Akt regulates the basal and stimulated
production of NO in endothelial cells. In FIG. 4A, BLMVEC were
infected with adenoviral constructs (.beta.-gal as control, myr Akt
and AA-Akt) and the amount of NO.sub.2.sup.- produced over 24 hrs
determined (n=3). The inset shows the expression of eNOS and Akt.
In FIG. 4B, lysates from adenoviral infected BLMVEC were examined
for NOS activity. Equal amounts of protein (50 mg) were incubated
with various concentrations of free calcium and NOS activity
determined (n=3 experiments). In FIG. 4C, BLMVEC were infected with
adenoviruses as above followed by stimulation with VEGF (40 ng/ml)
for 30 min and NO.sub.2.sup.- release quantified by
chemiluminescence. Data are presented as VEGF stimulated release of
NO.sub.2.sup.- after subtraction of basal levels. Data are
mean.+-.SEM, n=4; * represent significant differences
(p<0.05).
[0020] FIGS. 5A and 5B. Purity and dimer/monomer ratio of wild type
and eNOS S1179D. In A and B, SDS-PAGE analysis was performed on
7.5% polyacrylamide gels stained with Coomassie Blue. Molecular
mass standards (lane 1) and their size in kDa are indicated at the
left. Wild type eNOS (lane 2) and eNOS S1179D (lane 3) (1 .mu.g of
each) were resolved as indicated by arrowheads. In B, proteins (2
.mu.g of each) were resolved on SDS-PAGE run at 4.degree. C.
Molecular mass standards were in lane 1. Nonboiled samples of wild
type and eNOS S1179D were resolved in lanes 2 and 3, respectively.
In lane 4, wild type-eNOS was boiled in SDS sample buffer.
[0021] FIGS. 6A and 6B. eNOS S1179D has higher rates of NO
production (A) and reductase activity (B) than does wild type eNOS.
In A, the rate of NO generated from wild type (.circle-solid.) and
S1179D (.largecircle.) eNOS was determined, using the hemoglobin
capture assay, as a function of L-arginine concentration, and data
are presented in a double reciprocal plot. In B, DCIP and
cytochrome c assays were performed in the presence or absence of
CaM. Values are mean.+-.S.E., n=46 determinations. Similar results
were obtained with at least three enzyme preparations. Significant
differences (p<0.05) between the wild type and S1179D eNOS are
indicated by the asterisks.
[0022] FIGS. 7A and 7B. NOS activities (A) and NADPH-dependent
reductase (B) are increased with eNOS S1179D compared with the wild
type enzyme. Hemoglobin capture (A) and NADPH-dependent cytochrome
c reduction (B) assays were performed on both wild type and S1179D
eNOS. In A, the rate of NO production was determined in the
presence of all NOS cofactors (wild type (filled symbols) and
S1179D (open symbols) eNOS). The rate of cytochrome c reduction was
performed in the absence of arginine and BH4 (A) for wild type
(circles) and S1179D (triangles) in the presence (filled symbols)
or absence of 120 nM calmodulin (open symbols). Values are
mean.+-.S.E., n=3-6 determinations from at least three enzyme
preparations.
[0023] FIGS. 8A-8D. Calmodulin- and calcium-dependent activation of
NOS and reductase activities are slightly enhanced for S1179D eNOS.
Calmodulin-dependent hemoglobin capture (A) and cytochrome c
reduction (B) were performed on both wild type (filled symbols) and
S1179D eNOS (open symbols). The rate of NO production detected by
hemoglobin capture method is in the presence of all NOS cofactors,
whereas cytochrome c reduction was performed in the absence of
arginine and BH4. In C and D, identical experiments were performed
in the presence of increasing concentrations of free calcium. The
insets in C and D depict the calcium-dependent turnover of S1179D
and wild type eNOS in both NO production and cytochrome c assays.
The maximal turnover rates were as follows for wild type and S1179D
eNOS, respectively: A, 22 and 43 min.sup.-1; B, 620 and 1400 min1;
C, 58 and 100 min1; and D, 1930 and 3810 min.sup.-1. Values are
mean.+-.S.E., n=3-6 determinations from at least three enzyme
preparations.
[0024] FIGS. 9A and 9B. EGTA-initiated inactivation of NOS is
reduced in S1179D eNOS. Hemoglobin capture (A) and reductase assays
(B) were performed as described earlier, with the following
modifications. The reaction was monitored for 1 min to determine
the initial rate; then, EGTA was added to the reaction mixture, and
the rate was monitored for an additional 1 min. The free calcium
concentration in the reaction was 200 .mu.M, and the amount of EGTA
added resulted in final concentrations of 0, 200, 400, and 600
.mu.M chelator. The specific activities are normalized to 100% for
wild type and S1179D eNOS. Values are mean.+-.S.E., n=3-6
determinations from at least three enzyme preparations. nd, no
detectable activity for wild type eNOS.
DETAILED DESCRIPTION OF THE INVENTION
A. General Description
[0025] The present inventions are based, in part, upon the
discovery that the serine/threonine protein kinase, Akt (protein
kinase B), can directly phosphorylate eNOS on serine 1179 (serine
1177 in human eNOS), and activate the enzyme leading to NO
production while mutant eNOS (S1179A) is resistant to Akt
phosphorylation and activation. Moreover, using adenoviral mediated
gene transfer activated Akt increases basal NO release from
endothelial cells and activation deficient Akt attenuates VEGF
stimulated NO production. Thus, eNOS is a newly described Akt
substrate linking signal transduction via Akt to the release of the
gaseous second messenger, NO. The present inventions are also based
in part on the findings that mutant eNOS, for instance, S1179D,
exhibits an increase in the rate of NO production and an increase
in reductase activity.
[0026] The demonstration that NO production is regulated by Akt
dependent phosphorylation of eNOS provides novel constitutively
active eNOS mutants for use in gene therapy aimed at improving
endothelial function in cardiovascular diseases associated with
dysfunction in the synthesis or biological activity of NO. Such
diseases include post angioplasty restenosis, hypertension,
atherosclerosis, heart failure including myocardial infarction,
diabetes, and diseases with defective angiogenesis. This discovery
also provides a novel therapeutic target useful for the design of
drugs useful for treating diseases associated with dysfunction in
the synthesis or biological activity of NO.
[0027] The present invention also provides novel constitutively
active nNOS mutants which have a substituted amino acid
corresponding to residue 1412 of rat nNOS or 1415 or human nNOS for
use in gene therapy aimed at the treatment of diseases.
B. Specific Embodiments
[0028] Production of NOS Mutant Proteins or Polypeptides
[0029] The present invention provides NOS proteins or polypeptides,
allelic variants of NOS proteins, and conservative amino acid
substitutions of NOS proteins, all of which contain a mutation of a
serine residue which is the site of Akt mediated phosphorylation.
For instance, the proteins or polypeptides of the invention include
but are not limited to: (1) human eNOS proteins which comprise a
mutation of residue 1177 (Janssens et al. (1992) J. Biol. Chem.
267:14519-14522 which is herein incorporated by reference in its
entirety) from a serine to another amino acid, such as alanine, and
are resistant to Akt mediated phosphorylation; (2) bovine eNOS
proteins which comprise a mutation of residue 1179 (SEQ ID NO: 2 of
U.S. Pat. No. 5,498,539, which is herein incorporated by reference
in its entirety) from a serine to another amino acid, such as
alanine, and are resistant to Akt mediated phosphorylation; (3)
human nNOS proteins which comprise a mutation of residue 1415 from
a serine to another amino acid, such as alanine, and are resistant
to Akt mediated phosphorylation; (4) rat nNOS proteins which
comprise a mutation of residue 1412 from a serine to another amino
acid, such as alanine, and are resistant to Akt mediated
phosphorylation; (5) human eNOS proteins which comprise a mutation
of residue 1177 from a serine to an amino acid containing a
negatively charged R group, such as aspartic or glutamic acid, and
are constitutively active and exhibit increased NO production and
increased reductase activity; (6) bovine eNOS proteins which
comprise a mutation of residue 1179 from a serine to an amino acid
containing a negatively charged R group, such as aspartic or
glutamic acid, and are constitutively active and exhibit increased
NO production and increased reductase activity; (7) human nNOS
proteins which comprise a mutation of residue 1415 from a serine to
an amino acid containing a negatively charged R group, such as
aspartic or glutamic acid, and are constitutively active and
exhibit increased NO production and increased reductase activity;
(8) rat nNOS proteins which comprise a mutation of residue 1412
from a serine to an amino acid containing a negatively charged R
group, such as aspartic or glutamic acid, and are constitutively
active and exhibit increased NO production and increased reductase
activity; and (9) NOS proteins from species other than humans,
cows, or rat which are modified to contain an amino acid other than
serine at a position corresponding to the serine at position 1177
in the human eNOS or position 1179 in the bovine eNOS, position
1412 in the rat nNOS, and position 1415 in the human nNOS and which
are either resistant to Akt phosphorylation, are constitutively
active, or exhibit increased NO production and increased reductase
activity. NOS mutants may also be produced by mutating other amino
acids in the phosphorylation motif RXRXXS/T.
[0030] The present invention provides constitutively active NOS
polypeptides, preferably eNOS or nNOS, exhibiting increased NO
production and reductase activity and comprising a mutation at the
serine residue at the site of Akt mediated phosphorylation. It is
also within the skill of the artisan to obtain conservative
variants such as substitutions, deletions, and insertions mutants
of these NOS polypeptides exhibiting increased NO production and
reductase activity. As used herein, a conservative variant refers
to alterations in the amino acid sequence that do not adversely
affect the ability of constitutively active NOS, preferably eNOS or
nNOS, to produce NO or the reductase activity of constitutively
active NOS, preferably eNOS or nNOS. A substitution, insertion, or
deletion is said to adversely affect constitutively active NOS
polypeptide, when the altered sequence affects the ability of
constitutive NOS, such that it does not produce NO at an increased
level and does not have increased reductase activity as compared to
the wild-type NOS. For example, the overall charge, structure or
hydrophobic/hydrophilic properties of constitutive NOS can be
altered without adversely affecting the activity of constitutive
NOS. Accordingly, the amino acid sequence of NOS polypeptide can be
altered, for instance to render the polypeptide more hydrophobic or
hydrophilic, without adversely affecting the activity of NOS.
[0031] As used herein, a "constitutively active" mutant or variant
of NOS, whether modified or isolated from a natural source, refers
to a NOS protein, preferably an eNOS or a nNOS, which produces NO
at a rate higher than native NOS containing a serine in its
unphosphorylated form at an amino acid residue corresponding to
residue 1177 in human NOS or residue 1179 in bovine NOS. Preferred
constitutively active variants comprise an amino acid with a
negatively charged R group, such as aspartic or glutamic acid, at
the amino acid residue corresponding to the serine at position 1177
in the human eNOS or position 1179 in the bovine NOS.
[0032] The present invention provides NOS proteins or polypeptides,
allelic variants of NOS proteins, and conservative amino acid
substitutions of NOS proteins that contain a substituted amino acid
residue corresponding to residue 1177 of bovine eNOS, to residue
1179 of human eNOS, to residue 1412 of rat nNOS, and to residue
1415 of human nNOS, wherein the substituted amino acid residue
comprises a non-negatively charged R group, such as alanine.
[0033] The NOS proteins, preferably eNOS or nNOS proteins, of the
present invention may be in isolated form. As used herein, a
protein is said to be isolated when physical, mechanical or
chemical methods are employed to remove the protein from cellular
constituents that are normally associated with the protein. A
skilled artisan can readily employ standard purification methods to
obtain an isolated protein.
[0034] Also included in the invention are NOS peptides which span
the Akt phosphorylation site corresponding to residue 1179 in
bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS
or residue 1415 in human nNOS. Peptides may contain a serine at the
phosphorylation site or, preferably, may contain a substitution of
the serine at position corresponding to residue 1179 in bovine
eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, or
residue 1415 in human nNOS. Such substitutions include, but are not
limited to, amino acids with an R group that mimics serine in its
phosphorylated state, such as aspartic acid or glutamic acid. Such
substitutions also include, amino acids with a non-negative R
group, such as alanine. Peptides spanning this site may be about 3,
5, 7, 10, 12, 15, 17, 20, 25, 30, 40, 50 or more amino acids in
length.
[0035] NOS proteins, polypeptides or peptides of the invention may
be prepared by any means available, including recombinant
expression from an NOS cDNA which has been modified to replace or
alter the nucleotide triplet encoding a serine corresponding to the
serine at position 1177 in the human eNOS, position 1179 in the
bovine eNOS, residue 1412 in rat nNOS, or residue 1415 in human
nNOS. Any available technique may be used to mutate the nucleotide
triplet encoding the serine residue, such as homologous
recombination, site-directed mutagenesis or PCR mutagenesis (see,
Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory
Press, 1989). Starting cDNAs may include the human and bovine eNOS
cDNAs as well as cDNAs encoding eNOS proteins of other animal
species, including but not limited to rabbit, rat, murine, porcine,
ovine, equine and non-human primate species.
[0036] As used herein, a nucleic acid molecule encoding a NOS
protein or polypeptide, preferably eNOS or nNOS protein or
polypeptide, of the invention is said to be "isolated" when the
nucleic acid molecule is substantially separated from contaminant
nucleic acid encoding other polypeptides from the source of nucleic
acid.
[0037] The present invention further provides fragments of the
encoding nucleic acid molecule. As used herein, a fragment of an
encoding nucleic acid molecule refers to a small portion of the
entire protein encoding sequence. The size of the fragment will be
determined by the intended use. For example, if the fragment is
chosen so as to encode an active portion of the protein, the
fragment will need to be large enough to encode the functional
region(s) of the protein, including the Akt phosphorylation site.
If the fragment is to be used as a nucleic acid probe or PCR
primer, then the fragment length is chosen so as to obtain a
relatively small number of false positives during probing/priming
to a region which spans or flanks the NOS Akt phosphorylation
site.
[0038] Fragments of the encoding nucleic acid molecules of the
present invention (i.e., synthetic oligonucleotides) that are used
as probes or specific primers for the polymerase chain reaction
(PCR), or to synthesize gene sequences encoding proteins of the
invention can easily be synthesized by chemical techniques, for
example, the phosphotriester method of Matteucci, et al. 1981 J.
Am. Chem. Soc. 103:3185-3191) or using synthesis methods. In
addition, larger DNA segments can readily be prepared by well known
methods, such as synthesis of a group of oligonucleotides that
define various modular segments of the gene, followed by ligation
of oligonucleotides to build the complete modified gene.
[0039] The encoding nucleic acid molecules of the present invention
may further be modified so as to contain a detectable label for
diagnostic and probe purposes. A variety of such labels are known
in the art and can readily be employed with the encoding molecules
herein described. Suitable labels include, but are not limited to,
biotin, radiolabeled nucleotides and the like. A skilled artisan
can employ any of the art known labels to obtain a labeled encoding
nucleic acid molecule.
[0040] The present invention further provides recombinant DNA
molecules (rDNAs) that contain an NOS coding sequence as described
above. As used herein, a rDNA molecule is a DNA molecule that has
been subjected to molecular manipulation. Methods for generating
rDNA molecules are well known in the art, for example, see Sambrook
et al., Molecular Cloning (1989). In the preferred rDNA molecules,
a coding DNA sequence is operably linked to expression control
sequences and/or vector sequences.
[0041] The choice of vector and/or expression control sequences to
which one of the protein family encoding sequences of the present
invention is operably linked depends directly, as is well known in
the art, on the functional properties desired, e.g., protein
expression, and the host cell to be transformed. A vector
contemplated by the present invention is at least capable of
directing the replication or insertion into the host chromosome,
and preferably also expression, of the structural gene included in
the rDNA molecule.
[0042] Expression control elements that are used for regulating the
expression of an operably linked protein encoding sequence are
known in the art and include, but are not limited to, inducible
promoters, constitutive promoters, secretion signals, and other
regulatory elements. Preferably, the inducible promoter is readily
controlled, such as being responsive to a nutrient in the host
cell's medium.
[0043] In one embodiment, the vector containing a coding nucleic
acid molecule will include a prokaryotic replicon, i.e., a DNA
sequence having the ability to direct autonomous replication and
maintenance of the recombinant DNA molecule extrachromosomally in a
prokaryotic host cell, such as a bacterial host cell transformed
therewith. Such replicons are well known in the art. In addition,
vectors that include a prokaryotic replicon may also include a gene
whose expression confers a detectable marker such as a drug
resistance. Typical bacterial drug resistance genes are those that
confer resistance to ampicillin or tetracycline.
[0044] Vectors that include a prokaryotic replicon can further
include a prokaryotic or bacteriophage promoter capable of
directing the expression (transcription and translation) of the
coding gene sequences in a bacterial host cell, such as E. coli. A
promoter is an expression control element formed by a DNA sequence
that permits binding of RNA polymerase and transcription to occur.
Promoter sequences compatible with bacterial hosts are typically
provided in plasmid vectors containing convenient restriction sites
for insertion of a DNA segment of the present invention. Typical of
such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available
from Biorad Laboratories, (Richmond, Calif.), pPL and pKK223
available from Pharmacia, Piscataway, N.J.
[0045] Expression vectors compatible with eukaryotic cells,
preferably those compatible with vertebrate cells, can also be used
to form a rDNA molecules that contains a coding sequence.
Eukaryotic cell expression vectors are well known in the art and
are available from several commercial sources. Typically, such
vectors are provided containing convenient restriction sites for
insertion of the desired DNA segment Typical of such vectors are
pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d (International
Biotechnologies, Inc.), pTDT1 (ATCC, #31255), and the like
eukaryotic expression vectors.
[0046] Eukaryotic cell expression vectors used to construct the
rDNA molecules of the present invention may further include a
selectable marker that is effective in an eukaryotic cell,
preferably a drug resistance selection marker. A preferred drug
resistance marker is the gene whose expression results in neomycin
resistance, i.e., the neomycin phosphotransferase (neo) gene.
(Southern et al. (1982) J. Mol. Anal. Genet. 1:327-341)
Alternatively, the selectable marker can be present on a separate
plasmid, and the two vectors are introduced by co-transfection of
the host cell and selected by culturing the appropriate drug for
the selectable marker.
[0047] The present invention further provides host cells
transformed or transfected with a nucleic acid molecule that
encodes an NOS protein, preferably eNOS or nNOS protein, of the
present invention. The host cell can be either prokaryotic or
eukaryotic. Eukaryotic cells useful for expression of a protein of
the invention are not limited, so long as the cell line is
compatible with cell culture methods and compatible with the
propagation of the expression vector and expression of the gene
product. Preferred eukaryotic host cells include, but are not
limited to, yeast, insect and mammalian cells, preferably
vertebrate cells such as those from a mouse, rat, monkey or human
cell line. Preferred eukaryotic host cells include Chinese hamster
ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse
embryo cells NIH/3T3 available from the ATCC as CRL 1658, baby
hamster kidney cells (BHK), and the like eukaryotic tissue culture
cell lines. Any prokaryotic host can be used to express a rDNA
molecule encoding a protein of the invention. The preferred
prokaryotic host is E. coli, particularly for the constitutively
active NOS mutants.
