U.S. patent number RE39,062 [Application Number 09/717,095] was granted by the patent office on 2006-04-11 for ingap protein involved in pancreatic islet neogenesis.
This patent grant is currently assigned to Eastern Virginia Medical School of the Medical College of Hampton Roads, McGill Unviersity. Invention is credited to Jean T. S. Duguid, William P. Duguid, Gary L. Pittenger, Ronit Rafaeloff-Phail, Lawrence Rosenberg, Aaron I. Vinik.
United States Patent |
RE39,062 |
Vinik , et al. |
April 11, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
INGAP protein involved in pancreatic islet neogenesis
Abstract
Cellophane wrapping (CW) of hamster pancreas induces
proliferation of duct epithelial cells followed by endocrine cell
differentiation and islet neogenesis. Using the mRNA differential
display technique a cDNA clone expressed in cellophane wrapped but
not in control pancreata was identified. Using this cDNA as a
probe, a cDNA library was screened and a gene not previously
described was identified and named INGAP.
Inventors: |
Vinik; Aaron I. (Norfolk,
VA), Pittenger; Gary L. (Virginia Beach, VA),
Rafaeloff-Phail; Ronit (Indianapolis, IN), Rosenberg;
Lawrence (Montreal, CA), Duguid; Jean T. S.
(Quebec, CA), Duguid; William P. (Montreal,
CA) |
Assignee: |
McGill Unviersity
(CA)
Eastern Virginia Medical School of the Medical College of
Hampton Roads (Norfolk, VA)
|
Family
ID: |
26675411 |
Appl.
No.: |
09/717,095 |
Filed: |
November 22, 2000 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
08401530 |
Feb 22, 1995 |
5834590 |
|
|
|
60006271 |
Nov 7, 1995 |
|
|
|
Reissue of: |
08709662 |
Sep 9, 1996 |
05840531 |
Nov 24, 1998 |
|
|
Current U.S.
Class: |
435/69.1;
435/320.1; 435/325; 530/350; 536/23.5; 536/24.1; 536/23.1; 435/7.1;
435/252.3; 435/20 |
Current CPC
Class: |
A61P
1/00 (20180101); C07K 14/474 (20130101); C07K
14/47 (20130101); A61P 3/08 (20180101); C07K
14/4733 (20130101); A61P 43/00 (20180101); C07K
2319/00 (20130101); A61K 38/00 (20130101); A01K
2217/05 (20130101) |
Current International
Class: |
C12P
21/06 (20060101) |
Field of
Search: |
;435/69.1,7.1,320.1,325,252.3,6 ;536/23.1,23.5,24.1 ;530/350 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4965188 |
October 1990 |
Mullis et al. |
5834214 |
November 1998 |
Iovanna et al. |
|
Foreign Patent Documents
|
|
|
|
|
|
|
94/15218 |
|
Jul 1994 |
|
WO |
|
WO96/26215 |
|
Aug 1996 |
|
WO |
|
Other References
Lu et al. (1996) Nature, vol. 380, pp. 544-547. cited by examiner
.
Dagorn et al. (1995) Accession No. Q69201, GenBank Database. cited
by examiner .
Hillier et al. (1996) Accession No. AA034395. EST database. cited
by examiner .
Hillier et al. (1995) Accession No. H20422, EST database. cited by
examiner .
Stein et al., "Antisense Oligonucleotides as Therapeutic Agents--Is
the Bullet Really Magical?", Science 261:1004-1012 (1993). cited by
examiner .
Bradley et al., "BoiTechnology. Modifying the Mouse", Design and
Desire 10:534-539 (1992). cited by examiner .
Miller et al., "Human Gene Therapy Comes of Age", Nature
357:455-460 (1992). cited by examiner .
Watanabe et al., "Pancreatic Beta-Cell Replication and Amelioration
of Surgical Diabetes by Reg Protein", Proc. Natl. Acad. Sci. USA
91:3589-3592 (1994). cited by examiner .
Liang et al., "Distribution and Cloning of Eukaryotic mRNAs by
Means of Differential Display: Refinements and Optimization",
Nucleic Acids Research 21(14):3269-3275 (1993). cited by examiner
.
Rosenberg et al., "Reversal of Diabetes by the Induction of Islet
Cell Neogenesis", Transplantation Proceedings 24(3):1027-1028
(1992). cited by examiner .
Rouquier et al., "Rat Pancreatic Stone Protein Messenger RNA", J.
Biol. Chem., 266(2):786-791 (1991). cited by examiner .
Lasserre et al., "A Novel Gene (HIP) Activated in Human Primary
Liver Cancer", Cancer Research 52:5089-5095 (1992). cited by
examiner .
Terazono et al., "A Novel Gene Activated in Regenerating Islets",
J. Biol. Chem., 263(5):211-2114 (1988). cited by examiner .
Vinik et al., "Factors Controlling Pancreatic Islet Neogenesis",
Yale Journal of Biology and Medicine 65: pp. 471-491 (1992). cited
by examiner .
Orelle et al., "Human Pancreatitis-associated Protein" J. Clin.
Invest. 90:2284-2291 (1992). cited by examiner .
Pittenger et al., "The Partial Isolation and Characterization of
Iltropin, a Novel Islet-Specific Growth Factor", Adv. Exp. Med.
Biol. 321:123-130 (1992) ABSTRACT. cited by other .
Rosenberg, et al., "Trophic Stimulatin of th eDuctular-Islet Cell
Axis: A New Approach to the Treatment of Diabetes", Pancreatic
Islet Cell Regeneration and Growth, edited by A. I. Vinik, Plenum
Press, New York, 1992. cited by other .
International Search Report dated Jun. 23, 2000, Appl. No. EP
96905368, pp. 1-3. cited by other .
Dusetti et al., Molecular Cloning, Genomic Organization, and
Chromosomal Localization of the Human Pancreatitis-Associated
Protein (PAP) Gene, Genomics, vol. 19, Jan. 1994, pp. 108-114.
cited by other .
Vinik, M.D. et al., "Factors Controlling Pancreatic Islet
Neogenesis", Tumor Biology, vol. 14, 1993. pp. 184-200. cited by
other.
|
Primary Examiner: Weber; Jon
Assistant Examiner: Robinson; Hope A.
Attorney, Agent or Firm: Banner & Witcoff, Ltd.
Parent Case Text
This application is a continuation-in-part of U.S. Ser. No.
08/401,530, filed Feb. 22, 1995, and claims the benefit of U.S.
Ser. No. 60/006,271, filed Nov. 7, 1995.
Claims
We claim:
1. An isolated DNA molecule encoding a mammalian islet cell
neogenesis associated protein (INGAP) .[.protein.]. , wherein
.[.the.]. INGAP .[.protein.]. has the amino acid sequence shown in
SEQ ID NO: 2.
2. The DNA molecule of claim 1 .[.wherein the INGAP protein.].
.Iadd.which .Iaddend.has the nucleotide sequence shown in SEQ ID
NO: 1.
3. A vector comprising the DNA of claim 1.
4. The vector of claim 3 further comprising expression control
sequences, whereby said DNA is expressed in a host cell.
5. The vector of claim 4 which comprises .[.a.]. .Iadd.an Epstein
Barr Nuclear Antigen-Histidine (.Iaddend.EBNA His.Iadd.)
.Iaddend.plasmid.
6. .[.A.]. .Iadd.An isolated .Iaddend.host cell transformed with
the DNA of claim 1.
7. .[.A.]. .Iadd.An isolated .Iaddend.host cell transformed with
the vector of claim 3.
8. The host cell of claim 6 which is a .[.cos7,African.].
.Iadd.cos7, African .Iaddend.Green Monkey kidney cell.
9. A nucleotide probe comprising at least 30 contiguous nucleotides
of a sequence encoding a mammalian islet cell neogenesis associated
protein (INGAP), wherein said protein has the sequence shown in SEQ
ID NO: 2.
