U.S. patent application number 10/663401 was filed with the patent office on 2005-01-20 for human glucose-6-phosphatase molecules and uses thereof.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Chen, Hong.
Application Number | 20050014241 10/663401 |
Document ID | / |
Family ID | 24346035 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050014241 |
Kind Code |
A1 |
Chen, Hong |
January 20, 2005 |
Human glucose-6-phosphatase molecules and uses thereof
Abstract
The invention provides isolated nucleic acids encoding human
pancreatic islet-specific glucose-6-phosphatase proteins and
nucleic acids having diagnostic, preventive, therapeutic, and other
uses. These nucleic acids and proteins are useful for diagnosis,
prevention, and therapy of a number of human and other animal
disorders. The invention also provides antisense nucleic acid
molecules, expression vectors containing the nucleic acid molecules
of the invention, host cells into which the expression vectors have
been introduced, and non-human transgenic animals in which a
nucleic acid molecule of the invention has been introduced or
disrupted. The invention still further provides isolated
polypeptides, fusion polypeptides, antigenic peptides, and
antibodies. Diagnostic, screening, and therapeutic methods
utilizing compositions of the invention are also provided. The
nucleic acids and polypeptides of the present invention are useful
as modulating agents in regulating a variety of cellular processes,
including those which are aberrant in diabetes and other disorders
associated with pancreatic dysfunction. The invention includes
methods of modulating secretion of pancreatic hormones such as
insulin and glucagon, and these methods can be used to alleviate
disorders (e.g., diabetes and hyperinsulinemia) associated with
aberrant secretion of these hormones.
Inventors: |
Chen, Hong; (Newton,
MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
Intellectual Property Department
75 Sidney Street
Cambridge
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
24346035 |
Appl. No.: |
10/663401 |
Filed: |
September 16, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10663401 |
Sep 16, 2003 |
|
|
|
09874132 |
Jun 4, 2001 |
|
|
|
6623947 |
|
|
|
|
09874132 |
Jun 4, 2001 |
|
|
|
09586611 |
Jun 2, 2000 |
|
|
|
6527356 |
|
|
|
|
Current U.S.
Class: |
435/196 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A01K 2217/05 20130101;
C12N 9/16 20130101; A61P 5/48 20180101; A61K 38/00 20130101 |
Class at
Publication: |
435/196 ;
435/069.1; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12N 009/16; C07H
021/04 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule having a nucleotide
sequence which is at least 91% identical to the nucleotide sequence
of SEQ ID NO: 1 or 2, the nucleotide sequence of the clone
deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282, or a complement of one of these; b) a nucleic acid
molecule comprising at least 35 nucleotide residues and having a
nucleotide sequence identical to at least 35 consecutive nucleotide
residues of SEQ ID NO: 1 or 2, the nucleotide sequence of the clone
deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282, or a complement of one of these; c) a nucleic acid
molecule which encodes a polypeptide having the amino acid sequence
of SEQ ID NO: 3 or the amino acid sequence encoded by the clone
deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282; d) a nucleic acid molecule which encodes a fragment of a
polypeptide having the amino acid sequence of SEQ ID NO: 3 or the
amino acid sequence encoded by the clone deposited with ATCC.RTM.
on Jul. 28, 2000 as accession number PTA-2282, wherein the fragment
comprises at least 15 consecutive amino acids of SEQ ID NO: 3 or
the amino acid sequence encoded by the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282; and e) a
nucleic acid molecule which encodes a naturally occurring allelic
variant of a polypeptide having the amino acid sequence of SEQ ID
NO: 3 or the amino acid sequence encoded by the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282,
wherein the nucleic acid molecule hybridizes with a nucleic acid
molecule having a sequence comprising SEQ ID NO: 1 or 2, or the
nucleotide sequence of the clone deposited with ATCC.RTM. on Jul.
28, 2000 as accession number PTA-2282, or with a complement of one
of these, under stringent conditions.
2. The isolated nucleic acid molecule of claim 1, which is selected
from the group consisting of: a) a nucleic acid having the
nucleotide sequence of SEQ ID NO: 1 or 2, or the nucleotide
sequence of the clone deposited with ATCC.RTM. on Jul. 28, 2000 as
accession number PTA-2282; and b) a nucleic acid molecule which
encodes a polypeptide having the amino acid sequence of SEQ ID NO:
3 or the amino acid sequence encoded by the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282.
3. The nucleic acid molecule of claim 1 further comprising vector
nucleic acid sequences.
4. The nucleic acid molecule of claim 1 further comprising nucleic
acid sequences encoding a heterologous polypeptide.
5. A host cell which contains the nucleic acid molecule of claim
1.
6. The host cell of claim 5 which is a mammalian host cell.
7. A non-human mammalian host cell containing the nucleic acid
molecule of claim 1.
8. An isolated polypeptide selected from the group consisting of:
a) a fragment of a polypeptide having the amino acid sequence SEQ
ID NO: 3 or the amino acid sequence encoded by the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282,
wherein the sequence of the fragment comprises at least 15
consecutive amino acid residues of SEQ ID NO: 3 or the amino acid
sequence encoded by the clone deposited with ATCC.RTM. on Jul. 28,
2000 as accession number PTA-2282. b) a naturally occurring allelic
variant of a polypeptide having the amino acid sequence SEQ ID NO:
3 or the amino acid sequence encoded by the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, wherein
the polypeptide is encoded by a nucleic acid molecule which
hybridizes with a nucleic acid molecule having a sequence
comprising SEQ ID NO: 1 or 2, the nucleotide sequence of the clone
deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282, or a complement of one of these, under stringent
conditions; and c) a polypeptide which is encoded by a nucleic acid
molecule having a nucleotide sequence which is at least 91%
identical to a nucleic acid molecule having a sequence comprising
SEQ ID NO: 1 or 2, the nucleotide sequence of the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or a
complement of one of these.
9. The isolated polypeptide of claim 8 having the amino acid
sequence SEQ ID NO: 3 or the amino acid sequence encoded by the
clone deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282.
10. The polypeptide of claim 8 further comprising heterologous
amino acid sequences.
11. An antibody which selectively binds with the polypeptide of
claim 8.
12. A method for producing a polypeptide selected from the group
consisting of: a) a polypeptide having the amino acid sequence SEQ
ID NO: 3 or the amino acid sequence encoded by the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282; b) a
polypeptide comprising a fragment of the amino acid sequence of SEQ
ID NO: 3 or the amino acid sequence encoded by the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282,
wherein the fragment comprises at least 15 consecutive residues of
SEQ ID NO: 3 or the amino acid sequence encoded by the clone
deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282; and c) a naturally occurring allelic variant of a
polypeptide having the amino acid sequence of SEQ ID NO: 3 or the
amino acid sequence encoded by the clone deposited with ATCC.RTM.
on Jul. 28, 2000 as accession number PTA-2282, wherein the
polypeptide is encoded by a second nucleic acid molecule which
hybridizes with a third nucleic acid molecule having a sequence
comprising SEQ ID NO: 1 or 2, the nucleotide sequence of the clone
deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282, or a complement of one of these, under stringent
conditions; the method comprising culturing the host cell of claim
5 under conditions in which the nucleic acid molecule is
expressed.
13. A method for detecting the presence of the polypeptide of claim
8 in a sample, comprising: a) contacting the sample with a compound
which selectively binds with the polypeptide; and b) determining
whether the compound binds with the polypeptide in the sample.
14. The method of claim 13, wherein the compound which binds with
the polypeptide is an antibody.
15. A kit comprising a compound which selectively binds with the
polypeptide of claim 8 and instructions for use.
16. A method for detecting the presence of the nucleic acid
molecule of claim 1 in a sample, comprising the steps of: a)
contacting the sample with a nucleic acid probe or primer which
selectively hybridizes with the nucleic acid molecule; and b)
determining whether the nucleic acid probe or primer binds to the
nucleic acid molecule in the sample.
17. The method of claim 16, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
18. A kit comprising a compound which selectively hybridizes with
the nucleic acid molecule of claim 1 and instructions for use.
19. A method for identifying a compound which binds with the
polypeptide of claim 8, the method comprising the steps of: a)
contacting the polypeptide, or a cell expressing the polypeptide
with a test compound; and b) determining whether the polypeptide
binds with the test compound.
20. The method of claim 19, wherein binding of the test compound
with the polypeptide is detected by a method selected from the
group consisting of: a) detection of binding by direct detecting of
test compound/polypeptide binding; b) detection of binding using a
competition binding assay; c) detection of binding using an assay
for h-ig6p-mediated signal transduction.
21. A method for modulating the activity of the polypeptide of
claim 8, the method comprising contacting the polypeptide or a cell
expressing the polypeptide with a compound which binds with the
polypeptide, at a concentration sufficient to modulate the activity
of the polypeptide.
22. A method for identifying a compound which modulates the
activity of the polypeptide of claim 8, the method comprising: a)
contacting the polypeptide with a test compound; and b) determining
the effect of the test compound on the activity of the polypeptide
to thereby identify a compound which modulates the activity of the
polypeptide.
23. The method of claim 22, wherein the activity is conversion of
glucose-6-phosphate to glucose.
24. An antibody substance which selectively binds with the
polypeptide of claim 8.
25. A method of modulating a function of a pancreatic islet cell
that is attributable to the activity of h-ig6p protein, the method
comprising contacting the cell with a compound which modulates one
of expression of a gene encoding h-ig6p protein and activity of
h-ig6p protein, whereby the function is modulated.
26. The method of claim 25, wherein the function is insulin
secretion by the cell.
27. A method of assessing whether a compound is useful for
modulating insulin secretion, the method comprising contacting a
test cell which expresses h-ig6p with the compound and comparing
one of expression of h-ig6p protein and activity of h-ig6p protein
in the test cell with expression or activity of h-ig6p protein in a
control cell of the same type, whereby a difference between
expression or activity of h-ig6p protein in the test and control
cells is an indication that the compound is useful for modulating
insulin secretion.
28. The method of claim 27, wherein the test and control cells are
pancreatic cells.
29. The method of claim 27, wherein the test and control cells are
cells that have been transformed with an expression vector encoding
h-ig6p.
30. A method of alleviating diabetes in a human patient, the method
comprising administering to the patient a compound that inhibits
one of expression of a gene encoding h-ig6p protein and activity of
h-ig6p protein in pancreatic islet cells of the patient, whereby
diabetes is alleviated in the patient.
31. A method of alleviating hyperinsulinemia in a human patient,
the method comprising administering to the patient a compound that
enhances one of expression of a gene encoding h-ig6p protein and
activity of h-ig6p protein in pancreatic islet cells of the
patient, whereby hyperinsulinemia is alleviated in the patient.
38. A method of enhancing secretion of insulin in a human patient,
the method comprising administering to the patient a compound that
inhibits one of expression of a gene encoding h-ig6p protein and
activity of h-ig6p protein in pancreatic islet cells of the
patient, whereby secretion of insulin is enhanced in the
patient.
39. A method of inhibiting secretion of insulin in a human patient,
the method comprising administering to the patient a compound that
enhances one of expression of a gene encoding h-ig6p protein and
activity of h-ig6p protein in pancreatic islet cells of the
patient, whereby secretion of insulin is inhibited in the patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of co-pending
U.S. application Ser. No. 09/586,511, filed on Jun. 2, 2000.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
REFERENCE TO A MICROFICHE APPENDIX
[0003] Not applicable.
BACKGROUND OF THE INVENTION
[0004] Glucose-6-phosphatase (G6 Pase; EC 3.1.3.9) catalyzes
hydrolysis of glucose-6-phosphate (G6P), yielding glucose. This
reaction is the terminal step in the gluconeogenic and
glycogenolytic pathways.
[0005] Most cells of the body are able to convert glucose absorbed
from the blood stream to G6P, thereby preventing facilitated
diffusion of the glucose moiety out of the cell. Some cells, such
as liver cells, possess G6 Pase activity, whereby G6P can be
converted to glucose and released to the bloodstream or used by the
cell for metabolism. For example, formation of glucose in the liver
from hepatically-stored glycogen (i.e., involving intermediate
hydrolysis of G6P by G6 Pase) is an important mechanism by which
blood glucose is maintained at a normal level between meals.
[0006] Maintenance of normal blood glucose levels is important for
nutrition of certain tissues (e.g., brain and other nervous system
tissues and gonadal germinal epithelium) which are substantially
incapable of metabolizing other energy sources such as fatty acids
or amino acids. Lipid and protein metabolism can be undesirable, in
that such metabolism depletes bodily stores of lipids and proteins,
and in that the by-products of such metabolism (e.g., certain
lipoprotein-containing particles) can cause or contribute to
pathological conditions (e.g., deposition of lipoprotein plaque in
arteries). Thus, in addition to providing nutrition to tissues
which metabolize glucose almost exclusively, maintenance of normal
blood glucose levels prevents physiologically inappropriate
reliance of the body on non-carbohydrate catabolic routes.
[0007] In diabetic patients, in whom aberrantly diminished
secretion of insulin leads to defects in carbohydrate metabolism,
fat metabolism is abnormally increased, leading to
greater-than-normal levels of circulating fatty acids, which in
turn cause greater-than-normal deposition of cholesterol and other
plaque materials in arteries. Indeed, abnormalities in fat and
protein metabolism are common in diabetics, and account for much of
the morbidity and mortality experienced by such patients, including
acidosis, arteriosclerosis, coronary artery disease and other
circulatory disorders, and wasting disease conditions (i.e.,
attributable to aberrant protein degradation).
[0008] In normal patients, blood insulin level during fasting is
relatively constant, but increases in a two-stage manner upon
influx of glucose, certain amino acids (e.g., lysine, arginine, and
alanine), or particularly both, into the blood stream. A rapid
increase in insulin, attributable to release of pre-formed insulin
stored in secretory granules of pancreas islet of Langerhans beta
cells occurs in the first stage, followed by more gradual and
pronounced release of presumably newly-synthesized insulin in a
second stage. Secretion of glucagon, a hormone secreted by alpha
cells of pancreas islet of Langerhans, is also closely regulated in
coordination with blood levels of glucose and amino acids.
[0009] Although it is known that secretion of insulin and secretion
of glucagon are tightly regulated, and that modulation of secretion
of these molecules occurs rapidly, the mechanisms by which such
secretions are modulated are not fully understood. More
particularly, the mechanism by which blood glucose level, blood
amino acid levels, or both, affect production, processing, and
release of hormones like insulin and glucagon has not been fully
elucidated. Further knowledge of the physiological mechanisms by
which these processes are regulated would enable medical
practitioners to more predictably and efficaciously prognosticate,
diagnose, inhibit, prevent, alleviate, or even cure both
hormone-associated metabolic disorders (e.g., diabetes and
hyperinsulinism) and undesirable physiological phenomena (e.g.
atherosclerosis, tissue wasting) that accompany such disorders.
[0010] Previously characterized G6 Pase enzymes isolated from liver
and kidney tissues are believed to be localized at the membrane of
the endoplasmic reticulum (Ebert et al., 1999, Diabetes 48:543-554;
Burchell, 1990, FASEB J. 4:2978-2988; Mithieux, 1997, Eur. J.
Endocrinol. 136:137-145; Foster et al., 1997, Proc. Soc. Exp. Biol.
Med. 215:314-332) and are also believed to be associated with one
or more proteins which facilitate transmembrane transport of
glucose-6-phostphate, glucose, or both (Gerin et al., 1997, FEBS
Lett. 419:235-238; Waddell et al., 1992, Biochem. J. 286:173-177).
Genes encoding G6 Pase enzymes and catalytic subunits thereof have
been cloned in humans and mice (Lei et al., 1993, Science
262:580-583; Shelly et al., 1993, J. Biol. Chem. 268:21482-21485).
Pancreatic G6 Pase is distinct from the hepatic and kidney forms of
this enzyme, and has been described as being present in the
endoplasmic reticulum of murine pancreatic islet of Langerhans
cells of the alpha and beta types, likely in the form of a
multi-protein complex. (Arden et al., 1999, Diabetes 48:531-542;
Trinh et al., 1997, J. Biol. Chem. 272:24837-24842).
[0011] Human pancreatic G6 Pase has not previously been isolated,
nor has its sequence been determined. G6 Pase activity has been
reported in pancreatic islet cells (Ashcroft et al., 1968, Nature
219:857-858; Waddell et al, 1988, Biochem. J. 255:471-476).
However, what, if any, role the pancreatic form of this enzyme
might have physiologically was not known. A need remains for
isolation and sequencing of the human gene encoding the catalytic
subunit of human pancreatic islet cell-specific G6 Pase. The
present invention satisfies this need.
SUMMARY OF THE INVENTION
[0012] The present invention is based, at least in part, on the
discovery of a cDNA molecule encoding the catalytic subunit of
human pancreatic islet cell-specific G6 Pase. This protein and
fragments, derivatives, and variants thereof are collectively
referred to as polypeptides of the invention or proteins of the
invention. Nucleic acid molecules encoding polypeptides of the
invention are among those collectively referred to as nucleic acid
molecules of the invention.
[0013] Polypeptides of the invention include the catalytic subunit
of human pancreatic islet cell-specific G6 Pase ("h-ig6p") and
proteins which exhibit significant homology therewith (i.e.,
proteins having an amino acid sequence that is at least 85%, 90%,
95%, 98%, or 99% or more identical to SEQ ID NO: 3). Other
polypeptides of the invention include those which comprise (or
consist of) a biologically active portion of h-ig6p (e.g., a
portion which exhibits a catalytic activity of h-ig6p), a
structural feature (e.g., an epitope or secondary structural
domain) of h-ig6p, a functional portion of h-ig6p (e.g., a portion
which binds a physiological substrate), or some combination of
these.
[0014] Nucleic acid molecules of the invention include those which
encode any of the polypeptides of the invention (e.g., a nucleic
acid molecule that encodes the entire catalytic subunit of human
pancreatic islet cell-specific G6 Pase). By way of example, such
nucleic acid molecules can have a nucleotide sequence that
comprises (or consists of) all, or a portion, of one of SEQ ID NO:
1, SEQ ID NO: 2, and the nucleotide sequence of the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or a
complement of one of these sequences. Nucleic acids of the
invention can, alternatively, have a nucleotide sequence that is at
least 91% (or 92%, 95%, 98%, or 99% or more) identical to one of
these sequences, particularly where the sequence identity is such
that some or all of the amino acid residues described herein as
having structural, functional, or catalytic relevance are
preserved.
[0015] The invention also includes nucleic acid molecules which do
not necessarily encode a polypeptide of the invention, but which
are nonetheless suitable, for example, as a hybridization probe for
detection of a nucleic acid encoding a polypeptide of the invention
or as a primer for amplifying (or replicating) all or a portion of
such a nucleic acid.
[0016] Also included within the scope of the invention are
modulators of polypeptides and nucleic acid molecules of the
invention and methods for making and identifying such modulators.
Examples of such modulators include antibodies which bind
specifically with h-ig6p (i.e., with an epitope of h-ig6p) and
h-ig6p-binding fragments of such antibodies. Other examples of
modulators of the invention include anti-sense nucleic acid
molecules which are capable of hybridizing with a nucleic acid
molecule of the invention (particularly including those which
hybridize under stringent binding conditions) and inhibiting (or
even preventing) expression thereof.
[0017] The nucleic acid molecules, polypeptides, and modulators of
the invention are useful as modulating agents for regulating a
variety of cellular processes, particularly including cellular
processes which occur in human pancreatic islet of Langerhans cells
(e.g., in alpha cells, in beta cells, or both). Examples of these
cellular processes include interaction of blood glucose with alpha
and beta cells and sub-cellar components thereof, secretion of
insulin by beta cells, secretion of glucagon by alpha cells, and
transmembrane transport of glucose (optionally coupled with
phosphorylation of glucose) or of G6P (optionally coupled with
de-phosphorylation of G6P) by alpha or beta cells of the pancreas.
The membrane across which the hormone is transported can be, for
example, the cytoplasmic membrane or the membrane surrounding the
endoplasmic reticulum. These cellular processes are involved in
homeostasis in humans not afflicted with a pancreatic disorder, and
can also be involved in development or manifestation of a
pancreatic disease state.
[0018] The invention thus includes methods of inhibiting,
preventing, prognosticating, diagnosing, or treating disorders
which are associated with aberrant expression or activity of
h-ig6p, including pancreatic disorders. Examples of such disorders
include diabetes (e.g., type 2 diabetes, maturity-onset diabetes of
the young, and the like), hyperinsulinism, and glycogen storage
diseases. Pharmaceutical compositions comprising a polypeptide of
the invention, a nucleic acid molecule of the invention, a
modulator of the invention, and combinations of these are included
within the scope of the invention.
