U.S. patent application number 11/880894 was filed with the patent office on 2008-09-25 for tgf-beta type receptor cdnas encoded products and uses therefor.
Invention is credited to Herbert Y. Lin, Harvey F. Lodish, Xiao-Fan Wang, Robert A. Weinberg.
Application Number | 20080234216 11/880894 |
Document ID | / |
Family ID | 25137484 |
Filed Date | 2008-09-25 |
United States Patent
Application |
20080234216 |
Kind Code |
A1 |
Lin; Herbert Y. ; et
al. |
September 25, 2008 |
TGF-beta type receptor cDNAs encoded products and uses therefor
Abstract
DNA encoding TGF-.beta. type III receptor of mammalian origin,
DNA encoding TGF-.beta. type II receptor of mammalian origin,
TGF-.beta. type III receptor, TGF-.beta. type II receptor and uses
therefor.
Inventors: |
Lin; Herbert Y.; (Cambridge,
MA) ; Wang; Xiao-Fan; (Cambridge, MA) ;
Weinberg; Robert A.; (Brookline, MA) ; Lodish; Harvey
F.; (Brookline, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD, P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
25137484 |
Appl. No.: |
11/880894 |
Filed: |
July 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11402226 |
Apr 10, 2006 |
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11880894 |
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10420192 |
Apr 18, 2003 |
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11402226 |
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09584929 |
Jun 1, 2000 |
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10420192 |
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08446936 |
May 23, 1995 |
6201108 |
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09584929 |
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08311703 |
Sep 23, 1994 |
6010872 |
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08446936 |
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07786063 |
Oct 31, 1991 |
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08311703 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 48/00 20130101;
A61P 29/00 20180101; C07K 14/71 20130101; A61K 38/00 20130101; A61P
35/00 20180101; A61P 43/00 20180101; C07K 16/2863 20130101; C07K
14/70575 20130101; A61P 37/06 20180101 |
Class at
Publication: |
514/44 |
International
Class: |
A61K 31/711 20060101
A61K031/711; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] Work described herein was funded by National Cancer
Institute Grant No. R35-CA39826; National Heart, Lung and Blood
Institute Centers of excellence Grant HL-41484; the Damon
Runyon-Walter Winchell Cancer Research Fund; National Institutes of
Health predoctoral training grant number T 32 BM07287-16; and the
American Cancer Society. The United States government has certain
rights in the invention.
Claims
1. A method of treating a disease or condition associated with
overproduction of TGF-.beta. in an individual comprising
administering to said individual an expression vector comprising
DNA encoding a soluble TGF-.beta. receptor selected from the group
consisting of: soluble TGF-.beta. type II receptor and soluble
TGF-.beta. type III receptor, wherein said encoded soluble
TGF-.beta. receptor is expressed in an amount sufficient to reduce
the effects of said overproduction of TGF-.beta. in said
individual.
2. The method of claim 1 wherein the soluble TGF-.beta. type II
receptor has a nucleotide sequence comprising a contiguous fragment
of a) a cDNA insert contained in a plasmid deposited under ATCC
accession number 209455, b) SEQ ID NO: 7, or c) a nucleotide
sequence which encodes an amino acid of SEQ ID NO: 8, wherein the
fragment encodes a soluble TGF-.beta. type II receptor polypeptide
capable of specifically binding TGF-.beta..
3. The method of claim 1 wherein the soluble TGF-.beta. type III
receptor has a nucleotide sequence comprising a contiguous fragment
of a) a cDNA insert contained in a plasmid deposited under ATCC
accession number 75127, b) SEQ ID NO: 5, or c) a nucleotide
sequence which encodes an amino acid of SEQ ID NO: 6, wherein the
fragment encodes a soluble TGF-.beta. type III receptor polypeptide
capable of specifically binding TGF-.beta..
4. A method of treating fibrosis in an individual comprising
administering to said individual an expression vector comprising
DNA encoding a soluble TGF-.beta. receptor selected from the group
consisting of: soluble TGF-.beta. type II receptor and soluble
TGF-.beta. type III receptor, wherein said encoded soluble
TGF-.beta. receptor is expressed in an amount sufficient to reduce
the effects of said fibrosis in said individual.