10. The nucleotide probe of claim 9 wherein the .Iadd.nucleotide
sequence encoding a .Iaddend.mammalian INGAP .[.gene.]. has the
sequence shown in SEQ ID NO: 1.
11. The nucleotide probe of claim 9 wherein said probe is labeled
with a detectable moiety.
12. .[.A.]. .Iadd.An isolated .Iaddend.DNA molecule comprising at
least 30 contiguous nucleotides of a sequence encoding a mammalian
islet cell neogenesis associated protein (INGAP), wherein said
protein has the sequence shown in SEQ ID NO: 2.Iadd., wherein said
DNA molecule encodes a polypeptide which stimulates islet cell
neogenesis.Iaddend..
13. The DNA molecule of claim 12 wherein the .Iadd.sequence
encoding the .Iaddend.mammalian INGAP .[.gene.]. has the sequence
shown in SEQ ID NO: 1.
14. The DNA molecule of claim 12 wherein said molecule is labeled
with a detectable moiety.
15. A method of producing a mammalian INGAP .[.protein.]. ,
comprising the steps of: providing a host cell according to claim
6; culturing the host cell in a nutrient medium so that the INGAP
.[.protein.]. is expressed; and harvesting the INGAP .[.protein.].
from the host cells or the nutrient medium.
16. A method of producing a mammalian INGAP .[.protein.]. ,
comprising the steps of: providing a host cell comprising the DNA
molecule of claim 1; culturing the host cell in a nutrient medium
so that the mammalian INGAP .[.protein.]. is expressed; and
harvesting the mammalian INGAP .[.protein.]. from the host cells or
the nutrient medium.
17. An antisense construct of a mammalian islet cell neogenesis
associated protein (INGAP) gene comprising: a promoter, a
terminator, and a nucleotide sequence .[.consisting of a mammalian
INGAP gene, wherein the gene.]. .Iadd.which .Iaddend.encodes
.Iadd.all or a portion of .Iaddend.a protein as shown in SEQ ID NO:
2, said nucleotide sequence being between said promoter and said
terminator, said nucleotide sequence being inverted with respect to
said promoter, whereby upon expression from said promoter an mRNA
complementary to native mammalian INGAP mRNA is produced.Iadd.,
wherein said mRNA complementary to native mammalian INGAP mRNA
prevents translation of the native mammalian INGAP
mRNA.Iaddend..
18. The DNA molecule of claim 1 wherein the INGAP .[.protein.]. is
from human.
19. The DNA molecule of claim 1 which comprises nucleotides 4 to
268 and 389 to 629 of SEQ ID NO: 1.
.Iadd.20. A vector comprising the DNA of claim 2..Iaddend.
.Iadd.21. An isolated host cell transformed with the vector of
claim 20..Iaddend.
.Iadd.22. The DNA molecule of claim 1 which is a cDNA
molecule..Iaddend.
.Iadd.23. The DNA molecule of claim 12 which is a cDNA
molecule..Iaddend.
.Iadd.24. The DNA molecule of claim 12 which encodes a portion of
INGAP, wherein said DNA molecule encodes a polypeptide which
stimulates islet cell neogenesis..Iaddend.
Description
BACKGROUND OF THE INVENTION
Pancreatic islets of Langerhans are the only organ of insulin
production in the body. However, they have a limited capacity for
regeneration. This limited regeneration capacity predisposes
mammals to develop diabetes mellitus. Thus there is a need in the
art of endocrinology for products which can stimulate the
regeneration of islets of Langerhans to prevent or ameliorate the
symptoms of diabetes mellitus.
One model of pancreatic islet cell regeneration involves
cellophane-wrapping of the pancreas in the Syrian golden hamster
(1). Wrapping of the pancreas induces the formation of new
endocrine cells which appear to arise from duct epithelium (2-4).
There is a need in the art to identify and isolate the factor(s)
which is responsible for islet cell regeneration.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a preparation of a
mammalian protein or polypeptide portions thereof involved in islet
cell neogenesis.
It is another object of the invention to provide a DNA molecule
encoding a mammalian protein involved in islet cell neogenesis.
It is yet another object of the invention to provide a preparation
of a mammalian INGAP (islet neogenesis associated protein)
protein.
It is still another object of the invention to provide nucleotide
probes for detecting mammalian genes involved in islet cell
neogenesis.
It is an object of the invention to provide a method for isolation
of INGAP genes from a mammal.
It is another object of the invention to provide an antibody
preparation which is specifically immunoreactive with an INGAP
protein.
It is yet another object of the invention to provide methods of
producing INGAP proteins.
It is an object of the invention to provide methods for treating
diabetic mammals.
It is another object of the invention to provide methods for
growing pancreatic islet cells in culture.
It is still another object of the invention to provide methods of
enhancing the life span of pancreatic islet cells encapsulated in
polycarbon shells.
It is an object of the invention to provide methods of enhancing
the number of pancreatic islet cells in a mammal.
It is an object of the invention to provide transgenic mammals.
It is another object of the invention to provide genetically
engineered mammals.
It is yet another object of the invention to provide methods of
identifying individual mammals at risk for diabetes.
It is an object of the invention to provide methods of detecting
INGAP protein in a sample from a mammal.
It is still another object of the invention to provide a method of
treating isolated islet cells to avoid apoptosis.
It is another object of the invention to provide methods of
treating mammals receiving islet cell transplants.
It is an object of the invention to provide a method of inducing
differentiation of .beta. cell progenitors.
It is an object of the invention to provide a method of identifying
.beta. cell progenitors.
It is another object of the invention to provide a method of
treating a mammal with pancreatic endocrine failure.
It is an object of the invention to provide antisense constructs
for regulating the expression of INGAP.
It is yet another object of the invention to provide a method for
treating nesidioblastosis.
It is still another object of the invention to provide kits for
detecting mammalian INGAP proteins.
It is an object of the invention to provide pharmaceutical
compositions for treatment of pancreatic insufficiency.
These and other objects of the invention are provided by one or
more of the embodiments described below.
In one embodiment a preparation of a mammalian INGAP protein is
provided. The preparation is substantially free of other mammalian
proteins.
In another embodiment an isolated cDNA molecule is provided. The
cDNA molecule encodes a mammalian INGAP protein.
In still another embodiment of the invention a preparation of a
mammalian INGAP protein is provided. The preparation is made by the
process of: inducing mammalian pancreatic cells to express INGAP
protein by cellophane-wrapping; and purifying said INGAP protein
from said induced mammalian pancreatic cells.
In yet another embodiment of the invention a nucleotide probe is
provided. The probe comprises at least 20 contiguous nucleotides of
the sequence shown in SEQ ID NO: 1.
In another embodiment of the invention a preparation of INGAP
protein of a mammal is provided. The preparation is substantially
purified from other proteins of the mammal. The INGAP protein is
inducible upon cellophane-wrapping of pancreas of the mammal.
In yet another embodiment of the invention a method of isolating an
INGAP gene from a mammal is provided. The method comprises:
hybridizing one or more oligonucleotides comprising at least 10
contiguous nucleotides of the sequence shown in SEQ ID NO: 1 to
genomic DNA or cDNA of said mammal; identifying DNA molecules from
said genomic DNA or cDNA which hybridize to said one or more
oligonucleotides.
In still another embodiment of the invention an isolated cDNA
molecule is provided. The cDNA molecule is obtained by the process
of: hybridizing one or more oligonucleotides comprising at least 10
contiguous nucleotides of the sequence shown in SEQ ID NO: 1 to
genomic DNA or cDNA of said mammal; identifying DNA molecules from
said genomic DNA or cDNA which hybridize to said one or more
oligonucleotides.
In another embodiment of the invention an antibody is provided. The
antibody is specifically immunoreactive with a mammalian INGAP
protein.
According to still another embodiment of the invention a method of
producing a mammalian INGAP protein is provided. The method
comprises the steps of: providing a host cell transformed with a
cDNA encoding a mammalian INGAP protein; culturing the host cell in
a nutrient medium so that the INGAP protein is expressed; and
harvesting the INGAP protein from the host cell or the nutrient
medium.