[0019] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is the nucleotide sequence (SEQ ID NO: 1) of the cDNA
described herein which encodes h-ig6p.
[0021] FIG. 2, comprising FIGS. 2A-2C, is the amino acid sequence
(SEQ ID NO: 3) of h-ig6p.
[0022] FIG. 3, comprising FIGS. 3A and 3B, is an alignment (made
using the ALIGN software {Myers and Miller (1989) CABIOS, ver.
2.0}; BLOSUM62 scoring matrix, gap penalties 12/4) of the amino
acid sequence (SEQ ID NO: 3) of h-ig6p ("H") and the amino acid
sequence (SEQ ID NO: 4) of the murine pancreatic islet-specific
glucose-6-phosphatase described by Arden et al., (1999, Diabetes
48:531-542).
[0023] FIG. 4 is an image that depicts expression of mRNA encoding
h-ig6p in human pancreas tissue, particularly in pancreatic islet
cells.
[0024] FIG. 5 is an image which depicts detection of RNA encoding
h-ig6p in pancreatic tissue samples obtained from wild type ("WT")
and obese/diabetic mouse strains ob ("OB"), db ("DB"), agouti
("AY"), and tubby ("TUB").
[0025] FIG. 6, comprising FIGS. 6A to 6C, is an alignment of the
amino acid sequences of h-ig6p ("Hp"; SEQ ID NO: 3), murine
pancreatic islet-specific glucose-6-phosphatase ("Mp"; SEQ ID NO:
4), cichlid fish liver glucose-6-phosphatase ("FI"; SEQ ID NO: 24),
murine liver glucose-6-phosphatase ("MI"; SEQ ID NO: 25), canine
liver glucose-6-phosphatase ("CI"; SEQ ID NO: 26), and human liver
glucose-6-phosphatase ("HI"; SEQ ID NO: 27). Bold, underlined
residues indicate predicted transmembrane regions, as indicated in
Arden et al. (1999, Diabetes 48:531-542).
DETAILED DESCRIPTION
[0026] The present invention is based on the discovery of a cDNA
molecule encoding the catalytic subunit of human pancreatic
glucose-6-phosphatase (G6 Pase) that is expressed specifically in
islet of Langerhans cells. This subunit is herein designated
h-ig6p.
[0027] A cDNA clone encoding h-ig6p was isolated from a human islet
of Langerhans cell cDNA library. h-ig6p is predicted by structural
analysis to be a transmembrane protein that can be localized at the
endoplasmic reticulum. Cell fractionation experiments performed
using Cos-7 cells which had been transformed with an expression
vector encoding h-ig6p indicated that h-ig6p localizes to the
microsomal fraction of fractionated cells, further indicating that
h-ig6p associates with the endoplasmic reticulum under
physiological conditions.
[0028] The full length of the cDNA encoding h-ig6p (FIG. 1; SEQ ID
NO: 1) is 1138 nucleotide residues. The ORF of this cDNA,
nucleotide residues 54 to 1121 of SEQ ID NO: 1 (i.e. SEQ ID NO: 2),
encodes a 355-amino acid residue transmembrane protein (FIG. 2; SEQ
ID NO: 3). A clone comprising a nucleic acid having a sequence
comprising SEQ ID NO: 1 was deposited with the American Type
Culture Collection.RTM. (ATCC.RTM.; 10801 University Blvd.
Manassas, Va. 20110-2209) on Jul. 28, 2000, and was assigned
accession number PTA-2282.
[0029] In addition to purified h-ig6p protein, the invention
includes fragments, derivatives, and variants (e.g., allelic
variants and highly homologous polypeptides) of h-ig6p protein, as
described herein. These proteins, fragments, derivatives, and
variants are collectively referred to herein as polypeptides of the
invention or proteins of the invention. In addition to full-length
h-ig6p (i.e., the protein having the amino acid sequence SEQ ID NO:
3), the invention includes portions of h-ig6p which have
structural, functional, or catalytic significance, as described
herein.
[0030] The invention also includes nucleic acid molecules which
encode h-ig6p and fragments, derivatives, and variants thereof.
Such nucleic acids include, for example, a DNA molecule having the
nucleotide sequence listed in SEQ ID NO: 1 or some portion thereof,
such as a portion encoding a domain of h-ig6p described herein, and
a DNA molecule having the nucleotide sequence of the clone
deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282. Nucleic acid molecules of the invention also include
those which do not necessarily encode h-ig6p or a structurally-,
functionally-, or catalytically-relevant portion thereof, but which
hybridize with a nucleic acid which does encode such a portion
(particularly including nucleic acid molecules which hybridize with
such nucleic acids under stringent binding conditions). These
nucleic acids are collectively referred to as nucleic acid
molecules of the invention.
[0031] h-ig6p proteins typically comprise a variety of potential
post-translational modification sites (often within an
extracellular domain), such as those described herein in Table I,
as predicted by computerized sequence analysis of h-ig6p using
amino acid sequence comparison software (comparing the amino acid
sequence of h-ig6p with the information in the PROSITE database
{rel. 12.2; February, 1995} and the Hidden Markov Models database
{Rel. PFAM 3.3}). In certain embodiments, a protein of the
invention has at least 1, 2, 4, 6, 10, or all 16 of the
post-translational modification sites listed in Table I.
1TABLE I Type of Potential Modification Amino Acid Residues of
Amino Acid Site or Domain SEQ ID NO: 3 Sequence N-glycosylation
site 50 to 53 NQTY 92 to 95 NHSS 287 to 290 NYTL Protein Kinase C
phosphoryla- 281 to 283 SCR tion site 291 to 293 SFR 350 to 352 SGK
Casein kinase II phosphorylation 103 to 106 TTCE site 314 to 317
THEE N-myristoylation site 110 to 115 GSPSGH 182 to 187 GVIGGM 198
to 203 GIQTAS 258 to 263 GLVRNL 264 to 269 GVLFGL 285 to 290 GNNYTL
Amidation site 350 to 353 SGKK Endoplasmic reticulum mem- 351 to
355 KKSQ brane retention site
[0032] h-ig6p protein exhibits sequence similarity to a murine
pancreatic islet-specific G6 Pase catalytic subunit, as indicated
herein in FIG. 3. FIG. 3 is an alignment of the amino acid
sequences of h-ig6p (SEQ ID NO: 3) and the murine pancreatic
islet-specific G6 Pase catalytic subunit (SEQ ID NO: 23;
GENBANK.TM. accession number Z47787; Arden et al., 1999, Diabetes
48:531-542; Ebert et al., 1999, Diabetes 48:543-554). In this
alignment (made using the ALIGN software {Myers and Miller (1989)
CABIOS, ver. 2.0}; BLOSUM62 scoring matrix, gap penalties 12/4),
the amino acid sequences of the proteins are 84.2% identical and
87.0% similar.
[0033] In an alignment of the nucleotide sequences of cDNA encoding
human h-ig6p protein (SEQ ID NO: 1) and murine cDNA encoding
pancreatic islet-specific G6 Pase catalytic subunit (SEQ ID NO: 22;
the alignment made using the ALIGN software {Myers and Miller
(1989) CABIOS, ver. 2.0}; PAM120 scoring matrix, gap penalties
12/4), the nucleic acid sequences of the cDNAs are 90.0% identical.
The sequence similarity between h-ig6p and murine pancreatic
islet-specific G6 Pase catalytic subunit and the cDNAs which encode
them is an indication that these proteins have analogous, or
overlapping, physiological roles. h-ig6p is likely the human
ortholog of murine pancreatic islet-specific G6 Pase catalytic
subunit. The presence of an endoplasmic reticulum membrane
retention site at the carboxyl terminus of h-ig6p is another
indication that this protein is a catalytic subunit of pancreatic
islet-specific G6 Pase, having physiological function analogous to
the murine islet-specific G6 Pase catalytic subunit.
[0034] h-ig6p amino acid sequence also exhibits high homology with
murine pancreatic islet G6 Pase catalytic subunit amino acid
sequence reported by Arden et al., within membrane-spanning
regions, conservation of charged residues (e.g. residues 65, 72,
168, 191, 226, 261, 274, 293, 305, and 327 of each of SEQ ID NOs: 3
and 4) within those regions, and conservation of the putative
N-glycosylation site at residue 92 of SEQ ID NOs: 3 and 4. These
charged residues are also conserved among integral membrane
proteins which facilitate transmembrane transport of phosphorylated
glycolytic intermediates. Regions of h-ig6p (including the region
spanning residues 59-66 and 68-80 of SEQ ID NO: 3, the region
spanning residues 111-118 and 120-127 of SEQ ID NO: 3, and the
region spanning residues 160-188 of SEQ ID NO: 3) are conserved
among h-ig6p, murine pancreatic islet-specific G6 Pase, liver G6
Pases, bacterial vanadate-sensitive haloperoxidases, mammalian type
2 phosphatidic acid phosphatases, bacterial acid phosphatases, and
the Drosophila developmental protein designated Wunen, indicating
that these regions can be important to one or more of the
structure, function, and catalytic activity of h-ig6p.
[0035] Conservation of amino acid residues at positions
corresponding to residues in the catalytic sites of soluble
haloperoxidases and phosphatases among those enzymes, murine
pancreatic islet-specific G6 Pase, and h-ig6p (i.e., at residues
72, 79, 80, 112-115, 167, 168, and 174 of SEQ ID NO: 3) is an
indication that conservation of these residues can be important for
maintaining one or more of the structure, function, and catalytic
activity of a polypeptide of the invention.
[0036] Postulated catalytically-relevant residues identified in
liver G6 Pase are also conserved in murine pancreatic islet G6 Pase
catalytic subunit and in h-ig6p (e.g., residues 72, 80, 167, and
168 of SEQ ID NO: 3).
[0037] The transmembrane segment prediction program MEMSAT (Jones
et al., 1994, Biochemistry. 33:3038-3049) was used to predict the
location of likely transmembrane domains of h-ig6p. Transmembrane
domains were identified at about residues 57-76, 116-138, 148-170,
177-193, 212-234, 256-273, 294-310, and 319-343 of SEQ ID NO: 3. By
analogy with the topology predicted for the murine islet-specific
G6 Pase catalytic subunit, the transmembrane domain predicted at
residues 212-234 of SEQ ID NO: 3 may instead be two distinct
transmembrane domains, one at about residues 212-217 of SEQ ID NO:
3 and the other at about residues 221-234 of SEQ ID NO: 3. It is
believed that h-ig6p is normally oriented in the membrane of the
endoplasmic reticulum such that its amino terminus and the
non-transmembrane segments which include residues 79, 80, 112-115,
and 174 of SEQ ID NO: 3 are exposed on the lumenal side of the
endoplasmic reticular membrane, and that the carboxyl terminus of
the protein extends into the cytosol. This topology is consistent
with the topology observed for another endoplasmic
reticulum-associated G6 Pase subunit (van de Werve et al., 2000,
Eur. J. Biochem. 267:1533-1549). In another embodiment, h-ig6p is
oriented in the membrane of the endoplasmic reticulum such that its
amino terminus and the non-transmembrane segments which include
residues 79, 80, 112-115, and 174 of SEQ ID NO: 3 are exposed on
the cytosolic side of the endoplasmic reticular membrane, and that
the carboxyl terminus of the protein extends into the lumen of the
endoplasmic reticulum.
[0038] In yet another embodiment, h-ig6p can share the topology and
orientation predicted in Arden et al. (1999, Diabetes 48:531-542)
for the liver G6 Pases of cichlid fish, mouse, dog, and human and
for the murine pancreatic G6 Pase. FIG. 6 depicts an alignment of
the amino acid sequence of h-ig6p with the amino acid sequences of
the aforementioned enzymes. In this embodiment residues 1 to about
27 of h-ig6p are on the non-cytoplasmic face of the membrane and,
as shown in FIG. 6, transmembrane regions exist at about residues
28-47, 57-76, 116-136, 151-173, 179-193, 210-230, 256-278, 290-307,
and 319-343 of SEQ ID NO: 3.
[0039] Northern blot experiments performed using standard methods
demonstrated significant expression of mRNA encoding h-ig6p in
human pancreas tissue and, to a lesser extent, in testis tissue,
although the size of the mRNA detected in testis tissue differed
from that detected in pancreas tissue.
[0040] Expression could not be detected in Northern blot
experiments in heart, brain, placenta, lung, liver, muscle, kidney,
spleen, thymus, prostate, ovary, small intestine, colon, stomach,
thyroid, spinal cord, lymph node, trachea, adrenal gland, or bone
marrow tissues.
[0041] In reverse-transcription PCR (RT-PCR) experiments performed
using TAQMAN.RTM. reagents, expression of mRNA encoding h-ig6p was
detected in pancreas tissue, but could not be detected at a
significant level in, for example, normal artery, normal vein,
aorta smooth muscle, coronary artery smooth muscle, umbilical vein
epithelium (static or sheared), normal heart, congestive heart
failure heart, kidney, skeletal muscle, normal adipose, primary
osteoblast, differentiated osteoblast, normal skin, normal spinal
cord, normal brain cortex, normal brain hypothalamus, nerve, dorsal
root ganglion, glial cells (astrocytes), glioblastoma, normal
breast, breast tumor, normal ovarian, ovarian tumor, normal
prostate, prostate tumor, prostate epithelium, normal colon, colon
tumor, irritable bowel disease colon, normal lung, lung tumor,
chronic obstructive pulmonary disorder lung, normal liver, fibrosis
liver, dermal cells (fibroblasts), normal spleen, normal tonsil,
lymph node, small intestine, skin decubitus, synovium, bone marrow
mononuclear, and activated peripheral blood mononuclear tissues
under the conditions used.
[0042] In contrast, expression of mRNA encoding human liver G6 Pase
could be detected at the indicated relative levels in only the
following tissues in RT-PCR experiments:
[0043] Normal liver: 43
[0044] Fibrosed liver: 23.77
[0045] Kidney: 25.47
[0046] Small intestine: 6.46.
[0047] Expression of mRNA encoding human liver G6 Pase could not be
detected at significant levels in RT-PCR experiments in normal
artery, normal vein, aorta smooth muscle, coronary artery smooth
muscle, umbilical vein epithelium (static or sheared), normal
heart, congestive heart failure heart, skeletal muscle, normal
adipose, pancreas, primary osteoblast, differentiated osteoblast,
normal skin, normal spinal cord, normal brain cortex, normal brain
hypothalamus, nerve, dorsal root ganglion, glial cells
(astrocytes), glioblastoma, normal breast, breast tumor, normal
ovarian, ovarian tumor, normal prostate, prostate tumor, prostate
epithelium, normal colon, colon tumor, irritable bowel disease
colon, normal lung, lung tumor, chronic obstructive pulmonary
disorder lung, dermal cells (fibroblasts), normal spleen, normal
tonsil, lymph node, skin decubitus, synovium, bone marrow
mononuclear, and activated peripheral blood mononuclear tissues
under the conditions used.
[0048] In situ hybridization experiments, performed using standard
methods, demonstrated that h-ig6p is expressed in human pancreatic
islet cells, as illustrated in FIG. 4. Expression of mRNA encoding
murine islet-specific G6 Pase was up-regulated (i.e., relative to
wild type mice) in the ob, db, agouti, and tubby strains of mice.
These strains are art-accepted mouse models of obesity, diabetes,
or both. Overexpression of the murine islet-specific G6 Pase was
demonstrated to be correlated with the degree of hyperinsulinemia
in the mouse models, indicating a correlation between h-ig6p
overexpression and human hyperinsulinemia. This observation
suggests that inhibition of h-ig6p expression (or inhibition of the
activity of h-ig6p protein) can prevent or reverse abnormally low
insulin secretion in humans, alleviating or preventing disorders
associated with abnormally low insulin levels (e.g., diabetes).
Conversely, this observation also suggests that enhancement of
h-ig6p expression (or enhancement of the activity of h-ig6p
protein) can prevent or reverse abnormally high insulin secretion
in humans, alleviating or preventing disorders associated with
abnormally high insulin levels.
[0049] The h-ig6p protein described herein can bind with one or
more phosphorylated or non-phosphorylated carbohydrates (e.g.
glucose) and can catalyze one or both of interconversion between
the phosphorylated and non-phosphorylated forms of the carbohydrate
(e.g., glucose G6P) and transmembrane transport of the
phosphorylated or non-phosphorylated form of the carbohydrate.
Homology of h-ig6p with liver and kidney G6 Pases suggests that,
like these enzymes, h-ig6p can participate in multimeric
membrane-associated protein complexes including, for example,
proteins which bind one or more of the phosphorylated or
non-phosphorylated form of the carbohydrate, proteins which
facilitate or catalyze transmembrane transport (i.e., across the
cytoplasmic or endoplasmic reticular membrane) of one or more of
the phosphorylated or non-phosphorylated form of the carbohydrate,
enzymes which catalyze interconversion of the phosphorylated and
non-phosphorylated forms of the carbohydrate, enzymes which
catalyze glycolytic reactions, and enzymes which catalyze
gluconeogenic reactions.
[0050] Because G6P (and xyulose-5-phosphate) mediate gene
transcription induced by glucose or carbohydrate feeding (Massillon
et al., 1998, J. Biol., Chem. 273:228-234), the ability of h-ig6p
to modulate intracellular G6P levels indicates that h-ig6p can act
as a regulator of gene transcription that is responsive to
intracellular (e.g. cytosolic or endoplasmic reticular) glucose
concentration. When h-ig6p is present, optionally in association
with other proteins, in the cytoplasmic membrane of a pancreatic
islet cell, it can also regulate gene expression within the cell in
response to blood (or other extracellular) glucose
concentration.
[0051] When h-ig6p is present, optionally in association with other
proteins, in the membrane surrounding the endoplasmic reticulum in
a cell, it can regulate one or both of expression of protein
secreted into the endoplasmic reticulum and processing of protein
(e.g., a hormone, a prohormone, or a pre-prohormone) present with
the endoplasmic reticulum. Without being bound by any particular
theory of operation, it is believed that h-ig6p can interact with
one or more proteins (e.g., ribosomal proteins or prohormone
processing enzymes) associated with expression, processing, or
secretion of hormone polypeptides, and that the manner in which
h-ig6p interacts with such proteins can be altered upon or
following interaction of h-ig6p with G6P. Thus, h-ig6p can act as
an intracellular `sensor` of cellular G6P content, and can modulate
(i.e., enhance or retard) hormone expression, processing, or
secretion in response to the cellular G6P level.
[0052] For example, because h-ig6p can associate with the
endoplasmic reticulum in pancreas alpha and beta cells, this
protein can modulate expression of hormones (e.g., insulin and
glucagon) which are transported into the endoplasmic reticulum of
these cells, and post-translational processing (e.g., cleavage of
pre-proinsulin to form proinsulin and cleavage of proinsulin to
form insulin) of such hormones. Ability of h-ig6p to modulate
production and activation of pancreatic hormones is an indication
that h-ig6p proteins, nucleic acids, and modulators thereof can be
used to prognosticate, diagnose, inhibit, prevent, or treat
disorders with which aberrant function of these hormones is
associated. Examples of these disorders include carbohydrate
metabolism disorders (e.g., diabetes).
[0053] Because insulin and glucagon can modulate the metabolic rate
of one or more tissues in a mammal (e.g., a human), expression of
h-ig6p in cells which produce these hormones, and association of
h-ig6p with the endoplasmic reticulum (within which these hormones
are produced and processed) in these cells indicates that h-ig6p
proteins, nucleic acids, and modulators thereof can be administered
to pancreatic cells (or systemically) in order to modulate (i.e.,
increase or decrease) the rate of metabolism in other (i.e.,
non-pancreas) cells or tissues. For example, homology of h-ig6p
with the murine G6 Pase catalytic subunit (and presumably with the
rat G6 Pase catalytic subunit) indicates that overexpression of
h-ig6p in pancreatic cells can uncouple glucose-stimulated insulin
secretion, leading to increased hepatic glucose production and
impaired glucose-stimulated insulin secretion. The ability of the
polypeptides, nucleic acid molecules, and modulators of the
invention to affect metabolic rate indicates that these agents can
be used to control body weight in humans (i.e., to enhance or
inhibit weight gain or to enhance or inhibit weight loss).