5. The method of claim 4 wherein the soluble TGF-.beta. type II
receptor has a nucleotide sequence comprising a contiguous fragment
of a) a cDNA insert contained in a plasmid deposited under ATCC
accession number 209455, b) SEQ ID NO: 7, or c) a nucleotide
sequence which encodes an amino acid of SEQ ID NO: 8, wherein the
fragment encodes a soluble TGF-.beta. type II receptor polypeptide
capable of specifically binding TGF-.beta..
6. The method of claim 4 wherein the soluble TGF-.beta. type III
receptor has a nucleotide sequence comprising a contiguous fragment
of a) a cDNA insert contained in a plasmid deposited under ATCC
accession number 75127, b) SEQ ID NO: 5, or c) a nucleotide
sequence which encodes an amino acid of SEQ ID NO: 6, wherein the
fragment encodes a soluble TGF-.beta. type III receptor polypeptide
capable of specifically binding TGF-.beta..
7. The method of claim 4 wherein the fibrosis occurs in an organ of
the individual selected from the group consisting of: lung, kidney,
liver and combinations thereof.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation Application of U.S.
application Ser. No. 11/402,226, filed Apr. 10, 2006, which is a
Continuation Application of U.S. application Ser. No. 10/420,192,
filed Apr. 18, 2003 (Abandoned), which is a Continuation
Application of U.S. application Ser. No. 09/584,929, filed Jun. 1,
2000 (Abandoned), which is a Continuation Application of U.S.
application Ser. No. 08/446,936, filed May 23, 1995 (Issued as U.S.
Pat. No. 6,201,108), which is a Divisional Application of
08/311,703, filed Sep. 23, 1994 (Issued as U.S. Pat. No.
6,010,872), which is a File Wrapper Continuation of Ser. No.
07/786,063, filed Oct. 31, 1991 (Abandoned). The entire teachings
of each application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Transforming growth factor-beta (TGF-.beta.) is a member of
a family of structurally related cytokines that elicit a variety of
responses, including growth, differentiation, morphogenesis, in
many different cell types. (Roberts, A. B. and M. B. Spron, In:
Peptide Growth Factors and Their Receptors, Springer-Verlag,
Heidelberg, pp. 421-472 (1990); Massague, J., Annu. Rev. Cell.
Biol. 6: 597-641 (1990)). In vertebrates at least five different
forms of TGF-.beta., termed TGF-.beta.1 to TGF-.beta.5, have been
identified; they all share a high degree (60%-80%) of amino-acid
sequence identity. While TGF-.beta.1 was initially characterized by
its ability to induce anchorage-independent growth of normal rat
kidney cells, its effects on most cell types are anti-mitogenic.
(Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990); Andres,
J. L. et al., J. Cell. Biol. 109:3137-3145 (1989)). It is strongly
growth-inhibitory for many types of cells, including both normal
and transformed epithelial, endothelial, fibroblast, neuronal,
lymphoid, and hematopoietic cells. In addition, TGF-.beta. plays a
central role in regulating the formation of extracellular matrix
and cell-matrix adhesion processes.
[0004] In spite of its widespread effects on cell phenotype and
physiology, little is known about the biochemical mechanisms that
enable TGF-.beta. family members to elicit these varied responses.
Three distinct high-affinity cell-surface TGF-.beta. binding
proteins, termed type I, II, and III, have been identified by
incubating cells with radiolabelled TGF-.beta.1, cross-linking
bound TGF-.beta.1 to cell surface molecules, and analyzing the
labeled complexes by polyacrylamide gel electrophoresis. (Massague,
J. and B. Like, J. Biol. Chem. 260:2636-2645 (1985); Cheifetz, S.
et al. J. Biol. Chem. 261:9972-9978 (1986)). The binding constants
are about 5-50 pM for the type I and II receptor and 30-300 pM for
the type III receptor. (Boyd, F. T. and J. Massague, J. Biol. Chem.
264:2272-2278 (1989)).
[0005] The type I and II receptors, of estimated 53 and 70-100
kilodaltons mass respectively, are N-glycosylated transmembrane
proteins that are similar in many respects. Each of these receptors
has a distinct affinity for each member of the TGF-.beta. family of
ligands. (Boyd, F. T. and J. Massague, J. Biol. Chem. 264:2272-2278
(1989)). In contrast, the type III receptor shows comparable
affinities for all TGF-.beta. isotypes; the type III receptor is
the most abundant cell-surface receptor for TGF-.beta. in many cell
lines (upwards of 200,000 per cell), and is an integral membrane
proteoglycan. It is heavily modified by glycosaminoglycan (GAG)
groups, and migrates heterogeneously upon gel electrophoresis as
proteins of 280 to 330 kilodaltons. When deglycosylated with
heparitinase and chondrontinase, the protein core migrates as a
100-110 kilodalton protein. The TGF-.beta. binding site resides in
this protein core, as non-glycosylated forms of this receptor that
are produced in cell mutants defective in GAG synthesis are capable
of ligand binding with affinities comparable to those of the
natural receptor. (Cheifetz, S. and J. Massague, J. Biol. Chem.