According to yet another embodiment of the invention a method of
producing a mammalian INGAP protein is provided. The method
comprises the steps of: providing a host cell comprising a DNA
molecule obtained by the process of: hybridizing one or more
oligonucleotides comprising at least 10 contiguous nucleotides of
the sequence shown in SEQ ID NO: 1 to genomic DNA or cDNA of said
mammal; identifying DNA molecules from said genomic DNA or cDNA
which hybridize to said one or more oligonucleotides; culturing the
host cell in a nutrient medium so that the mammalian INGAP protein
is expressed; and harvesting the mammalian INGAP protein from the
host cells or the nutrient medium.
According to another embodiment of the invention a method of
treating diabetic mammals is provided. The method comprises:
administering to a diabetic mammal a therapeutically effective
amount of an INGAP protein to stimulate growth of islet cells.
According to another embodiment of the invention a method of
growing pancreatic islet cells in culture is provided. The method
comprises: supplying an INGAP protein to a culture medium for
growing pancreatic islet cells; and growing islet cells in said
culture medium comprising INGAP protein.
According to another embodiment of the invention a method of
enhancing the life span of pancreatic islet cells encapsulated in a
polycarbon shell is provided. The method comprises: adding to
encapsulated pancreatic islet cells an INGAP protein in an amount
sufficient to enhance the survival rate or survival time of said
pancreatic islet cells.
According to another embodiment of the invention a method of
enhancing the number of pancreatic islet cells in a mammal is
provided. The method comprises: administering a DNA molecule which
encodes an INGAP protein to a pancreas in a mammal.
According to another embodiment of the invention a method of
enhancing the number of pancreatic islet cells in a mammal is
provided. The method comprises: administering an INGAP protein to a
pancreas in a mammal.
According to another embodiment of the invention a transgenic
mammal is provided. The mammal comprises an INGAP gene of a second
mammal.
According to another embodiment of the invention a non-human mammal
is provided. The mammal has been genetically engineered to contain
an insertion or deletion mutation of an INGAP gene of said
mammal.
According to another embodiment of the invention a method of
identifying individual mammals at risk for diabetes is provided.
The method comprises: identifying a mutation in an INGAP gene of a
sample of an individual mammal, said mutation causing a structural
abnormality in an INGAP protein encoded by said gene or causing a
regulatory defect leading to diminished or obliterated expression
of said INGAP gene.
According to another embodiment of the invention a method of
detecting INGAP protein in a sample from a mammal is provided. The
method comprises: contacting said sample with an antibody
preparation which is specifically immunoreactive with a mammalian
INGAP protein.
According to another embodiment of the invention a method of
treating isolated islet cells of a mammal to avoid apoptosis of
said cells is provided. The method comprises: contacting isolated
islet cells of a mammal with a preparation of a mammalian INGAP
protein, substantially purified from other mammalian proteins, in
an amount sufficient to increase the survival rate of said isolated
islet cells.
According to another embodiment of the invention a method of
treating a mammal receiving a transplant of islet cells is
provided. The method comprises: administering a preparation of a
mammalian INGAP protein to a mammal receiving a transplant of islet
cells, wherein said step of administering is performed before,
during, or after said transplant.
According to another embodiment of the invention a method of
inducing differentiation of .beta. cell progenitors is provided.
The method comprises: contacting a culture of pancreatic duct cells
comprising .beta. cell progenitors with a preparation of a
mammalian INGAP protein substantially free of other mammalian
proteins, to induce differentiation of said .beta. cell
progenitors.
In yet another embodiment of the invention a method is provided for
identification of .beta. cell progenitors. The method comprises:
contacting a population of pancreatic duct cells with a mammalian
INGAP protein; and detecting cells among said population to which
said INGAP protein specifically binds.
According to another embodiment of the invention a method of
treating a mammal with pancreatic endocrine failure is provided.
The method comprises: contacting a preparation of pancreatic duct
cells comprising .beta. cell progenitors isolated from a mammal
afflicted with pancreatic endocrine failure with a preparation of a
mammalian INGAP protein substantially free of other mammalian
proteins to induce differentiation of said .beta. cell progenitors;
and autologously transplanting said treated pancreatic duct cells
into said mammal.
According to another embodiment of the invention an antisense
construct of a mammalian INGAP gene is provided. The construct
comprises: a promoter, a terminator, and a nucleotide sequence
consisting of a mammalian INGAP gene, said nucleotide sequence
being between said promoter and said terminator, said nucleotide
sequence being inverted with respect to said promoter, whereby upon
expression from said promoter an mRNA complementary to native
mammalian INGAP mRNA is produced.
According to another embodiment of the invention a method of
treating nesidioblastosis is provided. The method comprises:
administering to a mammal with nesidioblastosis an anti-sense
construct as described above, whereby overgrowth of .beta. cells of
said mammal is inhibited.
According to another embodiment of the invention a kit for
detecting a mammalian INGAP protein in a sample from a mammal is
provided. The kit comprises: an antibody preparation which is
specifically immunoreactive with a mammalian INGAP protein; and a
polypeptide which comprises a sequence of at least 15 consecutive
amino acids of a mammalian INGAP protein.
According to another embodiment of the invention a pharmaceutical
composition for treatment of pancreatic insufficiency is provided.
The composition comprises: a mammalian INGAP protein in a
pharmaceutically acceptable diluent or carrier.
According to another embodiment of the invention a pharmaceutical
composition is provided. The composition comprises: a preparation
of a polypeptide which comprises a sequence of at least 15
consecutive amino acids of a mammalian INGAP protein and a
pharmaceutically acceptable diluent or carrier.
These and other embodiments of the invention provide the art with
means of stimulating and inhibiting islet cell neogenesis. Means of
diagnosis of subsets of diabetes mellitus are also provided by this
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B. Nucleotide sequence of hamster INGAP and deduced
sequence of encoded immature protein (SEQ ID NOS: 1 and 2). The
non-coding sequences are in lower case letters, and the
polyadenylation signal is underlined.
FIG. 2. Comparison of amino acid sequences of INGAP (SEQ ID NO: 2),
rat PAP-I (PAP-I)(18)(SEQ ID NO: 3), Human PAP/HIP
(PAP-H/HIP)(10,11)(SEQ ID NO: 4), rat PAP-III (PAP-III)(9)(SEQ ID
NO: 5), rat PAP-II (PAP-II) (8)(SEQ ID NO: 6), Rat
Reg/PSP/Lithostatine (REG/LITH) (13,15)(SEQ ID NO: 7) and the
invariable motif found by Drickamer in all members of C-type
lectins (Drickamer) (12). Six conserved cysteines are marked by
asterisks and the 2 putative N-glycosylation sites of INGAP are
underlined and in bold letters.
FIGS. 3A, 3B and 3C. Northern blot analysis of INGAP and amylase
gene expression in pancreatic tissue from control and wrapped
hamster pancreas. 30 g of heat denatured total RNA was separated by
electrophoresis on a 1.2% agarose, 0.6% formaldehyde/MOPS
denaturing gel, and transferred to nylon membrane. Membranes were
hybridized with a 747 bp hamster INGAP cDNA probe (cloned in our
lab) (A), a 1000 bp rat amylase cDNA probe (generously given by
Chris Newgard Dallas, Tex.) (B) and with an 18S ribosomal 24mer
synthetic oligonucleotide probe to control for RNA integrity and
loading (C).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
We now report the identification of a gene, INGAP, that shows
striking homology to the pancreatitis associated protein (PAP)
family of genes (7-11). The predicted protein shares the
carbohydrate recognition domain (CRD) of the calcium dependent
C-type lectins as defined by Drickamer (12). INGAP protein plays a
role in stimulation of islet neogenesis, in particular, in beta
cell regeneration from ductal cells.
The cDNA sequence of a mammalian INGAP is provided in SEQ ID NO: 1.