[0048] Transformation or transfection of appropriate cell hosts
with a rDNA molecule of the present invention is accomplished by
well known methods that typically depend on the type of vector used
and host system employed. With regard to transformation of
prokaryotic host cells, electroporation and salt treatment methods
are typically employed, see, for example, Cohen et al., (1972)
Proc. Natl. Acad. Sci. USA 69:2110; and Maniatis et al., Molecular
Cloning, A Laboratory Mammal, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1982). With regard to transformation of
vertebrate cells with vectors containing rDNAs, electroporation,
cationic lipid or salt treatment methods are typically employed,
see, for example, Graham et al. 1983) Virol. 52:456; Wigler et al.,
(1979) Proc. Natl. Acad. Sci. USA 76:1373-76. Successfully
transformed or transfected cells, i.e., cells that contain a rDNA
molecule of the present invention, can be identified by well known
techniques including the selection for a selectable marker. For
example, cells resulting from the introduction of an rDNA of the
present invention can be cloned to produce single colonies. Cells
from those colonies can be harvested, lysed and their DNA content
examined for the presence of the rDNA using a method such as that
described by Southern (1975) J. Mol. Biol. 98:503 or Berent et al.
(1985) Biotech. 3:208, or the proteins produced from the cell
assayed via an immunological method.
[0049] The present invention further provides methods for producing
a NOS protein, preferably an eNOS protein or a nNOS protein, of the
invention using nucleic acid molecules herein described. In general
terms, the production of a recombinant form of a protein typically
involves the following steps. A nucleic acid molecule is first
obtained that encodes a protein of the invention. If the encoding
sequence is uninterrupted by introns, it is directly suitable for
expression in any host. The nucleic acid molecule is then
preferably placed in operable linkage with suitable control
sequences, as described above, to form an expression unit
containing the protein open reading frame. The expression unit is
used to transform or transfect a suitable host and the transformed
or transfected host is cultured under conditions that allow the
production of the recombinant protein. Optionally the recombinant
protein is isolated from the medium or from the cells; recovery and
purification of the protein may not be necessary in some instances
where some impurities may be tolerated.
[0050] Each of the foregoing steps can be done in a variety of
ways. For example, the desired coding sequences may be obtained
from genomic fragments and used directly in appropriate hosts. The
construction of expression vectors that are operable in a variety
of hosts is accomplished using appropriate replicons and control
sequences, as set forth above. The control sequences, expression
vectors, and transformation or transfection methods are dependent
on the type of host cell used to express the gene and were
discussed in detail earlier. Suitable restriction sites can, if not
normally available, be added to the ends of the coding sequence so
as to provide an excisable gene to insert into these vectors. A
skilled artisan can readily adapt any host/expression system known
in the art for use with the nucleic acid molecules of the invention
to produce recombinant protein.
[0051] Gene Therapy
[0052] Any appropriate gene delivery system combined with a
suitable gene expression system using the most appropriate route of
delivery is encompassed by the present invention. For instance, NOS
mutant or variant genes, preferably eNOS or nNOS mutant or variant
genes, of the invention or Akt genes may be transferred to the
heart (or skeletal muscle), including cardiac myocytes (and
skeletal myocytes), in vitro or in vivo to direct production of the
encoded protein. Particularly useful are human Akt genes and NOS
mutants, preferably human eNOS, containing an amino acid with a
negatively charged R group, such as aspartic or glutamic acid, at a
position corresponding to serine 1177 in human eNOS. Routes of
administering NOS mutant or variant genes include, but are not
limited to, intravascular, intramuscular, intraperitoneal,
intradermal, and intraarterial injection.
[0053] The adenovirus gene delivery system offers several
advantages: adenovirus can (i) accommodate relatively large DNA
inserts; (ii) be grown to high-titer; (iii) infect a broad range of
cell types; and (iv) be used with a large number of available
vectors containing different promoters. Also, because adenoviruses
are stable in the bloodstream, they can be administered by
intravenous injection. A preferred delivery vector is a
helper-independent replication deficient human adenovirus 5,
although other delivery means are available and may be used,
including delivery of nucleic acids directly to the cells of
interest (see Sawa et al. (1998) Gene Ther. 5(11):1472-80;
Labhasetwar et al. (1998) J. Pharm. Sci. 87(11):1347-50; Lin et al.
(1997) Hypertension 30:307-313; Chen et al. (1997) Circ. Res.
80(3):327-335; Channon et al. (1996) Cardiovasc. Res. 32:962-972;
Harv Heart Lett. (1999) 9(8):5-6; and Nabel et al. (1999) Nat. Med.
5(2):141-2.
[0054] Using the adenovirus 5 system, transfection frequencies of
greater than 60% have been demonstrated in myocardial cells in vivo
by a single intracoronary injection (Giordano and Hammond (1994)
Clin. Res. 42: 123A). Non-replicative recombinant adenoviral
vectors are particularly useful in transfecting coronary
endothelium and cardiac myocytes resulting in highly efficient
transfection after intracoronary injection. Non-replicative
recombinant adenoviral vectors are also useful for transfecting
desired cells of the peripheral vascular system (see U.S. Pat. No.
5,792,453, which is herein incorporated by reference in its
entirety).
[0055] Adenoviral vectors used in the present invention can be
constructed by the rescue recombination technique described in
Graham et al. (1988) Virology 163:614-617. Briefly, the eNOS
transgene is cloned into a shuttle vector that contains a promoter,
polylinker and partial flanking adenoviral sequences from which
E1A/E1B genes have been deleted. As the shuttle vector, plasmid
pAC1 (Virology 163:614-617, 1988) (or an analog) which encodes
portions of the left end of the human adenovirus 5 genome (Virology
163:614-617, 1988) minus the early protein encoding E1A and E1B
sequences that are essential for viral replication, and plasmid
ACCMVPLPA (J. Biol. Chem. 267:25129-25134, 1992) which contains
polylinker, the CMV promoter and SV40 polyadenylation signal
flanked by partial adenoviral sequences from which the EA/E1B genes
have been deleted can be exemplified. The use of plasmid PAC1 or
ACCMVPLA facilitates the cloning process. The shuttle vector is
then co-transfected with a plasmid which contains the entire human
adenoviral 5 genome with a length too large to be encapsidated,
into 293 cells. Co-transfection can be conducted by calcium
phosphate precipitation or lipofection (Biotechniques 15:868-872,
1993). Plasmid JM17 encodes the entire human adenovirus 5 genome
plus portions of the vector pBR322 including the gene for
ampicillin resistance (4.3 kb). Although JM17 encodes all of the
adenoviral proteins necessary to make mature viral particles, it is
too large to be encapsidated (40 kb versus 36 kb for wild type). In
a small subset of co-transfected cells, rescue recombination
between the transgene containing the shuttle vector such as plasmid
pAC1 and the plasmid having the entire adenoviral 5 genome such as
plasmid pJM17 provides a recombinant genome that is deficient in
the E1A/E1B sequences, and that contains the transgene of interest
but secondarily loses the additional sequence such as the pBR322
sequences during recombination, thereby being small enough to be
encapsidated. The CMV driven beta-galactosidase encoding adenovirus
HCMVSP1lacZ (Clin. Res. 42:123A, 1994) can be used to evaluate
efficiency of gene transfer using X-gal treatment.
[0056] In another embodiment, the gene encoding NOS, preferably
eNOS or nNOS, may be introduced in vivo via an attenuated or
defective DNA virus, such as but not limited to herpes simplex
virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus,
and adeno-associated virus (AAV). Defective viruses, which entirely
or almost entirely lack viral genes, are preferred. Defective virus
is not infective after introduction into a cell. Use of defective,
vital vectors allows for administration to cells in a specific,
localized area, without concern that the vector can infect other
cells. Thus, a particular locus, e.g., in the brain or spinal
chord, can be specifically targeted with the vector. In a specific
embodiment, a defective herpes virus 1 (HSV1) vector may be used
(Kaplitt et al. (1991) Molec. Cell. Neurosci. 2:320-330). In yet
another embodiment, the viral vector is an attenuated adenovirus
vector, such as the vector described by Stratford-Perricaudet et
al. (J. Clin. Invest. 90:626-630 (1992)). In a yet a further
embodiment, the vector is a defective adeno-associated virus vector
(Samulski et al. (1987) J. Virol. 61:3096-3101; Samulski et al.
(1989) J. Virol. 63:3822-3828).
[0057] The present invention also contemplates the use of cell
targeting not only by delivery of the transgene into the coronary
artery, or femoral artery, for example, but also the use of
tissue-specific promoters. By fusing, for example, tissue-specific
transcriptional control sequences of left ventricular myosin light
chain-2 (MLC[2V]) or myosin heavy chain (MHC) to a transgene such
as the NOS genes of the invention within the adenoviral construct,
transgene expression is limited to ventricular cardiac myocytes.
The efficacy of gene expression and degree of specificity provided
by MLC[2V ] and MHC promoters with lacZ have been determined, using
the recombinant adenoviral system of the present invention.
Cardiac-specific expression has been reported previously by Lee et
al. (J. Biol. Chem. 267:15875-15885 (1992)). The MLC[2V] promoter
is comprised of 250 bp, and fits easily within the adenoviral-5
packaging constraints. The myosin heavy chain promoter, known to be
a vigorous promoter of transcription, provides a reasonable
alternative cardiac-specific promoter and is comprised of less than
300 bp. Smooth muscle cell promoters such as SM22 alpha promoter
(Kemp et al., (1995) Biochem J 310 (Pt 3):1037-43) and SM alpha
actin promoter (Shimizu et al. (1995) J Biol Chem 270(13):763-143)
are also available. Other promoters, such as the troponin-C
promoter, while highly efficacious and sufficiently small, lacks
adequate tissue specificity. By using the MLC[2V ] or MHC promoters
and delivering the transgene in vivo, it is believed that the
cardiac myocyte alone (that is without concomitant expression in
endothelial cells, smooth muscle cells, and fibroblasts within the
heart) will provide adequate expression of the NOS protein.
[0058] Limiting expression to the cardiac myocyte also has
advantages regarding the utility of gene transfer for the treatment
of clinical myocardial ischemia. By limiting expression to the
heart, one avoids the potentially harmful effect of angiogenesis in
non-cardiac tissues such as the retina. In addition, of the cells
in the heart, the myocyte would likely provide the longest
transgene expression since the cells do not undergo rapid turnover;
expression would not therefore be decreased by cell division and
death as would occur with endothelial cells. Endothelial-specific
promoters are already available for this purpose. Examples of
endothelial specific promoters include the Tie-2 promoter
(Schlaeger et al. (1997) Proc Natl Acad Sci 1; 94(7):3058-63), the
endothelin promoter (Lee et al. (1990) J. Biol. Chem.
265:10446-10450), and the eNOS promoter (Zhang et al. (1995) J
Biol. Chem 270(25):15320-6).
[0059] The present invention includes, with regard to the treatment
of heart disease, targeting the heart by intracoronary or
intramuscular injection with a high titer of the vector and
transfecting all cell types is presently preferred. Diseases such
as erectile dysfunction and cardiovascular diseases including,
myocardial infarction, myocardial ischemia, heart failure,
restenosis, stent stenosis, post-angioplasty stenosis, and by-pass
graft failure may be treated as described using the NOS transgenes,
preferably eNOS or nNOS transgenes.
[0060] Successful recombinant vectors can be plaque purified
according to standard methods. The resulting viral vectors are
propagated on 293 cells which provide E1A and E1B functions to in
trans to titers in the preferred about 10.sup.10-about 10.sup.12
viral particles/ml range. Cells can be infected at 80% confluence
and harvested 48 hours later. After 3 freeze-thaw cycles the
cellular debris is pelleted by centrifugation and the virus
purified by CsCl gradient ultracentrifugation (double CsCl gradient
ultracentrifugation is preferred). Prior to in vivo injection, the
viral stocks are desalted by gel filtration through Sepharose
columns such as G25 Sephadex. The product is then filtered through
a 30 micron filter, thereby reducing deleterious effects of
intracoronary injection of unfiltered virus (life threatening
cardiac arrhythmias) and promoting efficient gene transfer. The
resulting viral stock has a final viral titer in the range of
10.sup.10-10.sup.12 viral particles/ml. The recombinant adenovirus
must be highly purified, with no wild-type (potentially
replicative) virus. Impure constructs can cause an intense immune
response in the host animal. From this point of view, propagation
and purification may be conducted to exclude contaminants and
wild-type virus by, for example, identifying successful
recombinants with PCR using appropriate primers, conducting two
rounds of plaque purification, and double CsCl gradient
ultracentrifugation. Additionally, the problems associated with
cardiac arrhythmias induced by adenovirus vector injection into
patients can be avoided by filtration of the recombinant adenovirus
through an appropriately-sized filter prior to intracoronary
injection. This strategy also appears to substantially improve gene
transfer and expression.
[0061] The viral stock can be in the form of an injectable
preparation containing pharmaceutically acceptable carrier such as
saline, for example, as necessary. The final titer of the vector in
the injectable preparation is preferably in the range of about
10.sup.7-about 10.sup.13 viral particles which allows for effective
gene transfer. Other pharmaceutical carriers, formulations and
dosages are described below. The adenovirus transgene constructs
are delivered to the myocardium by direct intracoronary (or graft
vessel) injection using standard percutaneous catheter based
methods under fluoroscopic guidance, at an amount sufficient for
the transgene to be expressed to a degree which allows for highly
effective therapy. The injection may be made deeply into the lumen
(about 1 cm within the arterial lumen) of the coronary arteries (or
graft vessel), and preferably be made in both coronary arteries, as
the growth of collateral blood vessels is highly variable within
individual patients. By injecting the material directly into the
lumen of the coronary artery by coronary catheters, it is possible
to target the gene rather effectively, and to minimize loss of the
recombinant vectors to the proximal aorta during injection. It is
known that gene expression when delivered in this manner does not
occur in hepatocytes and viral RNA cannot be found in the urine at
any time after intracoronary injection. Any variety of coronary
catheter, or a Stack perfusion catheter, for example, can be used
in the present invention. In addition, other techniques known to
those having ordinary skill in the art can be used for transfer of
NOS genes, preferably eNOS or nNOS, to the arterial wall.
[0062] For the treatment of peripheral vascular disease, a disease
characterized by insufficient blood supply to the legs, recombinant
adenovirus expressing a NOS, preferably an eNOS or a nNOS, peptide
or protein of the invention may be delivered by a catheter inserted
into the proximal portion of the femoral artery or arteries,
thereby effecting gene transfer into the cells of the skeletal
muscles receiving blood flow from the femoral arteries.
[0063] In instances wherein a transgene or nucleic acid encoding an
NOS, preferably an eNOS or a nNOS, or Akt protein of the invention
is first transferred to endothelial or vascular smooth muscle cells
in vitro, including the patients own cells, DNA may be transfected
into the cells directly (see U.S. Pat. No. 5,658,565). Generally,
to transfect target cells, a plasmid vector comprising a DNA
sequence encoding an Akt or NOS of the invention or a biologically
active fragment thereof may be utilized in liposome-mediated
transfection of the target cell. The stability of liposomes,
coupled with the impermeable nature of these vesicles, makes them
useful vehicles for the delivery of therapeutic DNA sequences (for
a review, see Mannino and Gould-Forgerite (1988) BioTechniques
6(7): 682-690). Liposomes are known to be absorbed by many cell
types by fusion. In one embodiment, a cationic liposome containing
cationic cholesterol derivatives, such as SF-chol or DC-chol, may
be utilized. The DC-chol molecule includes a tertiary amino group,
a medium length spacer arm and a carbamoyl linker bond as described
by Gao and Huang (Biochem. Biophys. Res. Comm. 179: 280-285,
1991).
[0064] In another embodiment regarding the use of liposome
technology, the viral or nonviral based vector comprising the DNA
sequence encoding a biologically active NOS protein fragment,
preferably eNOS or nNOS protein fragment, is delivered to the
target cell by transfection of the target cell with lipofectamine
(Bethesda Research Laboratory). Lipofectamine is a 3:1 Liposome
formulation of the polycationic lipid 2,3
dioleyloxy-N-[2(sperminecarboxymido)ethyl]-N,N-dimethyl-1-propanaminiumtr-
ic fluoroacetate (DOPSA) and the neutral lipid
dioleoly-phosphatidylethano- lamine (DOPE).
[0065] Other non-viral modes of gene delivery include, but are not
limited to: (a) direct injection of naked DNA; (b) calcium
phosphate [Ca.sub.3(PO.sub.4).sub.2] mediated cell transfection;
(c) mammalian host cell transfection by electroporation; (d)
DEAE-dextran mediated cell transfection; (e) polybrene mediated
delivery; (f) protoplast fusion; (g) microinjection; and (h)
polylysine mediated transformation, with the genetically engineered
cells transferred back to the mammalian host.
[0066] Production of Transgenic Animals
[0067] Transgenic animals containing a mutant NOS gene, preferably
a mutant eNOS or nNOS gene, as described herein are also included
in the invention. Transgenic animals are genetically modified
animals into which recombinant, exogenous or cloned genetic
material has been experimentally transferred. Such genetic material
is often referred to as a "transgene". The nucleic acid sequence of
the transgene, in this case a form of NOS, may be integrated either
at a locus of a genome where that particular nucleic acid sequence
is not otherwise normally found or at the normal locus for the
transgene. The transgene may consist of nucleic acid sequences
derived from the genome of the same species or of a different
species than the species of the target animal.
[0068] The term "germ cell line transgenic animal" refers to a
transgenic animal in which the genetic alteration or genetic
information was introduced into a germ line cell, thereby
conferring the ability of the transgenic animal to transfer the
genetic information to offspring. If such offspring in fact possess
some or all of that alteration or genetic information, then they
too are transgenic animals.
[0069] The alteration or genetic information may be foreign to the
species of animal to which the recipient belongs, foreign only to
the particular individual recipient, or may be genetic information
already possessed by the recipient. In the last case, the altered
or introduced gene may be expressed differently than the native
gene.
[0070] Transgenic animals can be produced by a variety of different
methods including transfection, electroporation, microinjection,
gene targeting embryonic stem cells and recombinant viral and
retroviral infection (see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat.
No. 5,602,307; Mullins et al. (1993) Hypertension 22(4):630-633;
Brenin et al. (1997) Surg. Oncol. 6(2)99-110; Tuan (ed.),
Recombinant Gene Expression Protocols, Methods in Molecular Biology
No. 62, Humana Press (1997)).