[0054] The activities which can be attributed to h-ig6p indicate
that this protein modulates glucose-stimulated insulin secretion by
pancreatic beta cells, and can also modulate glucose-regulated
glucagon secretion by pancreatic alpha cells. Thus, agents
including polypeptides, nucleic acid molecules, and modulators of
the invention, can be used to prognosticate, prevent, diagnose, or
treat one or more of disorders associated with aberrant insulin or
glucagon secretion. Examples of such disorders include diabetes
(including diabetes of various types, such as type 2 diabetes or
maturity-onset diabetes of the young) and hyperinsulinism. The
agents can also be used to prevent, alleviate, or eliminate
symptoms associated with such disorders (e.g. hepatic glucose
production or defects in insulin-dependent peripheral glucose
utilization associated with type 2 diabetes, diabetic ketoacidosis,
non-ketotic hyperglycemic-hyperosmolar coma, hyperglycemia,
hypoglycemia, atherosclerosis, coronary artery disease, wasting
disorders, diabetic retinopathy, and the like). Because insulin and
glucagon are involved in regulating uptake and storage of glucose
by the body, these agents can also be used to modulate body weight
(i.e. maintenance of body weight or modulation of weight gain or
loss) in humans.
[0055] Correlation in mouse models of obesity and diabetes of the
obese or diabetic phenotype with overexpression of the gene
encoding h-ig6p is an indication that enhancement of the activity
or level of expression of h-ig6p inhibits insulin secretion by
pancreatic islet beta cells and, conversely, that inhibition of
h-ig6p activity or expression enhances insulin secretion by those
cells. In addition, these data indicate that modulating expression
or activity of h-ig6p can inhibit obesity and lead to weight gain
and, conversely, that enhancing expression or activity of h-ig6p
can enhance weight gain in a human. Thus, the results presented in
this disclosure demonstrate that modulation of h-ig6p expression or
activity can be useful for alleviation, inhibition, or prevention
of various pancreatic disorders, including metabolic disorders
(e.g., carbohydrate metabolism disorders such as diabetes, obesity,
and hyperinsulinemia).
[0056] Localization of pancreatic islet G6 Pase catalytic subunits,
including h-ig6p, in the membrane of the endoplasmic reticulum
indicates that these enzymes can have a physiological role in other
processes which involve hydrolysis of phosphorylated carbohydrates.
By way of example, these enzymes can modulate glycolysis of
proteins (e.g. by trimming core-glycosylated proteins). These
enzymes can also modulate accumulation of physiologically relevant
ions (e.g. phosphate or calcium ions) within the endoplasmic
reticulum (or another membrane-bound compartment, such as the
cytosol or the nucleus) by mediating transport of a phosphorylated
carbohydrate into or out of the compartment. Phosphohydrolysis of a
phosphorylated carbohydrate within the compartment can increase the
phosphate concentration within the compartment which, in turn, can
be used to drive import from, export to, or exchange of another ion
with the extra-compartmental milieu.
[0057] Various aspects of the invention are described in further
detail in the following subsections.
[0058] I. Isolated Nucleic Acid Molecules
[0059] The invention includes a variety of isolated nucleic acid
molecules including:
[0060] (i) isolated nucleic acid molecules which encode a
polypeptide of the invention (e.g., an isolated nucleic acid
molecule that encodes full length h-ig6p or a portion of h-ig6p
that includes a structural, functional, or catalytic feature
described herein);
[0061] (ii) isolated nucleic acid molecules which have a sequence
that is sufficiently identical to, or sufficiently complementary
to, all or part of SEQ ID NO: 1 that they can be used as
hybridizable probes or as primers for amplification of a nucleic
acid encoding all or a portion of h-ig6p;
[0062] (iii) isolated nucleic acid molecules which have a sequence
that is sufficiently identical to, or sufficiently complementary
to, all or part of SEQ ID NO: 1 that they can be used to inhibit
expression of the gene encoding h-ig6p (i.e., either by inhibiting
DNA transcription or RNA translation); and
[0063] (iv) isolated nucleic acid molecules (e.g., ribozymes) which
comprise a portion having a sequence that is sufficiently identical
to, or sufficiently complementary to, all or part of SEQ ID NO: 1
that they hybridize with a substrate nucleic acid having the
sequence SEQ ID NO: 1, and which further comprise a portion that
catalyzes modification (e.g., cleavage) of the substrate nucleic
acid.
[0064] Further details of these isolated nucleic acid molecules and
how they can be made and used are described in the remainder of
this subsection.
[0065] As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g.,
mRNA) and analogs of the DNA or RNA generated using nucleotide
analogs. The nucleic acid molecule can be single-stranded or
double-stranded, and can also include those nucleic acid molecules
which form triple helical structures. See generally Helene (1991)
Anticancer Drug Des. 6(6):569-584; Helene (1992) Ann. N.Y. Acad.
Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-815.
[0066] Nucleic acid molecules also include those which comprise one
or more nucleotide residues having a modified purine or pyrimidine
moiety, a modified sugar-phosphate backbone. Such modified base
moieties and modified backbones are known in the art. Examples of
modified base moieties include 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Examples of nucleic acid molecules which have
modified sugar-phosphate backbones include peptide nucleic acids
("PNAs"; see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4(1): 5-23; Perry-O'Keefe et al. (1996) Proc. Natl. Acad.
Sci. USA 93: 14670-14675), and nucleic acids having one or more
internucleoside linkages selected from the group consisting of
phosphotriester, phosphoramidate, siloxane, carbonate,
carboxymethylester, acetamidate, carbamate, thioether, bridged
phosphoramidate, bridged methylene phosphonate, bridged
phosphoramidate, bridged phosphoramidate, bridged methylene
phosphonate, phosphorothioate, methylphosphonate,
phosphorodithioate, bridged phosphorothioate, phosphonate
phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl
phosphoramidate, formacetal, thioformacetal, diisopropylsilyl,
acetamidate, carbamate, dimethylene-sulfide
(--CH.sub.2--S--C.sub.1H.sub.2--), dimethylene-sulfoxide
(--CH.sub.2--SO--CH.sub.2--), dimethylene-sulfone
(--CH.sub.2--SO.sub.2--- CH.sub.2--), 2'-O-alkyl, 2'-deoxy2'-fluoro
phosphorothioate, and sulfone linkages (see, e.g., Uhlmann et al.,
1990, Chem. Rev. 90:543-584; Schneider et al., 1990, Tetrahedron
Lett. 31:335).
[0067] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. Preferably, an
"isolated" nucleic acid molecule is free of regions (preferably
regions encoding protein) which naturally flank the nucleic acid
(i.e., polynucleotides located at the 5' and 3' ends of the nucleic
acid) in the genomic DNA of the organism from which the nucleic
acid molecule is derived. For example, the invention includes
isolated nucleic acid molecule which comprise one or two
polynucleotide regions flanking the isolated nucleic acid molecule,
wherein the flanking polynucleotides together comprise fewer than
about 5000, 4000, 3000, 2000, 1000, 500, or 100 nucleotide
residues.
[0068] The terms "nucleotides" and "nucleotide residues" are used
interchangeably herein to refer to individual ribonucleotide or
deoxyribonucleotide moieties of a polymeric nucleic acid. Thus, A,
C, G, and T each represent an individual nucleotide residue of the
nucleic acid having the sequence 5'-ACGT-3'.
[0069] The nucleotide sequences of the isolated nucleic acid
molecules of the invention are based on the nucleotide sequence
(SEQ ID NO: 1) of the cDNA described herein which encodes h-ig6p. A
nucleic acid clone having a sequence comprising SEQ ID NO: 1 was
deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282, and the nucleotide sequences of the isolated nucleic acid
molecules of the invention can be based on the nucleotide sequence
of this deposited clone as well.
[0070] The invention includes nucleic acid molecules which have a
sequence which comprises, or consists of, all or a portion of SEQ
ID NO: 1, the nucleotide sequence of the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or the
complement thereof. The portion can comprise 20, 25, 30, 35, 40,
50, 75, 100, 150, 200, 350, 500, 750, 1000, 1138, or any
intermediate number of consecutive residues of SEQ ID NO: 1, the
nucleotide sequence of the clone deposited with ATCC.RTM. on Jul.
28, 2000 as accession number PTA-2282, or the complement thereof.
Alternatively, the isolated nucleic acid molecule can comprise, or
consist of, a nucleic acid having a sequence that is at least 91%
(or 92%, 95%, 98%, or 99% or more) identical to all or a portion
(e.g., 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 350, 500, 750,
1000, 1138 or any intermediate number of consecutive residues) of
SEQ ID NO: 1, of the nucleotide sequence of the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or of
the complement thereof.
[0071] The nucleic acid molecule having the sequence SEQ ID NO: 1
(and the clone deposited with ATCC.RTM. on Jul. 28, 2000 as
accession number PTA-2282) encode h-ig6p protein, which has the
amino acid sequence SEQ ID NO: 3. The isolated nucleic acid
molecules of the invention include nucleic acid molecules which do
not have the sequence SEQ ID NO: 1, but nonetheless encode a
protein having the amino acid sequence SEQ ID NO: 3 or a protein
having an amino acid sequence that is at least 85% (or 90%, 95%,
98%, or 99% or more) identical to all or a portion of SEQ ID NO: 3.
Such nucleic acid molecules include those which encode all or a
portion of SEQ ID NO: 3 and which have a nucleotide sequence that
differs from the corresponding portion of SEQ ID NO: 1 owing to the
degeneracy of the genetic code (i.e., the nucleotide sequence
includes at least one codon synonymous with, but not identical to,
the corresponding codon of SEQ ID NO: 1). When the amino acid
sequence encoded by the nucleic acid molecule differs from the
corresponding portion of SEQ ID NO: 3, it is preferred that the
amino acid sequence not differ at residues described herein as
having structural, functional, or catalytic significance.
[0072] Isolated nucleic acid molecules which encode h-ig6p having
one or more amino acid substitutions, insertions, or deletions at
selected positions can be made using known methods (e.g.,
site-directed mutagenesis or PCR-mediated mutagenesis). Amino acid
residue substitutions are preferably not made at residues with
structural, functional, or catalytic significance, and insertions
and deletions are preferably not made within structural,
functional, or catalytic domains identified herein. Alternatively,
mutations can be introduced randomly along all or part of the
coding sequence, such as by saturation mutagenesis, and the
resulting mutants can be screened for biological activity in order
to identify mutants that retain functional or catalytic activity of
h-ig6p.
[0073] Other isolated nucleic acid molecules of the invention
encode allelic variants of h-ig6p (i.e., including both allelic
variants wherein both the amino acid sequence and the nucleotide
sequence corresponding to h-ig6p differ from SEQ ID NOs: 3 and 1
and allelic variants wherein h-ig6p has the amino acid sequence SEQ
ID NO: 3, but wherein the nucleotide sequence that encodes this
protein differs from SEQ ID NO: 1). These allelic variants include
those commonly referred to as polymorphisms, including single
nucleotide polymorphisms (i.e., allelic variants wherein the
nucleotide sequence encoding h-ig6p differs from SEQ ID NO: 1 only
at one or more non-adjacent nucleotide residues). Typically,
allelic variants are encoded by the same genetic locus among
different individuals in a species of organism.
[0074] The degree of sequence identity between two nucleic acid
sequences can be assessed by aligning the sequences and comparing
the number of identical residues to the number of total residues in
the overlapping region (i.e., making no allowance for insertions
and deletions). Alternatively, and preferably, the degree of
sequence identity can be assessed using an algorithm that accounts
for the possibility of inserted and deleted residues. An example of
such an algorithm is that incorporated into the NBLAST computer
program (see Karlin et al., 1990, Proc. Natl. Acad. Sci. USA
87:2264-2268; Karlin et al., 1993, Proc. Natl. Acad. Sci. USA
90:5873-5877; Altschul et al., 1990, J. Mol. Biol. 215:403-410;
Altschul et al., 1997, Nucl. Acids Res. 25:3389-3402; States et
al., 1991, Methods 3:66-70), which can be obtained, for example at
the World Wide Web site having the universal resource locator
http://www.ncbi.nlm.nih.gov. Preferred parameters used in the
NBLAST program are wordlength=12, PAM 120 weight residue table, gap
existence penalty=12, gap length penalty=4 per residue, expectation
value=10.0, mismatch penalty=-3, and match reward=1.
[0075] The degree of sequence homology between two nucleic acid
molecules can also be assessed by determining the ability of the
molecules to hybridize with one another. Nucleic acid molecules of
the invention include those which hybridize under stringent
conditions with a nucleic acid having a sequence comprising SEQ ID
NO: 1, with the clone deposited with ATCC.RTM. on Jul. 28, 2000 as
accession number PTA-2282, or with the complement thereof. An
example of stringent hybridization conditions is hybridization in
6.times. sodium chloride/sodium citrate buffer at pH 7.4 (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0076] Isolated nucleic acid molecules which encode full length
h-ig6p or a biologically active portion thereof (i.e., a portion of
h-ig6p that includes a structural, functional, or catalytic feature
described herein) can be isolated or synthesized using standard
molecular biology techniques, in view of the sequence information
provided herein. For example, using a nucleic acid probe having a
sequence comprising about 20-500 nucleotide residues of SEQ ID NO:
1, the nucleotide sequence of the clone deposited with ATCC.RTM. on
Jul. 28, 2000 as accession number PTA-2282, or the complement
thereof, nucleic acid molecules of the invention can be isolated
from, for example, human tissue samples (e.g., as described in
Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989). Alternatively, such nucleic
acid molecules can be prepared by standard synthetic techniques
(e.g., using an automated DNA synthesizer).
[0077] Nucleic acid molecules which encode a polypeptide of the
invention can be used to express that polypeptide, either in vitro
or in vivo, using methods known in the art. Briefly, the portion of
the nucleic acid molecule which encodes the polypeptide must be
operably linked with the control/regulatory sequences necessary for
translation and, if necessary (i.e., if the nucleic acid molecule
is DNA), transcription of the nucleic acid molecule. Such
control/regulatory sequences are known in the art and include, for
example, promoter sequences, ribosome binding sites, and the like.
It is not necessary that the control/regulatory sequences of the
gene encoding h-ig6p be used. Substantially any control/regulatory
sequences that can be operably linked with the coding region of the
nucleic acid molecule of the invention can be used. By way of
example, control/regulatory sequences that render expression of
h-ig6p inducible or tissue-specific are known and can be used.
[0078] Uses of oligonucleotides as probes for detecting or as
primers for amplifying nucleic acids with which they hybridize are
known in the art. Accordingly, nucleic acid molecules of the
invention can be used as probes for detecting a nucleic acid
encoding all or a portion of h-ig6p or for amplifying such a
nucleic acid. Such probes can be isolated from naturally-occurring
nucleic acids or synthesized, as described above. When used as
probes or primers, the nucleic acid molecules of the invention
preferably have a length of at least 20, 25, 30, 35, 40, 50, or
more nucleic acid residues, and a sequence that is at least 91% (or
92%, 95%, 98%, or 99% or more) identical to the corresponding
portion of SEQ ID NO: 1, the nucleotide sequence of the clone
deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282, or the complement thereof, or at least being more
identical to one of these human sequences than to an analogous
portion of SEQ ID NO: 22. Such probes and primers can, optionally,
have other moieties bound therewith, such as a detectable label
(e.g., a radionuclide or an enzyme which catalyzes a chromogenic
reaction), a relatively fixed substrate (e.g., a nylon membrane),
or another polynucleotide (e.g., a portion of a plasmid or virus
vector, or a polynucleotide adapted for insertion into a multiple
restriction site of a vector). These probes and primers can be used
as part of a diagnostic test kit for identifying cells or tissues
which express h-ig6p (i.e., normally or aberrantly), or for
assessing levels of h-ig6p expression.
[0079] The invention includes antisense nucleic acid molecules,
i.e., molecules which are complementary to a nucleic acid molecule
encoding a polypeptide of the invention (e.g., nucleic acid
molecules which are complementary to at least a portion of the
coding strand of a double-stranded cDNA molecule or complementary
to at least a portion of an mRNA sequence encoding a polypeptide of
the invention). Accordingly, an antisense nucleic acid can
hybridize with a nucleic acid which encodes all or a portion of
h-ig6p. Alternatively, an antisense nucleic acid molecule of the
invention can be antisense with respect to all or part of a
non-coding region of a nucleic acid encoding a polypeptide of the
invention. The non-coding regions (e.g., 5' and 3' un-translated
regions) include the 5' and 3' sequences which flank the coding
region, and are not translated into amino acids. Antisense nucleic
acid molecules can be, for example, about 5, 10, 15, 20, 25, 30,
35, 40, 45 or 50 nucleotides in length, and preferably have a
sequence that is 91% (or 92%, 95%, 98%, or 99% or more) identical
to a portion of SEQ ID NO: 1, to a portion of the nucleotide
sequence of the clone deposited with ATCC.RTM. on Jul. 28, 2000 as
accession number PTA-2282, or to the complement thereof.
[0080] The antisense nucleic acid molecules of the invention can be
administered to a subject (or generated in situ, e.g., by
transcribing a DNA) so that the antisense molecule hybridizes with
or binds with cellular mRNA and/or genomic DNA encoding h-ig6p to
thereby inhibit expression (i.e., by inhibiting transcription
and/or translation). Inhibition in a cell of expression of a gene
encoding h-ig6p (e.g., by inhibiting transcription of the gene or
translation of the corresponding mRNA) inhibits h-ig6p activity in
the cell, leading, for example, to enhanced insulin secretion
(e.g., if the cell is a pancreatic islet beta cell). Furthermore,
inhibition of h-ig6p activity in a cell can modulate (i.e.,
activate or inhibit) the cell's transmembrane signaling systems for
detecting extracellular glucose levels. For example, the antisense
nucleic acid molecule can be directly injected at a tissue site or
modified to target selected cells following systemic administration
(e.g., by modifying the antisense molecule {e.g., by attaching it
to an antibody} so that it specifically binds with a receptor or
antigen expressed on a selected cell surface). Any of the
modifications of antisense molecules that are known in the art can
be used to make antisense nucleic acid molecules of the invention
(see, e.g., Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641;
Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148; Inoue et al.
(1987) FEBS Lett. 215:327-330).
[0081] The invention also includes nucleic acid molecules (e.g.,
ribozymes) which have a portion which hybridizes with all or a
portion of a substrate nucleic acid having the sequence of SEQ ID
NO: 1, of the nucleotide sequence of the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or of the
complement thereof and a second portion which modifies (e.g.,
cleaves) the substrate nucleic acid. Ribozymes, for example, are
catalytic RNA molecules with ribonuclease activity which are
capable of cleaving a single-stranded nucleic acid, such as an
mRNA, to which they have a complementary region. Thus, ribozymes
(e.g., hammerhead ribozymes as described in Haselhoff and Gerlach
(1988) Nature 334:585-591) can be used to catalytically cleave mRNA
transcripts encoding h-ig6p to thereby translation thereof. A
ribozyme having specificity for a nucleic acid molecule encoding
h-ig6p (or another polypeptide of the invention) can be designed,
based on 1-5 the nucleotide sequences disclosed herein. For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved, as
described in Cech et al. U.S. Pat. No. 4,987,071; and Cech et al.
U.S. Pat. No. 5,116,742. Alternatively, an mRNA encoding a
polypeptide of the invention can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel and Szostak (1993) Science
261:1411-1418. Expression of such a ribozyme in a cell which
expresses h-ig6p can inhibit expression of h-ig6p in the cell,
leading to a decrease in the enzymatic activity of h-ig6p in the
cell. As a result, insulin secretion can be enhanced, the
sensitivity of the cell's signaling mechanism(s) for systemic
glucose can be decreased, or some combination of the effects can
occur.
[0082] Nucleic acid molecules of the invention can comprise
appended groups such as peptides (e.g., antibodies or other
specifically-binding proteins for targeting host cell receptors in
vivo) or agents for facilitating transport across the cell membrane
(see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA
86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA
84:648-652; PCT Publication No. WO 88/09810) or the blood-brain
barrier (see, e.g., PCT Publication No. WO 89/10134). In addition,
oligonucleotides can be modified with hybridization-triggered
cleavage agents (see, e.g., Krol et al. (1988) Bio/Techniques
6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm.
Res. 5:539-549). To this end, the nucleic acid molecules of the
invention can be conjugated with another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent,
hybridization-triggered cleavage agent, etc., using methods known
in the art.
[0083] II. Isolated Proteins and Antibodies
[0084] One aspect of the invention pertains to isolated proteins,
and biologically active portions thereof, as well as polypeptide
fragments suitable for use as immunogens to raise antibodies
directed against a polypeptide of the invention. In one embodiment,
the native polypeptide can be isolated from cells or tissue sources
by an appropriate purification scheme using standard protein
purification techniques. In another embodiment, polypeptides of the
invention are produced by recombinant DNA techniques. Alternative
to recombinant expression, a polypeptide of the invention can be
synthesized chemically using standard peptide synthesis techniques.
Proteins isolated from microsomal preparations of cells transfected
with an expression vector encoding h-ig6p exhibited catalytic
reactions that are characteristic of G6 Pases (e.g., conversion of
G6P to glucose), confirming that the isolated proteins are, in
fact, a pancreas-specific form of G6 Pase.