The predicted amino acid sequence is shown in SEQ ID NO:2. These
sequences were determined from nucleic acids isolated from hamster,
but it is believed that other mammalian species will contain INGAP
genes which are quite similar. For example, human INGAP cDNA shares
the entire sequence in SEQ ID NO:1 with the hamster. The predicted
amino acid sequence of human INGAP protein is from 1 to 174 in SEQ
ID NO:2. One would expect homologous genes to contain at least
about 70% identity. Closer species would be expected to have at
least about 75%, 80%, 85%, 90%, 95%, or even 99% identity. In
contrast, other family members of the calcium dependent C-type
lectins contain at most 60% identity with INGAP.
The DNA sequence provided herein can be used to form vectors which
will replicate the gene in a host cell, and may also express INGAP
protein. DNA sequences which encode the same amino acid sequence as
shown in SEQ ID NO:2 can also be used, without departing from the
contemplation of the invention. DNA sequences coding for other
mammalian INGAPs are also within the contemplation of the
invention. Suitable vectors, for both prokaryotic and eukaryotic
cells, are known in the art. Some vectors are specifically designed
to effect expression of inserted DNA segments downstream from a
transcriptional and translational control site. One such vector for
expression in eukaryotic cells employs EBNA His, a plasmid which is
available commercially from InVitrogen Corp. The loaded vector
produces a fusion protein comprising a portion of a histidine
biosynthetic enzyme and INGAP. Another vector, which is suitable
for use in prokaryotic cells, is pcDNA3. Selection of a vector for
a particular purpose may be made using knowledge of the properties
and features of the vectors, such as useful expression control
sequences. Vectors may be used to transform or transfect host
cells, either stably or transiently. Methods of transformation and
transfection are known in the art, and may be used according to
suitability for a particular host cell. Host cells may be selected
according to the purpose of the transfection. A suitable
prokaryotic host is E. coli DH5.alpha.. A suitable eukaryotic host
is cos7, an African Green Monkey kidney cell line. For some
purposes, proper glycosylation of INGAP may be desired, in which
case a suitable host cell should be used which recognizes the
glycosylation signal of INGAP.
Probes comprising at least 10, 15, 20, or 30 nucleotides of
contiguous sequence according to SEQ ID NO:1 can be used for
identifying INGAP genes in particular individuals or in members of
other species. Appropriate conditions for hybridizations to same or
different species' DNA are known in the art as high stringency and
low stringency, respectively. These can be used in a variety of
formats according to the desired use. For example, Southern blots,
Northern blots, and in situ colony hybridization, can be used as
these are known in the art. Probes typically are DNA or RNA
oligomers of at least 10, 15, 20, or 30 nucleotides. The probe may
be labeled with any detectable moiety known in the art, including
radiolabels, fluorescent labels, enzymes, etc. Probes may also be
derived from other mammalian INGAP gene sequences.
INGAP genes can be isolated from other mammals by utilizing the
nucleotide sequence information provided herein. (More laboriously,
they can be isolated using the same method described in detail
below for isolation of the hamster INGAP gene.) Oligonucleotides
comprising at least 10 contiguous nucleotides of the disclosed
nucleotide sequence of INGAP are hybridized to genomic DNA or cDNA
of the mammal. The DNA may conveniently be in the form of a library
of clones. The oligonucleotides may be labelled with any convenient
label, such as a radiolabel or an enzymatic or fluorescence label.
DNA molecules which hybridize to the probe are isolated. Complete
genes can be constructed by isolating overlapping DNA segments, for
example using the first isolated DNA as a probe to contiguous DNA
in the library or preparation of the mammal's DNA. Confirmation of
the identity of the isolated DNA can be made by observation of the
pattern of expression of the gene in the pancreas when subjected to
cellophane wrapping, for example. Similarly, the biological effect
of the encoded product upon pancreatic ductal cells will also serve
to identify the gene as an INGAP gene.
If two oligonucleotides are hybridized to the genomic DNA or cDNA
of the mammal then they can be used as primers for DNA synthesis,
for example using the polymerase chain reaction or the ligase chain
reaction. Construction of a full-length gene and confirmation of
the identity of the isolated gene can be performed as described
above.
INGAP protein may be isolated according to the invention by
inducing mammalian pancreatic cells to express INGAP protein by
means of cellophane-wrapping. This technique is described in detail
in reference no. 1 which is expressly incorporated herein. INGAP
protein so produced may be purified from other mammalian proteins
by means of immunoaffinity techniques, for example, or other
techniques known in the art of protein purification. An antibody
specific for a mammalian INGAP is produced using all, or fragments
of, the amino acid sequence of an INGAP protein, such as shown in
SEQ ID NO: 2, as immunogens. The immunogens can be used to identify
and purify immunoreactive antibodies. Monoclonal or polyclonal
antibodies can be made as is well known in the art. The antibodies
can be conjugated to other moieties, such as detectable labels or
solid support materials. Such antibodies can be used to purify
proteins isolated form mammalian pancreatic cells or from
recombinant cells. Hybridomas which secrete specific antibodies for
an INGAP protein are also within the contemplation of the
invention.
Host cells as described above can be used to produce a mammalian
INGAP protein. The host cells comprise a DNA molecule encoding a
mammalian INGAP protein. The DNA can be according to SEQ ID NO:1,
or isolated from other mammals according to methods described
above. Host cells can be cultured in a nutrient medium under
conditions where INGAP protein is expressed. INGAP protein can be
isolated from the host cells or the nutrient medium, if the INGAP
protein is secreted from the host cells.
It has now been found that INGAP and fragments thereof are capable
of inducing and stimulating islet cells to grow. Moreover, they are
capable of inducing differentiation of pancreatic duct cells, and
of allowing such cells to avoid the apoptotic pathway. Thus many
therapeutic modalities are now possible using INGAP, fragments
thereof, and nucleotide sequences encoding INGAP. Therapeutically
effective amounts of INGAP are supplied to patient pancreata, to
isolated islet cells, and to encapsulated pancreatic islet cells,
such as in a polycarbon shell. Suitable amounts of INGAP for
therapeutic purposes range from 1-150 .mu.g/kg of body weight or in
vitro from 1-10,000 .mu.g/ml. Optimization of such dosages can be
ascertained by routine testing. Methods of administering INGAP to
mammals can be any that are known in the art, including
subcutaneous, via the portal vein, by local perfusion, etc.
Conditions which can be treated according to the invention by
supplying INGAP include diabetes mellitus, both insulin dependent
and non-insulin dependent, pancreatic insufficiency, pancreatic
failure, etc. Inhibition of INGAP expression can be used to treat
nesidioblastosis.
According to the present invention, it has now been found that a
small portion of INGAP is sufficient to confer biological activity.
A fragment of 20 amino acids of the sequence of SEQ ID NO: 2, from
amino acid #103-#122 is sufficient to stimulate pancreatic ductal
cells to grow and proliferate. The effect has been seen on a rat
tumor duct cell line, a hamster duct cell line, a hamster
insulinoma cell line, and a rat insulinoma cell line. The analogous
portions of other mammalian INGAP proteins are quite likely to have
the same activity. This portion of the protein is not similar to
other members of the pancreatitis associated protein (PAP) family
of proteins. It contains a glycosylation site and it is likely to
be a primary antigenic site of the protein as well. This fragment
has been used to immunize mice to generate monoclonal
antibodies.
The physiological site of expression of INGAP has been determined.
INGAP is expressed in acinar tissue, in the exocrine portion of the
pancreas. It is not expressed in ductal or islet cells, i.e., the
paracrine portion of the pancreas. Expression occurs within 24-48
hours of induction by means of cellophane wrapping.
Transgenic animals according to the present invention are mammals
which carry an INGAP gene from a different mammal. The transgene
can be expressed to a higher level than the endogenous INGAP genes
by judicious choice of transcription regulatory regions. Methods
for making transgenic animals are well known in the art, and any
such method can be used. Animals which have been genetically
engineered to carry insertions, deletions, or other mutations which
alter the structure of the INGAP protein or regulation of
expression of INGAP are also contemplated by this invention. The
techniques for effecting these mutations are known in the art.