[0071] A number of recombinant or transgenic mice have been
produced, including those which express an activated oncogene
sequence (U.S. Pat. No. 4,736,866); express simian SV 40 T-antigen
(U.S. Pat. No. 5,728,915); lack the expression of interferon
regulatory factor 1 (IRF-1) (U.S. Pat. No. 5,731,490); exhibit
dopaminergic dysfunction (U.S. Pat. No. 5,723,719); express at
least one human gene which participates in blood pressure control
(U.S. Pat. No. 5,731,489); display greater similarity to the
conditions existing in naturally occurring Alzheimer's disease
(U.S. Pat. No. 5,720,936); have a reduced capacity to mediate
cellular adhesion (U.S. Pat. No. 5,602,307); possess a bovine
growth hormone gene (Clutter et al. (1996) Genetics
143(4):1753-1760); or, are capable of generating a fully human
antibody response (McCarthy (1997) The Lancet 349(9049):405).
[0072] While mice and rats remain the animals of choice for most
transgenic experimentation, in some instances it is preferable or
even necessary to use alternative animal species. Transgenic
procedures have been successfully utilized in a variety of
non-murine animals, including sheep, goats, pigs, dogs, cats,
monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see
e.g., Kim et al. (1997) Mol. Reprod. Dev. 46(4):515-526; Houdebine
(1995) Reprod. Nutr. Dev. 35(6):609-617; Petters (1994) Reprod.
Fertil. Dev. 6(5):643-645; Schnieke et al. (1997) Science
278(5346):2130-2133; and Amoah (1997) J. Animal Science
75(2):578-585).
[0073] The method of introduction of nucleic acid fragments into
recombination competent mammalian cells can be by any method which
favors co-transformation of multiple nucleic acid molecules.
Detailed procedures for producing transgenic animals are readily
available to one skilled in the art, including the disclosures in
U.S. Pat. No. 5,489,743 and U.S. Pat. No. 5,602,307. Furthermore,
the production of NOS transgenic animals is well developed. For
instance, transgenic mice which inducibly express or overexpress
wild type eNOS have been produced (see Ohashi et al. (1998) J.
Clin. Invest. 102(12):2061-71; and Drummond et al. (1998) J. Clin.
Invest. 102(12):2033-4). These methods may be used to produce
transgenic mice which express the NOS mutants of the invention.
[0074] Therapeutic Screening Assays
[0075] The discovery that phosphorylation of eNOS regulates its
activity allows for the development of screening assays to identify
agents which modulate Akt regulated NOS, preferably eNOS or nNOS,
activity or expression. Any available format may be used, including
in vivo transgenic animal assays, in vitro protein based assays,
cell culture assays and high-throughput formats.
[0076] In many drug screening programs which test libraries of
compounds, high throughput assays are desirable in order to
maximize the number of compounds surveyed in a given period of
time. Assays which are performed in cell-free systems, such as may
be derived with purified or semi-purified proteins, are often
preferred as "primary" screens in that they can be generated to
permit rapid development and relatively easy detection of an
alteration in a molecular target which is mediated by a test
compound. Moreover, the effects of cellular toxicity and/or
bioavailability of the test compound can be generally ignored in
the in vitro system, the assay instead being focused primarily on
the effect of the drug on the molecular target as may be manifest
in an inhibition of, for instance, binding between molecules.
[0077] Cell or tissue culture based assays may be performed, for
example, by plating COS-7 cells (100 mm dish) and transfecting with
NOS (7.5-30 mg) and Akt (1 mg) plasmids using calcium phosphate. To
balance all transfections, an expression vector for
.beta.-galactosidase cDNA may be cotransfected. Twenty-four to
forty-eight hours after transfection, the expression of appropriate
proteins (40-80 mg) may be confirmed by Western blot analysis using
NOS mAb (9D10, Zymed), HA mAb (12CA5, Boehringer Mannheim), iNOS
pAb (Zymed Laboratories) or nNOS mAb (Zymed Laboratories).
[0078] Twenty-four to forty-eight hours after transfection, media
may be processed for the measurement of nitrite (NO.sub.2.sup.-),
the stable breakdown product of NO in aqueous solution, by NO
specific chemiluminescence as described (Sessa et al., 1995). Media
is deproteinized and samples containing NO.sub.2.sup.- are refluxed
in glacial acetic acid containing sodium iodide. Under these
conditions, NO.sub.2.sup.- is quantitatively reduced to NO which is
quantified by a chemiluminescence detector after reaction with
ozone in a NO analyzer (Sievers, Boulders, Colo.). In all
experiments, controls may be prepared by inhibiting NO.sub.2.sup.-
release by the use of a NOS inhibitor. In addition,
NO.sub.2.sup.--release from cells transfected with the
.beta.-galactosidase cDNA may subtracted to control for background
levels of NO.sub.2.sup.- found in serum or media. cGMP accumulation
in COS may also be used as a bioassay for the production of NO as
described. In an alternative format, the conversion of
.sup.3H-L-arginine to .sup.3H-L-citrulline may be used to
determined NOS activity in COS cell or endothelial cell lysates as
previously described (Garcia-Cardena et al., 1998).
[0079] For in vivo phosphorylation studies, COS cells may be
transfected with the cDNAs for wild-type or S635 (control), bovine
1179A, D, or E eNOS, human 1177 D or E eNOS, rat 1412D or E nNOS,
human 1415D or E nNOS and HA-Akt overnight. 36 hrs after
transfection, cells are placed into dialyzed serum replete,
phosphate-free Dulbecco's minimum essential medium supplemented
with 80 .mu.Ci/ml of .sup.32P orthophosphoric acid for 3 hr. A cell
aliquot may be pretreated with wortmannin (500 nM) in the
phosphate-free media for 1 hr and during the labeling. Lysates are
then harvested, NOS solubilized and partially purified by ADP
sepharose affinity chromatography as previously described and the
.sup.32P incorporation into NOS visualized after SDS-PAGE (7.5%) by
autoradiography and the amount of NOS protein verified by Western
blotting for NOS.
[0080] For in vitro phosphorylation studies, recombinant NOS
purified from E. coli, eNOS purified from another source, or NOS
peptides spanning the Akt phosphorylation site are incubated with
wild-type or kinase inactive Akt immunoprecipitated from
transfected COS cells. Briefly, the NOS proteins or peptides are
incubated with .sup.32P g-ATP (2 ml, specific activity 3000
Ci/mmol), ATP (50 mM), DTT (1 mM), in a buffer containing HEPES (20
mM, pH=7.4), MnCl.sub.2 (10 mM), MgCl.sub.2 (10 mM) and
immunoprecipitated Akt for 20 min at room temperature.
[0081] In experiments examining the in vitro phosphorylation of
wild-type and mutant NOS, recombinant Akt (1 mg) purified from
baculovirus infected SF9 cells, is incubated with wild-type, S1179A
bovine eNOS, S1177A human eNOS, S1179 D or E bovine eNOS, S1177D or
E human eNOS, S1412D or E rat nNOS, or S1415D or E human nNOS using
essentially the same conditions as above. Proteins may be resolved
by SDS-PAGE and .sup.32P incorporation and the amount of protein
determined by Coomassie staining as above.
[0082] The above screening assays which assay the Akt dependent
phosphorylation or activation of NOS, preferably eNOS or nNOS, may
be used to screen for a wide-variety of agents. For instance,
agents which inhibit the dephosphorylation of NOS (phosphatase
inhibitors) at an amino acid corresponding to serine 1179 in bovine
eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, or 1415
in human nNOS may be useful therapeutic molecules. Similarly,
agents which activate Akt or which mimic the Akt phosphorylation
site on eNOS may be useful therapeutic molecules.
[0083] Agents that are assayed in the above methods can be randomly
selected or rationally selected or designed. As used herein, an
agent is said to be randomly selected when the agent is chosen
randomly without considering the specific sequences involved in the
association of the a protein of the invention alone or with its
associated substrates, binding partners, etc. An example of
randomly selected agents is the use a chemical library or a peptide
combinatorial library, or a growth broth of an organism.
[0084] As used herein, an agent is said to be rationally selected
or designed when the agent is chosen on a nonrandom basis which
takes into account the sequence of the target site and/or its
conformation in connection with the agent's action. For example, a
rationally selected peptide agent can be a peptide whose amino acid
sequence is similar to the Akt phosphorylation site in NOS,
particularly, peptides or small molecules that mimic the NOS
phosphorylation state.
[0085] The agents of the present invention can be, as examples,
peptides, small molecules, vitamin derivatives, as well as
carbohydrates. A skilled artisan can readily recognize that there
is no limit as to the structural nature of the agents of the
present invention.
[0086] The peptide agents of the invention can be prepared using
standard solid phase (or solution phase) peptide synthesis methods,
as is known in the art. In addition, the DNA encoding these
peptides may be synthesized using commercially available
oligonucleotide synthesis instrumentation and produced
recombinantly using standard recombinant production systems. The
production using solid phase peptide synthesis is necessitated if
non-gene-encoded amino acids are to be included.
[0087] Another class of agents of the present invention are
antibodies immunoreactive with critical positions of proteins of
the invention. Antibody agents are obtained by immunization of
suitable mammalian subjects with peptides, containing as antigenic
regions, those portions of the protein intended to be targeted by
the antibodies.
[0088] Use of Agents Identified as Modulating eNOS Activity
[0089] The agents of the present invention, such as agents that
inhibit the dephosphorylation of NOS (phosphatase inhibitors) at an
amino acid corresponding to serine 1179 in bovine eNOS, residue
1177 in human eNOS, residue 1412 in rat nNOS, or residue 1415 in
human nNOS, as well as agents which activate Akt or which mimic the
Akt phosphorylation site on NOS, can be administered via
parenteral, subcutaneous, intravenous, intramuscular,
intraperitoneal, transdermal, or buccal routes. Alternatively, or
concurrently, administration may be by the oral route. The dosage
administered will be dependent upon the age, health, and weight of
the recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired. As described
below, there are many methods that can readily be adapted to
administer such agents.
[0090] The present invention further provides compositions
containing one or more agents of the invention. While individual
needs vary, a determination of optimal ranges of effective amounts
of each component is within the skill of the art. Typical dosages
comprise 0.1 to 100 mg/kg body wt. The preferred dosages comprise
0.1 to 10 mg/kg body wt. The most preferred dosages comprise 0.1 to
1 mg/kg body wt.
[0091] In addition to the pharmacologically active agent, the
compositions of the present invention may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically for delivery
to the site of action. Suitable formulations for parenteral
administration include aqueous solutions of the active compounds in
water-soluble form, for example, water-soluble salts. In addition,
suspensions of the active compounds as appropriate oily injection
suspensions may be administered. Suitable lipophilic solvents or
vehicles include fatty oils, for example, sesame oil, or synthetic
fatty acid esters, for example, ethyl oleate or triglycerides.
Aqueous injection suspensions may contain substances which increase
the viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol and/or dextran. Optionally, the
suspension may also contain stabilizers. Liposomes can also be used
to encapsulate the agent for delivery into the cell.
[0092] The pharmaceutical formulation for systemic administration
according to the invention may be formulated for enteral,
parenteral or topical administration. Indeed, all three types of
formulations may be used simultaneously to achieve systemic
administration of the active ingredient.
[0093] Suitable formulations for oral administration include hard
or soft gelatin capsules, pills, tablets, including coated tablets,
elixirs, suspensions, syrups or inhalations and controlled release
forms thereof.
[0094] In practicing the methods of this invention, the compounds
of this invention may be used alone or in combination, or in
combination with other therapeutic or diagnostic agents. In certain
preferred embodiments, the compounds of this invention may be
coadministered along with other compounds typically prescribed for
these conditions according to generally accepted medical practice,
such as anticoagulant agents, thrombolytic agents, or other
antithrombotics, including platelet aggregation inhibitors, tissue
plasminogen activators, urokinase, prourokinase, streptokinase,
heparin, aspirin, or warfarin. The compounds of this invention can
be utilized in vivo, ordinarily in mammals, such as humans, sheep,
horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
[0095] The following working examples specifically point out
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the disclosure.
Other generic configurations will be apparent to one skilled in the
art.
EXAMPLES
[0096] The following procedures were employed in Examples 1-2
[0097] Cell Transfections: The bovine eNOS, human iNOS, rat nNOS
cDNAs in pcDNA3 and HA-tagged wild-type Akt, Akt (K179M) or myr-Akt
in pCMV6 were generated by standard cloning methods. The myr-nNOS
in pcDNA3 was generated by PCR, incorporating a new amino terminus
containing the eNOS N-myristoylation consensus site (MGNLKSVG, SEQ
ID NO:1) fused in frame to the second amino acid of the nNOS coding
sequence. In preliminary experiments in COS cells, this construct
was N-myristoylated based on incorporation of .sup.3H-myristic acid
whereas native nNOS is not and resulted in approximately 60% of the
total protein targeted into the membrane fraction of cells whereas
only 5-10% of nNOS was membrane associated in COS cells. Mutation
of the putative Akt phosphorylation sites in eNOS were generated
using the Quick Change site-directed mutagenesis kit (Stratagene)
according to the manufacturers instructions. All mutants were
verified by DNA sequencing. COS-7 cells were plated (100 mm dish)
and transfected with the NOS (7.5-30 mg) and Akt (1 mg) plasmids
using calcium phosphate. To balance all transfections, the
expression vector for .beta.-galactosidase cDNA was used.
Twenty-four to forty-eight hours after transfection, the expression
of appropriate proteins (40-80 mg) were confirmed by Western blot
analysis using eNOS mAb (9D10, Zymed), HA mAb (12CA5, Boehringer
Mannheim), iNOS pAb (Zymed Laboratories) or nNOS mAb (Zymed
Laboratories).
[0098] NO release from transfected COS cells: 24-48 hrs after
transfection, media was processed for the measurement of nitrite
(NO.sub.2.sup.-), the stable breakdown product of NO in aqueous
solution, by NO specific chemiluminescence as described (Sessa et
al., 1995). Media was deproteinized and samples containing
NO.sub.2.sup.- were refluxed in glacial acetic acid containing
sodium iodide. Under these conditions, NO.sub.2.sup.- was
quantitatively reduced to NO which was quantified by a
chemiluminescence detector after reaction with ozone in a NO
analyzer (Sievers, Boulders, Colo.). In all experiments,
NO.sub.2.sup.- release was inhibitable by a NOS inhibitor. In
addition, NO.sub.2.sup.--release from cells transfected with the
.beta.-galactosidase cDNA was subtracted to control for background
levels of NO.sub.2.sup.- found in serum or media. In some
experiments, cGMP accumulation in COS was used as a bioassay for
the production of NO as described.
[0099] NOS activity assays: The conversion of .sup.3H -L-arginine
to .sup.3H-L-citrulline was used to determine NOS activity in COS
cell or endothelial cell lysates as previously described by
Garcia-Cardena et al. (1998).
[0100] Phosphorylation studies in vivo and in vitro: For in vivo
phosphorylation studies, COS cells were transfected with the cDNAs
for wild-type or S635, 1179A eNOS and HA-Akt overnight. 36 hrs
after transfection, cells were placed into dialyzed serum replete,
phosphate-free Dulbecco's minimum essential medium supplemented
with 80 .mu.Ci/ml of .sup.32P orthophosphoric acid for 3 hr. Some
cells were pretreated with wortmannin (500 nM) in the
phosphate-free media for 1 hr and during the labeling. Lysates were
harvested, eNOS solubilized and partially purified by ADP sepharose
affinity chromatography as previously described and the .sup.32P
incorporation into eNOS visualized after SDS-PAGE (7.5%) by
autoradiography and the amount of eNOS protein verified by Western
blotting for eNOS. For in vitro phosphorylation studies,
recombinant eNOS purified from E. coli was incubated with wild-type
or kinase inactive Akt immunoprecipitated from transfected COS
cells. eNOS was incubated with .sup.32P g-ATP (2 ml, specific
activity 3000 Ci/mmol), ATP (50 mM), DTT (1 mM), in a buffer
containing HEPES (20 mM, pH=7.4), MnCl.sub.2 (10 mM), MgCl.sub.2
(10 mM) and immunoprecipitated Akt for 20 min at room
temperature.
[0101] In experiments examining the in vitro phosphorylation of
wild-type and mutant eNOS, recombinant Akt (1 mg) purified from
baculovirus infected SF9 cells, was incubated with wild-type or
S1179A eNOS (2.4 mg, purified from E. coli) using essentially the
same conditions as above. Proteins were resolved by SDS-PAGE and
.sup.32P incorporation and the amount of protein determined by
Coomassie staining as above.
[0102] In studies identifying the labeled eNOS peptide,
immunoprecipitated Akt was incubated with recombinant eNOS as
above. The sample was run on SDS-PAGE, and the eNOS band digested
in gel, and the resultant tryptic fragments purified by RP-HPLC.
Peptide mass and .sup.32P incorporation were monitored and the
prominent labeled peak further analyzed by mass spectrometry. In
other experiments, peptides corresponding to the potential Akt
phosphorylation site were synthesized, purified by HPLC and
verified by mass spectrometry (W. M. Keck Biotechnology Resource
Center, Yale University School of Medicine). The wild-type peptide
was 1174RIRTQSFSLQERHLRGAVPWA1194 (SEQ ID NO:2) and the mutant
peptide was identical except S1179 was changed to an alanine. In
vitro kinase reactions were essentially as described above
incubating peptides (25 mg) with recombinant Akt (1 mg). Reactions
were then spotted onto phosphocellulose filters and the amount of
phosphate incorporated measured by Cerenkov counting.
[0103] Adenoviral infections and NO release in endothelial cells:
Bovine lung microvascular endothelial cells (BLMVEC) were cultured
in either in 100 mm dishes (for basal NO release and NOS activity
assays) or C6 well plates (for stimulated NO) as previously
described (Garcia-Cardena et al., 1996a). BLMVEC were infected with
200 MOI of adenovirus containing the .beta.-galactosidase 29,
HA-tagged, inactive phosphorylation mutant Akt (AA-Akt; Alessi et
al., 1996) or carboxyl terminal HA-tagged constitutively active Akt
(myr-Akt) for 4 hrs. The virus was removed and cells left to
recover for 18 hrs in complete medium. In preliminary experiments
with the .beta.-galactosidase virus, these conditions were optimal
for infecting 100% of the cultures. For measurement of basal NO
production, media was collected for NO release 24 hrs after the
initial infection with virus. For measurement of stimulated NO
release, cells were then washed with serum-free medium followed by
stimulation with VEGF (40 ng/ml) for 30 min. In some experiments
the calcium dependency of NOS was determined 24 hrs after
adenoviral infection. Infected cells were lysed in NOS assay buffer
containing 1% NP40, and detergent soluble material used for
activity. Lysates were incubated with EGTA buffered calcium to
yield appropriate amounts of free calcium in the incubation.