[0085] Biologically active portions of h-ig6p are able to catalyze
conversion of glucose-6-phosphate to glucose in vitro, in vivo, or
both. Biologically active portions preferably include the residues
conserved among G6 Pases, as noted above.
[0086] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein"). When the
protein or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the protein is
produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals, i.e., it is separated
from chemical precursors or other chemicals which are involved in
the synthesis of the protein. Accordingly such preparations of the
protein have less than about 30%, 20%, 10%, 5% (by dry weight) of
chemical precursors or compounds other than the polypeptide of
interest.
[0087] Biologically active portions of a polypeptide of the
invention include polypeptides comprising amino acid sequences
sufficiently identical to or derived from the amino acid sequence
of the protein (e.g., the amino acid sequence of SEQ ID NO: 3, or
the amino acid sequence encoded by the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or of 15
or more {e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 250, 300, 350, or 355} consecutive residues thereof),
which include fewer amino acids than the full length protein, and
exhibit at least one activity of the corresponding full-length
protein. Typically, biologically active portions comprise a domain
or motif with at least one activity of the corresponding protein. A
biologically active portion of a protein of the invention can be a
polypeptide which is, for example, 10, 25, 50, 100 or more amino
acids in length. Moreover, other biologically active portions, in
which other regions of the protein are deleted, can be prepared by
recombinant techniques and evaluated for one or more of the
functional activities of the native form of a polypeptide of the
invention.
[0088] Preferred polypeptides have the amino acid sequence of SEQ
ID NO: 3, or the amino acid sequence encoded by the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or of
15 or more {e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120, 140,
160, 180, 200, 250, 300, 350, or 355} consecutive residues thereof.
Other useful proteins are substantially identical (e.g., at least
about 85%, 90%, 95%, 98%, or 99% or more) to SEQ ID NO: 3, or the
amino acid sequence encoded by the clone deposited with ATCC.RTM.
on Jul. 28, 2000 as accession number PTA-2282, or of 15 or more
{e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120, 140, 160, 180,
200, 250, 300, 350, or 355} consecutive residues thereof and retain
the functional activity of the protein of the corresponding
naturally-occurring protein yet differ in amino acid sequence due
to natural allelic variation or mutagenesis.
[0089] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), non-polar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0090] In a preferred embodiment, a mutant polypeptide that is a
variant of a polypeptide of the invention can be assayed for: (1)
the ability to form protein:protein interactions with proteins in a
signaling pathway of the polypeptide of the invention; (2) the
ability to bind a ligand of the polypeptide of the invention; or
(3) the ability to bind to an intracellular target protein of the
polypeptide of the invention. In yet another preferred embodiment,
the mutant polypeptide can be assayed for the ability to modulate
cellular proliferation, cellular migration or chemotaxis, or
cellular differentiation.
[0091] To determine the percent identity of two amino acid
sequences, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in the sequence of a first
amino acid for optimal alignment with a second amino). The amino
acid residues at corresponding amino acid positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
identity=# of identical positions/total # of positions (e.g.,
overlapping positions).times.100). In one embodiment the two
sequences are the same length.
[0092] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm used for the
comparison of two amino acid sequences is the algorithm of Karlin
and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268,
modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci.
USA 90:5873-5877. Such an algorithm is incorporated into the XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST amino acid sequence identity assessments can be performed
using the XBLAST program, score=50, wordlength=3, BLOSUM62 scoring
matrix, gap existence penalty=12, gap extension penalty=4 per
residue, expectance=10.0, mismatch penalty=-3, reward for match=1,
to identify identity of amino acid sequences. To obtain gapped
alignments for comparison purposes, Gapped BLAST can be used as
described in Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402. In calculating percent identity, only exact matches
are counted.
[0093] The invention also provides chimeric or fusion proteins. As
used herein, a "chimeric protein" or "fusion protein" comprises all
or part (preferably biologically active) of a polypeptide of the
invention operably linked to a heterologous polypeptide (i.e., a
polypeptide other than the same polypeptide of the invention).
Within the fusion protein, the term "operably linked" is intended
to indicate that the polypeptide of the invention and the
heterologous polypeptide are fused in-frame to each other. The
heterologous polypeptide can be fused to the N-terminus or
C-terminus of the polypeptide of the invention.
[0094] One useful fusion protein is a GST fusion protein in which
the polypeptide of the invention is fused to the C-terminus of GST
sequences. Such fusion proteins can facilitate the purification of
a recombinant polypeptide of the invention.
[0095] In another embodiment, the fusion protein contains a
heterologous signal sequence at its N-terminus. For example, a
signal sequence from another protein can be fused at the N-terminus
of a polypeptide of the invention. For example, the gp67 secretory
sequence of the baculovirus envelope protein can be used as a
heterologous signal sequence (Current Protocols in Molecular
Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other
examples of eukaryotic heterologous signal sequences include the
secretory sequences of melittin and human placental alkaline
phosphatase (Stratagene; La Jolla, Calif.). In yet another example,
useful prokaryotic heterologous signal sequences include the phoA
secretory signal (Sambrook et al., supra) and the protein A
secretory signal (Pharmacia Biotech; Piscataway, N.J.).
[0096] In yet another embodiment, the fusion protein is an
immunoglobulin fusion protein in which all or part of a polypeptide
of the invention is fused to sequences derived from a member of the
immunoglobulin protein family. The immunoglobulin fusion proteins
of the invention can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between a ligand (soluble or membrane-bound) and a
protein on the surface of a cell (receptor), to thereby suppress
signal transduction in vivo. The immunoglobulin fusion protein can
be used to affect the bioavailability of a cognate ligand of a
polypeptide of the invention. Inhibition of ligand/receptor
interaction can be useful therapeutically, both for treating
proliferative and differentiative disorders and for modulating
(e.g. promoting or inhibiting) cell survival. Moreover, the
immunoglobulin fusion proteins of the invention can be used as
immunogens to produce antibodies directed against a polypeptide of
the invention in a subject, to purify ligands and in screening
assays to identify molecules which inhibit the interaction of
receptors with ligands. For example, antibodies described herein
can be used to inhibit association between h-ig6p and an
extracellular ligand thereof (e.g., glucose) if the antibody is
contacted with the non-cytoplasmic face of h-ig6p. An
intracellularly-expressed antibody can be used to inhibit
association between h-ig6p and either an intracellular small
molecular ligand (e.g., glucose-6-phosphate) or a cellular protein
with which h-ig6p normally associates. Thus, either extracellular
or intracellular antibodies can be used to inhibit an activity
catalyzed or mediated by h-ig6p, and such antibodies can also be
used to inhibit interactions between h-ig6p and other proteins.
[0097] Chimeric and fusion proteins of the invention can be
produced by standard recombinant DNA techniques. In another
embodiment, the fusion gene can be synthesized by conventional
techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor
primers which give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be annealed and
re-amplified to generate a chimeric gene sequence (see, e.g.,
Ausubel et al., supra). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A nucleic acid encoding a polypeptide of the
invention can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the polypeptide of the
invention.
[0098] The present invention also pertains to variants of the
polypeptides of the invention. Such variants have an altered amino
acid sequence which can function as either agonists (mimetics) or
as antagonists. Variants can be generated by mutagenesis, e.g.,
discrete point mutation or truncation. An agonist can retain
substantially the same, or a subset, of the biological activities
of the naturally occurring form of the protein. An antagonist of a
protein can inhibit one or more of the activities of the naturally
occurring form of the protein by, for example, competitively
binding to a downstream or upstream member of a cellular signaling
cascade which includes the protein of interest. Thus, specific
biological effects can be elicited by treatment with a variant of
limited function. Treatment of a subject with a variant having a
subset of the biological activities of the naturally occurring form
of the protein can have fewer side-effects in a subject relative to
treatment with the naturally occurring form of the protein.
[0099] Variants of a protein of the invention which function as
either agonists (mimetics) or as antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the protein of the invention for agonist or antagonist
activity. In one embodiment, a variegated library of variants is
generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A variegated library
of variants can be produced by, for example, enzymatically ligating
a mixture of synthetic oligonucleotides into gene sequences such
that a degenerate set of potential protein sequences is expressible
as individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display). There are a variety of
methods which can be used to produce libraries of potential
variants of the polypeptides of the invention from a degenerate
oligonucleotide sequence. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang (1983)
Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;
Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic
Acid Res. 11:477).
[0100] In addition, libraries of fragments of the coding sequence
of a polypeptide of the invention can be used to generate a
variegated population of polypeptides for screening and subsequent
selection of variants. For example, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of the coding sequence of interest with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, re-naturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes
N-terminal and internal fragments of various sizes of the protein
of interest.
[0101] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. The most widely used techniques, which
are amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a technique
which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify variants of a protein of the invention (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.
(1993) Protein Engineering 6(3):327-331).
[0102] An isolated polypeptide of the invention, or a fragment
thereof, can be used as an immunogen to generate antibodies using
standard techniques for polyclonal and monoclonal antibody
preparation. The full-length polypeptide or protein can be used or,
alternatively, the invention provides antigenic peptide fragments
for use as immunogens. The antigenic peptide of a protein of the
invention comprises at least 8 (preferably 10, 15, 20, or 30) amino
acid residues of the amino acid sequence of SEQ ID NO: 3, or the
amino acid sequence encoded by the clone deposited with ATCC.RTM.
on Jul. 28, 2000 as accession number PTA-2282, and encompasses an
epitope of the protein such that an antibody raised against the
peptide forms a specific immune complex with the protein.
[0103] An immunogen typically is used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse or other
mammal). An appropriate immunogenic preparation can contain, for
example, recombinantly expressed or chemically synthesized
polypeptide. The preparation can further include an adjuvant, such
as Freund's complete or incomplete adjuvant, or similar
immunostimulatory agent.
[0104] Accordingly, another aspect of the invention pertains to
antibodies directed against a polypeptide of the invention. The
terms "antibody" and "antibody substance" as used interchangeably
herein refer to immunoglobulin molecules and immunologically active
portions of immunoglobulin molecules, i.e., molecules that contain
an antigen binding site which specifically binds an antigen, such
as a polypeptide of the invention (e.g., an epitope of a
polypeptide of the invention). A molecule which specifically binds
to a given polypeptide of the invention is a molecule which binds
the polypeptide, but does not substantially bind other molecules in
a sample, e.g., a biological sample, which naturally contains the
polypeptide. Examples of immunologically active portions of
immunoglobulin molecules include F(ab) and F(ab').sub.2 fragments
which can be generated by treating the antibody with an enzyme such
as pepsin. The invention provides polyclonal and monoclonal
antibodies. The term "monoclonal antibody" or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one species of an antigen binding site
capable of immunoreacting with a particular epitope.
[0105] Polyclonal antibodies can be prepared as described above by
immunizing a suitable subject with a polypeptide of the invention
as an immunogen. Preferred polyclonal antibody compositions are
ones that have been selected for antibodies directed against a
polypeptide or polypeptides of the invention. Particularly
preferred polyclonal antibody preparations are ones that contain
only antibodies directed against a polypeptide or polypeptides of
the invention. Particularly preferred immunogen compositions are
those that contain no other human proteins such as, for example,
immunogen compositions made using a non-human host cell for
recombinant expression of a polypeptide of the invention. In such a
manner, the only human epitope or epitopes recognized by the
resulting antibody compositions raised against this immunogen will
be present as part of a polypeptide or polypeptides of the
invention.
[0106] The antibody titer in the immunized subject can be monitored
over time by standard techniques, such as with an enzyme linked
immunosorbent assay (ELISA) using immobilized polypeptide. If
desired, the antibody molecules can be isolated from the mammal
(e.g., from the blood) and further purified by well-known
techniques, such as protein A chromatography to obtain the IgG
fraction. Alternatively, antibodies specific for a protein or
polypeptide of the invention can be selected for (e.g., partially
purified) or purified by, e.g., affinity chromatography. For
example, a recombinantly expressed and purified (or partially
purified) protein of the invention is produced as described herein,
and covalently or non-covalently coupled to a solid support such
as, for example, a chromatography column. The column can then be
used to affinity purify antibodies specific for the proteins of the
invention from a sample containing antibodies directed against a
large number of different epitopes, thereby generating a
substantially purified antibody composition, i.e., one that is
substantially free of contaminating antibodies. By a substantially
purified antibody composition is meant, in this context, that the
antibody sample contains at most only 30% (by dry weight) of
contaminating antibodies directed against epitopes other than those
on the desired protein or polypeptide of the invention, and
preferably at most 20%, yet more preferably at most 10%, and most
preferably at most 5% (by dry weight) of the sample is
contaminating antibodies. A purified antibody composition means
that at least 99% of the antibodies in the composition are directed
against the desired protein or polypeptide of the invention.
[0107] At an appropriate time after immunization, e.g., when the
specific antibody titers are highest, antibody-producing cells can
be obtained from the subject and used to prepare monoclonal
antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975) Nature
256:495-497, the human B cell hybridoma technique (Kozbor et al.
(1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et
al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
hybridomas is well known (see generally Current Protocols in
Immunology (1994) Coligan et al. (eds.) John Wiley & Sons,
Inc., New York, N.Y.). Hybridoma cells producing a monoclonal
antibody of the invention are detected by screening the hybridoma
culture supernatants for antibodies that bind the polypeptide of
interest, e.g., using a standard ELISA assay.
[0108] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody directed against a polypeptide of
the invention can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (e.g., an antibody
phage display library) with the polypeptide of interest. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SURFZAP.TM. Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods
and reagents particularly amenable for use in generating and
screening antibody display library can be found in, for example,
U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT
Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT
Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT
Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT
Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology
9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85;
Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993)
EMBO J. 12:725-734.
[0109] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are within the scope of the invention. A chimeric
antibody is a molecule in which different portions are derived from
different animal species, such as those having a variable region
derived from a murine monoclonal antibody and a human
immunoglobulin constant region. (See, e.g., Cabilly et al., U.S.
Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which
are incorporated herein by reference in their entirety.) Humanized
antibodies are antibody molecules from non-human species having one
or more complementarity determining regions (CDRs) from the
non-human species and a framework region from a human
immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No.
5,585,089, which is incorporated herein by reference in its
entirety.) Such chimeric and humanized monoclonal antibodies can be
produced by recombinant DNA techniques known in the art, for
example using methods described in PCT Publication No. WO 87/02671;
European Patent Application 184,187; European Patent Application
171,496; European Patent Application 173,494; PCT Publication No.
WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science
229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0110] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced, for example, using transgenic mice which are incapable of
expressing endogenous immunoglobulin heavy and light chains genes,
but which can express human heavy and light chain genes. The
transgenic mice are immunized in the normal fashion with a selected
antigen, e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
using conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA and IgE antibodies. For an
overview of this technology for producing human antibodies, see
Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S.
Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No.
5,661,016; and U.S. Pat. No. 5,545,806. In addition, companies such
as Abgenix, Inc. (Fremont, Calif.), can be engaged to provide human
antibodies directed against a selected antigen using technology
similar to that described above.
[0111] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a murine antibody, is used to guide the selection
of a completely human antibody recognizing the same epitope.
(Jespers et al. (1994) Bio/technology 12:899-903).
[0112] An antibody directed against a polypeptide of the invention
(e.g., monoclonal antibody) can be used to isolate the polypeptide
by standard techniques, such as affinity chromatography or
immunoprecipitation. Moreover, such an antibody can be used to
detect the protein (e.g., in a cellular lysate or cell supernatant)
in order to evaluate the abundance and pattern of expression of the
polypeptide. The antibodies can also be used diagnostically to
monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, beta-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.1311, .sup.35S or .sup.3H.
[0113] Further, an antibody (or fragment thereof) can be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0114] The conjugated antibody substances of the invention can be
used for modifying a given biological response, and the drug moiety
conjugated with the antibody substance is not limited to classical
chemical therapeutic agents. For example, the drug moiety can be a
protein or polypeptide possessing a desired biological activity.
Such proteins can include, for example, a toxin such as abrin,
ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such
as tumor necrosis factor, alpha-interferon, beta-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator; or, biological response modifiers such as, for example,
lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"),
or other growth factors.
[0115] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
[0116] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980.
[0117] Accordingly, in one aspect, the invention provides
substantially purified antibodies or fragment thereof, and
non-human antibodies or fragments thereof, which antibodies or
fragments specifically bind with
[0118] (i) a polypeptide having the amino acid sequence of SEQ ID
NO: 3 or the amino acid sequence encoded by the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282;
[0119] (ii) a polypeptide fragment having the amino acid sequence
of at least 15 (e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120,
140, 160, 180, 200, 250, 300, 350, or 355) consecutive amino acid
residues of SEQ ID NO: 3 or the amino acid sequence encoded by the
clone deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282;
[0120] (iii) a polypeptide having an amino acid sequence which is
at least 95% identical to the amino acid sequence of SEQ ID NO: 3,
or the amino acid sequence encoded by the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or of 15
or more (e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 250, 300, 350, or 355) consecutive residues thereof,
wherein the percent identity is determined using the ALIGN program
of the GCG software package with a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4; or
[0121] (iv) a polypeptide encoded by a nucleic acid molecule which
hybridizes with a nucleic acid molecule having a nucleotide
sequence complementary to SEQ ID NO: 1 or 2, or the nucleotide
sequence of the clone deposited with ATCC.RTM. on Jul. 28, 2000 as
accession number PTA-2282, under conditions of hybridization of
6.times.SSC at 45.degree. C. and washing in 0.2.times.SSC, 0.1% SDS
at 65.degree. C.
[0122] In various embodiments, the substantially purified
antibodies of the invention, or fragments thereof, can be human,
non-human, chimeric and/or humanized antibodies.
[0123] In another aspect, the invention provides non-human
antibodies or fragments thereof, which antibodies or fragments
specifically bind with:
[0124] (i) a polypeptide having the amino acid sequence of SEQ ID
NO: 3 or the amino acid sequence encoded by the clone deposited
with ATCC.RTM. on July-28, 2000 as accession number PTA-2282;
[0125] (ii) a polypeptide fragment having the amino acid sequence
of at least 15 (e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120,
140, 160, 180, 200, 250, 300, 350, or 355) consecutive amino acid
residues of SEQ ID NO: 3 or the amino acid sequence encoded by the
clone deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282;
[0126] (iii) a polypeptide having an amino acid sequence which is
at least 95% identical to the amino acid sequence of SEQ ID NO: 3,
or the amino acid sequence encoded by the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or of 15
or more (e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 250, 300, 350, or 355) consecutive residues thereof,
wherein the percent identity is determined using the ALIGN program
of the GCG software package with a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4; or
[0127] (iv) a polypeptide encoded by a nucleic acid molecule which
hybridizes with a nucleic acid molecule having a nucleotide
sequence complementary to SEQ ID NO: 1 or 2, or the nucleotide
sequence of the clone deposited with ATCC.RTM. on Jul. 28, 2000 as
accession number PTA-2282, under conditions of hybridization of
6.times.SSC at 45.degree. C. and washing in 0.2.times.SSC, 0.1% SDS
at 65.degree. C.
[0128] Such non-human antibodies can be goat, mouse, sheep, horse,
chicken, rabbit, or rat antibodies. Alternatively, the non-human
antibodies of the invention can be chimeric and/or humanized
antibodies. In addition, the non-human antibodies of the invention
can be polyclonal antibodies or monoclonal antibodies.
[0129] In still a further aspect, the invention provides monoclonal
antibodies or fragments thereof, which antibodies or fragments
specifically bind with:
[0130] (i) a polypeptide having the amino acid sequence of SEQ ID
NO: 3 or the amino acid sequence encoded by the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282;
[0131] (ii) a polypeptide fragment having the amino acid sequence
of at least 15 (e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120,
140, 160, 180, 200, 250, 300, 350, or 355) consecutive amino acid
residues of SEQ ID NO: 3 or the amino acid sequence encoded by the
clone deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282;
[0132] (iii) a polypeptide having an amino acid sequence which is
at least 95% identical to the amino acid sequence of SEQ ID NO: 3,
or the amino acid sequence encoded by the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or of 15
or more (e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 250, 300, 350, or 355) consecutive residues thereof,
wherein the percent identity is determined using the ALIGN program
of the GCG software package with a PAM 120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4; or
[0133] (iv) a polypeptide encoded by a nucleic acid molecule which
hybridizes with a nucleic acid molecule having a nucleotide
sequence complementary to SEQ ID NO: 1 or 2, or the nucleotide
sequence of the clone deposited with ATCC.RTM. on Jul. 28, 2000 as
accession number PTA-2282, under conditions of hybridization of
6.times.SSC at 45.degree. C. and washing in 0.2.times.SSC, 0.1% SDS
at 65.degree. C.
[0134] The monoclonal antibodies can be human, humanized, chimeric
and/or non-human antibodies.