Diagnostic assays are also contemplated within the scope of the
present invention. Mutations in INGAP can be ascertained in samples
such as blood, amniotic fluid, chorionic villus, blastocyst, and
pancreatic cells. Such mutations identify individuals who are at
risk for diabetes. Mutations can be identified by comparing the
nucleotide sequence to a wild-type sequence of an INGAP gene. This
can be accomplished by any technique known in the art, including
comparing restriction fragment length polymorphisms, comparing
polymerase chain reaction products, nuclease protection assays,
etc. Alternatively, altered proteins can be identified, e.g.,
immunologically or biologically.
The present invention also contemplates the use of INGAP antisense
constructs for treating nesidioblastosis, a condition characterized
by overgrowth of .beta. cells. The antisense construct is
administered to a mammal having nesidioblastosis, thereby
inhibiting the overgrowth of .beta. cells. An antisense construct
typically comprises a promoter, a terminator, and a nucleotide
sequence consisting of a mammalian INGAP gene. The INGAP sequence
is between the promoter and the terminator and is inverted with
respect to the promoter as it is expressed naturally. Upon
expression from the promoter, an mRNA complementary to native
mammalian INGAP is produced.
Immunological methods for assaying INGAP in a sample from a mammal
are useful, for example, to monitor the therapeutic administration
of INGAP. Typically an antibody specific for INGAP will be
contacted with the sample and the binding between the antibody and
any INGAP in the sample will be detected. This can be by means of a
competitive binding assay, in which the incubation mixture is
spiked with a known amount of a standard INGAP preparation, which
may conveniently be detectably labeled. Alternatively, a
polypeptide fragment of INGAP may be used as a competitor. In one
particular assay format, the antibodies are bound to a solid phase
or support, such as a bead, polymer matrix, or a microtiter
plate.
According to the present invention, pancreatic duct cells of a
mammal with pancreatic endocrine failure can be removed from the
body and treated in vitro. The duct cells typically comprise .beta.
cell progenitors. Thus treatment with a preparation of a mammalian
INGAP protein will induce differentiation of the .beta. cell
progenitors. The duct cells are contacted with a preparation of a
mammalian INGAP protein substantially free of other mammalian
proteins. The treated cells can then used as an autologous
transplant into the mammal from whom they were derived. Such an
autologous treatment minimizes adverse host versus graft reactions
involved in transplants.
INGAP protein can also be used to identify those cells which bear
receptors for INGAP. Such cells are likely to be the .beta. cell
progenitors, which are sensitive to the biological effects of
INGAP. INGAP protein can be detectably labeled, such as with a
radiolabel or a fluorescent label, and then contacted with a
population of cells from the pancreatic duct. Cells which bind to
the labeled protein will be identified as those which bear
receptors for INGAP, and thus are .beta. cell progenitors.
Fragments of INGAP can also be used for this purpose, as can
immobilized INGAP which can be used to separate cells from a mixed
population of cells to a solid support. INGAP can be immobilized to
solid phase or support by adsorption to a surface, by means of an
antibody, or by conjugation. Any other means as is known in the art
can also be used.
Kits are provided by the present invention for detecting a
mammalian INGAP protein in a sample. This may be useful, inter
alia, for monitoring metabolism of INGAP during therapy which
involves administration of INGAP to a mammal. The kit will
typically contain an antibody preparation which is specifically
immunoreactive with a mammalian INGAP protein. The antibodies may
be polyclonal or monoclonal. If polyclonal they may be affinity
purified to render them monospecific. The kit will also typically
contain a polypeptide which has at least 15 consecutive amino acids
of a mammalian INGAP protein. The polypeptide is used to compete
with the INGAP protein in a sample for binding to the antibody.
Desirably the polypeptide will be detectably labeled. The
polypeptide will contain the portion of INGAP to which the antibody
binds. Thus if the antibody is monoclonal, the polypeptide will
successfully compete with INGAP by virtue of it containing the
epitope of the antibody. It may also be desirable that the
antibodies be bound to a solid phase or support, such as polymeric
beads, sticks, plates, etc.
Pharmaceutical compositions containing a mammalian INGAP protein
may be used for treatment of pancreatic insufficiency. The
composition may alternatively contain a polypeptide which contains
a sequence of at least 15 consecutive amino acids of a mammalian
INGAP protein. The polypeptide will contain a portion of INGAP
which is biologically active in the absence of the other portions
of the protein. The polypeptide may be part of a larger protein,
such as a genetic fusion with a second portion or polypeptide.
Alternatively, the polypeptide may be conjugated to a second
protein, for example, by means of a cross-linking agent. Suitable
portions of INGAP proteins may be determined by homology with amino
acids #103 to #122 of SEQ ID NO:2, or by the ability of test
polypeptides to stimulate pancreatic duct cells to grow and
proliferate. As is known in the art, it is often the case that a
relatively small number of amino acids can be removed from either
end of a protein without destroying activity. Thus it is
contemplated within the scope of the invention that up to about 10%
of the protein can be deleted, and still provide essentially all
functions of INGAP. Such proteins have at least about 130 amino
acids, in the case of hamster INGAP.
The pharmaceutical composition will contain a pharmaceutically
acceptable diluent or carrier. A liquid formulation is generally
preferred. INGAP may be formulated at different concentrations or
using different formulants. For example, these formulants may
include oils, polymers, vitamins, carbohydrates, amino acids,
salts, buffers, albumin, surfactants, or bulking agents. Preferably
carbohydrates include sugar or sugar alcohols such as mono-, di-,
or polysaccharides, or water soluble glucans. The saccharides or
glucans can include fructose, dextrose, lactose, glucose, mannose,
sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin,
alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and
carboxymethylcellulose, or mixtures thereof. Sucrose is most
preferred. Sugar alcohol is defined as a C.sub.4 to C.sub.8
hydrocarbon having an --OH group and includes galacitol, inositol,
mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is
most preferred. These sugars or sugar alcohols mentioned above may
be used individually or in combination. There is no fixed limit to
amount used as long as the sugar or sugar alcohol is soluble in the
aqueous preparation. Preferably, the sugar or sugar alcohol
concentration is between 1.0 w/v % and 7.0 w/v %, more preferable
between 2.0 and 6.0 w/v %. Preferably amino acids include
levorotary (L) forms of carnitine, arginine, and betaine; however,
other amino acids may be added. Preferred polymers include
polyvinylpyrrolidone (PVP) with an average molecular weight between
2,000 and 3,000, or polyethylene glycol (PEG) with an average
molecular weight between 3,000 and 5,000. It is also preferred to
use a buffer in the composition to minimize pH changes in the
solution before lyophilization or after reconstitution, if these
are used. Most any physiological buffer may be used, but citrate,
phosphate, succinate, and glutamate buffers or mixtures thereof are
preferred. Preferably, the concentration is from 0.01 to 0.3 molar.
Surfactants can also be added to the formulation.
Additionally, INGAP or polypeptide portions thereof can be
chemically modified by covalent conjugation to a polymer to
increase its circulating half-life, for example. Preferred
polymers, and methods to attach them to peptides, are shown in U.S.
Pat. Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546. Preferred
polymers are polyoxyethylated polyols and polyethylene glycol
(PEG). PEG is soluble in water at room temperature and has the
general formula: R(O--CH.sub.2--CH.sub.2).sub.nO--R where R can be
hydrogen, or a protective group such as an alkyl or alkanol group.
Preferably, the protective group has between 1 and 8 carbons, more
preferably it is methyl. The symbol n is a positive integer,
preferably between 1 and 1,000, more preferably between 2 and 500.
The PEG has a preferred average molecular weight between 1000 and
40,000, more preferably between 2000 and 20,000, most preferably
between 3,000 and 12,000. Preferably, PEG has at least one hydroxy
group, more preferably it is a terminal hydroxy group. It is this
hydroxy group which is preferably activated to react with a free
amino group on the inhibitor.