[0104] Statistics: Data are expressed as mean.+-.SEM. Comparison's
were made using a two-tailed, Student's t-test or ANOVA with a
post-hoc test where appropriate. Differences were considered to be
significant at p<0.05.
Example 1
Akt Modulates NO Production from eNOS
[0105] To explore the possibility that a downstream effector of
PI-3 kinase, Akt, could directly influence the production of NO,
COS-7 cells (which do not express NOS) were co-transfected with
eNOS and wild type Akt (HA-Akt), or kinase inactive Akt (HA-Akt
K179M) and the accumulation of nitrite (NO.sub.2.sup.-) measured by
NO specific chemiluminescence. Transfection of eNOS results in an
increase in NO.sub.2.sup.- accumulation, an effect that is markedly
enhanced by co-transfection of wild-type Akt, but not the kinase
inactive variant (FIG. 1A). Identical results were obtained using
cGMP as a bioassay for biologically active NO. Under these
experimental conditions, Akt was catalytically active as determined
by Western blotting with a phospho-Akt specific Ab (which
recognizes serine 473; not shown) and Akt activity assays (see FIG.
2A). Transfection of a constitutively active form of Akt (myr-Akt)
increases cGMP accumulation (assayed in COS cells) from 5.5.+-.0.8
to 11.6.+-.0.9 pmol cGMP/mg protein (in cells transfected with eNOS
alone or eNOS with myr-Akt, respectively) whereas the kinase
inactive Akt did not influence cGMP accumulation (5.8.+-.0.8 pmol
cGMP, n=4 experiments). As seen in the inset, equal levels of eNOS
and Akt were expressed in COS cell lysates suggesting that Akt
modulates eNOS thereby increasing NO production under basal
conditions.
[0106] eNOS is a dually acylated peripheral membrane protein that
targets into the Golgi region and plasma membrane of endothelial
cells (Liu et al., 1997; Garcia-Cardena et al., 1996a; Shaal et
al., 1996) and compartmentalization is required for efficient
production of NO in response to agonist challenge (Sessa et al.,
1995; Liu et al., 1996; Kantor et al., 1996). To examine if eNOS
activation by Akt requires membrane compartmentalization, COS-7
cells were co-transfected with cDNAs for Akt and a myristoylation,
palmitoylation defective mutant of eNOS (G2A eNOS) and the release
of NO quantified. As seen in FIG. 1B, Akt did not activate the
non-acylated form of eNOS suggesting that compartmentalization of
both proteins to the membrane is required for their functional
interaction. (Downward, et al., 1998). Next, it was determined if
Akt could activate structurally similar but distinct soluble NOS
isoforms, neuronal and inducible NOS (nNOS and iNOS, respectively).
Co-transfection Akt with nNOS and iNOS did not result in a further
increase in NO release demonstrating the specificity of Akt for
eNOS. However, the addition of an N-myristoylation site to nNOS, in
order to enhance its interactions with biological membranes,
results in Akt stimulation of nNOS in a manner analogous to that
seen with eNOS, suggesting that both isoforms may be susceptible to
activation by Akt kinase when membrane anchored.
Example 2
Production of eNOS Mutations
[0107] The above experiments imply that Akt, perhaps via
phosphorylation of eNOS, can modulate NO release from intact cells.
Indeed, two putative Akt phosphorylation motifs (RXRXXS/T) are
present in eNOS (serines 635 and 1179 in bovine eNOS or serines 633
and 1177 in human eNOS) and one motif present in nNOS (serines 1412
in rat and 1415 human nNOS), with no obvious motifs found in iNOS.
To examine if eNOS is a potential substrate for Akt phosphorylation
in vitro, COS cells were transfected with HA-Akt or HA-Akt (K179M)
and kinase activity assessed using recombinant eNOS as a substrate.
As seen in FIG. 2A, the active kinase phosphorylates histone 2B and
eNOS (69.3.+-.2.9 and 115.4.+-.3.8 pmol of ATP/nmol substrate,
respectively, n=3), whereas the inactive Akt did not significantly
increase histone or eNOS phosphorylation. To elucidate if the
putative Akt phosphorylation sites in eNOS were responsible for the
incorporation of .sup.32P, the two serines were mutated to alanine
residues and the ability of Akt to stimulate wild-type and mutant
eNOS phosphorylation examined in intact COS cells. Transfected
cells were labeled with .sup.32P -orthophosphate, eNOS partially
purified by ADP-sepharose affinity chromatography, and the
phosphorylation state and protein levels quantified. As seen in
FIG. 2B, co-expression of Akt results in a 2 fold enhancement in
the phosphorylation of eNOS relative to non-stimulated cells.
Pretreatment of eNOS/Akt transfected cells with wortmannin
abolished the Akt induced increase in phosphorylation. Moreover,
mutation of serine 635 and 1179 to alanine residues abolished Akt
dependent phosphorylation of eNOS suggesting that these residues
could serve as potential phosphorylation sites in intact cells.
[0108] To directly identify the residues phosphorylated by Akt,
wild-type eNOS was incubated with immunopurified Akt and the sites
of phosphorylation determined by HPLC followed by MALDi-mass
spectrometry (MALDi-MS). As seen in FIG. 2C, the primary
.sup.32P-labeled tryptic phosphopeptide co-elutes with a synthetic
phosphopeptide (amino acids 1177-1185 with phosphoserine at
position 1179) and has the identical mass ion as determined by
linear mode MS. Using reflectron mode MALDi-MS monitoring, both the
labeled tryptic peptide and the standard phosphopeptide
demonstrated a loss of HP.sub.3O.sub.4 indicating that the tryptic
peptide was phosphorylated. In addition, mutation of S1179 to A
markedly reduces Akt-dependent phosphorylation of eNOS compared to
the wild-type protein (FIG. 2D). Identical results were obtained
utilizing peptides (amino acids 1174-1194) derived from wild-type
or eNOS S1179A as substrates for recombinant Akt (wild-type peptide
incorporated 24.6.+-.3.7 nmol phosphate/mg compared to the alanine
mutant peptide which incorporated 0.22.+-.0.02 nmol phosphate/mg,
n=5). Collectively, these data demonstrate that eNOS is a substrate
for Akt and that the primary site of phosphorylation is serine 1179
(serine 1177 in human eNOS).
[0109] Next we examined the functional significance of the putative
Akt phosphorylation site at serine 635 and the identified site at
serine 1179. Transfection of COS cells with the double mutant eNOS
S635/1179A abolishes Akt dependent NO release. Mutation of serine
635 to alanine did not attenuate NO release whereas eNOS S1179A
abolishes Akt dependent activation of eNOS (FIG. 3). These results
suggest that serine 1179 is functionally important for NO release.
Mutation of serine 1179 into aspartic acid (eNOS S1179D) to
substitute for the negative charge afforded by the addition of
phosphate, partially mimics the activation state induced by Akt
(S1177D in human eNOS). All site directed mutants were amply
expressed (see inset Western blots) and retained NOS catalytic
activity in cell lysates (in COS cells transfected with eNOS only,
NOS activity was 85.3.+-.27.0, 71.9.+-.2.9, 80.8.+-.23.2 and
131.8.+-.36.7 pmol L-citrulline generated/mg protein from lysates
of COS cells expressing wild type, S1179A, S635, 1179A and eNOS
S1179D, respectively, n=3 experiments).
[0110] To examine if Akt mediates NO release from endothelial
cells, bovine lung microvascular endothelial cells (BLMVEC) were
infected with adenoviruses expressing activated Akt (myr-Akt),
activation deficient Akt (AA-Akt) or .beta.-galactosidase as a
control and the accumulation of NO measured. As seen in FIG. 4A,
myr-Akt stimulates the basal production of NO from BLMVEC, whereas
cells infected with .beta.-galactosidase or activation deficient
Akt released low levels of NO that were close to the limits of
detection. These data in conjunction with similar results in COS
cells suggests that Akt phosphorylation of eNOS is sufficient to
regulate NO production at resting levels of calcium. Indeed, NOS
activity measured in lysates from myr-Akt infected BLMVEC
demonstrates that the sensitivity of the enzyme to activation by
calcium, assayed at a fixed calmodulin concentration, is enhanced
relative to NOS activity seen in BLMVEC infected with the
.beta.-galactosidase virus (FIG. 4B). Interestingly, the calcium
sensitivity of NOS activity in cells infected with activation
deficient Akt was greatly suppressed relative to both myr-Akt and
.beta.-galactosidase infected cells.
[0111] Treatment of endothelial cells with VEGF is known to
activate Akt 23 and the release of NO through a mechanism partially
blocked by inhibitors of PI-3 kinase (Papapetropoulos et al.,
1997). To examine the functional link between VEGF as an agonist
for NO release and Akt activation BLMVEC were infected with
adenoviruses for myr-Akt, AA-Akt or .beta.-galactosidase and VEGF
stimulated NO release quantified. As seen in FIG. 4C, infection of
endothelial cells with myr-Akt enhances VEGF driven NO production
while the AA-Akt attenuates NO release. These results imply that
Akt participates in the signal transduction events required for
both basal and stimulated NO production in endothelial cells.
[0112] Collectively these data demonstrate that Akt can
phosphorylate eNOS on serine 1179 (serine 1177 in human eNOS) and
that phosphorylation enhances the ability of the enzyme to generate
NO.
Example 3
Materials and Methods
[0113] eNOS Constructs and Protein Purification--Wild type bovine
eNOS in the plasmid pCW was expressed as described previously with
groELS in E. coli BL21 cells (Martasek et al., 1996). The S1179D
mutant eNOS for expression in E. coli was generated as follows.
eNOS S1179D in pcDNA 3 (Fulton et al., 1999) was digested with
XhoI/XbaI, subcloned into the identical sites of eNOS in pCW, and
co-expressed with groELS. Isolation of recombinant eNOS was
preformed as reported previously (Roman et al., 1995; Martasek et
al., 1999), with the following modifications. eNOS was eluted from
2'5'-ADP Sepharose with either 10 mM NADPH or 10 mM 2'-AMP. The
amount of eNOS was quantitated using the peak absorbance at 409-412
nm, with an extinction coefficient for heme content of 0.1
.mu.M.sup.-1 cm.sup.-1. The purity of eNOS was determined by 7.5%
SDS-PAGE followed by Coomassie staining. Low temperature SDS-PAGE
was performed identically, except that samples were not boiled and
the electrophoresis was carried out at 4.degree. C. in a slurry of
ice/water (Klatt et al., 1995). In experiments in which NOS
cofactors (L-arginine, calmodulin, and NADPH) were titrated, they
were omitted from the purification and storage of the enzymes and
were incubated as described below.
[0114] Assay for NOS Activity--NO production was measured by the
hemoglobin capture assay as described (Kelm et al., 1988). Briefly,
the reaction mixture contained eNOS (0.5-2.5 .mu.g), oxyhemoglobin
(8 .mu.M), L-arginine (100 .mu.M), BH4 (5 .mu.M), CaCl2 (120
.mu.M), calmodulin (120-200 nM), and NADPH (100 .mu.M) in HEPES
buffer (50 mM), pH 7.4. In the determination of calcium EC50 value
for eNOS, the above reaction mixture was modified as follows: MOPS
buffer (10 mM, pH 7.6), KCl (100 mM) and CaM (250 nM) were
substituted. Under these conditions, free calcium was calculated
using the WinMAXC program, version 1.8 (Stanford University), with
a Kd of 2.2.times.10.sup.-8 M. The precise free calcium
concentration was achieved by mixing an appropriate proportion of
10 mM K.sub.2EGTA and 10 mM CaEGTA stock solutions (Molecular
Probes). NOS activity was monitored for linearity over 2 min at 401
nm, and NO production was calculated based on the change of
absorbance using the extinction coefficient of 60 mM.sup.-1
cm.sup.-1. All reactions were carried out at 23.degree. C., and
each data point represents 3-8 observations. The extinction
coefficient of 0.0033 .mu.M.sup.-1 cm.sup.-1 at 276 nm was used for
determination of calmodulin concentration. The production of NO
using this method was completely blocked by the addition of
nitro-L-arginine (1 mM). When the inactivation of eNOS was
determined by the addition of EGTA (200-800 .mu.M) to the reaction
mixture, chelator was added 1 min after initiation of the reaction
by NADPH. Identical conditions were used when NADPH-cytochrome c
reductase activity was examined. These reactions contained CaM (120
nM) and CaCl.sub.2 (200 .mu.M) in a 0.5-ml volume with eNOS (0.5
.mu.g).
[0115] The conversion of L-arginine to L-citrulline was assayed as
described previously by Bredt et al. (1990). Briefly, eNOS (0.25-2
.mu.g) was incubated for 3-10 min at 23.degree. C. in the following
reaction mixture: 3 pmol of L-[3H ]arginine (55 Ci/mmol), 10-300
.mu.M arginine, 1 mM NADPH, 120-200 nM calmodulin, 2 mM CaCl2, and
30 .mu.M BH4 in a final reaction volume of 50-100 .mu.l. The
reaction was terminated by the addition of 0.5 ml of 20 mM HEPES,
pH 5.5, containing 2 mM EGTA and EDTA. The reaction mixture was
placed over Dowex AG50WX8, and the flow-through was counted on a
Packard 1500 liquid scintillation analyzer.
[0116] Assays for Reductase Activity--NADPH-cytochrome c reductase
activity and 2,6-dichlorophenolindophenol (DCIP) reduction were
measured as a change in absorbance at 550nm as described previously
by Martasek et al. (1999) and Masters et al. (1967) using an
extinction coefficient of 0.021 .mu.M.sup.-1 cm.sup.-1 for both
cytochrome c and DCIP. Briefly, a reaction mixture (1 ml) contained
either cytochrome c (90 .mu.M); DCIP (36 .mu.M), HEPES buffer (50
mM) at pH 7.6, NaCl (250 mM), NADPH (100 .mu.M), calmodulin (120
nM), and CaCl2 (200 .mu.M); or other substances as indicated. The
reaction was monitored for 60 s (at 23.degree. C.) after the
addition of eNOS. When inactivation of reductase activity was
determined by the addition of EGTA (200-800 .mu.M), chelator was
added 1 min after initiation of the reaction and monitored for an
additional 1 min. The reaction contained HEPES buffer (50 mM) at pH
7.6, CaM (120 nM), and CaCl2 (200 .mu.M) and was initiated with
NADPH (100 .mu.M. No NaCl was added in experiments that examined
EGTA inactivation of eNOS to mimic the conditions used in the
hemoglobin capture experiments. The addition of NOS inhibitors did
not influence the rate of cytochrome c reduction (not shown).
Determination of the calcium EC50 for eNOS was performed as
described above for the hemoglobin capture assay.
[0117] Data Analysis and Statistics--All data were expressed as
mean.+-.S.E. At least triplicate determinations were performed with
a minimum of three different batches of enzymes for each data set.
Wild type and mutant enzymes were purified simultaneously to
control for activity variations between preparations. Statistical
significance was determined using Student's t test, and p<0.05
was considered statistically significant.
Results
[0118] Expression and Purification of eNOS--Both wild type and
S1179D eNOS were expressed and purified from E. coli. In a culture
of 1.6 liters, approximately 2.5-4.0 mg of eNOS was typically
recovered using 2'5'-ADP Sepharose 4B chromatography. As seen in
FIG. 5A, both enzymes were >90% pure based on Coomassie
staining. These results are typical, as seen from seven independent
preparations of both wild type and S1179D eNOS prepared
side-by-side. Both enzymes were primarily in their dimeric form, as
shown by low temperature SDS-PAGE (FIG. 5B).
[0119] eNOS S1179D Has Greater NO Synthase and Reductase Activities
Than Does Wild Type eNOS--Next, the activities of wild type and
S1179D eNOS were compared by measuring the rate of NO production.
S1179D eNOS exhibited a higher turnover number (under optimal
conditions) as compared with wild type enzyme (84.+-.6 versus
27.+-.1 min.sup.-1, n=6 separate and paired preparations of
enzymes). The Km values with L-arginine were similar for wild type
and S1179D eNOS (FIG. 6A, 1.8 versus 2.5 .mu.M, respectively; see
Table I).
1TABLE I Kinetic parameters for wild type and S1179D eNOS Catalytic
Substrate/cofactors Assay determination Wild type S1179D Arginine
Hemoglobin K.sub.cat 27 .+-. 1 min.sup.-1 84 .+-. 6 min.sup.-1
capture K.sub.m 1.8 .mu.M 2.5 .mu.M Hemoglobin K.sub.cat 17 .+-. 1
min.sup.-1 53 .+-. 3 min.sup.-1 capture K.sub.m 8 .mu.M 36 .mu.M
Cytochrome c K.sub.cat + CaM 460 .+-. 18 min.sup.-1 840 .+-. 59
min.sup.-1 K.sub.cat - CaM 70 .+-. 5 min.sup.-1 290 .+-. 9
min.sup.-1 K.sub.m + CaM 0.75 .mu.M 1.9 .mu.M NADPH -CaM 0.40 .mu.M
2.0 .mu.M L-Citrulline K.sub.cat 22 .+-. 2 min.sup.-1 43 .+-. 2
min.sup.-1 EC.sub.50 8 nM 7 nM Cytochrome c K.sub.cat 620 .+-. 78
min.sup.-1 1140 .+-. 75 min.sup.-1 CaM EC.sub.50 13 nM 21 mM
Hemoglobin K.sub.cat 58 .+-. 1 min.sup.-1 100 .+-. 3 min.sup.-1
capture (100 mM EC.sub.50 310 nM 250 nM KCl) Cytochrome c K.sub.cat
1909 .+-. 33 min.sup.-1 3798 .+-. 54 min.sup.-1 Ca2+ (100 mM
EC.sub.50 290 nM 220 nM KCl)
[0120] Because the rate of electron flux from the reductase domain
to the oxygenase domain is critical for NOS catalysis, the increase
in S1179D eNOS activity was examined to determine whether it could
be attributed to enhanced reductase activity. When both DCIP and
cytochrome c reduction were examined, a significant increase in
activity for S1179D compared with wild type eNOS was observed (FIG.
6B). Furthermore, this increase was accentuated by the presence of
CaM, which increased the overall activity for both enzymes. Basal
cytochrome c reduction, in the absence of CaM, was 4-fold higher
for S1179D compared with wild type eNOS. The magnitude of
CaM-stimulated cytochrome c reduction was higher for S1179D eNOS
(749.+-.35 versus 1272.+-.55 min.sup.-1 for wild type and S1179D
eNOS, respectively, n=3-5); however, the level of stimulation by
CaM was 8-fold for wild type eNOS compared with only 3-fold for
S1179D eNOS.