[0135] Any of the antibodies of the invention can be conjugated
with a therapeutic moiety or to a detectable substance.
Non-limiting examples of detectable substances that can be
conjugated with the antibodies of the invention are an enzyme, a
prosthetic group, a fluorescent material, a luminescent material, a
bioluminescent material, and a radioactive material.
[0136] The invention also provides a kit containing an antibody of
the invention conjugated with a detectable substance, and
instructions for use. Still another aspect of the invention is a
pharmaceutical composition comprising an antibody of the invention
and a pharmaceutically acceptable carrier. In preferred
embodiments, the pharmaceutical composition contains an antibody of
the invention, a therapeutic moiety, and a pharmaceutically
acceptable carrier.
[0137] Still another aspect of the invention is a method of making
an antibody that specifically recognizes h-ig6p, the method
comprising immunizing a mammal with a polypeptide of the invention.
The polypeptide used as an immunogen comprises an amino acid
sequence selected from the group consisting of:
[0138] (i) a polypeptide having the amino acid sequence of SEQ ID
NO: 3 or the amino acid sequence encoded by the clone deposited
with ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282;
[0139] (ii) a polypeptide fragment having the amino acid sequence
of at least 15 (e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120,
140, 160, 180, 200, 250, 300, 350, or 355) consecutive amino acid
residues of SEQ ID NO: 3 or the amino acid sequence encoded by the
clone deposited with ATCC.RTM. on Jul. 28, 2000 as accession number
PTA-2282;
[0140] (iii) a polypeptide having an amino acid sequence which is
at least 95% identical to the amino acid sequence of SEQ ID NO: 3,
or the amino acid sequence encoded by the clone deposited with
ATCC.RTM. on Jul. 28, 2000 as accession number PTA-2282, or of 15
or more (e.g., 30, 56, 57, 58, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 250, 300, 350, or 355) consecutive residues thereof,
wherein the percent identity is determined using the ALIGN program
of the GCG software package with a PAM 120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4; or
[0141] (iv) a polypeptide encoded by a nucleic acid molecule which
hybridizes with a nucleic acid molecule having a nucleotide
sequence complementary to SEQ ID NO: 1 or 2, or the nucleotide
sequence of the clone deposited with ATCC.RTM. on Jul. 28, 2000 as
accession number PTA-2282, under conditions of hybridization of
6.times.SSC at 45.degree. C. and washing in 0.2.times.SSC, 0.1% SDS
at 65.degree. C.
[0142] After immunization, a sample is collected from the mammal
that contains an antibody that specifically recognizes h-ig6p.
Preferably, the polypeptide is recombinantly produced using a
non-human host cell. Optionally, the antibodies can be further
purified from the sample using techniques well known to those of
skill in the art. The method can further comprise producing a
monoclonal antibody-producing cell from the cells of the mammal.
Optionally, antibodies are collected from the antibody-producing
cell.
[0143] III. Recombinant Expression Vectors and Host Cells
[0144] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
polypeptide of the invention (or a portion thereof). As used
herein, the term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid", which refers to a circular
double stranded DNA loop into which additional DNA segments can be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors, expression vectors, are capable of
directing the expression of genes to which they are operably
linked. In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids (vectors).
However, the invention is intended to include such other forms of
expression vectors, such as viral vectors (e.g., replication
defective retroviruses, adenoviruses and adeno-associated viruses),
which serve equivalent functions.
[0145] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell. This means that the recombinant
expression vectors include one or more regulatory sequences,
selected on the basis of the host cells to be used for expression,
which is operably linked to the nucleic acid sequence to be
expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory sequence(s) in a manner which
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel, Gene
Expression Technology: Methods in Enzymology, vol. 185, Academic
Press, San Diego, Calif. (1990). Regulatory sequences include those
which direct constitutive expression of a nucleotide sequence in
many types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, etc. The
expression vectors of the invention can be introduced into host
cells to thereby produce proteins or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described
herein.
[0146] The recombinant expression vectors of the invention can be
designed for expression of a polypeptide of the invention in
prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells
(using baculovirus expression vectors), yeast cells or mammalian
cells). Suitable host cells are discussed further in Goeddel,
supra. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0147] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:3140),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0148] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology, vol. 185, Academic Press, San Diego, Calif. (1990)
60-89). Target gene expression from the pTrc vector relies on host
RNA polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
co-expressed viral RNA polymerase (T7 gnl). This viral polymerase
is supplied by host strains BL21(DE3) or HMS174(DE3) from a
resident lambda prophage harboring a T7 gnl gene under the
transcriptional control of the lacUV 5 promoter.
[0149] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, Gene Expression Technology: Methods in Enzymology, vol.
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid
to be inserted into an expression vector so that the individual
codons for each amino acid are those preferentially used in E. coli
(Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[0150] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J.
6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corp, San
Diego, Calif.).
[0151] Alternatively, the expression vector is a baculovirus
expression vector. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
[0152] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO
J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook et al.,
supra.
[0153] Expression of h-ig6p in mammalian cells (e.g., pancreatic
cells, including alpha and beta cells of pancreatic islets) can
affect metabolic disorders, including various pancreatic disorders.
For example, expression of h-ig6p in mammalian cells can affect
carbohydrate metabolism in the cells. Expression of h-ig6p in
pancreatic cells (e.g., pancreatic islet beta cells) can inhibit
secretion of insulin. Furthermore, expression of h-ig6p in
pancreatic cells (e.g., pancreatic islet alpha cells) can enhance
glucagon secretion. Because h-ig6p can be involved in modulating
intracellular levels of small molecules (e.g., glucose and G6P)
that are involved in transmembrane signaling of extracellular and
intracellular glucose, expression of h-ig6p in cells can affect the
sensitivity of the signaling apparatus. The role of h-ig6p in
intracellular G6P-mediated signaling indicates that expression of
this protein from an expression vector can also influence
intracellular G6P-mediated signaling. These properties of h-ig6p
make expression vectors which encode this protein useful for
treatment of pancreatic aid metabolic disorders, such as
carbohydrate metabolism disorders including diabetes,
hyperinsulinemia, and obesity. Even in the absence of a disease
state, such vectors can be used to affect carbohydrate metabolism
and weight gain in humans.
[0154] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the alpha-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0155] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operably linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to the mRNA encoding a
polypeptide of the invention. Regulatory sequences operably linked
to a nucleic acid cloned in the antisense orientation can be chosen
which direct the continuous expression of the antisense RNA
molecule in a variety of cell types, for instance viral promoters
and/or enhancers, or regulatory sequences can be chosen which
direct constitutive, tissue specific or cell type specific
expression of antisense RNA. The antisense expression vector can be
in the form of a recombinant plasmid, phagemid or attenuated virus
in which antisense nucleic acids are produced under the control of
a high efficiency regulatory region, the activity of which can be
determined by the cell type into which the vector is introduced.
For a discussion of the regulation of gene expression using
antisense genes see Weintraub et al. (Reviews--Trends in Genetics,
Vol. 1(1) 1986).
[0156] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications can
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0157] A host cell can be any prokaryotic (e.g., E. coli) or
eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
[0158] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (supra), and other
laboratory manuals.
[0159] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells can integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
for resistance to antibiotics) is generally introduced into the
host cells along with the gene of interest. Preferred selectable
markers include those which confer resistance to drugs, such as
G418, hygromycin and methotrexate. Cells stably transfected with
the introduced nucleic acid can be identified by drug selection
(e.g., cells that have incorporated the selectable marker gene will
survive, while the other cells die).
[0160] In another embodiment, the expression characteristics of an
endogenous (e.g., h-ig6p) nucleic acid within a cell, cell line or
microorganism can be modified by inserting a DNA regulatory element
heterologous to the endogenous gene of interest into the genome of
a cell, stable cell line or cloned microorganism such that the
inserted regulatory element is operatively linked with the
endogenous gene and controls, modulates or activates the endogenous
gene. For example, endogenous h-ig6p which is normally
"transcriptionally silent" or is expressed only at very low levels
in a cell line or microorganism, can be activated by inserting a
regulatory element which is capable of promoting the expression of
a normally expressed gene product in that cell line or
microorganism. Alternatively, transcriptionally silent, endogenous
h-ig6p genes can be activated by insertion of a promiscuous
regulatory element that works across cell types.
[0161] A heterologous regulatory element can be inserted into a
stable cell line or cloned microorganism, such that it is
operatively linked with and activates expression of endogenous
h-ig6p genes, using techniques, such as targeted homologous
recombination, which are well known to those of skill in the art,
and described e.g., in Chappel, U.S. Pat. No. 5,272,071; PCT
publication No. WO 91/06667, published May 16, 1991.
[0162] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce a
polypeptide of the invention. Accordingly, the invention further
provides methods for producing a polypeptide of the invention using
the host cells of the invention. In one embodiment, the method
comprises culturing the host cell of invention (into which a
recombinant expression vector encoding a polypeptide of the
invention has been introduced) in a suitable medium such that the
polypeptide is produced. In another embodiment, the method further
comprises isolating the polypeptide from the medium or the host
cell. Such host cells can be used, for example, in screening assays
described herein.
[0163] By way of example, the host cell can be a cos-7 cell. Of
course, numerous other mammalian cell lines (e.g., CHO cell line)
can be used as host cells, depending on the particular
characteristics desired, as is understood by the skilled artisan.
In experiments described herein, cos-7 cells were transfected with
no DNA (a control), with 10 micrograms of pcDNA3.1 (vector
control), with 10 micrograms of human liver G6 Pase-pcDNA3.1
(vector for expressing liver G6 Pase), or with 10 micrograms of
h-ig6p-pcDNA3.1 (vector for expressing h-ig6p). The cells were
co-transfected with 2 micrograms of pCMV-beta-galactosidase vector
so that expression of beta-galactosidase could be used to confirm
transfection efficiency. Transfected cells were harvested about a
day later, using a non-enzymatic harvesting method. The harvested
cells were suspended in homogenization buffer (0.3 molar sucrose,
10 millimolar potassium MES buffer, pH adjusted to 6.5, 2
millimolar EGTA, and 1 millimolar MgSO.sub.4) and homogenized on
use using a POLYTRAN.TM. homogenizer. The homogenate was
centrifuged at 2000.times.g for 3 minutes, and then at 6000.times.g
for 3 minutes. The supernatant was collected and centrifuged at
234,000.times.g for 1 hour. The microsomal fraction was
re-suspended in 200 to 300 microliters of homogenization buffer to
yield microsomal samples.
[0164] The host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which a sequences encoding a polypeptide of the
invention have been introduced. Such host cells can then be used to
create non-human transgenic animals in which exogenous sequences
encoding a polypeptide of the invention have been introduced into
their genome or homologous recombinant animals in which endogenous
encoding a polypeptide of the invention sequences have been
altered. Such animals are useful for studying the function and/or
activity of the polypeptide and for identifying and/or evaluating
modulators of polypeptide activity. As used herein, a "transgenic
animal" is a non-human animal, preferably a mammal, more preferably
a rodent such as a rat or mouse, in which one or more of the cells
of the animal includes a transgene. Other examples of transgenic
animals include non-human primates, sheep, dogs, cows, goats,
chickens, amphibians, etc. A transgene is exogenous DNA which is
integrated into the genome of a cell from which a transgenic animal
develops and which remains in the genome of the mature animal,
thereby directing the expression of an encoded gene product in one
or more cell types or tissues of the transgenic animal. As used
herein, an "homologous recombinant animal" is a non-human animal,
preferably a mammal, more preferably a mouse, in which an
endogenous gene has been altered by homologous recombination
between the endogenous gene and an exogenous DNA molecule
introduced into a cell of the animal, e.g., an embryonic cell of
the animal, prior to development of the animal.
[0165] A transgenic animal of the invention can be created by
introducing nucleic acid encoding a polypeptide of the invention
(or a homologue thereof) into the male pronuclei of a fertilized
oocyte, e.g., by microinjection, retroviral infection, and allowing
the oocyte to develop in a pseudopregnant female foster animal.
Intronic sequences and polyadenylation signals can also be included
in the transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be operably
linked to the transgene to direct expression of the polypeptide of
the invention to particular cells. Methods for generating
transgenic animals via embryo manipulation and microinjection,
particularly animals such as mice, have become conventional in the
art and are described, for example, in U.S. Pat. Nos. 4,736,866 and
4,870,009, U.S. Pat. No. 4,873,191 and in Hogan, Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986) and Wakayama et al., (1999), Proc. Natl. Acad.
Sci. USA, 96:14984-14989. Similar methods are used for production
of other transgenic animals. A transgenic founder animal can be
identified based upon the presence of the transgene in its genome
and/or expression of mRNA encoding the transgene in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying the transgene can further be bred to
other transgenic animals carrying other transgenes.
[0166] To create an homologous recombinant animal, a vector is
prepared which contains at least a portion of a gene encoding a
polypeptide of the invention into which a deletion, addition or
substitution has been introduced to thereby alter, e.g.,
functionally disrupt, the gene. In a preferred embodiment, the
vector is designed such that, upon homologous recombination, the
endogenous gene is functionally disrupted (i.e., no longer encodes
a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous protein). In the homologous
recombination vector, the altered portion of the gene is flanked at
its 5' and 3' ends by additional nucleic acid of the gene to allow
for homologous recombination to occur between the exogenous gene
carried by the vector and an endogenous gene in an embryonic stem
cell. The additional flanking nucleic acid sequences are of
sufficient length for successful homologous recombination with the
endogenous gene. Typically, several kilobases of flanking DNA (both
at the 5' and 3' ends) are included in the vector (see, e.g.,
Thomas and Capecchi (1987) Cell 51:503 for a description of
homologous recombination vectors). The vector is introduced into an
embryonic stem cell line (e.g., by electroporation) and cells in
which the introduced gene has homologously recombined with the
endogenous gene are selected (see, e.g., Li et al. (1992) Cell
69:915). The selected cells are then injected into a blastocyst of
an animal (e.g., a mouse) to form aggregation chimeras (see, e.g.,
Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A
chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term.
Progeny harboring the homologously recombined DNA in their germ
cells can be used to breed animals in which all cells of the animal
contain the homologously recombined DNA by germline transmission of
the transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in
PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO
93/04169.
[0167] In another embodiment, transgenic non-human animals can be
produced which contain selected systems which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a
cre/loxP recombinase system is used to regulate expression of the
transgene, animals containing transgenes encoding both the Cre
recombinase and a selected protein are required. Such animals can
be provided through the construction of "double" transgenic
animals, e.g., by mating two transgenic animals, one containing a
transgene encoding a selected protein and the other containing a
transgene encoding a recombinase.
[0168] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al. (1997) Nature 385:810-813 and PCT Publication Nos. WO
97/07668 and WO 97/07669.
[0169] IV. Pharmaceutical Compositions
[0170] The nucleic acid molecules, polypeptides, and antibodies
(also referred to herein as "active compounds") of the invention
can be incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the nucleic
acid molecule, protein, or antibody and a pharmaceutically
acceptable carrier. As used herein the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0171] The invention includes methods for preparing pharmaceutical
compositions for modulating the expression or activity of a
polypeptide or nucleic acid of the invention. Such methods comprise
formulating a pharmaceutically acceptable carrier with an agent
which modulates expression or activity of a polypeptide or nucleic
acid of the invention. Such compositions can further include
additional active agents. Thus, the invention further includes
methods for preparing a pharmaceutical composition by formulating a
pharmaceutically acceptable carrier with an agent which modulates
expression or activity of a polypeptide or nucleic acid of the
invention and one or more additional active compounds.
[0172] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediamine-tetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0173] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, CREMOPHOR.TM. EL (BASF; Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0174] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a polypeptide or antibody)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0175] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
[0176] Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, PRIMOGEL.TM., or corn starch; a lubricant such as
magnesium stearate or STEROTES.TM.; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
[0177] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
[0178] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0179] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0180] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0181] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0182] For antibodies, the preferred dosage is 0.1 to 100 milligram
per kilogram body weight (generally 10 to 20 milligram per kilogram
body weight). If the antibody is to act in the brain, a dosage of
50 to 100 milligram per kilogram body weight is usually
appropriate. Generally, partially human antibodies and fully human
antibodies have a longer half-life within the human body than other
antibodies. Accordingly, lower dosages and less frequent
administration is often possible. Modifications such as lipidation
can be used to stabilize antibodies and to enhance uptake and
tissue penetration (e.g., into the brain). A method for lipidation
of antibodies is described by Cruikshank et al. ((1997) J. Acquired
Immune Deficiency Syndromes and Human Retrovirology 14:193).
[0183] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (U.S. Pat. No. 5,328,470) or by
stereotactic injection (see, e.g., Chen et a! (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g. retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0184] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0185] V. Uses and Methods of the Invention
[0186] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) detection assays (e.g.,
chromosomal mapping, tissue typing, forensic biology); c)
predictive medicine (e.g., diagnostic assays, prognostic assays,
monitoring clinical trials, and pharmacogenomics); and d) methods
of treatment (e.g., therapeutic and prophylactic). For example,
polypeptides of the invention can to used to (i) modulate cellular
proliferation; (ii) modulate cellular differentiation; and/or (iii)
modulate cellular adhesion. The isolated nucleic acid molecules of
the invention can be used to express proteins (e.g., via a
recombinant expression vector in a host cell in gene therapy
applications), to detect mRNA (e.g., in a biological sample) or a
genetic lesion, and to modulate activity of a polypeptide of the
invention. In addition, the polypeptides of the invention can be
used to screen drugs or compounds which modulate activity or
expression of a polypeptide of the invention as well as to treat
disorders characterized by insufficient or excessive production of
a protein of the invention or production of a form of a protein of
the invention which has decreased or aberrant activity compared to
the wild type protein. In addition, the antibodies of the invention
can be used to detect and isolate a protein of the and modulate
activity of a protein of the invention.
[0187] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0188] A. Screening Assays
[0189] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) which bind to polypeptide of the
invention or have a stimulatory or inhibitory effect on, for
example, expression or activity of a polypeptide of the invention.
By way of example, these assays include assays for identifying
compounds which enhance expression of h-ig6p, compounds which
inhibit its expression, and compounds which act as agonists or
antagonists of h-ig6p enzymatic activity. The assays can also be
used to identify compounds which inhibit or enhance interaction of
h-ig6p with one or more normal cellular proteins. As such, these
screening assays can be used to identify compounds that are useful
for alleviation, inhibition, or prevention of carbohydrate
metabolism disorders described herein, such as diabetes and
obesity. Compounds which enhance h-ig6p activity or expression can,
for example, be used to alleviate or inhibit conditions (e.g.,
hyperinsulinemia or aberrant weight loss) associated with
aberrantly high insulin secretion. Compounds which inhibit h-ig6p
activity or expression can be used to alleviate or inhibit
conditions (e.g., diabetes or obesity) associated with aberrantly
low insulin secretion.
[0190] In one embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of the membrane-bound form of a polypeptide of the
invention or biologically active portion thereof. The test
compounds of the present invention can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is
limited to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam (1997) Anticancer Drug Des.
12:145).
[0191] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew.
Chem. Int. Ed. Engi. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem.
37:1233.
[0192] Libraries of compounds can be presented in solution (e.g.,
Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos.
5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992)
Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith
(1990) Science 249:386-390; Devlin (1990) Science 249:404-406;
Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and
Felici (1991) J. Mol. Biol. 222:301-310).
[0193] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a membrane-bound form of a polypeptide of the
invention, or a biologically active portion thereof, on the cell
surface is contacted with a test compound and the ability of the
test compound to bind to the polypeptide determined. The cell, for
example, can be a yeast cell or a cell of mammalian origin.
Determining the ability of the test compound to bind to the
polypeptide can be accomplished, for example, by coupling the test
compound with a radioisotope or enzymatic label such that binding
of the test compound to the polypeptide or biologically active
portion thereof can be determined by detecting the labeled compound
in a complex. For example, test compounds can be labeled with
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radio emission or by scintillation counting. Alternatively, test
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product. In a preferred embodiment, the
assay comprises contacting a cell which expresses a membrane-bound
form of a polypeptide or the invention, or a biologically active
portion thereof, on the cell surface with a known compound which
binds the polypeptide to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with the polypeptide, wherein
determining the ability of the test compound to interact with the
polypeptide comprises determining the ability of the test compound
to preferentially bind to the polypeptide or a biologically active
portion thereof as compared to the known compound.
[0194] Substantially any known or hereafter-developed method of
assaying G6 Pase activity can be used to assess the effect of a
test compound on h-ig6p activity (including whether, or to what
degree, h-ig6p is expressed). The activity can be assessed, for
example, according to the methods disclosed in Arden et al., 1999,
Diabetes 48:531-542). Colorimetric, radiometric, and other
detection technologies can be used in the assay.