After the liquid pharmaceutical composition is prepared, it is
preferably lyophilized to prevent degradation and to preserve
sterility. Methods for lyophilizing liquid compositions are known
to those of ordinary skill in the art. Just prior to use, the
composition may be reconstituted with a sterile diluent (Ringer's
solution, distilled water, or sterile saline, for example) which
may include additional ingredients. Upon reconstitution, the
composition is preferably administered to subjects using those
methods that are known to those skilled in the art.
The following examples are not intended to limit the scope of the
invention, but merely to exemplify that which is taught above.
EXAMPLES
Example 1
This example describes the cloning and isolation of a cDNA encoding
a novel, developmentally regulated, pancreatic protein.
We hypothesized that a unique locally produced factor(s) is
responsible for islet cell regeneration. Using the recently
developed mRNA differential display technique (5,6) to compare
genes differentially expressed in cellophane wrapped (CW) versus
control pancreata (CP) allowed us to identify a cDNA clone (RD19-2)
which was uniquely expressed in cellophane wrapped pancreas.
A cDNA library was constructed from mRNA isolated from cellophane
wrapped hamster pancreas using oligo d(T) primed synthesis, and
ligation into pcDNA3 vector (Invitrogen). The number of primary
recombinants in the library was 1.2.times.10.sup.6 with an average
size of 1.1 kb. The cDNA library was screened for clones of
interest using high density colony plating techniques. Colonies
were lifted onto nylon membranes (Schleicher & Schuell) and
further digested with proteinase K (50(g/ml). Treated membranes
were baked at 80.degree. C. for 1 hour and hybridized at 50.degree.
C. for 16-18 hours with 1-5.times.10.sup.6 cpm/ml of
[(.sup.32P]-dCTP(Dupont-New England Nuclear) radiolabeled RD 19-2
probe. Colonies with a positive hybridization signal were isolated,
compared for size with Northern mRNA transcript, and sequenced to
confirm identity with the RD 19-2 sequence.
Example 2
This example compares the sequence of INGAP to other proteins with
which it shares homology.
The nucleotide sequence of the hamster INGAP clone with the longest
cDNA insert was determined. As shown in FIG. 1 the hamster cDNA
comprises 747 nucleotides (nt), exclusive of the poly(A) tail and
contains a major open reading frame encoding a 175 amino acid
protein. The open reading frame is followed by a 3'-untranslated
region of 206nt. A typical polyadenylation signal is present 11nt
upstream of the poly(A) tail. The predicted INGAP protein shows
structural homology to both the PAP/HIP family of genes which is
associated with pancreatitis or liver adenocarcinoma (7-11) and the
Reg/PSP/lithostatine family of genes (13,15) which has been shown
to stimulate pancreatic beta-cell growth (14) and might play a role
in pancreatic islet regeneration. Comparison of the nucleotide
sequence and their deduced amino acids between hamster INGAP and
rat PAP-I shows a high degree of homology in the coding region (60
and 58% in nucleotide and amino acid sequences, respectively). The
predicted amino acid sequence of the hamster INGAP reveals 45%
identity to PAP II and 50% to PAP III both of which have been
associated with acute pancreatitis, and 54% to HIP which was found
in a hepatocellular carcinoma. INGAP also shows 40% identity to the
rat Reg/PSP/lithostatine protein (FIG. 2). Reg is thought to be
identical to the pancreatic stone protein (PSP) (15,16) or
pancreatic thread protein (PTP) (17). The N-terminus of the
predicted sequence of INGAP protein is highly hydrophobic which
makes it a good candidate for being the signal peptide which would
allow the protein to be secreted. Similar to PAP/HIP but different
from the Reg/PSP/lithostatine proteins a potential N-glycosylation
site is situated at position 135 of the INGAP sequence. Unique to
INGAP is another potential N-glycosylation site situated at
position 115. INGAP also shows a high degree of homology (12/18)
(FIG. 2) with a consensus motif in members of the calcium-dependent
(C-type) animal lectin as determined by Drickamer including four
perfectly conserved cysteines which form two disulfide bonds (12).
Two extra cysteines found at the amino-terminus of INGAP (FIG. 2)
are also present in Reg/PSP and PAP/HIP. However, it is not clear
what the biological significance might be.
Example 3
This example demonstrates the temporal expression pattern of INGAP
upon cellophane-wrapping.
In order to determine the temporal expression of the INGAP gene,
total RNA extracted from CP and CW pancreas was probed with the
hamster INGAP cDNA clone in Northern blot analysis. A strong single
transcript of 900 bp was detected (FIGS. 3A, 3B and 3C) 1 and 2
days after cellophane wrapping which disappeared by 6 through 42
days and was absent from CP. INGAP mRNA is associated with CW
induced pancreatic islet neogenesis, since it is present only after
CW. It is not likely that the increased expression of INGAP is
associated with acute pancreatitis as is the case with the PAP
family of genes. During the acute phase of pancreatitis the
concentrations of most mRNAs encoding pancreatic enzymes including
amylase are decreased significantly (16,18). In contrast, in the CW
model of islet neogenesis in which high expression of INGAP has
been detected, amylase gene expression was simultaneously increased
above normal (FIGS. 3A, 3B and 3C) rather than decreased,
suggesting that INGAP expression is not associated with
pancreatitis but rather with islet neogenesis. The cause of
increased amylase gene expression 1 and 2 days after CW is as yet
unclear, and more studies need to be done to elucidate this issue.
It is unlikely though, that the increase is associated with
exocrine cell regeneration which occurs at a later time after CW
(19). Thus, INGAP protein plays a role in stimulation of islet
neogenesis, in particular, in beta cell regeneration from ductal
cells.
Example 4
This example describes the cloning and partial sequence of a human
cDNA encoding INGAP protein.
Human polyA.sup.+ RNA was isolated from a normal human pancreas
using a commercially available polyA.sup.+ extraction kit from
Qiagen. Subsequently, 500 ng polyA.sup.+ RNA was used as a template
for reverse transcription and polymerase chain reaction (RT-PCR).
The experimental conditions were set according to the instructions
in the RT-PCR kit from Perkin Elmer. Oligo d(T) was used as the
primer in reverse transcription. Primers corresponding to
nucleotides 4 to 23 and 610 to 629 in SEQ ID NO:1 were used as the
specific primers in the polymerase chain reaction. A 626 bp PCR
fragment was cloned using a TA cloning kit from Invitrogen. The
human INGAP cDNA is 100% identical to the hamster INGAP cDNA
sequence in SEQ ID NO:1.
Example 5
This example demonstrates that synthetic peptides from INGAP play a
role in stimulation of islet neogenesis, and that at least one
epitope coded by the as yet unsequenced 120 bp segment of human
INGAP is shared with hamster INGAP.
A synthetic peptide corresponding to amino acids 104-118 in SEQ ID
NO:2 of the deduced hamster INGAP protein was used as an immunogen
to raise polyclonal antibodies in a rabbit. The antiserum was
subsequently used in immunohistochemistry assays using the
avidin-biotin complex (ABC) method. Cells in the peri-islet region
in humans with neoislet formation stained positively for INGAP
demonstrating that human and hamster INGAP share a common epitope
between amino acids 104 to 118 in SEQ ID NO:2.
The same synthetic peptide was tested for its ability to stimulate
.sup.3H-thymidine incorporation into rat pancreatic tumor duct
cells (ARIP) and hamster insulinoma tumor cells (HIT). 10 .mu.Ci of
.sup.3H-thymidine at 80.4 Ci/mmole concentration was added to
approximate 10.sup.6 cells cultured in Ham's F-12K media. After 24
hrs, the cells were harvested and solubilized. Differential
precipitation of the nucleic acids with trichloroacetic acid (TCA)
was performed according to the procedure modified by Rosenberg et
al. and the .sup.3H-thymidine proportion incorporated was
calculated. Addition of the synthetic peptide to ARIP in culture
resulted in a 2.4-fold increase in .sup.3H-thymidine incorporation
comparing to the absence of the synthetic peptide in the culture.