[0121] Next, it was determined whether S1179D eNOS produces more
superoxide than wild type eNOS, which could reduce cytochrome c. As
expected, no superoxide dismutase inhibitable cytochrome c
reduction was observed (as an index of superoxide anion generation)
in the absence of CaM, as reported earlier for wild type eNOS
(86.+-.6 versus 95.+-.8 min.sup.-1) and for S1179D eNOS (278.+-.9
versus 288.+-.7 min.sup.-1, n=3-5). However, in the presence of
CaM, superoxide dismutase reduced the rate of cytochrome c
reduction for both wild type (610.+-.51 versus 866.+-.8 min.sup.-1)
and S1179D (1179.+-.43 versus 1518.+-.19 min.sup.-1) eNOS. The
relative decrease in cytochrome c activity in the presence of
superoxide dismutase was similar with both enzymes (30% for wild
type and 22% for S1179D eNOS), suggesting that the enhanced
reductase activity of S1179D compared with wild type eNOS (assayed
by cytochrome c reduction) was not due to uncoupling of the
enzyme.
[0122] NADPH-dependent NO Formation and Reductase Activities Are
Not Different for Wild Type and S1179D eNOS--The NADPH dependence
of NO production and cytochrome c reduction were examined because
the NADPH binding site lies close in proximity to the Ser-1179 in
eNOS. S1179D eNOS had a higher maximum turnover number (k.sub.cat)
than did wild type enzyme based on NO production, assayed in the
presence of CaM (FIG. 7A). The increased k.sub.cat was associated
with a 4-fold increase in the Km for NADPH for S1179D eNOS compared
with wild type eNOS (36 versus 8 .mu.M, respectively). The
k.sub.cat for NADPH-dependent cytochrome c reduction in the absence
of CaM was greater for eNOS S1179D than wild type eNOS (290 versus
70 min.sup.-1, respectively; FIG. 7B). In the presence of CaM, the
k.sub.cat for cytochrome c reduction was considerably higher for
S1179D compared with wild type eNOS (840 versus 460 min.sup.-1,
respectively). The Km values for NADPH were unchanged in the
absence or presence of CaM (0.40 and 0.75 .mu.M for wild type eNOS
and 2.0 and 1.9 .mu.M for S1179D eNOS in the absence and presence
of CaM, respectively).
[0123] EC50 Values for CaM Are Unchanged between Wild Type and
S1179D eNOS--To assess whether the increased activity of S1179D
eNOS was attributable to changes in the affinity of the enzyme for
CaM, the kinetics of NOS activity and cytochrome c reduction
assayed in the presence of all NOS cofactors in excess as a
function of CaM concentration were examined. The kcat for CaM
activation of NOS activity was 22 min.sup.-1 for wild type and 43
min.sup.-1 for S1179D eNOS. Transformation of the data, normalizing
for the differences in k.sub.cat, revealed a slight shift in the
curve to the left for S1179D eNOS but little difference in the EC50
values for CaM, consistent with reported data on phospho-eNOS
(Mitchell et al., 1999). The EC50 values were 8 nM for wild type
and 7 nM S1179D eNOS (FIG. 8A). NADPH-mediated cytochrome c
reduction was measured. The k.sub.cat for CaM activation of
cytochrome c reduction was about 2-fold higher for S1179D eNOS
compared with wild type enzyme (1140 and 620 min.sup.-1 for S1179D
and wild type eNOS, respectively). Transformation of the data
normalized for the differences in k.sub.cat, revealed small
differences in the EC50 values for CaM between wild type and S1179D
eNOS (21 versus 13 nM; FIG. 8B).
[0124] Comparison of Calcium Activation and Inactivation of
eNOS--In previous experiments, it was demonstrated that the
"apparent calcium sensitivity" of eNOS was enhanced in cells
expressing either a majority of phospho-eNOS or S1179D eNOS,
suggesting that phosphorylation changed the affinity of calcium/CaM
activation (Dimmeler et al., 1999; Fulton et al. 1999). As seen in
FIG. 8C, after normalization for the differences in maximal
activity, the calcium dependence was slightly increased for S1179D
eNOS (p<0.05, two-way analysis of variance). The EC50 values for
calcium with wild type and S1179D eNOS were slightly different also
(310 and 250 nM calcium, respectively), as determined by NO
production (in the presence of 250 nM CaM). As seen in the inset to
FIG. 8C, increasing concentrations of free calcium did indeed
enhance S1179D eNOS turnover to a greater extent then that seen
with wild type enzyme. Furthermore, the EC50 value for calcium
assaying cytochrome c reduction were similar to those obtained
measuring NO production (FIG. 8D; 290 and 220 nM for wild type and
S1179D eNOS, respectively). Again, the Vmax for calcium activation
of S1179D eNOS turnover was greater than wild type (FIG. 4D,
inset).
[0125] To examine whether the inactivation of S1179D eNOS was
different than that of wild type enzyme, the decay in eNOS activity
after chelation of calcium with EGTA was measured. In these
experiments, all NOS cofactors were added in the presence of
calcium (200 .mu.M), and NO production was monitored for 1 min,
followed by the addition of different concentrations of EGTA and
monitoring for an additional 1 min. As seen in FIG. 9A, the
addition of EGTA to wild type and S1179D eNOS reduced NO production
in a concentration-dependent manner. However, NO production from
S1179D eNOS was less sensitive to the addition of EGTA; i.e. wild
type eNOS activity declined more rapidly at lower concentrations of
EGTA than did S1179D eNOS activity. The greatest difference in
activity between the enzymes was seen at 400 .mu.M EGTA.
Furthermore, at 600 .mu.M EGTA, no activity was detected for wild
type eNOS, whereas residual activity was still detected for S1179D
eNOS. Similar results were obtained using cytochrome c reduction
(FIG. 9B), with significant differences in activity seen with 400
and 600 .mu.M chelator added to the reaction. However, at the
highest concentration of EGTA, residual reductase activity was
found for both wild type and S1179D eNOS.
[0126] In summary, bovine endothelial nitric oxide synthase (eNOS)
is phosphorylated directly by the protein kinase Akt at serine 1179
(Fulton et al., 1999) and human endothelial nitric oxide synthease
is phosphorylated directly by the protein kinase Akt at serine 1177
(Dimmeler et al., 1999). Mutation of residue 1179 in bovine eNOS to
the negatively charged aspartate increases nitric oxide (NO)
production constitutively, in the absence of agonist challenge.
[0127] It should be understood that the foregoing discussion and
examples merely present a detailed description of certain preferred
embodiments. It therefore should be apparent to those of ordinary
skill in the art that various modifications and equivalents can be
made without departing from the spirit and scope of the invention.
All references, articles and patents identified above or below are
herein incorporated by reference in their entirety.
References
[0128] Additional references for which the entire citation is not
given in the text are herein incorporated in their entirety.
[0129] Alessi et al., Embo J 15, 6541-6551 (1996).
[0130] Bredt et al., Proc. Natl. Acad. Sci. U.S.A. 87, 682-685
(1990).
[0131] Corson et al., Circulation Research 79, 984-991 (1996).
[0132] Dimmeler et al., Nature 399, 601-605 (1999).
[0133] Downward, Curr Opin Cell Biol 10, 262-267 (1998).
[0134] Fulton et al., Nature 399, 597-601 (1999).
[0135] Garcia-Cardena et al., Nature 392, 821-824 (1998).
[0136] Garcia-Cardena et al., J Biol Chem 271, 27237-27240
(1996).
[0137] Garca-Cardea et al., Proc. Natl. Acad. Sci. USA 93,
6448-6453 (1996a).
[0138] Huang et al., Nature 377, 239-242 (1995).
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[0140] Kelm et al., Biochem. Biophys. Res. Commun. 154, 236-244
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[0141] Klatt et al., EMBO J. 14, 3687-3695 (1995).
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[0143] Liu et al., J. Cell Biol. 137, 1525-1535 (1997).
[0144] Martasek et al., Methods Enzymol. 301, 70-78 (1999).
[0145] Martasek et al., Biochem Biophys Res Commun 219, 359-365
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[0146] Masters et al., Methods Enzymol. 10, 565-573 (1967)
[0147] Michel et al., Proc. Natl. Acad. Sci. 90, 6252-6256
(1993).
[0148] Michell et al., Curr. Biol. 9, 845-848 (1999).
[0149] Murohara et al., J Clin Invest 101, 2567-2578 (1998).
[0150] Papapetropoulos et al., J Clin Invest 100, 3131-3139
(1997).
[0151] Roman et al., Proc. Natl. Acad. Sci. U.S.A. 92, 8428-8432
(1995).
[0152] Rudic et al., J Clin Invest 101, 731-736 (1998).
[0153] Sessa et al., J Biol Chem 270, 17641-17644 (1995).
[0154] Shaul et al., J Biol Chem 271, 6518-6522 (1996).
[0155] Shesely et al., Proc Natl Acad Sci USA 93, 13176-13181
(1996).
[0156] Yano et al., Nature 396, 584-587 (1998).
Sequence CWU 1
1
9 1 8 PRT Bos sp. 1 Met Gly Asn Leu Lys Ser Val Gly 1 5 2 21 PRT
Bos sp. 2 Arg Ile Arg Thr Gln Ser Phe Ser Leu Gln Glu Arg His Leu
Arg Gly 1 5 10 15 Ala Val Pro Trp Ala 20 3 1203 PRT Homo sapiens 3
Met Gly Asn Leu Lys Ser Val Ala Gln Glu Pro Gly Pro Pro Cys Gly 1 5
10 15 Leu Gly Leu Gly Leu Gly Leu Gly Leu Cys Gly Lys Gln Gly Pro
Ala 20 25 30 Thr Pro Ala Pro Glu Pro Ser Arg Ala Pro Ala Ser Leu
Leu Pro Pro 35 40 45 Ala Pro Glu His Ser Pro Pro Ser Ser Pro Leu
Thr Gln Pro Pro Glu 50 55 60 Gly Pro Lys Phe Pro Arg Val Lys Asn
Trp Glu Val Gly Ser Ile Thr 65 70 75 80 Tyr Asp Thr Leu Ser Ala Gln
Ala Gln Gln Asp Gly Pro Cys Thr Pro 85 90 95 Arg Arg Cys Leu Gly
Ser Leu Val Phe Pro Arg Lys Leu Gln Gly Arg 100 105 110 Pro Ser Pro
Gly Pro Pro Ala Pro Glu Gln Leu Leu Ser Gln Ala Arg 115 120 125 Asp
Phe Ile Asn Gln Tyr Tyr Ser Ser Ile Lys Arg Ser Gly Ser Gln 130 135
140 Ala His Glu Gln Arg Leu Gln Glu Val Glu Ala Glu Val Ala Ala Thr
145 150 155 160 Gly Thr Tyr Gln Leu Arg Glu Ser Glu Leu Val Phe Gly
Ala Lys Gln 165 170 175 Ala Trp Arg Asn Ala Pro Arg Cys Val Gly Arg
Ile Gln Trp Gly Lys 180 185 190 Leu Gln Val Phe Asp Ala Arg Asp Cys
Arg Ser Ala Gln Glu Met Phe 195 200 205 Thr Tyr Ile Cys Asn His Ile
Lys Tyr Ala Thr Asn Arg Gly Asn Leu 210 215 220 Arg Ser Ala Ile Thr
Val Phe Pro Gln Arg Cys Pro Gly Arg Gly Asp 225 230 235 240 Phe Arg
Ile Trp Asn Ser Gln Leu Val Arg Tyr Ala Gly Tyr Arg Gln 245 250 255
Gln Asp Gly Ser Val Arg Gly Asp Pro Ala Asn Val Glu Ile Thr Glu 260
265 270 Leu Cys Ile Gln His Gly Trp Thr Pro Gly Asn Gly Arg Phe Asp
Val 275 280 285 Leu Pro Leu Leu Leu Gln Ala Pro Asp Glu Pro Pro Glu
Leu Phe Leu 290 295 300 Leu Pro Pro Glu Leu Val Leu Glu Val Pro Leu
Glu His Pro Thr Leu 305 310 315 320 Glu Trp Phe Ala Ala Leu Gly Leu
Arg Trp Tyr Ala Leu Pro Ala Val 325 330 335 Ser Asn Met Leu Leu Glu
Ile Gly Gly Leu Glu Phe Pro Ala Ala Pro 340 345 350 Phe Ser Gly Trp
Tyr Met Ser Thr Glu Ile Gly Thr Arg Asn Leu Cys 355 360 365 Asp Pro
His Arg Tyr Asn Ile Leu Glu Asp Val Ala Val Cys Met Asp 370 375 380
Leu Asp Thr Arg Thr Thr Ser Ser Leu Trp Lys Asp Lys Ala Ala Val 385
390 395 400 Glu Ile Asn Val Ala Val Leu His Ser Tyr Gln Leu Ala Lys
Val Thr 405 410 415 Ile Val Asp His His Ala Ala Thr Ala Ser Phe Met
Lys His Leu Glu 420 425 430 Asn Glu Gln Lys Ala Arg Gly Gly Cys Pro
Ala Asp Trp Ala Trp Ile 435 440 445 Val Pro Pro Ile Ser Gly Ser Leu
Thr Pro Val Phe His Gln Glu Met 450 455 460 Val Asn Tyr Phe Leu Ser
Pro Ala Phe Arg Tyr Gln Pro Asp Pro Trp 465 470 475 480 Lys Gly Ser
Ala Ala Lys Gly Thr Gly Ile Thr Arg Lys Lys Thr Phe 485 490 495 Lys
Glu Val Ala Asn Ala Val Lys Ile Ser Ala Ser Leu Met Gly Thr 500 505
510 Val Met Ala Lys Arg Val Lys Ala Thr Ile Leu Tyr Gly Ser Glu Thr
515 520 525 Gly Arg Ala Gln Ser Tyr Ala Gln Gln Leu Gly Arg Leu Phe
Arg Lys 530 535 540 Ala Phe Asp Pro Arg Val Leu Cys Met Asp Glu Tyr
Asp Val Val Ser 545 550 555 560 Leu Glu His Glu Thr Leu Val Leu Val
Val Thr Ser Thr Phe Gly Asn 565 570 575 Gly Asp Pro Pro Glu Asn Gly
Glu Ser Phe Ala Ala Ala Leu Met Glu 580 585 590 Met Ser Gly Pro Tyr
Asn Ser Ser Pro Arg Pro Glu Gln His Lys Ser 595 600 605 Tyr Lys Ile