[0195] By way of example, G6 Pase activities were assayed in
microsomal samples in experiments described herein by incubating 20
microliters of a microsomal sample for 30 minutes at 30.degree. C.
with 40 microliters of assay buffer (100 millimolar
dimethylglutarate, 20 millimolar sodium tartrate, 10 millimolar
EDTA, and 20-40 millimolar glucose-6-phosphate, pH adjusted to
6.5). The reaction was terminated by adding 40 microliters of 10%
(w/v) trichloroacetic acid to the assay mixture, and then the assay
mixture was centrifuged for 5 minutes at 2000.times.g. 25
Microliters of the supernatant was transferred to a well on a
96-well plate, and 250 microliters of color reagent (1 part 4.2%
(w/v) ammonium molybdate in 4 molar HCl, mixed with 3 parts 0.2%
(w/v) malachite green, and then filtered after 30 minutes) was
added to the well. The optical density at 650 nanometers (using
sodium phosphate buffer as a standard) was measured in order to
detect the reaction product. The concentration of protein in
microsomal samples was determined using the Pierce Chemical Co.
BCA.TM. bicinchoninic acid reagent. These assays demonstrated that
the h-ig6p protein obtained from mammalian cell microsomal
fractions described herein exhibits activity that is characteristic
of a G6 Pase enzyme.
[0196] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a membrane-bound form of a
polypeptide of the invention, or a biologically active portion
thereof, on the cell surface with a test compound and determining
the ability of the test compound to modulate (e.g., stimulate or
inhibit) the activity of the polypeptide or biologically active
portion thereof. Determining the ability of the test compound to
modulate the activity of the polypeptide or a biologically active
portion thereof can be accomplished, for example, by determining
the ability of the polypeptide protein to bind to or interact with
a target molecule.
[0197] Determining the ability of a polypeptide of the invention to
bind to or interact with a target molecule can be accomplished by
one of the methods described above for determining direct binding.
As used herein, a "target molecule" is a molecule with which a
selected polypeptide (e.g., a polypeptide of the invention binds or
interacts with in nature, for example, a molecule on the surface of
a cell which expresses the selected protein, a molecule on the
surface of a second cell, a molecule in the extracellular milieu,
or a molecule associated with the internal surface of a cell
membrane or a cytoplasmic molecule). A target molecule can be a
polypeptide of the invention or some other polypeptide or protein.
For example, a target molecule can be a component of a signal
transduction pathway which facilitates transduction of an
extracellular signal (e.g., a signal generated by binding of a
compound to a polypeptide of the invention) through the cell
membrane and into the cell or a second intercellular protein which
has catalytic activity or a protein which facilitates the
association of downstream signaling molecules with a polypeptide of
the invention. Determining the ability of a polypeptide of the
invention to bind to or interact with a target molecule can be
accomplished by determining the activity of the target molecule.
For example, the activity of the target molecule can be determined
by detecting induction of a cellular second messenger of the target
(e.g., G6P, intracellular Ca.sup.2+, diacylglycerol, IP3, etc.),
detecting catalytic/enzymatic activity of the target on an
appropriate substrate, detecting the induction of a reporter gene
(e.g., a regulatory element that is responsive to a polypeptide of
the invention operably linked to a nucleic acid encoding a
detectable marker, e.g. luciferase), or detecting a cellular
response, for example, cellular differentiation, or cell
proliferation.
[0198] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting a polypeptide of the
invention or biologically active portion thereof with a test
compound and determining the ability of the test compound to bind
to the polypeptide or biologically active portion thereof. Binding
of the test compound to the polypeptide can be determined either
directly or indirectly as described above. In a preferred
embodiment, the assay includes contacting the polypeptide of the
invention or biologically active portion thereof with a known
compound which binds the polypeptide to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with the polypeptide,
wherein determining the ability of the test compound to interact
with the polypeptide comprises determining the ability of the test
compound to preferentially bind to the polypeptide or biologically
active portion thereof as compared to the known compound.
[0199] In another embodiment, an assay is a cell-free assay
comprising contacting a polypeptide of the invention or
biologically active portion thereof with a test compound and
determining the ability of the test compound to modulate (e.g.,
stimulate or inhibit) the activity of the polypeptide or
biologically active portion thereof. Determining the ability of the
test compound to modulate the activity of the polypeptide can be
accomplished, for example, by determining the ability of the
polypeptide to bind to a target molecule by one of the methods
described above for determining direct binding. In an alternative
embodiment, determining the ability of the test compound to
modulate the activity of the polypeptide can be accomplished by
determining the ability of the polypeptide of the invention to
further modulate the target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as described herein.
[0200] In yet another embodiment, the cell-free assay comprises
contacting a polypeptide of the invention or biologically active
portion thereof with a known compound which binds the polypeptide
to form an assay mixture, contacting the assay mixture with a test
compound, and determining the ability of the test compound to
interact with the polypeptide, wherein determining the ability of
the test compound to interact with the polypeptide comprises
determining the ability of the polypeptide to preferentially bind
to or modulate the activity of a target molecule.
[0201] The cell-free assays of the present invention are amenable
to use of both a soluble form or the membrane-bound form of a
polypeptide of the invention. In the case of cell-free assays
comprising the membrane-bound form of the polypeptide, it can be
desirable to use a solubilizing agent such that the membrane-bound
form of the polypeptide is maintained in solution. Examples of such
solubilizing agents include non-ionic detergents such as
n-octylglucoside, n-dodecylglucoside, n-octylmaltoside,
octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton
X-100, Triton X-114, Thesit, Isotridecypoly(ethylene glycol
ether).sub.n, 3-{(3-cholamidopropyl)dimethylamminio}-1-propane
sulfonate (CHAPS),
3-{(3-cholamidopropyl)dimethylamminio}-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0202] In more than one embodiment of the above assay methods of
the present invention, it can be desirable to immobilize either the
polypeptide of the invention or its target molecule to facilitate
separation of complexed from non-complexed forms of one or both of
the proteins, as well as to accommodate automation of the assay.
Binding of a test compound to the polypeptide, or interaction of
the polypeptide with a target molecule in the presence and absence
of a candidate compound, can be accomplished in any vessel suitable
for containing the reactants. Examples of such vessels include
microtiter plates, test tubes, and micro-centrifuge tubes. In one
embodiment, a fusion protein can be provided which adds a domain
that allows one or both of the proteins to be bound to a matrix.
For example, glutathione-S-transferase fusion proteins or
glutathione-S-transferase fusion proteins can be adsorbed onto
glutathione SEPHAROSE.TM. beads (Sigma Chemical; St. Louis, Mo.) or
glutathione derivatized microtiter plates, which are then combined
with the test compound or the test compound and either the
non-adsorbed target protein or A polypeptide of the invention, and
the mixture incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads or microtiter plate wells are
washed to remove any unbound components and complex formation is
measured either directly or indirectly, for example, as described
above. Alternatively, the complexes can be dissociated from the
matrix, and the level of binding or activity of the polypeptide of
the invention can be determined using standard techniques.
[0203] Other techniques for immobilizing proteins on matrices can
also be used in the screening-assays of the invention. For example,
either the polypeptide of the invention or its target molecule can
be immobilized using conjugation of biotin and streptavidin.
Biotinylated polypeptide of the invention or target molecules can
be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques
well known in the art (e.g., biotinylation kit, Pierce Chemicals;
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with the polypeptide of the
invention or target molecules but which do not interfere with
binding of the polypeptide of the invention to its target molecule
can be derivatized to the wells of the plate, and unbound target or
polypeptide of the invention trapped in the wells by antibody
conjugation. Methods for detecting such complexes, in addition to
those described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the
polypeptide of the invention or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the polypeptide of the invention or target
molecule.
[0204] In another embodiment, modulators of expression of a
polypeptide of the invention are identified in a method in which a
cell is contacted with a candidate compound and the expression of
the selected mRNA or protein (i.e., the mRNA or protein
corresponding to a polypeptide or nucleic acid of the invention) in
the cell is determined. The level of expression of the selected
mRNA or protein in the presence of the candidate compound is
compared to the level of expression of the selected mRNA or protein
in the absence of the candidate compound. The candidate compound
can then be identified as a modulator of expression of the
polypeptide of the invention based on this comparison. For example,
when expression of the selected mRNA or protein is greater
(statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of the selected mRNA or protein
expression. Alternatively, when expression of the selected mRNA or
protein is less (statistically significantly less) in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of the selected mRNA or
protein expression. The level of the selected mRNA or protein
expression in the cells can be determined by methods described
herein.
[0205] In yet another aspect of the invention, a polypeptide of the
inventions can be used as "bait proteins" in a two-hybrid assay or
three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication
No. WO 94/10300), to identify other proteins, which bind to or
interact with the polypeptide of the invention and modulate
activity of the polypeptide of the invention. Such binding proteins
are also likely to be involved in the propagation of signals by the
polypeptide of the inventions as, for example, upstream or
downstream elements of a signaling pathway involving the
polypeptide of the invention.
[0206] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0207] B. Detection Assays
[0208] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (i) map their respective genes on a
chromosome and, thus, locate gene regions associated with genetic
disease; (ii) identify an individual from a minute biological
sample (tissue typing); and (iii) aid in forensic identification of
a biological sample. These applications are described in the
subsections below.
[0209] 1. Chromosome Mapping
[0210] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. Accordingly, nucleic acid molecules
described herein or fragments thereof, can be used to map the
location of the corresponding genes on a chromosome. The mapping of
the sequences to chromosomes is an important first step in
correlating these sequences with genes associated with disease. By
way of example, the gene encoding h-ig6p maps to human chromosome
2, as assessed by alignment of the h-ig6p cDNA sequence with known
human genomic sequence fragments. Aberrant expression or activity
of h-ig6p can thus be associated with a disorder attributable to a
genomic lesion that maps to this region of chromosome 2.
[0211] Briefly, genes can be mapped to chromosomes by preparing PCR
primers (preferably 15-25 base pairs in length) from the sequence
of a gene of the invention. Computer analysis of the sequence of a
gene of the invention can be used to rapidly select primers that do
not span more than one exon in the genomic DNA, thus complicating
the amplification process. These primers can then be used for PCR
screening of somatic cell hybrids containing individual human
chromosomes. Only those hybrids containing the human gene
corresponding to the gene sequences will yield an amplified
fragment. For a review of this technique, see D'Eustachio et al.
((1983) Science 220:919-924).
[0212] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the nucleic acid sequences of the invention to design
oligonucleotide primers, sub-localization can be achieved with
panels of fragments from specific chromosomes. Other mapping
strategies which can similarly be used to map a gene to its
chromosome include in situ hybridization (described in Fan et al.
(1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening with
labeled flow-sorted chromosomes, and pre-selection by hybridization
to chromosome specific cDNA libraries. Fluorescence in situ
hybridization (FISH) of a DNA sequence to a metaphase chromosomal
spread can further be used to provide a precise chromosomal
location in one step. For a review of this technique, see Verma et
al. (Human Chromosomes: A Manual of Basic Techniques (Pergamon
Press, New York, 1988)).
[0213] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to non-coding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0214] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, e.g.,
Egeland et al. (1987) Nature 325:783-787.
[0215] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a gene of the invention can be determined. If a mutation is
observed in some or all of the affected individuals but not in any
unaffected individuals, then the mutation is likely to be the
causative agent of the particular disease. Comparison of affected
and unaffected individuals generally involves first looking for
structural alterations in the chromosomes such as deletions or
translocations that are visible from chromosome spreads or
detectable using PCR based on that DNA sequence. Ultimately,
complete sequencing of genes from several individuals can be
performed to confirm the presence of a mutation and to distinguish
mutations from polymorphisms.
[0216] Furthermore, the nucleic acid sequences disclosed herein can
be used to perform searches against "mapping databases", e.g.,
BLAST-type search, such that the chromosome position of the gene is
identified by sequence homology or identity with known sequence
fragments which have been mapped to chromosomes.
[0217] A polypeptide and fragments and sequences thereof and
antibodies specific thereto can be used to map the location of the
gene encoding the polypeptide on a chromosome. This mapping can be
carried out by specifically detecting the presence of the
polypeptide in members of a panel of somatic cell hybrids between
cells of a first species of animal from which the protein
originates and cells from a second species of animal and then
determining which somatic cell hybrid(s) expresses the polypeptide
and noting the chromosome(s) from the first species of animal that
it contains. For examples of this technique, see Pajunen et al.
(1988) Cytogenet. Cell Genet. 47:37-41 and Van Keuren et al. (1986)
Hum. Genet. 74:34-40. Alternatively, the presence of the
polypeptide in the somatic cell hybrids can be determined by
assaying an activity or property of the polypeptide, for example,
enzymatic activity, as described in Bordelon-Riser et al. (1979)
Somatic Cell Genetics 5:597-613 and Owerbach et al. (1978) Proc.
Natl. Acad. Sci. USA 75:5640-5644.
[0218] 2. Tissue Typing
[0219] The nucleic acid sequences of the present invention can also
be used to identify individuals from minute biological samples. The
United States military, for example, is considering the use of
restriction fragment length polymorphism (RFLP) for identification
of its personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "dog tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0220] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected portions of an
individual's genome. Thus, the nucleic acid sequences described
herein can be used to prepare two PCR primers from the 5' and 3'
ends of the sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0221] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The nucleic acid
sequences of the invention uniquely represent portions of the human
genome. Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
non-coding regions. It is estimated that allelic variation between
individual humans occurs with a frequency of about once per each
500 bases. Each of the sequences described herein can, to some
degree, be used as a standard against which DNA from an individual
can be compared for identification purposes. Because greater
numbers of polymorphisms occur in the non-coding regions, fewer
sequences are necessary to differentiate individuals. The
non-coding sequences of SEQ ID NO: 1 (or the nucleotide sequence of
the clone deposited with ATCC.RTM. on Jul. 28, 2000 as accession
number PTA-2282) can comfortably provide positive individual
identification with a panel of perhaps 10 or more primers which
each yield a non-coding amplified sequence of 100 bases. If
predicted coding sequences, such as those in SEQ ID NO: 2, are
used, a more appropriate number of primers for positive individual
identification would be higher.
[0222] If a panel of reagents from the nucleic acid sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
[0223] 3. Use of Partial Gene Sequences in Forensic Biology
[0224] DNA-based identification techniques can also be used in
forensic biology. Forensic biology is a scientific field employing
genetic typing of biological evidence found at a crime scene as a
means for positively identifying, for example, a perpetrator of a
crime. To make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological samples
such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[0225] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
non-coding regions are particularly appropriate for this use as
greater numbers of polymorphisms occur in the non-coding regions,
making it easier to differentiate individuals using this technique.
Examples of polynucleotide reagents include the nucleic acid
sequences of the invention or portions thereof, e.g., fragments
derived from non-coding regions having a length of at least 20 or
30 bases.
[0226] The nucleic acid sequences described herein can further be
used to provide polynucleotide reagents, e.g., labeled or labelable
probes which can be used in, for example, an in situ hybridization
technique, to identify a specific tissue, e.g., brain tissue. This
can be very useful in cases where a forensic pathologist is
presented with a tissue of unknown origin. Panels of such probes
can be used to identify tissue by species and/or by organ type.
[0227] C. Predictive Medicine:
[0228] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining h-ig6p protein and/or nucleic
acid expression as well as h-ig6p activity, in the context of a
biological sample (e.g., blood, serum, cells, tissue) to thereby
determine whether an individual is afflicted with a disease or
disorder, or is at risk of developing a disorder, associated with
aberrant or unwanted h-ig6p expression or activity. The invention
also provides for prognostic (or predictive) assays for determining
whether an individual is at risk of developing a disorder
associated with h-ig6p protein, nucleic acid expression or
activity. For example, mutations in a h-ig6p gene can be assayed in
a biological sample. Such assays can be used for prognostic or
predictive purpose to thereby prophylactically treat an individual
prior to the onset of a disorder characterized by or associated
with h-ig6p protein, nucleic acid expression or activity.
[0229] As an alternative to making determinations based on the
absolute expression level of selected genes, determinations can be
based on the normalized expression levels of these genes.
Expression levels are normalized by correcting the absolute
expression level of a h-ig6p gene by comparing its expression to
the expression of a gene that is not a h-ig6p gene, e.g., a
housekeeping gene that is constitutively expressed. Suitable genes
for normalization include housekeeping genes such as the actin
gene. This normalization allows the comparison of the expression
level in one sample, e.g., a patient sample, to another sample,
e.g., a sample obtained from a non-diseased source, or between
samples from different sources.
[0230] Alternatively, the expression level can be provided as a
relative expression level. To determine a relative expression level
of a gene, the level of expression of the gene is determined for 10
or more samples of different pancreatic islet of Langerhans cell
isolates, preferably 50 or more samples, prior to the determination
of the expression level for the sample in question. The mean
expression level of each of the genes assayed in the larger number
of samples is determined and this is used as a baseline expression
level for the gene(s) in question. The expression level of the gene
determined for the test sample (absolute level of expression) is
then divided by the mean expression value obtained for that gene.
This provides a relative expression level and aids in identifying
extreme cases of a disorder associated with aberrant activity or
expression of h-ig6p.
[0231] Preferably, the samples used in the baseline determination
are obtained from diseased or from non-diseased cells of pancreatic
tissue (preferably islet of Langerhans cells or isolated alpha or
beta islet cells). The choice of the cell source is dependent on
the use of the relative expression level. Using expression found in
normal tissues as a mean expression score aids in validating
whether the h-ig6p gene assayed is diseased cell-specific (versus
normal cells). Such a use is particularly important in identifying
whether a h-ig6p gene can serve as a target gene. In addition, as
more data is accumulated, the mean expression value can be revised,
providing improved relative expression values based on accumulated
data. Expression data from pancreatic islet cells provides a means
for grading the severity of the disease state.
[0232] Another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs, compounds) on the expression or
activity of h-ig6p in clinical trials.
[0233] These and other agents are described in further detail in
the following sections.
[0234] 1. Diagnostic Assays
[0235] An exemplary method for detecting the presence or absence of
a polypeptide or nucleic acid of the invention in a biological
sample involves obtaining a biological sample from a test subject
and contacting the biological sample with a compound or an agent
capable of detecting a polypeptide or nucleic acid (e.g., mRNA,
genomic DNA) of the invention such that the presence of a
polypeptide or nucleic acid of the invention is detected in the
biological sample. A preferred agent for detecting mRNA or genomic
DNA encoding a polypeptide of the invention is a labeled nucleic
acid probe capable of hybridizing to mRNA or genomic DNA encoding a
polypeptide of the invention. The nucleic acid probe can be, for
example, a full-length cDNA, such as the nucleic acid of SEQ ID NO:
1, the nucleotide sequence of the clone deposited with ATCC.RTM. on
Jul. 28, 2000 as accession number PTA-2282, or a portion thereof,
such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions with a mRNA or genomic DNA encoding a
polypeptide of the invention. Other suitable probes for use in the
diagnostic assays of the invention are described herein.
[0236] A preferred agent for detecting a polypeptide of the
invention is an antibody capable of binding to a polypeptide of the
invention, preferably an antibody with a detectable label.
Antibodies can be polyclonal, or more preferably, monoclonal. An
intact antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2)
can be used. The term "labeled", with regard to the probe or
antibody, is intended to encompass direct labeling of the probe or
antibody by coupling (i.e., physically linking) a detectable
substance to the probe or antibody, as well as indirect labeling of
the probe or antibody by reactivity with another reagent that is
directly labeled. Examples of indirect labeling include detection
of a primary antibody using a fluorescently labeled secondary
antibody and end-labeling of a DNA probe with biotin such that it
can be detected with fluorescently labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method of the invention can be used to detect mRNA, protein, or
genomic DNA in a biological sample in vitro as well as in vivo. For
example, in vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detection of a polypeptide of the invention include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations
and immunofluorescence. In vitro techniques for detection of
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of a polypeptide of the invention include
introducing into a subject a labeled antibody directed against the
polypeptide. For example, the antibody can be labeled with a
radioactive marker whose presence and location in a subject can be
detected by standard imaging techniques.
[0237] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a peripheral blood leukocyte sample isolated by conventional
means from a subject.
[0238] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting a
polypeptide of the invention or mRNA or genomic DNA encoding a
polypeptide of the invention, such that the presence of the
polypeptide or mRNA or genomic DNA encoding the polypeptide is
detected in the biological sample, and comparing the presence of
the polypeptide or mRNA or genomic DNA encoding the polypeptide in
the control sample with the presence of the polypeptide or mRNA or
genomic DNA encoding the polypeptide in the test sample.