The synthetic peptide had no effect on the control cell line HIT.
This result strongly suggests that INGAP plays a role in
stimulating islet neogenesis.
References
1. Rosenberg, L., Brown, R. A. and Duguid, W. P. (1982). Surg.
Forum 33, 227-230. 2. Rosenberg, L., Brown, R. A. and Duguid, W. P.
(1983). J. Surg. Res. 35, 63-72. 3. Rosenberg, L., Duguid, W. P.
and Vinik, A. I. (1987). Dig. Dis. Sci. 32, 1185. 4. Clas, D.,
Rosenberg, L. and Duguid, W. P. (1989). Pancreas 4, 613 (Abstract).
5. Liang, P. and Pardee, B. A. (1992). Science 257, 967-971. 6.
Liang, P., Averboukh, L. and Pardee, B. A. (1993). Nucleic Acid
Res. 21, 3269-3275. 7. Iovanna, J., Orelle, B., Keim, V. and
Dagorn, J. C. (1991). J. Biol. Chem. 266, 24664-24669. 8. Frigerio,
J. M., Dusetti, N., Keim, V., Dagorn, J. C. and Iovanna, J. (1993).
Biochemistry 32, 9236-9241. 9. Frigerio, J. M., Dusetti, N.,
Garrido, P., Dagorn, J. C. and Iovanna, J. (1993). Biochim.
Biophys. Acta 1216, 329-331. 10. Orelle, B., Keim, V., Masciotra,
L., Dagorn, J. C. and Iovanna, J. (1992). J. Clin. Invest. 90,
2284-2291. 11. Lasserre, C., Christa, L., Simon, M. T., Vernier, P.
and Brechot, C. (1992). Cancer Res. 52, 5089-5095. 12. Drickamer,
K. (1988). J. Biol. Chem. 263, 9557-9560. 13. Terazono, K.,
Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y.
and Okamoto, H. (1988). J. Biol. Chem. 263, 2111-2114. 14.
Watanabe, T., Yutaka, Y., Yonekura, H., Suzuki, Y., Miyashita, H.,
Sugiyama, K., Morizumi, S., Unno, M., Tanaka, O., Kondo, H., Bone,
A. J., Takasawa, S. and Okamoto, H. (1994). Proc. Natl. Acad. Sci.
USA 91, 3589-3592. 15. Rouquier, S., Giorgi, D., Iovanna, J. and
Dagorn, J. C. (1989). Biochem. J. 264, 621-624. 16. Rouquier, S.,
Verdier, J., Iovanna, J., Dagorn, J. C. and Giorgi, D. (1991) J.
Biol. Chem. 266, 786-791. 17. Gross, J., Carlson, R. I., Brauer, A.
W., Margolies, M. N., Warshaw, A. L. and Wands, J. R. (1985). J.
Clin. Invest. 76, 2115-2126. 18. Iovanna, J., Keim, V., Michael, R.
and Dagorn, J. C. (1991). Am. J. Physiol. 261, G485-G489. 19.
Rosenberg, L. and Vinik, A. I. (1989). J. Lab. Clin. Med. 114,
75-83.
SEQUENCE LISTINGS
1
7747 base pairsnucleic acidsinglelinearcDNANONOCricetulusCDS
20..541 1CTGCAAGACA GGTACCATG ATG CTT CCC ATG ACC CTC TGT AGG ATG
TCT TGG 52 Met Leu Pro Met Thr Leu Cys Arg Met Ser Trp 1 5 10ATG
CTG CTT TCC TGC CTG ATG TTC CTT TCT TGG GTG GAA GGT GAA GAA 100Met
Leu Leu Ser Cys Leu Met Phe Leu Ser Trp Val Glu Gly Glu Glu 15 20
25TCT CAA AAG AAA CTG CCT TCT TCA CGT ATA ACC TGT CCT CAA GGC TCT
148Ser Gln Lys Lys Leu Pro Ser Ser Arg Ile Thr Cys Pro Gln Gly Ser
30 35 40GTA GCC TAT GGG TCC TAT TGC TAT TCA CTG ATT TTG ATA CCA CAG
ACC 196Val Ala Tyr Gly Ser Tyr Cys Tyr Ser Leu Ile Leu Ile Pro Gln
Thr 45 50 55TGG TCT AAT GCA GAA CTA TCC TGC CAG ATG CAT TTC TCA GGA
CAC CTG 244Trp Ser Asn Ala Glu Leu Ser Cys Gln Met His Phe Ser Gly
His Leu 60 65 70 75GCA TTT CTT CTC AGT ACT GGT GAA ATT ACC TTC GTG
TCC TCC CTT GTG 292Ala Phe Leu Leu Ser Thr Gly Glu Ile Thr Phe Val
Ser Ser Leu Val 80 85 90AAG AAC AGT TTG ACG GCC TAC CAG TAC ATC TGG
ATT GGA CTC CAT GAT 340Lys Asn Ser Leu Thr Ala Tyr Gln Tyr Ile Trp
Ile Gly Leu His Asp 95 100 105CCC TCA CAT GGT ACA CTA CCC AAC GGA
AGT GGA TGG AAG TGG AGC AGT 388Pro Ser His Gly Thr Leu Pro Asn Gly
Ser Gly Trp Lys Trp Ser Ser 110 115 120TCC AAT GTG CTG ACC TTC TAT
AAC TGG GAG AGG AAC CCC TCT ATT GCT 436Ser Asn Val Leu Thr Phe Tyr
Asn Trp Glu Arg Asn Pro Ser Ile Ala 125 130 135GCT GAC CGT GGT TAT
TGT GCA GTT TTG TCT CAG AAA TCA GGT TTT CAG 484Ala Asp Arg Gly Tyr
Cys Ala Val Leu Ser Gln Lys Ser Gly Phe Gln140 145 150 155AAG TGG
AGA GAT TTT AAT TGT GAA AAT GAG CTT CCC TAT ATC TGC AAA 532Lys Trp
Arg Asp Phe Asn Cys Glu Asn Glu Leu Pro Tyr Ile Cys Lys 160 165
170TTC AAG GTC TAGGGCAGTT CTAATTTCAA CAGCTTGAAA ATATTATGAA 581Phe
Lys ValGCTCACATGG ACAAGGAAGC AAGTATGAGG ATTCACTCAG GAAGAGCAAG
CTCTGCCTAC 641ACACCCACAC CAATTCCCTT ATATCATCTC TGCTGTTTTT
CTATCAGTAT ATTCTGTGGT 701GGCTGTAACC TAAAGGCTCA GAGAACAAAA
ATAAAATGTC ATCAAC 747174 amino acidsamino acidlinearprotein 2Met
Leu Pro Met Thr Leu Cys Arg Met Ser Trp Met Leu Leu Ser Cys 1 5 10
15Leu Met Phe Leu Ser Trp Val Glu Gly Glu Glu Ser Gln Lys Lys Leu
20 25 30Pro Ser Ser Arg Ile Thr Cys Pro Gln Gly Ser Val Ala Tyr Gly
Ser 35 40 45Tyr Cys Tyr Ser Leu Ile Leu Ile Pro Gln Thr Trp Ser Asn
Ala Glu 50 55 60Leu Ser Cys Gln Met His Phe Ser Gly His Leu Ala Phe
Leu Leu Ser 65 70 75 80Thr Gly Glu Ile Thr Phe Val Ser Ser Leu Val
Lys Asn Ser Leu Thr 85 90 95Ala Tyr Gln Tyr Ile Trp Ile Gly Leu His
Asp Pro Ser His Gly Thr 100 105 110Leu Pro Asn Gly Ser Gly Trp Lys