Arg Phe Asn Ser Ile Ser Cys Ser Asp Pro Leu Val Ser 610 615 620 Ser
Trp Arg Arg Lys Arg Lys Glu Ser Ser Asn Thr Asp Ser Ala Gly 625 630
635 640 Ala Leu Gly Thr Leu Arg Phe Cys Val Phe Gly Leu Gly Ser Arg
Ala 645 650 655 Tyr Pro His Phe Cys Ala Phe Ala Arg Ala Val Asp Thr
Arg Leu Glu 660 665 670 Glu Leu Gly Gly Glu Arg Leu Leu Gln Leu Gly
Gln Gly Asp Glu Leu 675 680 685 Cys Gly Gln Glu Glu Ala Phe Arg Gly
Trp Ala Gln Ala Ala Phe Gln 690 695 700 Ala Ala Cys Glu Thr Phe Cys
Val Gly Glu Asp Ala Lys Ala Ala Ala 705 710 715 720 Arg Asp Ile Phe
Ser Pro Lys Arg Ser Trp Lys Arg Gln Arg Tyr Arg 725 730 735 Leu Ser
Ala Gln Ala Glu Gly Leu Gln Leu Leu Pro Gly Leu Ile His 740 745 750
Val His Arg Arg Lys Met Phe Gln Ala Thr Ile Arg Ser Val Glu Asn 755
760 765 Leu Gln Ser Ser Lys Ser Thr Arg Ala Thr Ile Leu Val Arg Leu
Asp 770 775 780 Thr Gly Gly Gln Glu Gly Leu Gln Tyr Gln Pro Gly Asp
His Ile Gly 785 790 795 800 Val Cys Pro Pro Asn Arg Pro Gly Leu Val
Glu Ala Leu Leu Ser Arg 805 810 815 Val Glu Asp Pro Pro Ala Pro Thr
Glu Pro Val Ala Val Glu Gln Leu 820 825 830 Glu Lys Gly Ser Pro Gly
Gly Pro Pro Pro Gly Trp Val Arg Asp Pro 835 840 845 Arg Leu Pro Pro
Cys Thr Leu Arg Gln Ala Leu Thr Phe Phe Leu Asp 850 855 860 Ile Thr
Ser Pro Pro Ser Pro Gln Leu Leu Arg Leu Leu Ser Thr Leu 865 870 875
880 Ala Glu Glu Pro Arg Glu Gln Gln Glu Leu Glu Ala Leu Ser Gln Asp
885 890 895 Pro Arg Arg Tyr Glu Glu Trp Lys Trp Phe Arg Cys Pro Thr
Leu Leu 900 905 910 Glu Val Leu Glu Gln Phe Pro Ser Val Ala Leu Pro
Ala Pro Leu Leu 915 920 925 Leu Thr Gln Leu Pro Leu Leu Gln Pro Arg
Tyr Tyr Ser Val Ser Ser 930 935 940 Ala Pro Ser Thr His Pro Gly Glu
Ile His Leu Thr Val Ala Val Leu 945 950 955 960 Ala Tyr Arg Thr Gln
Asp Gly Leu Gly Pro Leu His Tyr Gly Val Cys 965 970 975 Ser Thr Trp
Leu Ser Gln Leu Lys Pro Gly Asp Pro Val Pro Cys Phe 980 985 990 Ile
Arg Gly Ala Pro Ser Phe Arg Leu Pro Pro Asp Pro Ser Leu Pro 995
1000 1005 Cys Ile Leu Val Gly Pro Gly Thr Gly Ile Ala Pro Phe Arg
Gly 1010 1015 1020 Phe Trp Gln Glu Arg Leu His Asp Ile Glu Ser Lys
Gly Leu Gln 1025 1030 1035 Pro Thr Pro Met Thr Leu Val Phe Gly Cys
Arg Cys Ser Gln Leu 1040 1045 1050 Asp His Leu Tyr Arg Asp Glu Val
Gln Asn Ala Gln Gln Arg Gly 1055 1060 1065 Val Phe Gly Arg Val Leu
Thr Ala Phe Ser Arg Glu Pro Asp Asn 1070 1075 1080 Pro Lys Thr Tyr
Val Gln Asp Ile Leu Arg Thr Glu Leu Ala Ala 1085 1090 1095 Glu Val
His Arg Val Leu Cys Leu Glu Arg Gly His Met Phe Val 1100 1105 1110
Cys Gly Asp Val Thr Met Ala Thr Asn Val Leu Gln Thr Val Gln 1115
1120 1125 Arg Ile Leu Ala Thr Glu Gly Asp Met Glu Leu Asp Glu Ala
Gly 1130 1135 1140 Asp Val Ile Gly Val Leu Arg Asp Gln Gln Arg Tyr
His Glu Asp 1145 1150 1155 Ile Phe Gly Leu Thr Leu Arg Thr Gln Glu
Val Thr Ser Arg Ile 1160 1165 1170 Arg Thr Gln Ser Phe Ser Leu Gln
Glu Arg Gln Leu Arg Gly Ala 1175 1180 1185 Val Pro Trp Ala Phe Asp
Pro Pro Gly Ser Asp Thr Asn Ser Pro 1190 1195 1200 4 1205 PRT
Bovine 4 Met Gly Asn Leu Lys Ser Val Gly Gln Glu Pro Gly Pro Pro
Cys Gly 1 5 10 15 Leu Gly Leu Gly Leu Gly Leu Gly Leu Cys Gly Lys
Gln Gly Pro Ala 20 25 30 Ser Pro Ala Pro Glu Pro Ser Arg Ala Pro
Ala Pro Ala Thr Pro His 35 40 45 Ala Pro Asp His Ser Pro Ala Pro
Asn Ser Pro Thr Leu Thr Arg Pro 50 55 60 Pro Glu Gly Pro Lys Phe
Pro Arg Val Lys Asn Trp Glu Leu Gly Ser 65 70 75 80 Ile Thr Tyr Asp
Thr Leu Cys Ala Gln Ser Gln Gln Asp Gly Pro Cys 85 90 95 Thr Pro
Arg Cys Cys Leu Gly Ser Leu Val Leu Pro Arg Lys Leu Gln 100 105 110
Thr Arg Pro Ser Pro Gly Pro Pro Pro Ala Glu Gln Leu Leu Ser Gln 115
120 125 Ala Arg Asp Phe Ile Asn Gln Tyr Tyr Ser Ser Ile Lys Arg Ser
Gly 130 135 140 Ser Gln Ala His Glu Glu Arg Leu Gln Glu Val Glu Ala
Glu Val Ala 145 150 155 160 Ser Thr Gly Thr Tyr His Leu Arg Glu Ser
Glu Leu Val Phe Gly Ala 165 170 175 Lys Gln Ala Trp Arg Asn Ala Pro
Arg Cys Val Gly Arg Ile Gln Trp 180 185 190 Gly Lys Leu Gln Val Phe
Asp Ala Arg Asp Cys Ser Ser Ala Gln Glu 195 200 205 Met Phe Thr Tyr
Ile Cys Asn His Ile Lys Tyr Ala Thr Asn Arg Gly 210 215 220 Asn Leu
Arg Ser Ala Ile Thr Val Phe Pro Gln Arg Ala Pro Gly Arg 225 230 235
240 Gly Asp Phe Arg Ile Trp Asn Ser Gln Leu Val Arg Tyr Ala Gly Tyr
245 250 255 Arg Gln Gln Asp Gly Ser Val Arg Gly Asp Pro Ala Asn Val
Glu Ile 260 265 270 Thr Glu Leu Cys Ile Gln His Gly Trp Thr Pro Gly
Asn Gly Arg Phe 275 280 285 Asp Val Leu Pro Leu Leu Leu Gln Ala Pro
Asp Glu Ala Pro Glu Leu 290 295 300 Phe Val Leu Pro Pro Glu Leu Val
Leu Glu Val Pro Leu Glu His Pro 305 310 315 320 Thr Leu Glu Trp Phe
Ala Ala Leu Gly Leu Arg Trp Tyr Ala Leu Pro 325 330 335 Ala Val Ser
Asn Met Leu Leu Glu Ile Gly Gly Leu Glu Phe Ser Ala 340 345 350 Ala
Pro Phe Ser Gly Trp Tyr Met Ser Thr Glu Ile Gly Thr Arg Asn 355 360
365 Leu Cys Asp Pro His Arg Tyr Asn Ile Leu Glu Asp Val Ala Val Cys
370 375 380 Met Asp Leu Asp Thr Arg Thr Thr Ser Ser Leu Trp Lys Asp
Lys Ala 385 390 395 400 Ala Val Glu Ile Asn Leu Ala Val Leu His Ser
Phe Gln Leu Ala Lys 405 410 415 Val Thr Ile Val Asp His His Ala Ala
Thr Val Ser Phe Met Lys His 420 425 430 Leu Asp Asn Glu Gln Lys Ala
Arg Gly Gly Cys Pro Ala Asp Trp Ala 435 440 445 Trp Ile Val Pro Pro
Ile Ser Gly Ser Leu Thr Pro Val Phe His Gln 450 455 460 Glu Met Val
Asn Tyr Ile Leu Ser Pro Ala Phe Arg Tyr Gln Pro Asp 465 470 475 480
Pro Trp Lys Gly Ser Ala Thr Lys Gly Ala Gly Ile Thr Arg Lys Lys 485
490 495 Thr Phe Lys Glu Val Ala Asn Ala Val Lys Ile Ser Ala Ser Leu
Met 500 505 510 Gly Thr Leu Met Ala Lys Arg Val Lys Ala Thr Ile Leu
Tyr Ala Ser 515 520 525 Glu Thr Gly Arg Ala Gln Ser Tyr Ala Gln Gln
Leu Gly Arg Leu Phe 530 535 540 Arg Lys Ala Phe Asp Pro Arg Val Leu
Cys Met Asp Glu Tyr Asp Val 545 550 555 560 Val Ser Leu Glu His Glu
Ala Leu Val Leu Val Val Thr Ser Thr Phe 565 570 575 Gly Asn Gly Asp
Pro Pro Glu Asn Gly Glu Ser Phe Ala Ala Ala Leu 580 585 590 Met Glu
Met Ser Gly Pro Tyr Asn Ser Ser Pro Arg Pro Glu Gln His 595 600 605
Lys Ser Tyr Lys Ile Arg Phe Asn Ser Val Ser Cys Ser Asp Pro Leu 610
615 620 Val Ser Ser Trp Arg Arg Lys Arg Lys Glu Ser Ser Asn Thr Asp
Ser 625 630 635 640 Ala Gly Ala Leu Gly Thr Leu Arg Phe Cys Gly Phe
Gly Leu Gly Ser 645 650 655 Arg Ala Tyr Pro His Phe Cys Ala Phe Ala
Arg Ala Val Asp Thr Arg 660 665 670 Leu Glu Glu Leu Gly Gly Glu Arg
Leu Leu Gln Leu Gly Gln Gly Asp 675 680 685 Glu Leu Cys Gly Gln Glu
Glu Ala Phe Arg Gly Trp Ala Lys Ala Ala 690 695 700 Phe Gln Ala Ser
Cys Glu Thr Phe Cys Val Gly Glu Glu Ala Lys Ala 705 710 715 720 Arg
Pro Gln Asp Ile Phe Ser Pro Lys Arg Ser Trp Lys Arg Gln Arg 725 730
735 Tyr Arg Leu Ser Thr Gln Ala Glu Gly Leu Gln Leu Leu Pro Gly Leu
740 745 750 Ile His Val His Arg Arg Lys Met Phe Gln Ala Thr Val Leu
Ser Val 755 760 765 Glu Asn Leu Gln Ser Ser Lys Ser Thr Arg Ala Thr
Ile Leu Val Arg 770 775 780 Leu Asp Thr Ala Gly Gln Glu Gly Leu Gln
Tyr Gln Pro Gly Asp His 785 790 795 800 Ile Gly Ile Cys Pro Pro Asn
Arg Pro Gly Leu Val Glu Ala Leu Leu 805 810 815 Ser Arg Val Glu Asp
Pro Pro Pro Pro Thr Glu Ser Val Ala Val Glu 820 825 830 Gln Leu Glu
Lys Gly Ser Pro Gly Gly Pro Pro Pro Ser Trp Val Arg 835 840 845 Asp
Pro Arg Leu Pro Pro Cys Thr Leu Arg Gln Ala Leu Thr Phe Phe 850 855
860 Leu Asp Ile Thr Ser Pro Pro Ser Pro Arg Leu Leu Arg Leu Leu Ser
865 870 875 880 Thr Leu Ala Glu Glu Pro Ser Glu Gln Gln Glu Leu Glu
Thr Leu Ser 885 890 895 Gln Asp Pro Arg Arg Tyr Glu Glu Trp Lys Trp
Phe Arg Cys Pro Thr 900 905 910 Leu Leu Glu Val Leu Glu Gln Phe Pro
Ser Val Ala Leu Pro Ala Pro 915 920 925 Leu Leu Leu Thr Gln Leu Pro
Leu Leu Gln Pro Arg Tyr Tyr Ser Val 930 935 940 Ser Ser Ala Pro Asn
Ala His Pro Gly Glu Val His Leu Thr Val Ala 945 950 955 960 Val Leu
Ala Tyr Arg Thr Gln Asp Gly Leu Gly Pro Leu His Tyr Gly 965 970 975
Val Cys Ser Thr Trp Leu Ser Gln Leu Lys Thr Gly Asp Pro Val Pro 980
985 990 Cys Phe Ile Arg Gly Ala Pro Ser Phe Arg Leu Pro Pro Asp Pro
Tyr 995 1000 1005 Val Pro Cys Ile Leu Val Gly Pro Gly Thr Gly Ile
Ala Pro Phe 1010 1015 1020 Arg Gly Phe Trp Gln Glu Arg Leu His Asp
Ile Glu Ser Lys Gly 1025 1030 1035 Leu Gln Pro Ala Pro Met Thr Leu
Val Phe Gly Cys Arg Cys Ser 1040 1045 1050 Gln Leu Asp His Leu Tyr
Arg Asp Glu Val Gln Asp Ala Gln Glu 1055 1060 1065 Arg Gly Val Phe
Gly Arg Val Leu Thr Ala Phe Ser Arg Glu Pro 1070 1075 1080 Asp Ser
Pro Lys Thr Tyr Val Gln Asp Ile Leu Arg Thr Glu Leu 1085 1090 1095
Ala Ala Glu Val His Arg Val Leu Cys Leu Glu Arg Gly His Met 1100
1105 1110 Phe Val Cys Gly Asp Val Thr Met Ala Thr Ser Val Leu Gln
Thr 1115 1120 1125 Val Gln Arg Ile Leu Ala Thr Glu Gly Asp Met Glu
Leu Asp Glu 1130 1135 1140 Ala Gly Asp Val Ile Gly Val Leu Arg Asp
Gln Gln Arg Tyr His 1145 1150 1155 Glu Asp Ile Phe Gly Leu Thr Leu
Arg Thr Gln Glu Val Thr Ser 1160 1165 1170 Arg Ile Arg Thr Gln Ser
Phe Ser Leu Gln Glu Arg His Leu Arg 1175 1180 1185 Gly Ala Val Pro
Trp Ala Phe Asp Pro Pro Gly Pro Asp Thr Pro 1190
1195 1200 Gly Pro 1205 5 1203 PRT Homo sapiens MUTAGEN
(1177)..(1177) S is substituted 5 Met Gly Asn Leu Lys Ser Val Ala
Gln Glu Pro Gly Pro Pro Cys Gly 1 5 10 15 Leu Gly Leu Gly Leu Gly
Leu Gly Leu Cys Gly Lys Gln Gly Pro Ala 20 25 30 Thr Pro Ala Pro
Glu Pro Ser Arg Ala Pro Ala Ser Leu Leu Pro Pro 35 40 45 Ala Pro
Glu His Ser Pro Pro Ser Ser Pro Leu Thr Gln Pro Pro Glu 50 55 60
Gly Pro Lys Phe Pro Arg Val Lys Asn Trp Glu Val Gly Ser Ile Thr 65
70 75 80 Tyr Asp Thr Leu Ser Ala Gln Ala Gln Gln Asp Gly Pro Cys
Thr Pro 85 90 95 Arg Arg Cys Leu Gly Ser Leu Val Phe Pro Arg Lys
Leu Gln Gly Arg 100 105 110 Pro Ser Pro Gly Pro Pro Ala Pro Glu Gln
Leu Leu Ser Gln Ala Arg 115 120 125 Asp Phe Ile Asn Gln Tyr Tyr Ser
Ser Ile Lys Arg Ser Gly Ser Gln 130 135 140 Ala His Glu Gln Arg Leu
Gln Glu Val Glu Ala Glu Val Ala Ala Thr 145 150 155 160 Gly Thr Tyr
Gln Leu Arg Glu Ser Glu Leu Val Phe Gly Ala Lys Gln 165 170 175 Ala
Trp Arg Asn Ala Pro Arg Cys Val Gly Arg Ile Gln Trp Gly Lys 180 185
190 Leu Gln Val Phe Asp Ala Arg Asp Cys Arg Ser Ala Gln Glu Met Phe
195 200 205 Thr Tyr Ile Cys Asn His Ile Lys Tyr Ala Thr Asn Arg Gly
Asn Leu 210 215 220 Arg Ser Ala Ile Thr Val Phe Pro Gln Arg Cys Pro
Gly Arg Gly Asp 225 230 235 240 Phe Arg Ile Trp Asn Ser Gln Leu Val
Arg Tyr Ala Gly Tyr Arg Gln 245 250 255 Gln Asp Gly Ser Val Arg Gly
Asp Pro Ala Asn Val Glu Ile Thr Glu 260 265 270 Leu Cys Ile Gln His
Gly Trp Thr Pro Gly Asn Gly Arg Phe Asp Val 275 280 285 Leu Pro Leu
Leu Leu Gln Ala Pro Asp Glu Pro Pro Glu Leu Phe Leu 290 295 300 Leu
Pro Pro Glu Leu Val Leu Glu Val Pro Leu Glu His Pro Thr Leu 305 310
315 320 Glu Trp Phe Ala Ala Leu Gly Leu Arg Trp Tyr Ala Leu Pro Ala
Val 325 330 335 Ser Asn Met Leu Leu Glu Ile Gly Gly Leu Glu Phe Pro
Ala Ala Pro 340 345 350 Phe Ser Gly Trp Tyr Met Ser Thr Glu Ile Gly
Thr Arg Asn Leu Cys 355 360 365 Asp Pro His Arg Tyr Asn Ile Leu Glu
Asp Val Ala Val Cys Met Asp 370 375 380 Leu Asp Thr Arg Thr Thr Ser
Ser Leu Trp Lys Asp Lys Ala Ala Val 385 390 395 400 Glu Ile Asn Val
Ala Val Leu His Ser Tyr Gln Leu Ala Lys Val Thr 405 410 415 Ile Val
Asp His His Ala Ala Thr Ala Ser Phe Met Lys His Leu Glu 420 425 430
Asn Glu Gln Lys Ala Arg Gly Gly Cys Pro Ala Asp Trp Ala Trp Ile 435
440 445 Val Pro Pro Ile Ser Gly Ser Leu Thr Pro Val Phe His Gln Glu
Met 450 455 460 Val Asn Tyr Phe Leu Ser Pro Ala Phe Arg Tyr Gln Pro
Asp Pro Trp 465 470 475 480 Lys Gly Ser Ala Ala Lys Gly Thr Gly Ile
Thr Arg Lys Lys Thr Phe 485 490 495 Lys Glu Val Ala Asn Ala Val Lys
Ile Ser Ala Ser Leu Met Gly Thr 500 505 510 Val Met Ala Lys Arg Val
Lys Ala Thr Ile Leu Tyr Gly Ser Glu Thr 515 520 525 Gly Arg Ala Gln
Ser Tyr Ala Gln Gln Leu Gly Arg Leu Phe Arg Lys 530 535 540 Ala Phe
Asp Pro Arg Val Leu Cys Met Asp Glu Tyr Asp Val Val Ser 545 550 555
560 Leu Glu His Glu Thr Leu Val Leu Val Val Thr Ser Thr Phe Gly Asn
565 570 575 Gly Asp Pro Pro Glu Asn Gly Glu Ser Phe Ala Ala Ala Leu
Met Glu 580 585 590 Met Ser Gly Pro Tyr Asn Ser Ser Pro Arg Pro Glu
Gln His Lys Ser 595 600 605 Tyr Lys Ile Arg Phe Asn Ser Ile Ser Cys
Ser Asp Pro Leu Val Ser 610 615 620 Ser Trp Arg Arg Lys Arg Lys Glu