[0239] The invention also encompasses kits for detecting the
presence of a polypeptide or nucleic acid of the invention in a
biological sample (a test sample). Such kits can be used to
determine if a subject is suffering from or is at increased risk of
developing a disorder associated with aberrant expression of a
polypeptide of the invention (e.g., a proliferative disorder, e.g.,
psoriasis or cancer). For example, the kit can comprise a labeled
compound or agent capable of detecting the polypeptide or mRNA
encoding the polypeptide in a biological sample and means for
determining the amount of the polypeptide or mRNA in the sample
(e.g., an antibody which binds the polypeptide or an
oligonucleotide probe which binds to DNA or mRNA encoding the
polypeptide). Kits can also include instructions for observing that
the tested subject is suffering from or is at risk of developing a
disorder associated with aberrant expression of the polypeptide if
the amount of the polypeptide or mRNA encoding the polypeptide is
above or below a normal level.
[0240] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a polypeptide of the invention; and, optionally, (2) a
second, different antibody which binds to either the polypeptide or
the first antibody and is conjugated to a detectable agent.
[0241] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence
encoding a polypeptide of the invention or (2) a pair of primers
useful for amplifying a nucleic acid molecule encoding a
polypeptide of the invention. The kit can also comprise, e.g., a
buffering agent, a preservative, or a protein stabilizing agent.
The kit can also comprise components necessary for detecting the
detectable agent (e.g., an enzyme or a substrate). The kit can also
contain a control sample or a series of control samples which can
be assayed and compared to the test sample contained. Each
component of the kit is usually enclosed within an individual
container and all of the various containers are within a single
package along with instructions for observing whether the tested
subject is suffering from or is at risk of developing a disorder
associated with aberrant expression of the polypeptide.
[0242] By way of example, abnormally high levels of expression of
h-ig6p in a human can be an indication that the human is afflicted
with, or is susceptible to, a disorder associated with aberrantly
low insulin secretion, such as diabetes or obesity. Alternatively,
abnormally low levels of expression of h-ig6p in a human can be an
indication that the human is afflicted with, or is susceptible to,
a disorder associated with aberrantly high insulin secretion, such
as hyperinsulinemia. Thus, compositions and kits as described
herein can be made and used for diagnosis and prognostication of
these and other disorders associated with pancreatic and metabolic
disorders.
[0243] 2. Prognostic Assays
[0244] The methods described herein can furthermore be used as
diagnostic or prognostic assays to identify subjects having or at
risk of developing a disease or disorder associated with aberrant
expression or activity of a polypeptide of the invention. For
example, the assays described herein, such as the preceding
diagnostic assays or the following assays, can be used to identify
a subject having or at risk of developing a disorder associated
with aberrant expression or activity of a polypeptide of the
invention, e.g., a proliferative disorder, e.g., psoriasis or
cancer, or an angiogenic disorder. Alternatively, the prognostic
assays can be used to identify a subject having or at risk for
developing such a disease or disorder. Thus, the present invention
provides a method in which a test sample is obtained from a subject
and a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the
invention is detected, wherein the presence of the polypeptide or
nucleic acid is diagnostic for a subject having or at risk of
developing a disease or disorder associated with aberrant
expression or activity of the polypeptide. As used herein, a "test
sample" refers to a biological sample obtained from a subject of
interest. For example, a test sample can be a biological fluid
(e.g., serum), cell sample, or tissue.
[0245] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant expression or activity
of a polypeptide of the invention. For example, such methods can be
used to determine whether a subject can be effectively treated with
a specific agent or class of agents (e.g., agents of a type which
decrease activity of the polypeptide). Thus, the present invention
provides methods for determining whether a subject can be
effectively treated with an agent for a disorder associated with
aberrant expression or activity of a polypeptide of the invention
in which a test sample is obtained and the polypeptide or nucleic
acid encoding the polypeptide is detected (e.g., wherein the
presence of the polypeptide or nucleic acid is diagnostic for a
subject that can be administered the agent to treat a disorder
associated with aberrant expression or activity of the
polypeptide).
[0246] The methods of the invention can also be used to detect
genetic lesions or mutations in a gene of the invention, thereby
determining if a subject with the lesioned gene is at risk for a
disorder characterized aberrant expression or activity of a
polypeptide of the invention. In preferred embodiments, the methods
include detecting, in a sample of cells from the subject, the
presence or absence of a genetic lesion or mutation characterized
by at least one of an alteration affecting the integrity of a gene
encoding the polypeptide of the invention, or the mis-expression of
the gene encoding the polypeptide of the invention. For example,
such genetic lesions or mutations can be detected by ascertaining
the existence of at least one of: 1) a deletion of one or more
nucleotides from the gene; 2) an addition of one or more
nucleotides to the gene; 3) a substitution of one or more
nucleotides of the gene; 4) a chromosomal rearrangement of the
gene; 5) an alteration in the level of a messenger RNA transcript
of the gene; 6) an aberrant modification of the gene, such as of
the methylation pattern of the genomic DNA; 7) the presence of a
non-wild type splicing pattern of a messenger RNA transcript of the
gene; 8) a non-wild type level of a the protein encoded by the
gene; 9) an allelic loss of the gene; and 10) an inappropriate
post-translational modification of the protein encoded by the gene.
As described herein, there are a large number of assay techniques
known in the art which can be used for detecting lesions in a
gene.
[0247] In certain embodiments, detection of the lesion involves the
use of a probe/primer in a polymerase chain reaction (PCR) (see,
e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR
or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran et al. (1988) Science 241:1077-1080; and
Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the
latter of which can be particularly useful for detecting point
mutations in a gene (see, e.g., Abravaya et al. (1995) Nucleic
Acids Res. 23:675-682). This method can include the steps of
collecting a sample of cells from a patient, isolating nucleic acid
(e.g., genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or more primers which
specifically hybridize to the selected gene under conditions such
that hybridization and amplification of the gene (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample. It is anticipated that
PCR and/or LCR can be desirable to use as a preliminary
amplification step in conjunction with any of the techniques used
for detecting mutations described herein.
[0248] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh, et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), or any
other nucleic acid amplification method, followed by the detection
of the amplified molecules using techniques well known to those of
skill in the art. These detection schemes are especially useful for
the detection of nucleic acid molecules if such molecules are
present in very low numbers.
[0249] In an alternative embodiment, mutations in a selected gene
from a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
e.g., U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0250] In other embodiments, genetic mutations can be identified by
hybridizing a sample and control nucleic acids, e.g., DNA or RNA,
to high density arrays containing hundreds or thousands of
oligonucleotides probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two-dimensional
arrays containing light-generated DNA probes as described in Cronin
et al., supra. Briefly, a first hybridization array of probes can
be used to scan through long stretches of DNA in a sample and
control to identify base changes between the sequences by making
linear arrays of sequential overlapping probes. This step allows
the identification of point mutations. This step is followed by a
second hybridization array that allows the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[0251] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
selected gene and detect mutations by comparing the sequence of the
sample nucleic acids with the corresponding wild-type (control)
sequence. Examples of sequencing reactions include those based on
techniques developed by Maxim and Gilbert ((1977) Proc. Natl. Acad.
Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA
74:5463). It is also contemplated that any of a variety of
automated sequencing procedures can be used when performing the
diagnostic assays ((1995) Bio/Techniques 19:448), including
sequencing by mass spectrometry (see, e.g., PCT Publication No. WO
94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and
Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
[0252] Other methods for detecting mutations in a selected gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the technique of
mismatch cleavage entails providing heteroduplexes formed by
hybridizing (labeled) RNA or DNA containing the wild-type sequence
with potentially mutant RNA or DNA obtained from a tissue sample.
The double-stranded duplexes are treated with an agent which
cleaves single-stranded regions of the duplex such as which will
exist due to base pair mismatches between the control and sample
strands. RNA/DNA duplexes can be treated with RNase to digest
mismatched regions, and DNA/DNA hybrids can be treated with S1
nuclease to digest mismatched regions.
[0253] In other embodiments, either DNA/DNA or RNA/DNA duplexes can
be treated with hydroxylamine or osmium tetroxide and with
piperidine in order to digest mismatched regions. After digestion
of the mismatched regions, the resulting material is then separated
by size on denaturing polyacrylamide gels to determine the site of
mutation. See, e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci.
USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In
a preferred embodiment, the control DNA or RNA can be labeled for
detection.
[0254] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called DNA mismatch repair enzymes) in
defined systems for detecting and mapping point mutations in cDNAs
obtained from samples of cells. For example, the mutY enzyme of E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase
from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994)
Carcinogenesis 15:1657-1662). According to an exemplary embodiment,
a probe based on a selected sequence, e.g., a wild-type sequence,
is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is treated with a DNA mismatch repair enzyme, and the
cleavage products, if any, can be detected from electrophoresis
protocols or the like. See, e.g., U.S. Pat. No. 5,459,039.
[0255] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in genes. For example,
single strand conformation polymorphism (SSCP) can be used to
detect differences in electrophoretic mobility between mutant and
wild type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci.
USA 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144;
Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded
DNA fragments of sample and control nucleic acids will be denatured
and allowed to re-nature. The secondary structure of
single-stranded nucleic acids varies according to sequence, and the
resulting alteration in electrophoretic mobility enables the
detection of even a single base change. The DNA fragments can be
labeled or detected with labeled probes. The sensitivity of the
assay can be enhanced by using RNA (rather than DNA), in which the
secondary structure is more sensitive to a change in sequence. In a
preferred embodiment, the subject method uses heteroduplex analysis
to separate double stranded heteroduplex molecules on the basis of
changes in electrophoretic mobility (Keen et al. (1991) Trends
Genet. 7:5).
[0256] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 base pairs of high-melting GC-rich DNA by PCR. In
a further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys.
Chem. 265:12753).
[0257] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonicleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers can be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163); Saikit
al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[0258] Alternatively, allele specific amplification technology
which depends on selective PCR amplification can be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification can carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent or reduce
polymerase extension (Prossner (1993) Tibtech 11:238). In addition,
it can be desirable to introduce a novel restriction site in the
region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification can also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci. USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0259] The methods described herein can be performed, for example,
by using pre-packaged diagnostic kits comprising at least one probe
nucleic acid or antibody reagent described herein, which can be
conveniently used, e.g., in clinical settings to diagnose patients
exhibiting symptoms or family history of a disease or illness
involving a gene encoding a polypeptide of the invention.
Furthermore, any cell type or tissue, preferably peripheral blood
leukocytes, in which the polypeptide of the invention is expressed
can be used in the prognostic assays described herein.
[0260] 3. Pharmacogenomics
[0261] Agents, or modulators which have a stimulatory or inhibitory
effect on activity or expression of a polypeptide of the invention
as identified by a screening assay described herein can be
administered to individuals to treat (prophylactically or
therapeutically) disorders associated with aberrant activity of the
polypeptide. In conjunction with such treatment, the
pharmacogenomics (i.e., the study of the relationship between an
individual's genotype and that individual's response to a foreign
compound or drug) of the individual can be considered. Differences
in metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, the
pharmacogenomics of the individual permits the selection of
effective agents (e.g., drugs) for prophylactic or therapeutic
treatments based on a consideration of the individual's genotype.
Such pharmacogenomics can further be used to determine appropriate
dosages and therapeutic regimens. Accordingly, the activity of a
polypeptide of the invention, expression of a nucleic acid of the
invention, or mutation content of a gene of the invention in an
individual can be determined to thereby select appropriate agent(s)
for therapeutic or prophylactic treatment of the individual.
[0262] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, e.g.,
Linder (1997) Clin. Chem. 43(2):254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body are referred to as "altered drug action." Genetic
conditions transmitted as single factors altering the way the body
acts on drugs are referred to as "altered drug metabolism". These
pharmacogenetic conditions can occur either as rare defects or as
polymorphisms. For example, glucose-6-phosphate dehydrogenase
deficiency (G6PD) is a common inherited enzymopathy in which the
main clinical complication is hemolysis after ingestion of oxidant
drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans.
[0263] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C 19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, a PM will show no therapeutic
response, as demonstrated for the analgesic effect of codeine
mediated by its CYP2D6-formed metabolite morphine. The other
extreme are the so called ultra-rapid metabolizers who do not
respond to standard doses. Recently, the molecular basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
[0264] Thus, the activity of a polypeptide of the invention,
expression of a nucleic acid encoding the polypeptide, or mutation
content of a gene encoding the polypeptide in an individual can be
determined to thereby select appropriate agent(s) for therapeutic
or prophylactic treatment of the individual. In addition,
pharmacogenetic studies can be used to apply genotyping of
polymorphic alleles encoding drug-metabolizing enzymes to the
identification of an individual's drug responsiveness phenotype.
This knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
a modulator of activity or expression of the polypeptide, such as a
modulator identified by one of the exemplary screening assays
described herein.
[0265] 4. Monitoring of Effects During Clinical Trials
[0266] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of a polypeptide of the invention
(e.g., the ability to modulate aberrant cell proliferation
chemotaxis, and/or differentiation) can be applied not only in
basic drug screening, but also in clinical trials. For example, the
effectiveness of an agent, as determined by a screening assay as
described herein, to increase gene expression, protein levels or
protein activity, can be monitored in clinical trials of subjects
exhibiting decreased gene expression, protein levels, or protein
activity. Alternatively, the effectiveness of an agent, as
determined by a screening assay, to decrease gene expression,
protein levels or protein activity, can be monitored in clinical
trials of subjects exhibiting increased gene expression, protein
levels, or protein activity. In such clinical trials, expression or
activity of a polypeptide of the invention and preferably, that of
other polypeptide that have been implicated in for example, a
cellular proliferation disorder, can be used as a marker of the
immune responsiveness of a particular cell.
[0267] For example, and not by way of limitation, genes, including
those of the invention, that are modulated in cells by treatment
with an agent (e.g., compound, drug or small molecule) which
modulates activity or expression of a polypeptide of the invention
(e.g., as identified in a screening assay described herein) can be
identified. Thus, to study the effect of agents on cellular
proliferation disorders, for example, in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of
expression of a gene of the invention and other genes implicated in
the disorder. The levels of gene expression (i.e., a gene
expression pattern) can be quantified by Northern blot analysis or
RT-PCR, as described herein, or alternatively by measuring the
amount of protein produced, by one of the methods as described
herein, or by measuring the levels of activity of a gene of the
invention or other genes. In this way, the gene expression pattern
can serve as a marker, indicative of the physiological response of
the cells to the agent. Accordingly, this response state can be
determined before, and at various points during, treatment of the
individual with the agent.
[0268] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of the polypeptide or nucleic acid of the invention in
the pre-administration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level the of the polypeptide or nucleic acid of the invention in
the post-administration samples; (v) comparing the level of the
polypeptide or nucleic acid of the invention in the
pre-administration sample with the level of the polypeptide or
nucleic acid of the invention in the post-administration sample or
samples; and (vi) altering the administration of the agent to the
subject accordingly. For example, increased administration of the
agent can be desirable to increase the expression or activity of
the polypeptide to higher levels than detected, i.e., to increase
the effectiveness of the agent. Alternatively, decreased
administration of the agent can be desirable to decrease expression
or activity of the polypeptide to lower levels than detected, i.e.,
to decrease the effectiveness of the agent.
[0269] C. Methods of Treatment
[0270] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant expression or activity of a polypeptide of the invention.
For example, disorders characterized by aberrant expression or
activity of the polypeptides of the invention include proliferative
disorders such as cancer. Aberrant expression or activity of h-ig6p
can be associated with disorders of the pancreas, including for
example, metabolic disorders such as disorders of carbohydrate
metabolism (e.g., diabetes, hyperinsulinemia, and obesity). By
inhibiting h-ig6p expression or activity, insulin secretion can be
enhanced, and disorders such as diabetes or obesity can be
alleviated, inhibited, or prevented. By enhancing h-ig6p expression
or activity, insulin secretion can be inhibited, and disorders such
as hyperinsulinemia can be alleviated, inhibited, or prevented.
Enhancing or inhibiting h-ig6p expression can also affect the
sensitivity of cell signaling mechanisms for intracellular and
extracellular glucose/G6P, and disorders associated with aberrant
cell signaling relating to these levels can be alleviated,
inhibited, or prevented.
[0271] 1. Prophylactic Methods
[0272] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant expression or activity of a polypeptide of the invention,
by administering to the subject an agent which modulates expression
or at least one activity of the polypeptide. Subjects at risk for a
disease which is caused or contributed to by aberrant expression or
activity of a polypeptide of the invention can be identified by,
for example, any or a combination of diagnostic or prognostic
assays as described herein. Administration of a prophylactic agent
can occur prior to the manifestation of symptoms characteristic of
the aberrance, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
aberrance, for example, an agonist or antagonist agent can be used
for treating the subject. The appropriate agent can be determined
based on screening assays described herein.
[0273] 2. Therapeutic Methods
[0274] Another aspect of the invention pertains to methods of
modulating expression or activity of a polypeptide of the invention
for therapeutic purposes. The modulatory method of the invention
involves contacting a cell with an agent that modulates one or more
of the activities of the polypeptide. An agent that modulates
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring cognate ligand of the
polypeptide, a peptide, a peptidomimetic, or other small molecule.
In one embodiment, the agent stimulates one or more of the
biological activities of the polypeptide. Examples of such
stimulatory agents include the active polypeptide of the invention
and a nucleic acid molecule encoding the polypeptide of the
invention that has been introduced into the cell. In another
embodiment, the agent inhibits one or more of the biological
activities of the polypeptide of the invention. Examples of such
inhibitory agents include antisense nucleic acid molecules and
antibodies. These modulatory methods can be performed in vitro
(e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject). As such, the
present invention provides methods of treating an individual
afflicted with a disease or disorder characterized by aberrant
expression or activity of a polypeptide of the invention. In one
embodiment, the method involves administering an agent (e.g., an
agent identified by a screening assay described herein), or
combination of agents that modulates (e.g., enhances or inhibits)
expression or activity. In another embodiment, the method involves
administering a polypeptide of the invention or a nucleic acid
molecule of the invention as therapy to compensate for reduced or
aberrant expression or activity of the polypeptide.
[0275] Stimulation of activity is desirable in situations in which
activity or expression is abnormally low or depressed and/or in
which increased activity is likely to have a beneficial effect.
Conversely, inhibition of activity is desirable in situations in
which activity or expression is abnormally high or enhanced and/or
in which decreased activity is likely to have a beneficial
effect.
[0276] The contents of all references, patents and published patent
applications cited throughout this application are hereby
incorporated by reference.