Trp Ser Ser Ser Asn Val Leu Thr 115 120 125Phe Tyr Asn Trp Glu Arg
Asn Pro Ser Ile Ala Ala Asp Arg Gly Tyr 130 135 140Cys Ala Val Leu
Ser Gln Lys Ser Gly Phe Gln Lys Trp Arg Asp Phe145 150 155 160Asn
Cys Glu Asn Glu Leu Pro Tyr Ile Cys Lys Phe Lys Val 165 170175
amino acidsamino acidlinearproteinNORattus rattus 3Met Leu His Arg
Leu Ala Phe Pro Val Met Ser Trp Met Leu Leu Ser1 5 10 15Cys Leu Met
Leu Leu Ser Gln Val Gln Gly Glu Asp Ser Pro Lys Lys 20 25 30Ile Pro
Ser Ala Arg Ile Ser Cys Pro Lys Gly Ser Gln Ala Tyr Gly 35 40 45Ser
Tyr Cys Tyr Ala Leu Phe Gln Ile Pro Gln Thr Trp Phe Asp Ala 50 55
60Glu Leu Ala Cys Gln Lys Arg Pro Glu Gly His Leu Val Ser Val Leu65
70 75 80Asn Val Ala Glu Ala Ser Phe Leu Ala Ser Met Val Lys Asn Thr
Gly 85 90 95Asn Ser Tyr Gln Tyr Ile Trp Ile Gly Leu His Asp Pro Thr
Leu Gly 100 105 110Gly Glu Pro Asn Gly Gly Gly Trp Glu Trp Ser Asn
Asn Asp Ile Met 115 120 125Asn Tyr Val Asn Trp Glu Arg Asn Pro Ser
Thr Ala Leu Asp Arg Gly 130 135 140Phe Cys Gly Ser Leu Ser Arg Ser
Ser Gly Phe Leu Arg Trp Arg Asp145 150 155 160Thr Thr Cys Glu Val
Lys Leu Pro Tyr Val Cys Lys Phe Thr Gly 165 170 175175 amino
acidsamino acidlinearproteinHomo sapiens 4Met Leu Pro Pro Met Ala
Leu Pro Ser Val Ser Trp Met Leu Leu Ser1 5 10 15Cys Leu Met Leu Leu
Ser Gln Val Gln Gly Glu Glu Pro Gln Arg Glu 20 25 30Leu Pro Ser Ala
Arg Ile Arg Cys Pro Lys Gly Ser Lys Ala Tyr Gly 35 40 45Ser His Cys
Tyr Ala Leu Phe Leu Ser Pro Lys Ser Trp Thr Asp Ala 50 55 60Asp Leu
Ala Cys Gln Lys Arg Pro Ser Gly Asn Leu Val Ser Val Leu65 70 75
80Ser Gly Ala Glu Gly Ser Phe Val Ser Ser Leu Val Lys Ser Ile Gly
85 90 95Asn Ser Tyr Ser Tyr Val Trp Ile Gly Leu His Asp Pro Thr Gln
Gly 100 105 110Thr Glu Pro Asn Gly Glu Gly Trp Glu Trp Ser Ser Ser
Asp Val Met 115 120 125Asn Tyr Phe Ala Trp Glu Arg Asn Pro Ser Thr
Ile Ser Ser Pro Gly 130 135 140His Cys Ala Ser Leu Ser Arg Ser Thr
Ala Phe Leu Arg Trp Lys Asp145 150 155 160Tyr Asn Cys Asn Val Arg
Leu Pro Tyr Val Cys Lys Phe Thr Asp 165 170 175174 amino acidsamino
acidlinearproteinRattus rattus 5Met Leu Pro Arg Val Ala Leu Thr Thr
Met Ser Trp Met Leu Leu Ser1 5 10 15Ser Leu Met Leu Leu Ser Gln Val
Gln Gly Glu Asp Ala Lys Glu Asp 20 25 30Val Pro Thr Ser Arg Ile Ser
Cys Pro Lys Gly Ser Arg Ala Tyr Gly 35 40 45Ser Tyr Cys Tyr Ala Leu
Phe Ser Val Ser Lys Ser Trp Phe Asp Ala 50 55 60Asp Leu Ala Cys Gln
Lys Arg Pro Ser Gly His Leu Val Ser Val Leu65 70 75 80Ser Gly Ser
Glu Ala Ser Phe Val Ser Ser Leu Ile Lys Ser Ser Gly 85 90 95Asn Ser
Gly Gln Asn Val Trp Ile Gly Leu His Asp Pro Thr Leu Gly 100 105
110Gln Glu Pro Asn Arg Gly Gly Trp Glu Trp Ser Asn Ala Asp Val Met
115 120 125Asn Tyr Phe Asn Trp Glu Thr Asn Pro Ser Ser Val Ser Gly
Ser His 130 135 140Cys Gly Thr Leu Thr Arg Ala Ser Gly Phe Leu Arg
Trp Arg Glu Asn145 150 155 160Asn Cys Ile Ser Glu Leu Pro Tyr Val
Cys Lys Phe Lys Ala 165 170174 amino acidsamino
acidlinearproteinRattus rattus 6Met Leu Pro Arg Leu Ser Phe Asn Asn
Val Ser Trp Thr Leu Leu Tyr1 5 10 15Tyr Leu Phe Ile Phe Gln Val Arg
Gly Glu Asp Ser Gln Lys Ala Val 20 25 30Pro Ser Thr Arg Thr Ser Cys
Pro Met Gly Ser Lys Ala Tyr Arg Ser 35 40 45Tyr Cys Tyr Thr Leu Val
Thr Thr Leu Lys Ser Trp Phe Gln Ala Asp 50 55 60Leu Ala Cys Gln Lys
Arg Pro Ser Gly His Leu Val Ser Ile Leu Ser65 70 75 80Gly Gly Glu
Ala Ser Phe Val Ser Ser Leu Val Thr Gly Arg Val Asn 85 90 95Asn Asn
Gln Asp Ile Trp Ile Trp Leu His Asp Pro Thr Met Gly Gln 100 105
110Gln Pro Asn Gly Gly Gly Trp Glu Trp Ser Asn Ser Asp Val Leu Asn
115 120 125Tyr Leu Asn Trp Asp Gly Asp Pro Ser Ser Thr Val Asn Arg
Gly Asn 130 135 140Cys Gly Ser Leu Thr Ala Thr Ser Glu Phe Leu Lys
Trp Gly Asp His145 150 155 160His Cys Asp Val Glu Leu Pro Phe Val
Cys Lys Phe Lys Gln 165 170165 amino acidsamino
acidlinearproteinRattus rattus 7Met Thr Arg Asn Lys Tyr Phe Ile Leu
Leu Ser Cys Leu Met Val Leu1 5 10 15Ser Pro Ser Gln Gly Gln Glu Ala
Glu Glu Asp Leu Pro Ser Ala Arg 20 25 30Ile Thr Cys Pro Glu Gly Ser
Asn Ala Tyr Ser Ser Tyr Cys Tyr Tyr 35 40 45Phe Met Glu Asp His Leu
Ser Trp Ala Glu Ala Asp Leu Phe Cys Gln 50 55 60Asn Met Asn Ser Gly
Tyr Leu Val Ser Val Leu Ser Gln Ala Glu Gly65 70 75 80Asn Phe Leu
Ala Ser Leu Ile Lys Glu Ser Gly Thr Thr Ala Ala Asn 85 90 95Val Trp
Ile Gly Leu His Asp Pro Lys Asn Asn Arg Arg Trp His Trp 100 105
110Ser Ser Gly Ser Leu Phe Leu Tyr Lys Ser Trp Asp Thr Gly Tyr Pro
115 120 125Asn Asn Ser Asn Arg Gly Tyr Cys Val Ser Val Thr Ser Asn
Ser Gly 130 135 140Tyr Lys Lys Trp Arg Asp Asn Ser Cys Asp Ala Gln
Leu Ser Phe Val145 150 155 160Cys Lys Phe Lys Ala 165
* * * * *