Ser Ser Asn Thr Asp Ser Ala Gly 625 630 635 640 Ala Leu Gly Thr Leu
Arg Phe Cys Val Phe Gly Leu Gly Ser Arg Ala 645 650 655 Tyr Pro His
Phe Cys Ala Phe Ala Arg Ala Val Asp Thr Arg Leu Glu 660 665 670 Glu
Leu Gly Gly Glu Arg Leu Leu Gln Leu Gly Gln Gly Asp Glu Leu 675 680
685 Cys Gly Gln Glu Glu Ala Phe Arg Gly Trp Ala Gln Ala Ala Phe Gln
690 695 700 Ala Ala Cys Glu Thr Phe Cys Val Gly Glu Asp Ala Lys Ala
Ala Ala 705 710 715 720 Arg Asp Ile Phe Ser Pro Lys Arg Ser Trp Lys
Arg Gln Arg Tyr Arg 725 730 735 Leu Ser Ala Gln Ala Glu Gly Leu Gln
Leu Leu Pro Gly Leu Ile His 740 745 750 Val His Arg Arg Lys Met Phe
Gln Ala Thr Ile Arg Ser Val Glu Asn 755 760 765 Leu Gln Ser Ser Lys
Ser Thr Arg Ala Thr Ile Leu Val Arg Leu Asp 770 775 780 Thr Gly Gly
Gln Glu Gly Leu Gln Tyr Gln Pro Gly Asp His Ile Gly 785 790 795 800
Val Cys Pro Pro Asn Arg Pro Gly Leu Val Glu Ala Leu Leu Ser Arg 805
810 815 Val Glu Asp Pro Pro Ala Pro Thr Glu Pro Val Ala Val Glu Gln
Leu 820 825 830 Glu Lys Gly Ser Pro Gly Gly Pro Pro Pro Gly Trp Val
Arg Asp Pro 835 840 845 Arg Leu Pro Pro Cys Thr Leu Arg Gln Ala Leu
Thr Phe Phe Leu Asp 850 855 860 Ile Thr Ser Pro Pro Ser Pro Gln Leu
Leu Arg Leu Leu Ser Thr Leu 865 870 875 880 Ala Glu Glu Pro Arg Glu
Gln Gln Glu Leu Glu Ala Leu Ser Gln Asp 885 890 895 Pro Arg Arg Tyr
Glu Glu Trp Lys Trp Phe Arg Cys Pro Thr Leu Leu 900 905 910 Glu Val
Leu Glu Gln Phe Pro Ser Val Ala Leu Pro Ala Pro Leu Leu 915 920 925
Leu Thr Gln Leu Pro Leu Leu Gln Pro Arg Tyr Tyr Ser Val Ser Ser 930
935 940 Ala Pro Ser Thr His Pro Gly Glu Ile His Leu Thr Val Ala Val
Leu 945 950 955 960 Ala Tyr Arg Thr Gln Asp Gly Leu Gly Pro Leu His
Tyr Gly Val Cys 965 970 975 Ser Thr Trp Leu Ser Gln Leu Lys Pro Gly
Asp Pro Val Pro Cys Phe 980 985 990 Ile Arg Gly Ala Pro Ser Phe Arg
Leu Pro Pro Asp Pro Ser Leu Pro 995 1000 1005 Cys Ile Leu Val Gly
Pro Gly Thr Gly Ile Ala Pro Phe Arg Gly 1010 1015 1020 Phe Trp Gln
Glu Arg Leu His Asp Ile Glu Ser Lys Gly Leu Gln 1025 1030 1035 Pro
Thr Pro Met Thr Leu Val Phe Gly Cys Arg Cys Ser Gln Leu 1040 1045
1050 Asp His Leu Tyr Arg Asp Glu Val Gln Asn Ala Gln Gln Arg Gly
1055 1060 1065 Val Phe Gly Arg Val Leu Thr Ala Phe Ser Arg Glu Pro
Asp Asn 1070 1075 1080 Pro Lys Thr Tyr Val Gln Asp Ile Leu Arg Thr
Glu Leu Ala Ala 1085 1090 1095 Glu Val His Arg Val Leu Cys Leu Glu
Arg Gly His Met Phe Val 1100 1105 1110 Cys Gly Asp Val Thr Met Ala
Thr Asn Val Leu Gln Thr Val Gln 1115 1120 1125 Arg Ile Leu Ala Thr
Glu Gly Asp Met Glu Leu Asp Glu Ala Gly 1130 1135 1140 Asp Val Ile
Gly Val Leu Arg Asp Gln Gln Arg Tyr His Glu Asp 1145 1150 1155 Ile
Phe Gly Leu Thr Leu Arg Thr Gln Glu Val Thr Ser Arg Ile 1160 1165
1170 Arg Thr Gln Ser Phe Ser Leu Gln Glu Arg Gln Leu Arg Gly Ala
1175 1180 1185 Val Pro Trp Ala Phe Asp Pro Pro Gly Ser Asp Thr Asn
Ser Pro 1190 1195 1200 6 1203 PRT Homo sapiens 6 Met Gly Asn Leu
Lys Ser Val Ala Gln Glu Pro Gly Pro Pro Cys Gly 1 5 10 15 Leu Gly
Leu Gly Leu Gly Leu Gly Leu Cys Gly Lys Gln Gly Pro Ala 20 25 30
Thr Pro Ala Pro Glu Pro Ser Arg Ala Pro Ala Ser Leu Leu Pro Pro 35
40 45 Ala Pro Glu His Ser Pro Pro Ser Ser Pro Leu Thr Gln Pro Pro
Glu 50 55 60 Gly Pro Lys Phe Pro Arg Val Lys Asn Trp Glu Val Gly
Ser Ile Thr 65 70 75 80 Tyr Asp Thr Leu Ser Ala Gln Ala Gln Gln Asp
Gly Pro Cys Thr Pro 85 90 95 Arg Arg Cys Leu Gly Ser Leu Val Phe
Pro Arg Lys Leu Gln Gly Arg 100 105 110 Pro Ser Pro Gly Pro Pro Ala
Pro Glu Gln Leu Leu Ser Gln Ala Arg 115 120 125 Asp Phe Ile Asn Gln
Tyr Tyr Ser Ser Ile Lys Arg Ser Gly Ser Gln 130 135 140 Ala His Glu
Gln Arg Leu Gln Glu Val Glu Ala Glu Val Ala Ala Thr 145 150 155 160
Gly Thr Tyr Gln Leu Arg Glu Ser Glu Leu Val Phe Gly Ala Lys Gln 165
170 175 Ala Trp Arg Asn Ala Pro Arg Cys Val Gly Arg Ile Gln Trp Gly
Lys 180 185 190 Leu Gln Val Phe Asp Ala Arg Asp Cys Arg Ser Ala Gln
Glu Met Phe 195 200 205 Thr Tyr Ile Cys Asn His Ile Lys Tyr Ala Thr
Asn Arg Gly Asn Leu 210 215 220 Arg Ser Ala Ile Thr Val Phe Pro Gln
Arg Cys Pro Gly Arg Gly Asp 225 230 235 240 Phe Arg Ile Trp Asn Ser
Gln Leu Val Arg Tyr Ala Gly Tyr Arg Gln 245 250 255 Gln Asp Gly Ser
Val Arg Gly Asp Pro Ala Asn Val Glu Ile Thr Glu 260 265 270 Leu Cys
Ile Gln His Gly Trp Thr Pro Gly Asn Gly Arg Phe Asp Val 275 280 285
Leu Pro Leu Leu Leu Gln Ala Pro Asp Glu Pro Pro Glu Leu Phe Leu 290
295 300 Leu Pro Pro Glu Leu Val Leu Glu Val Pro Leu Glu His Pro Thr
Leu 305 310 315 320 Glu Trp Phe Ala Ala Leu Gly Leu Arg Trp Tyr Ala
Leu Pro Ala Val 325 330 335 Ser Asn Met Leu Leu Glu Ile Gly Gly Leu
Glu Phe Pro Ala Ala Pro 340 345 350 Phe Ser Gly Trp Tyr Met Ser Thr
Glu Ile Gly Thr Arg Asn Leu Cys 355 360 365 Asp Pro His Arg Tyr Asn
Ile Leu Glu Asp Val Ala Val Cys Met Asp 370 375 380 Leu Asp Thr Arg
Thr Thr Ser Ser Leu Trp Lys Asp Lys Ala Ala Val 385 390 395 400 Glu
Ile Asn Val Ala Val Leu His Ser Tyr Gln Leu Ala Lys Val Thr 405 410
415 Ile Val Asp His His Ala Ala Thr Ala Ser Phe Met Lys His Leu Glu
420 425 430 Asn Glu Gln Lys Ala Arg Gly Gly Cys Pro Ala Asp Trp Ala
Trp Ile 435 440 445 Val Pro Pro Ile Ser Gly Ser Leu Thr Pro Val Phe
His Gln Glu Met 450 455 460 Val Asn Tyr Phe Leu Ser Pro Ala Phe Arg
Tyr Gln Pro Asp Pro Trp 465 470 475 480 Lys Gly Ser Ala Ala Lys Gly
Thr Gly Ile Thr Arg Lys Lys Thr Phe 485 490 495 Lys Glu Val Ala Asn
Ala Val Lys Ile Ser Ala Ser Leu Met Gly Thr 500 505 510 Val Met Ala
Lys Arg Val Lys Ala Thr Ile Leu Tyr Gly Ser Glu Thr 515 520 525 Gly
Arg Ala Gln Ser Tyr Ala Gln Gln Leu Gly Arg Leu Phe Arg Lys 530 535
540 Ala Phe Asp Pro Arg Val Leu Cys Met Asp Glu Tyr Asp Val Val Ser
545 550 555 560 Leu Glu His Glu Thr Leu Val Leu Val Val Thr Ser Thr
Phe Gly Asn 565 570 575 Gly Asp Pro Pro Glu Asn Gly Glu Ser Phe Ala
Ala Ala Leu Met Glu 580 585 590 Met Ser Gly Pro Tyr Asn Ser Ser Pro
Arg Pro Glu Gln His Lys Ser 595 600 605 Tyr Lys Ile Arg Phe Asn Ser
Ile Ser Cys Ser Asp Pro Leu Val Ser 610 615 620 Ser Trp Arg Arg Lys
Arg Lys Glu Ser Ser Asn Thr Asp Ser Ala Gly 625 630 635 640 Ala Leu
Gly Thr Leu Arg Phe Cys Val Phe Gly Leu Gly Ser Arg Ala 645 650 655
Tyr Pro His Phe Cys Ala Phe Ala Arg Ala Val Asp Thr Arg Leu Glu 660
665 670 Glu Leu Gly Gly Glu Arg Leu Leu Gln Leu Gly Gln Gly Asp Glu
Leu 675 680 685 Cys Gly Gln Glu Glu Ala Phe Arg Gly Trp Ala Gln Ala
Ala Phe Gln 690 695 700 Ala Ala Cys Glu Thr Phe Cys Val Gly Glu Asp
Ala Lys Ala Ala Ala 705 710 715 720 Arg Asp Ile Phe Ser Pro Lys Arg
Ser Trp Lys Arg Gln Arg Tyr Arg 725 730 735 Leu Ser Ala Gln Ala Glu
Gly Leu Gln Leu Leu Pro Gly Leu Ile His 740 745 750 Val His Arg Arg
Lys Met Phe Gln Ala Thr Ile Arg Ser Val Glu Asn 755 760 765 Leu Gln
Ser Ser Lys Ser Thr Arg Ala Thr Ile Leu Val Arg Leu Asp 770 775 780
Thr Gly Gly Gln Glu Gly Leu Gln Tyr Gln Pro Gly Asp His Ile Gly 785
790 795 800 Val Cys Pro Pro Asn Arg Pro Gly Leu Val Glu Ala Leu Leu
Ser Arg 805 810 815 Val Glu Asp Pro Pro Ala Pro Thr Glu Pro Val Ala
Val Glu Gln Leu 820 825 830 Glu Lys Gly Ser Pro Gly Gly Pro Pro Pro
Gly Trp Val Arg Asp Pro 835 840 845 Arg Leu Pro Pro Cys Thr Leu Arg
Gln Ala Leu Thr Phe Phe Leu Asp 850 855 860 Ile Thr Ser Pro Pro Ser
Pro Gln Leu Leu Arg Leu Leu Ser Thr Leu 865 870 875 880 Ala Glu Glu
Pro Arg Glu Gln Gln Glu Leu Glu Ala Leu Ser Gln Asp 885 890 895 Pro
Arg Arg Tyr Glu Glu Trp Lys Trp Phe Arg Cys Pro Thr Leu Leu 900 905
910 Glu Val Leu Glu Gln Phe Pro Ser Val Ala Leu Pro Ala Pro Leu Leu
915 920 925 Leu Thr Gln Leu Pro Leu Leu Gln Pro Arg Tyr Tyr Ser Val
Ser Ser 930 935 940 Ala Pro Ser Thr His Pro Gly Glu Ile His Leu Thr
Val Ala Val Leu 945 950 955 960 Ala Tyr Arg Thr Gln Asp Gly Leu Gly
Pro Leu His Tyr Gly Val Cys 965 970 975 Ser Thr Trp Leu Ser Gln Leu
Lys Pro Gly Asp Pro Val Pro Cys Phe 980 985 990 Ile Arg Gly Ala Pro
Ser Phe Arg Leu Pro Pro Asp Pro Ser Leu Pro 995 1000 1005 Cys Ile
Leu Val Gly Pro Gly Thr Gly Ile Ala Pro Phe Arg Gly 1010 1015 1020
Phe Trp Gln Glu Arg Leu His Asp Ile Glu Ser Lys Gly Leu Gln 1025
1030 1035 Pro Thr Pro Met Thr Leu Val Phe Gly Cys Arg Cys Ser Gln
Leu 1040 1045 1050 Asp His Leu Tyr Arg Asp Glu Val Gln Asn Ala Gln
Gln Arg Gly 1055 1060 1065 Val Phe Gly Arg Val Leu Thr Ala Phe Ser
Arg Glu Pro Asp Asn 1070 1075 1080 Pro Lys Thr Tyr Val Gln Asp Ile
Leu Arg Thr Glu Leu Ala Ala 1085 1090 1095 Glu Val His Arg Val Leu
Cys Leu Glu Arg Gly His Met Phe Val 1100 1105 1110 Cys Gly Asp Val
Thr Met Ala Thr Asn Val Leu Gln Thr Val Gln 1115 1120 1125 Arg Ile
Leu Ala Thr Glu Gly Asp Met Glu Leu Asp Glu Ala Gly 1130 1135 1140
Asp Val Ile Gly Val Leu Arg Asp Gln Gln Arg Tyr His Glu Asp 1145
1150 1155 Ile Phe Gly Leu Thr Leu Arg Thr Gln Glu Val Thr Ser Arg
Ile 1160 1165 1170 Arg Thr Gln Asp Phe Ser Leu Gln Glu Arg Gln Leu
Arg Gly Ala 1175 1180 1185 Val Pro Trp Ala Phe Asp Pro Pro Gly Ser
Asp Thr Asn Ser Pro 1190 1195 1200 7 1202 PRT Homo sapiens 7 Met
Gly Asn Leu Lys Ser Val Ala Gln Glu Pro Gly Pro Pro Cys Gly 1 5
10 15 Leu Gly Leu Gly Leu Gly Leu Gly Leu Cys Gly Lys Gln Gly Pro
Ala 20 25 30 Thr Pro Ala Pro Glu Pro Ser Arg Ala Pro Ala Ser Leu
Leu Pro Pro 35 40 45 Ala Pro Glu His Ser Pro Pro Ser Ser Pro Leu
Thr Gln Pro Pro Glu 50 55 60 Gly Pro Lys Phe Pro Arg Val Lys Asn
Trp Glu Val Gly Ser Ile Thr 65 70 75 80 Tyr Asp Thr Leu Ser Ala Gln
Ala Gln Gln Asp Gly Pro Cys Thr Pro 85 90 95 Arg Arg Cys Leu Gly
Ser Leu Val Phe Pro Arg Lys Leu Gln Gly Arg 100 105 110 Pro Ser Pro
Gly Pro Pro Ala Pro Glu Gln Leu Leu Ser Gln Ala Arg 115 120 125 Asp
Phe Ile Asn Gln Tyr Tyr Ser Ser Ile Lys Arg Ser Gly Ser Gln 130 135
140 Ala His Glu Gln Arg Leu Gln Glu Val Glu Ala Glu Val Ala Ala Thr
145 150 155 160 Gly Thr Tyr Gln Leu Arg Glu Ser Glu Leu Val Phe Gly
Ala Lys Gln 165 170 175 Ala Trp Arg Asn Ala Pro Arg Cys Val Gly Arg
Ile Gln Trp Gly Lys 180 185 190 Leu Gln Val Phe Asp Ala Arg Asp Cys
Arg Ser Ala Gln Glu Met Phe 195 200 205 Thr Tyr Ile Cys Asn His Ile
Lys Tyr Ala Thr Asn Arg Gly Asn Leu 210 215 220 Arg Ser Ala Ile Thr
Val Phe Pro Gln Arg Cys Pro Gly Arg Gly Asp 225 230 235 240 Phe Arg
Ile Trp Asn Ser Gln Leu Val Arg Tyr Ala Gly Tyr Arg Gln 245 250 255
Gln Asp Gly Ser Val Arg Gly Asp Pro Ala Asn Val Glu Ile Thr Glu 260
265 270 Leu Cys Ile Gln His Gly Trp Thr Pro Gly Asn Gly Arg Phe Asp
Val 275 280 285 Leu Pro Leu Leu Leu Gln Ala Pro Asp Glu Pro Pro Glu
Leu Phe Leu 290 295 300 Leu Pro Pro Glu Leu Val Leu Glu Val Pro Leu
Glu His Pro Thr Leu 305 310 315 320 Glu Trp Phe Ala Ala Leu Gly Leu
Arg Trp Tyr Ala Leu Pro Ala Val 325 330 335 Ser Asn Met Leu Leu Glu
Ile Gly Gly Leu Glu Phe Pro Ala Ala Pro 340 345 350 Phe Ser Gly Trp
Tyr Met Ser Thr Glu Ile Gly Thr Arg Asn Leu Cys 355 360 365 Asp Pro
His Arg Tyr Asn Ile Leu Glu Asp Val Ala Val Cys Met Asp 370 375 380
Leu Asp Thr Arg Thr Thr Ser Ser Leu Trp Lys Asp Lys Ala Ala Val 385
390 395 400 Glu Ile Asn Val Ala Val Leu His Ser Tyr Gln Leu Ala Lys
Val Thr 405 410 415 Ile Val Asp His His Ala Ala Thr Ala Ser Phe Met
Lys His Leu Glu 420 425 430 Asn Glu Gln Lys Ala Arg Gly Gly Cys Pro
Ala Asp Trp Ala Trp Ile 435 440 445 Val Pro Pro Ile Ser Gly Ser Leu
Thr Pro Val Phe His Gln Glu Met 450 455 460 Val Asn Tyr Phe Leu Ser
Pro Ala Phe Arg Tyr Gln Pro Asp Pro Trp 465 470 475 480 Lys Gly Ser
Ala Ala Lys Gly Thr Gly Ile Thr Arg Lys Lys Thr Phe 485 490 495 Lys
Glu Val Ala Asn Ala Val Lys Ile Ser Ala Ser Leu Met Gly Thr 500 505
510 Val Met Ala Lys Arg Val Lys Ala Thr Ile Leu Tyr Gly Ser Glu Thr
515 520 525 Gly Arg Ala Gln Ser Tyr Ala Gln Gln Leu Gly Arg Leu Phe
Arg Lys 530 535 540 Ala Phe Asp Pro Arg Val Leu Cys Met Asp Glu Tyr
Asp Val Val Ser 545 550 555 560 Leu Glu His Glu Thr Leu Val Leu Val
Val Thr Ser Thr Phe Gly Asn 565 570 575 Gly Asp Pro Pro Glu Asn Gly
Glu Ser Phe Ala Ala Ala Leu Met Glu 580 585 590 Met Ser Gly Pro Tyr
Asn Ser Ser Pro Arg Pro Glu Gln His Lys Ser 595 600 605 Tyr Lys Ile
Arg Phe Asn Ser Ile Ser Cys Ser Asp Pro Leu Val Ser 610 615 620 Ser
Trp Arg Arg Lys Arg Lys Glu Ser Ser Asn Thr Asp Ser Ala Gly 625 630
635 640 Ala Leu Gly Thr Leu Arg Phe Cys Val Phe Gly Leu Gly Ser Arg
Ala 645 650 655 Tyr Pro His Phe Cys Ala Phe Ala Arg Ala Val Asp Thr