[0277] Equivalents
[0278] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
27 1 1138 DNA Homo sapiens 1 aattcgccct tcagctccaa ttgctctatg
tttagaattg cctctttttc aagatggatt 60 tccttcacag gaatggagtg
ctcataattc agcatttgca gaaggactac cgagcttact 120 acacttttct
aaattttatg tccaatgttg gagaccccag gaatatcttt ttcatttatt 180
ttccactttg ttttcaattt aatcagacag ttggaaccaa gatgatatgg gtagcagtca
240 ttggggattg gttaaatctt atatttaaat ggatattatt tggtcatcga
ccttactggt 300 gggtccaaga aactcagatt tacccaaatc actcaagtcc
atgccttgaa cagttcccta 360 ctacatgtga aacaggtcca ggaagtccat
ctggccatgc aatgggcgca tcctgtgtct 420 ggtatgtcat ggtaaccgct
gccctgagcc acactgtctg tgggatggat aagttctcta 480 tcactctgca
cagactgacc tggtcatttc tttggagtgt tttttggttg attcaaatca 540
gtgtctgcat ctccagagta ttcatagcaa cacattttcc tcatcaagtt attcttggag
600 taattggtgg catgctggtg gcagaggcct ttgaacacac tccaggcatc
caaacggcca 660 gtctgggcac atacctgaag accaacctct ttctcttcct
gtttgcagtt ggcttttacc 720 tgcttcttag ggtgctcaac attgacctgc
tgtggtccgt gcccatagcc aaaaagtggt 780 gtgctaaccc cgactggatc
cacattgaca ccacgccttt tgctggactc gtgagaaacc 840 ttggggtcct
ctttggcttg ggctttgcaa tcaactcaga gatgttcctc ctgagctgcc 900
gagggggaaa taactacaca ctgagcttcc ggttgctctg tgccttgacc tcattgacaa
960 tactgcagct ctaccatttc ctccagatcc cgactcacga agagcattta
ttttatgtgc 1020 tgtctttttg taaaagtgca tccattcccc taactgtggt
tgctttcatt ccctactctg 1080 ttcatatgtt aatgaaacaa agcggaaaga
agagtcagta gaaaaaaaaa aaaaaaaa 1138 2 1065 DNA Homo sapiens 2
atggatttcc ttcacaggaa tggagtgctc ataattcagc atttgcagaa ggactaccga
60 gcttactaca cttttctaaa ttttatgtcc aatgttggag accccaggaa
tatctttttc 120 atttattttc cactttgttt tcaatttaat cagacagttg
gaaccaagat gatatgggta 180 gcagtcattg gggattggtt aaatcttata
tttaaatgga tattatttgg tcatcgacct 240 tactggtggg tccaagaaac
tcagatttac ccaaatcact caagtccatg ccttgaacag 300 ttccctacta
catgtgaaac aggtccagga agtccatctg gccatgcaat gggcgcatcc 360
tgtgtctggt atgtcatggt aaccgctgcc ctgagccaca ctgtctgtgg gatggataag
420 ttctctatca ctctgcacag actgacctgg tcatttcttt ggagtgtttt
ttggttgatt 480 caaatcagtg tctgcatctc cagagtattc atagcaacac
attttcctca tcaagttatt 540 cttggagtaa ttggtggcat gctggtggca
gaggcctttg aacacactcc aggcatccaa 600 acggccagtc tgggcacata
cctgaagacc aacctctttc tcttcctgtt tgcagttggc 660 ttttacctgc
ttcttagggt gctcaacatt gacctgctgt ggtccgtgcc catagccaaa 720
aagtggtgtg ctaaccccga ctggatccac attgacacca cgccttttgc tggactcgtg
780 agaaaccttg gggtcctctt tggcttgggc tttgcaatca actcagagat
gttcctcctg 840 agctgccgag ggggaaataa ctacacactg agcttccggt
tgctctgtgc cttgacctca 900 ttgacaatac tgcagctcta ccatttcctc
cagatcccga ctcacgaaga gcatttattt 960 tatgtgctgt ctttttgtaa
aagtgcatcc attcccctaa ctgtggttgc tttcattccc 1020 tactctgttc
atatgttaat gaaacaaagc ggaaagaaga gtcag 1065 3 355 PRT Homo sapiens
3 Met Asp Phe Leu His Arg Asn Gly Val Leu Ile Ile Gln His Leu Gln 1
5 10 15 Lys Asp Tyr Arg Ala Tyr Tyr Thr Phe Leu Asn Phe Met Ser Asn
Val 20 25 30 Gly Asp Pro Arg Asn Ile Phe Phe Ile Tyr Phe Pro Leu
Cys Phe Gln 35 40 45 Phe Asn Gln Thr Val Gly Thr Lys Met Ile Trp
Val Ala Val Ile Gly 50 55 60 Asp Trp Leu Asn Leu Ile Phe Lys Trp
Ile Leu Phe Gly His Arg Pro 65 70 75 80 Tyr Trp Trp Val Gln Glu Thr
Gln Ile Tyr Pro Asn His Ser Ser Pro 85 90 95 Cys Leu Glu Gln Phe
Pro Thr Thr Cys Glu Thr Gly Pro Gly Ser Pro 100 105 110 Ser Gly His
Ala Met Gly Ala Ser Cys Val Trp Tyr Val Met Val Thr 115 120 125 Ala
Ala Leu Ser His Thr Val Cys Gly Met Asp Lys Phe Ser Ile Thr 130 135
140 Leu His Arg Leu Thr Trp Ser Phe Leu Trp Ser Val Phe Trp Leu Ile
145 150 155 160 Gln Ile Ser Val Cys Ile Ser Arg Val Phe Ile Ala Thr
His Phe Pro 165 170 175 His Gln Val Ile Leu Gly Val Ile Gly Gly Met
Leu Val Ala Glu Ala 180 185 190 Phe Glu His Thr Pro Gly Ile Gln Thr
Ala Ser Leu Gly Thr Tyr Leu 195 200 205 Lys Thr Asn Leu Phe Leu Phe
Leu Phe Ala Val Gly Phe Tyr Leu Leu 210 215 220 Leu Arg Val Leu Asn
Ile Asp Leu Leu Trp Ser Val Pro Ile Ala Lys 225 230 235 240 Lys Trp
Cys Ala Asn Pro Asp Trp Ile His Ile Asp Thr Thr Pro Phe 245 250 255
Ala Gly Leu Val Arg Asn Leu Gly Val Leu Phe Gly Leu Gly Phe Ala 260
265 270 Ile Asn Ser Glu Met Phe Leu Leu Ser Cys Arg Gly Gly Asn Asn
Tyr 275 280 285 Thr Leu Ser Phe Arg Leu Leu Cys Ala Leu Thr Ser Leu
Thr Ile Leu 290 295 300 Gln Leu Tyr His Phe Leu Gln Ile Pro Thr His
Glu Glu His Leu Phe 305 310 315 320 Tyr Val Leu Ser Phe Cys Lys Ser
Ala Ser Ile Pro Leu Thr Val Val 325 330 335 Ala Phe Ile Pro Tyr Ser
Val His Met Leu Met Lys Gln Ser Gly Lys 340 345 350 Lys Ser Gln 355
4 355 PRT Mus musculus 4 Met Asp Phe Leu His Arg Ser Gly Val Leu
Ile Ile His His Leu Gln 1 5 10 15 Glu Asp Tyr Arg Thr Tyr Tyr Gly
Phe Leu Asn Phe Met Ser Asn Val 20 25 30 Gly Asp Pro Arg Asn Ile
Phe Ser Ile Tyr Phe Pro Leu Trp Phe Gln 35 40 45 Leu Asn Gln Asn
Val Gly Thr Lys Met Ile Trp Val Ala Val Ile Gly 50 55 60 Asp Trp
Phe Asn Leu Ile Phe Lys Trp Ile Leu Phe Gly His Arg Pro 65 70 75 80
Tyr Trp Trp Ile Gln Glu Thr Glu Ile Tyr Pro Asn His Ser Ser Pro 85
90 95 Cys Leu Glu Gln Phe Pro Thr Thr Cys Glu Thr Gly Pro Gly Ser
Pro 100 105 110 Ser Gly His Ala Met Gly Ser Ser Cys Val Trp Tyr Val
Met Val Thr 115 120 125 Ala Ala Leu Ser Tyr Thr Ile Ser Arg Met Glu
Glu Ser Ser Val Thr 130 135 140 Leu His Arg Leu Thr Trp Ser Phe Leu
Trp Ser Val Phe Trp Leu Ile 145 150 155 160 Gln Ile Ser Val Cys Ile
Ser Arg Val Phe Ile Ala Thr His Phe Pro 165 170 175 His Gln Val Ile
Leu Gly Val Ile Gly Gly Met Leu Val Ala Glu Ala 180 185 190 Phe Glu
His Thr Pro Gly Val His Met Ala Ser Leu Ser Val Tyr Leu 195 200 205
Lys Thr Asn Val Phe Leu Phe Leu Phe Ala Leu Gly Phe Tyr Leu Leu 210
215 220 Leu Arg Leu Phe Gly Ile Asp Leu Leu Trp Ser Val Pro Ile Ala
Lys 225 230 235 240 Lys Trp Cys Ala Asn Pro Asp Trp Ile His Ile Asp
Ser Thr Pro Phe 245 250 255 Ala Gly Leu Val Arg Asn Leu Gly Val Leu
Phe Gly Leu Gly Phe Ala 260 265 270 Ile Asn Ser Glu Met Phe Leu Arg
Ser Cys Gln Gly Glu Asn Gly Thr 275 280 285 Lys Pro Ser Phe Arg Leu
Leu Cys Ala Leu Thr Ser Leu Thr Thr Met 290 295 300 Gln Leu Tyr Arg
Phe Ile Lys Ile Pro Thr His Ala Glu Pro Leu Phe 305 310 315 320 Tyr
Leu Leu Ser Phe Cys Lys Ser Ala Ser Ile Pro Leu Met Val Val 325 330
335 Ala Leu Ile Pro Tyr Cys Val His Met Leu Met Arg Pro Gly Asp Lys
340 345 350 Lys Thr Lys 355 5 5 000 6 6 000 7 7 000 8 8 000 9 9 000
10 10 000 11 11 000 12 12 000 13 13 000 14 14 000 15 15 000 16 16
000 17 17 000 18 18 000 19 19 000 20 20 000 21 21 000 22 1901 DNA
Mus musculus 22 tagagacagt gggacacagg gccctgcagt tccacctgct
tcatgcttag acctgcatca 60 agatggattt ccttcatagg agtggagtgc
ttattattca tcatctgcag gaggactacc 120 ggacttacta tggttttcta
aattttatgt ccaatgttgg agacccccga aatatctttt 180 ctatttactt
cccactttgg tttcagttga atcagaatgt tggaaccaag atgatctggg 240
tagcggtcat aggggactgg ttcaatctca tatttaaatg gatattgttt ggccatcgtc
300 cttactggtg gatacaagaa actgagattt atccaaatca ttcaagccca
tgtcttgagc 360 agtttcctac tacgtgtgaa acaggcccag gaagtccatc
tggccacgca atgggctcat 420 cgtgcgtctg gtatgtcatg gtaacagctg
ccctaagcta caccatcagc cggatggagg 480 agtcctctgt cactctgcac
agactgacct ggtcctttct gtggagtgtt ttctggttga 540 ttcaaatcag
cgtctgcatc tcaagagtat tcatagccac acatttcccc catcaggtca 600
ttcttggagt gattggtggg atgctagtag ccgaggcctt tgaacacact ccaggagtcc
660 acatggccag cttgagtgtg tacctgaaga ccaacgtctt cctcttcctg
tttgccctcg 720 gcttttacct gcttctccga ctgttcggta ttgacctgct
gtggtccgtg cccatcgcca 780 aaaagtggtg tgccaaccca gactggatcc
acattgacag cacgcctttt gctggactcg 840 tgagaaacct cggggtcctc
tttggcttgg gtttcgccat caactcagaa atgttccttc 900 ggagctgcca
gggagaaaat ggcaccaagc cgagcttccg cttgctctgt gctctgacct 960
cactgaccac aatgcaactt tatcgcttca tcaagatccc gactcacgcg gaacctttat
1020 tttacctgtt gtctttctgt aaaagtgcgt ccatccccct gatggtggtg
gctctaattc 1080 cctactgtgt acatatgtta atgagacccg gtgacaagaa
gactaaatag agctgcagtg 1140 ccctgtggtc tgaggatcac ctactttctg
ttttcctcaa tagagccaca gcacagagac 1200 tgggagcgtc tctacagagg
tcacaccatg atgaccaaag gtcctgctcc acccacagac 1260 atgtttagtc
tgctttccaa gtggcattta aaaaataaca gtatttaacc agaaagtcca 1320
tattttcttg acaaaactga caatacggta acatatgaga gatggtataa cccatgtaaa
1380 gacagttgac aggggctgga tgcttacatt ccagttagca gaaagactcc
ttctaatcat 1440 agtatttagc agtcaacaaa acccccagga gctgatgttt
ctatcatctt aaagtctggc 1500 tacttcaggc tcctgtggac cacttagaag
tgaccacggt ctacttttac ttttaggagt 1560 caattctttc aaaattctca
tgtatcagat aaggaaatag aggtttgttc agatcaagta 1620 acttgactgt
aatagtgcag ggttgaaacc agagttggaa cacaaggctt ctgatacata 1680
tatctctata agaatgcttt ctttctttct ttttagggag ttaaaaaaaa agagcaaatg
1740 catgtattta aaatctatgt ttgccatcta aaacacccat cttttcagaa
atggcattgg 1800 aatgctacat tctgcttgac ttatgctcag agtacagtgt
cttttccagg ctagcaatgg 1860 ctgtatatat ttcaataaac gctgctgaaa
acaacccact g 1901 23 355 PRT Mus musculus 23 Met Asp Phe Leu His
Arg Ser Gly Val Leu Ile Ile His His Leu Gln 1 5 10 15 Glu Asp Tyr
Arg Thr Tyr Tyr Gly Phe Leu Asn Phe Met Ser Asn Val 20 25 30 Gly
Asp Pro Arg Asn Ile Phe Ser Ile Tyr Phe Pro Leu Trp Phe Gln 35 40
45 Leu Asn Gln Asn Val Gly Thr Lys Met Ile Trp Val Ala Val Ile Gly
50 55 60 Asp Trp Phe Asn Leu Ile Phe Lys Trp Ile Leu Phe Gly His
Arg Pro 65 70 75 80 Tyr Trp Trp Ile Gln Glu Thr Glu Ile Tyr Pro Asn
His Ser Ser Pro 85 90 95 Cys Leu Glu Gln Phe Pro Thr Thr Cys Glu
Thr Gly Pro Gly Ser Pro 100 105 110 Ser Gly His Ala Met Gly Ser Ser
Cys Val Trp Tyr Val Met Val Thr 115 120 125 Ala Ala Leu Ser Tyr Thr
Ile Ser Arg Met Glu Glu Ser Ser Val Thr 130 135 140 Leu His Arg Leu
Thr Trp Ser Phe Leu Trp Ser Val Phe Trp Leu Ile 145 150 155 160 Gln
Ile Ser Val Cys Ile Ser Arg Val Phe Ile Ala Thr His Phe Pro 165 170
175 His Gln Val Ile Leu Gly Val Ile Gly Gly Met Leu Val Ala Glu Ala
180 185 190 Phe Glu His Thr Pro Gly Val His Met Ala Ser Leu Ser Val
Tyr Leu 195 200 205 Lys Thr Asn Val Phe Leu Phe Leu Phe Ala Leu Gly
Phe Tyr Leu Leu 210 215 220 Leu Arg Leu Phe Gly Ile Asp Leu Leu Trp
Ser Val Pro Ile Ala Lys 225 230 235 240 Lys Trp Cys Ala Asn Pro Asp
Trp Ile His Ile Asp Ser Thr Pro Phe 245 250 255 Ala Gly Leu Val Arg
Asn Leu Gly Val Leu Phe Gly Leu Gly Phe Ala 260 265 270 Ile Asn Ser
Glu Met Phe Leu Arg Ser Cys Gln Gly Glu Asn Gly Thr 275 280 285 Lys
Pro Ser Phe Arg Leu Leu Cys Ala Leu Thr Ser Leu Thr Thr Met 290 295
300 Gln Leu Tyr Arg Phe Ile Lys Ile Pro Thr His Ala Glu Pro Leu Phe
305 310 315 320 Tyr Leu Leu Ser Phe Cys Lys Ser Ala Ser Ile Pro Leu
Met Val Val 325 330 335 Ala Leu Ile Pro Tyr Cys Val His Met Leu Met
Arg Pro Gly Asp Lys 340 345 350 Lys Thr Lys 355 24 352 PRT
Haplochromis nubilis 24 Met Asp Leu Leu His Ser Trp Gly Val Glu Leu
Ala Val Tyr Leu Gln 1 5 10 15 Thr Arg Tyr Gly Lys Tyr Glu Gly Leu
Phe Asp Leu Ala Ser Thr Val 20 25 30 Ala Asp Leu His Thr Thr Phe
Phe Trp Leu Phe Pro Ile Trp Phe His 35 40 45 Leu Arg Arg Asp Thr
Ala Leu Arg Leu Ile Trp Val Ala Val Ile Gly 50 55 60 Asp Trp Leu
Asn Leu Val Leu Lys Trp Val Leu Phe Gly Glu Arg Pro 65 70 75 80 Tyr
Trp Trp Val His Glu Thr Lys Phe Tyr Gly Ala Gly Pro Ala Pro 85 90
95 Ser Leu Gln Gln Phe Pro Ile Thr Cys Glu Thr Gly Pro Gly Ser Pro
100 105 110 Ser Gly His Ala Met Gly Ala Ala Gly Val Trp Tyr Val Met
Val Thr 115 120 125 Ala Leu Leu Ser Ile Ala Arg Glu Lys Gln Cys Pro
Pro Leu Leu Tyr 130 135 140 Arg Phe Leu Tyr Ile Gly Leu Trp Met Leu
Met Gly Leu Val Glu Leu 145 150 155 160 Val Val Cys Ile Ser Arg Val
Tyr Met Ala Ala His Phe Pro His Gln 165 170 175 Val Ile Ala Gly Ile
Ile Thr Gly Thr Leu Val Ala Glu Val Val Ser 180 185 190 Lys Glu Lys
Trp Ile Tyr Ser Ala Ser Leu Lys Lys Tyr Phe Leu Ile 195 200 205 Thr
Leu Phe Leu Thr Ser Phe Ala Val Gly Phe Tyr Val Leu Leu Lys 210 215
220 Ala Leu Asp Val Asp Leu Leu Trp Thr Met Glu Lys Ala Gln Lys Trp
225 230 235 240 Cys Ile Arg Pro Glu Trp Val His Leu Asp Ser Ala Pro
Phe Ala Ser 245 250 255 Leu Leu Arg Asn Met Gly Ser Leu Phe Gly Leu
Gly Leu Gly Leu His 260 265 270 Ser Pro Phe Tyr Lys Thr Thr Lys Met
Arg Ile Met Ser Ala Pro Leu 275 280 285 Arg Ile Gly Cys Ile Val Ile
Ser Val Ser Leu Leu His Leu Leu Asp 290 295 300 Gly Trp Thr Phe Ser
Pro Glu Asn His Met Thr Phe Tyr Ala Leu Ser 305 310 315 320 Phe Gly
Lys Ser Ala Val Ala Leu Leu Ile Pro Thr Thr Leu Val Pro 325 330 335
Trp Ala Leu Ser Lys Ile Tyr Pro Val Lys Thr Glu Gly Lys Asn Leu 340
345 350 25 357 PRT Mus sp. 25 Met Glu Glu Gly Met Asn Ile Leu His
Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 Arg Tyr Leu Gln Val Asn Tyr
Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala
Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe
His Leu Lys Glu Thr Val Gly Ile Asn Leu Leu Trp Val 50 55 60 Ala
Val Val Gly Asp Trp Phe Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70
75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser
Asn 85 90 95 Ser Ser Val Pro Ile Ile Lys Gln Phe Pro Val Thr Cys
Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Ala
Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ala Ile
Phe Arg Gly Lys Lys Lys Pro 130 135 140 Thr Tyr Gly Phe Arg Cys Leu
Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu
Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro
His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190
Glu Thr Phe Ser His Ile Arg Gly Ile Tyr Asn Ala Ser Leu Arg Lys 195
200 205 Tyr Cys Leu Ile Thr Ile Phe Leu Phe Gly Phe Ala Leu Gly Phe
Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr
Leu Glu Lys 225 230 235 240 Ala Lys Arg Trp Cys Glu Arg Pro Glu Trp
Val His Leu Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Phe Lys Asn
Leu Gly Thr Leu Leu Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser
Met Tyr Arg Lys Ser Cys Lys Gly Glu Leu 275 280 285 Ser Lys Ser Phe
Pro Phe Arg Phe Ala Cys Ile Val Ala Ser Leu Val 290 295 300 Leu
Leu
His Leu Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315
320 Ile Phe Tyr Ile Leu Ser Phe Cys Lys Ser Ala Thr Val Pro Phe Ala
325 330 335 Ser Val Ser Leu Ile Pro Tyr Cys Leu Ala Arg Ile Leu Gly
Gln Thr 340 345 350 His Lys Lys Ser Leu 355 26 357 PRT Canis
familiaris 26 Met Glu Lys Gly Met Asp Val Leu His Asp Phe Gly Ile
Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln
Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn
Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Arg Glu
Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp
Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg
Pro Tyr Trp Trp Val Met Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr
Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105
110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr
115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Arg Gly Arg Lys
Arg Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Ile Leu Leu Trp
Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser
Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala
Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Arg His
Ile Gln Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu
Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu
Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230
235 240 Ala Arg Arg Trp Cys Glu Arg Pro Glu Trp Val His Ile Asp Thr
Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Val Gly Thr Leu Phe
Gly Leu Gly 260 265 270 Val Thr Leu Asn Ser Ser Met Tyr Arg Glu Ser
Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Phe Pro Phe Arg Leu Ser
Cys Ile Val Val Ser Leu Ile 290 295 300 Leu Leu His Leu Phe Asp Ser
Leu Lys Pro Pro Ser Gln Thr Glu Leu 305 310 315 320 Ile Phe Tyr Thr
Leu Ser Phe Cys Lys Ser Ala Ala Val Pro Leu Ala 325 330 335 Ser Val
Ser Leu Ile Pro Tyr Cys Leu Ala Arg Val Phe Asp Gln Pro 340 345 350
Asp Lys Lys Ser Leu 355 27 357 PRT Homo sapiens 27 Met Glu Glu Gly
Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr
Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30
Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35
40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp
Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp
Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr
Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe
Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His
Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser
Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg
Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160
Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165
170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val
Thr 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser
Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe
Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp
Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu
Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser
Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala
Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285
Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290
295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu
Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val
Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala
Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355
* * * * *
References