Arg Leu Glu 660 665 670 Glu Leu Gly Gly Glu Arg Leu Leu Gln Leu Gly
Gln Gly Asp Glu Leu 675 680 685 Cys Gly Gln Glu Glu Ala Phe Arg Gly
Trp Ala Gln Ala Ala Phe Gln 690 695 700 Ala Ala Cys Glu Thr Phe Cys
Val Gly Glu Asp Ala Lys Ala Ala Ala 705 710 715 720 Arg Asp Ile Phe
Ser Pro Lys Arg Ser Trp Lys Arg Gln Arg Tyr Arg 725 730 735 Leu Ser
Ala Gln Ala Glu Gly Leu Gln Leu Leu Pro Gly Leu Ile His 740 745 750
Val His Arg Arg Lys Met Phe Gln Ala Thr Ile Arg Ser Val Glu Asn 755
760 765 Leu Gln Ser Ser Lys Ser Thr Arg Ala Thr Ile Leu Val Arg Leu
Asp 770 775 780 Thr Gly Gly Gln Glu Gly Leu Gln Tyr Gln Pro Gly Asp
His Ile Gly 785 790 795 800 Val Cys Pro Pro Asn Arg Pro Gly Leu Val
Glu Ala Leu Leu Ser Arg 805 810 815 Val Glu Asp Pro Pro Ala Pro Thr
Glu Pro Val Ala Val Glu Gln Leu 820 825 830 Glu Lys Gly Ser Pro Gly
Gly Pro Pro Pro Gly Trp Val Arg Asp Pro 835 840 845 Arg Leu Pro Pro
Cys Thr Leu Arg Gln Ala Leu Thr Phe Phe Leu Asp 850 855 860 Ile Thr
Ser Pro Pro Ser Pro Gln Leu Leu Arg Leu Leu Ser Thr Leu 865 870 875
880 Ala Glu Glu Pro Arg Glu Gln Gln Glu Leu Glu Ala Leu Ser Gln Asp
885 890 895 Pro Arg Arg Tyr Glu Glu Trp Lys Trp Phe Arg Cys Pro Thr
Leu Leu 900 905 910 Glu Val Leu Glu Gln Phe Pro Ser Val Ala Leu Pro
Ala Pro Leu Leu 915 920 925 Leu Thr Gln Leu Pro Leu Leu Gln Pro Arg
Tyr Tyr Ser Val Ser Ser 930 935 940 Ala Pro Ser Thr His Pro Gly Glu
Ile His Leu Thr Val Ala Val Leu 945 950 955 960 Ala Tyr Arg Thr Gln
Asp Gly Leu Gly Pro Leu His Tyr Gly Val Cys 965 970 975 Ser Thr Trp
Leu Ser Gln Leu Lys Pro Gly Asp Pro Val Pro Cys Phe 980 985 990 Ile
Arg Gly Ala Pro Ser Phe Arg Leu Pro Pro Asp Pro Ser Leu Pro 995
1000 1005 Cys Ile Leu Val Gly Pro Gly Thr Gly Ile Ala Pro Phe Arg
Gly 1010 1015 1020 Phe Trp Gln Glu Arg Leu His Asp Ile Glu Ser Lys
Gly Leu Gln 1025 1030 1035 Pro Thr Pro Met Thr Leu Val Phe Gly Cys
Arg Cys Ser Gln Leu 1040 1045 1050 Asp His Leu Tyr Arg Asp Glu Val
Gln Asn Ala Gln Gln Arg Gly 1055 1060 1065 Val Phe Gly Arg Val Leu
Thr Ala Phe Ser Arg Glu Pro Asp Asn 1070 1075 1080 Pro Lys Thr Tyr
Val Gln Asp Ile Leu Arg Thr Glu Leu Ala Ala 1085 1090 1095 Glu Val
His Arg Val Leu Cys Leu Glu Arg Gly His Met Phe Val 1100 1105 1110
Cys Gly Asp Val Thr Met Ala Thr Asn Val Leu Gln Thr Val Gln 1115
1120 1125 Arg Ile Leu Ala Thr Glu Gly Asp Met Glu Leu Asp Glu Ala
Gly 1130 1135 1140 Asp Val Ile Gly Val Leu Arg Asp Gln Gln Arg Tyr
His Glu Asp 1145 1150 1155 Ile Phe Gly Leu Thr Leu Arg Thr Gln Glu
Val Thr Ser Arg Ile 1160 1165 1170 Arg Thr Gln Glu Ser Leu Gln Glu
Arg Gln Leu Arg Gly Ala Val 1175 1180 1185 Pro Trp Ala Phe Asp Pro
Pro Gly Ser Asp Thr Asn Ser Pro 1190 1195 1200 8 3612 DNA Homo
sapiens 8 atgggcaact tgaagagcgt ggcccaggag cctgggccac cctgcggcct
ggggctgggg 60 ctgggccttg ggctgtgcgg caagcagggc ccagccaccc
cggcccctga gcccagccgg 120 gccccagcat ccctactccc accagcgcca
gaacacagcc ccccgagctc cccgctaacc 180 cagcccccag aggggcccaa
gttccctcgt gtgaagaact gggaggtggg gagcatcacc 240 tatgacaccc
tcagcgccca ggcgcagcag gatgggccct gcaccccaag acgctgcctg 300
ggctccctgg tatttccacg gaaactacag ggccggccct cccccggccc cccggcccct
360 gagcagctgc tgagtcaggc ccgggacttc atcaaccagt actacagctc
cattaagagg 420 agcggctccc aggcccacga acagcggctt caagaggtgg
aagccgaggt ggcagccaca 480 ggcacctacc agcttaggga gagcgagctg
gtgttcgggg ctaagcaggc ctggcgcaac 540 gctccccgct gcgtgggccg
gatccagtgg gggaagctgc aggtgttcga tgcccgggac 600 tgcaggtctg
cacaggaaat gttcacctac atctgcaacc acatcaagta tgccaccaac 660
cggggcaacc ttcgctcggc catcacagtg ttcccgcagc gctgccctgg ccgaggagac
720 ttccgaatct ggaacagcca gctggtgcgc tacgcgggct accggcagca
ggacggctct 780 gtgcgggggg acccagccaa cgtggagatc accgagctct
gcattcagca cggctggacc 840 ccaggaaacg gtcgcttcga cgtgctgccc
ctgctgctgc aggccccaga tgagccccca 900 gaactcttcc ttctgccccc
cgagctggtc cttgaggtgc ccctggagca ccccacgctg 960 gagtggtttg
cagccctggg cctgcgctgg tacgccctcc cggcagtgtc caacatgctg 1020
ctggaaattg ggggcctgga gttccccgca gcccccttca gtggctggta catgagcact
1080 gagatcggca cgaggaacct gtgtgaccct caccgctaca acatcctgga
ggatgtggct 1140 gtctgcatgg acctggatac ccggaccacc tcgtccctgt
ggaaagacaa ggcagcagtg 1200 gaaatcaacg tggccgtgct gcacagttac
cagctagcca aagtcaccat cgtggaccac 1260 cacgccgcca cggcctcttt
catgaagcac ctggagaatg agcagaaggc cagggggggc 1320 tgccctgcag
actgggcctg gatcgtgccc cccatctcgg gcagcctcac tcctgttttc 1380
catcaggaga tggtcaacta tttcctgtcc ccggccttcc gctaccagcc agacccctgg
1440 aaggggagtg ccgccaaggg caccggcatc accaggaaga agacctttaa
agaagtggcc 1500 aacgccgtga agatctccgc ctcgctcatg ggcacggtga
tggcgaagcg agtgaaggcg 1560 acaatcctgt atggctccga gaccggccgg
gcccagagct acgcacagca gctggggaga 1620 ctcttccgga aggcttttga
tccccgggtc ctgtgtatgg atgagtatga cgtggtgtcc 1680 ctcgaacacg
agacgctggt gctggtggta accagcacat ttgggaatgg ggatcccccg 1740
gagaatggag agagctttgc agctgccctg atggagatgt ccggccccta caacagctcc
1800 cctcggccgg aacagcacaa gagttataag atccgcttca acagcatctc
ctgctcagac 1860 ccactggtgt cctcttggcg gcggaagagg aaggagtcca
gtaacacaga cagtgcaggg 1920 gccctgggca ccctcaggtt ctgtgtgttc
gggctcggct cccgggcata cccccacttc 1980 tgcgcctttg ctcgtgccgt
ggacacacgg ctggaggaac tgggcgggga gcggctgctg 2040 cagctgggcc
agggcgacga gctgtgcggc caggaggagg ccttccgagg ctgggcccag 2100
gctgccttcc aggccgcctg tgagaccttc tgtgtgggag aggatgccaa ggccgccgcc
2160 cgagacatct tcagccccaa acggagctgg aagcgccaga ggtaccggct
gagcgcccag 2220 gccgagggcc tgcagttgct gccaggtctg atccacgtgc
acaggcggaa gatgttccag 2280 gctacaatcc gctcagtgga aaacctgcaa
agcagcaagt ccacgagggc caccatcctg 2340 gtgcgcctgg acaccggagg
ccaggagggg ctgcagtacc agccggggga ccacataggt 2400 gtctgcccgc
ccaaccggcc cggccttgtg gaggcgctgc tgagccgcgt ggaggacccg 2460
ccggcgccca ctgagcccgt ggcagtagag cagctggaga agggcagccc tggtggccct
2520 ccccccggct gggtgcggga cccccggctg cccccgtgca cgctgcgcca
ggctctcacc 2580 ttcttcctgg acatcacctc cccacccagc cctcagctct
tgcggctgct cagcaccttg 2640 gcagaagagc ccagggaaca gcaggagctg
gaggccctca gccaggatcc ccgacgctac 2700 gaggagtgga agtggttccg
ctgccccacg ctgctggagg tgctggagca gttcccgtcg 2760 gtggcgctgc
ctgccccact gctcctcacc cagctgcctc tgctccagcc ccggtactac 2820
tcagtcagct cggcacccag cacccaccca ggagagatcc acctcactgt agctgtgctg
2880 gcatacagga ctcaggatgg gctgggcccc ctgcactatg gagtctgctc
cacgtggcta 2940 agccagctca agcccggaga ccctgtgccc tgcttcatcc
ggggggctcc ctccttccgg 3000 ctgccacccg atcccagctt gccctgcatc
ctggtgggtc caggcactgg cattgccccc 3060 ttccggggat tctggcagga
gcggctgcat gacattgaga gcaaagggct gcagcccact 3120 cccatgactt
tggtgttcgg ctgccgatgc tcccaacttg accatctcta ccgcgacgag 3180
gtgcagaacg cccagcagcg cggggtgttt ggccgagtcc tcaccgcctt ctcccgggaa
3240 cctgacaacc ccaagaccta cgtgcaggac atcctgagga cggagctggc
tgcggaggtg 3300 caccgcgtgc tgtgcctcga gcggggccac atgtttgtct
gcggcgatgt taccatggca 3360 accaacgtcc tgcagaccgt gcagcgcatc
ctggcgacgg agggcgacat ggagctggac 3420 gaggccggcg acgtcatcgg
cgtgctgcgg gatcagcaac gctaccacga agacattttc 3480 gggctcacgc
tgcgcaccca ggaggtgaca agccgcatac gcacccagag cttttccttg 3540
caggagcgtc agttgcgggg cgcagtgccc tgggcgttcg accctcccgg ctcagacacc
3600 aacagcccct ga 3612 9 4077 DNA Homo sapiens 9 gaattcccac
tctgctgcct gctccagcag acggacgcac agtaacatgg gcaacttgaa 60
gagcgtggcc caggagcctg ggccaccctg cggcctgggg ctggggctgg gccttgggct
120 gtgcggcaag cagggcccag ccaccccggc ccctgagccc agccgggccc
cagcatccct 180 actcccacca gcgccagaac acagcccccc gagctccccg
ctaacccagc ccccagaggg 240 gcccaagttc cctcgtgtga agaactggga
ggtggggagc atcacctatg acaccctcag 300 cgcccaggcg cagcaggatg
ggccctgcac cccaagacgc tgcctgggct ccctggtatt 360 tccacggaaa
ctacagggcc ggccctcccc cggccccccg gcccctgagc agctgctgag 420
tcaggcccgg gacttcatca accagtacta cagctccatt aagaggagcg gctcccaggc
480 ccacgaacag cggcttcaag aggtggaagc cgaggtggca gccacaggca
cctaccagct 540 tagggagagc gagctggtgt tcggggctaa gcaggcctgg
cgcaacgctc cccgctgcgt 600 gggccggatc cagtggggga agctgcaggt
gttcgatgcc cgggactgca ggtctgcaca 660 ggaaatgttc acctacatct
gcaaccacat caagtatgcc accaaccggg gcaaccttcg 720 ctcggccatc
acagtgttcc cgcagcgctg ccctggccga ggagacttcc gaatctggaa 780
cagccagctg gtgcgctacg cgggctaccg gcagcaggac ggctctgtgc ggggggaccc
840 agccaacgtg gagatcaccg agctctgcat tcagcacggc tggaccccag
gaaacggtcg 900 cttcgacgtg ctgcccctgc tgctgcaggc cccagatgag
cccccagaac tcttccttct 960 gccccccgag ctggtccttg aggtgcccct
ggagcacccc acgctggagt ggtttgcagc 1020 cctgggcctg cgctggtacg
ccctcccggc agtgtccaac atgctgctgg aaattggggg 1080 cctggagttc
cccgcagccc ccttcagtgg ctggtacatg agcactgaga tcggcacgag 1140
gaacctgtgt gaccctcacc gctacaacat cctggaggat gtggctgtct gcatggacct
1200 ggatacccgg accacctcgt ccctgtggaa agacaaggca gcagtggaaa
tcaacgtggc 1260 cgtgctgcac agttaccagc tagccaaagt caccatcgtg
gaccaccacg ccgccacggc 1320 ctctttcatg aagcacctgg agaatgagca
gaaggccagg gggggctgcc ctgcagactg 1380 ggcctggatc gtgcccccca
tctcgggcag cctcactcct gttttccatc aggagatggt 1440 caactatttc
ctgtccccgg ccttccgcta ccagccagac ccctggaagg ggagtgccgc 1500
caagggcacc ggcatcacca ggaagaagac ctttaaagaa gtggccaacg ccgtgaagat
1560 ctccgcctcg ctcatgggca cggtgatggc gaagcgagtg aaggcgacaa
tcctgtatgg 1620 ctccgagacc ggccgggccc agagctacgc acagcagctg
gggagactct tccggaaggc 1680 ttttgatccc cgggtcctgt gtatggatga
gtatgacgtg gtgtccctcg aacacgagac 1740 gctggtgctg gtggtaacca
gcacatttgg gaatggggat cccccggaga atggagagag 1800 ctttgcagct
gccctgatgg agatgtccgg cccctacaac agctcccctc ggccggaaca 1860
gcacaagagt tataagatcc gcttcaacag catctcctgc tcagacccac tggtgtcctc
1920 ttggcggcgg aagaggaagg agtccagtaa cacagacagt gcaggggccc
tgggcaccct 1980 caggttctgt gtgttcgggc tcggctcccg ggcatacccc
cacttctgcg cctttgctcg 2040 tgccgtggac acacggctgg aggaactggg
cggggagcgg ctgctgcagc tgggccaggg 2100 cgacgagctg tgcggccagg
aggaggcctt ccgaggctgg gcccaggctg ccttccaggc 2160 cgcctgtgag
accttctgtg tgggagagga tgccaaggcc gccgcccgag acatcttcag 2220
ccccaaacgg agctggaagc gccagaggta ccggctgagc gcccaggccg agggcctgca
2280 gttgctgcca ggtctgatcc acgtgcacag gcggaagatg ttccaggcta
caatccgctc 2340 agtggaaaac ctgcaaagca gcaagtccac gagggccacc
atcctggtgc gcctggacac 2400 cggaggccag gaggggctgc agtaccagcc
gggggaccac ataggtgtct gcccgcccaa 2460 ccggcccggc cttgtggagg
cgctgctgag ccgcgtggag gacccgccgg cgcccactga 2520 gcccgtggca
gtagagcagc tggagaaggg cagccctggt ggccctcccc ccggctgggt 2580
gcgggacccc cggctgcccc cgtgcacgct gcgccaggct ctcaccttct tcctggacat
2640 cacctcccca cccagccctc agctcttgcg gctgctcagc accttggcag
aagagcccag 2700 ggaacagcag gagctggagg ccctcagcca ggatccccga
cgctacgagg agtggaagtg 2760 gttccgctgc cccacgctgc tggaggtgct
ggagcagttc ccgtcggtgg cgctgcctgc 2820 cccactgctc ctcacccagc
tgcctctgct ccagccccgg tactactcag tcagctcggc 2880 acccagcacc
cacccaggag agatccacct cactgtagct gtgctggcat acaggactca 2940
ggatgggctg ggccccctgc actatggagt ctgctccacg tggctaagcc agctcaagcc
3000 cggagaccct gtgccctgct tcatccgggg ggctccctcc ttccggctgc
cacccgatcc 3060 cagcttgccc tgcatcctgg tgggtccagg cactggcatt
gcccccttcc ggggattctg 3120 gcaggagcgg ctgcatgaca ttgagagcaa
agggctgcag cccactccca tgactttggt 3180 gttcggctgc cgatgctccc
aacttgacca tctctaccgc gacgaggtgc agaacgccca 3240 gcagcgcggg
gtgtttggcc gagtcctcac cgccttctcc cgggaacctg acaaccccaa 3300
gacctacgtg caggacatcc tgaggacgga gctggctgcg gaggtgcacc gcgtgctgtg
3360 cctcgagcgg ggccacatgt ttgtctgcgg cgatgttacc atggcaacca
acgtcctgca 3420 gaccgtgcag cgcatcctgg cgacggaggg cgacatggag
ctggacgagg ccggcgacgt 3480 catcggcgtg ctgcgggatc agcaacgcta
ccacgaagac attttcgggc tcacgctgcg 3540 cacccaggag gtgacaagcc
gcatacgcac ccagagcttt tccttgcagg agcgtcagtt 3600 gcggggcgca
gtgccctggg cgttcgaccc tcccggctca gacaccaaca gcccctgaga 3660
gccgcctggc tttcccttcc agttccggga gagcggctgc ccgactcagg tccgcccgac
3720 caggatcagc cccgctcctc ccctcttgag gtggtgcctt ctcacatctg
tccagaggct 3780 gcaaggattc agcattattc ctccaggaag gagcaaaacg
cctcttttcc ctctctaggc 3840 ctgttgcctc gggcctgggt ccgccttaat
ctggaaggcc cctcccagca gcggtacccc 3900 agggcctact gccacccgct
tcctgtttct tagtccgaat gttagattcc tcttgcctct 3960 ctcaggagta
tcttacctgt aaagtctaat ctctaaatca agtatttatt
attgaagatt 4020 taccataagg gactgtgcca gatgttagga gaactactaa
agtgcctacc ccagctc 4077
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