264:12025-12028 (1989)). A variant form of type III receptor is
secreted by some types of cells as a soluble molecule that
apparently lacks a membrane anchor. This soluble series is found in
low amounts in serum and in extracellular matrix.
[0006] The type III receptor, also called betaglycan, has a
biological function distinct from that of the type I and II
receptors. Some mutant mink lung epithelial cell (Mv1Lu) selected
for loss of TGF-.beta. responsiveness no longer express type I
receptors; others, similarly selected, lose expression of both the
type I and II receptors. However, all these variants continue to
express the type III receptor. (Boyd, F. T. and J. Massague, J.
Biol. Chem. 264:2272-2278 (1989); Laiho, M. et al., J. Biol. Chem.
265.18518-18524 (1990)). This has led to the proposal that types I
and II receptors are signal-transducing molecules while the type
III receptor, may subserve some other function, such as in
concentrating ligand before presentation to the bona fide
signal-transducing receptors. The secreted form of type III
receptor, on the other hand, may act as a reservoir or clearance
system for bioactive TGF-.beta..
[0007] Additional information about each of these TGF-.beta.
receptor types would enhance our understanding of their roles and
make it possible, if desired, to alter their functions.
SUMMARY OF THE INVENTION
[0008] The present invention relates to isolation, sequencing and
characterization of DNA encoding the TGF-.beta. type III receptor
of mammalian origin and DNA encoding the TGF-.beta. type II
receptor of mammalian origin. It also relates to the encoded
TGF-.beta. type III and type II receptors, as well as to the
soluble form of each; uses of the receptor-encoding genes and of
the receptors themselves; antibodies specific for TGF-.beta. type
III receptor and antibodies specific for TGF-.beta. type II
receptor. In particular, it relates to DNA encoding the TGF-.beta.
type III receptor of rat and human origin, DNA encoding the
TGF-.beta. type II receptor of human origin and homologues of
each.
[0009] The TGF-.beta. receptor-encoding DNA of the present
invention can be used to identify equivalent TGF-.beta. receptor
type III and type II genes from other sources, using, for example,
known hybridization-based methods or the polymerase chain reaction.
The type III receptor gene, the type II receptor gene or their
respective encoded products can be used to alter the effects of
TGF-.beta. (e.g., by altering receptivity of cells to TGF-.beta. or
interfering with binding of TGF-.beta. to its receptor), such as
its effects on cell proliferation or growth, cell adhesion and cell
phenotype. For example, the TGF-.beta. receptor type III gene, the
TGF-.beta. receptor type II gene, or a truncated gene which encodes
less than the entire receptor (e.g., soluble TGF-.beta. type III
receptor, soluble TGF-.beta. type II receptor or the TGF-.beta.
type III or type II binding site) can be administered to an
individual in whom TGF-.beta. effects care to be altered.
Alternatively, the TGF-.beta. type III receptor, the TGF-.beta.
type II receptor, a soluble form thereof (i.e., a form lacking the
membrane anchor) or an active binding site of the TGF-.beta. type
III or the type II receptor can be administered to an individual to
alter the effects of TGF-.beta..
[0010] Because of the many roles TGF-.beta. has in the body,
availability of the TGF-.beta. receptors described herein makes it
possible to further assess TGF-.beta. function and to alter
(enhance or diminish) its effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1A-1C are the DNA sequence (SEQ ID NO: 5) and the
translated amino acid sequence (SEQ ID NO: 6) of type III
TGF-.beta.1 receptor cDNA clone R3-OFF (full insert size 6 kb), in
which the open reading frame with flanking sequences of the clone
are shown. The transmembrane domain is indicated by a single
underline. Peptide sequences from purified type III receptor,
mentioned in text, that correspond to the derived sequence, are in
italics and underlined. Potential N-linked glycosylation sites are
indicated by #, and extracellular cysteines by &. A consensus
protein kinase C phosphorylation site is indicated by $. The last
non-vector encoded amino acid of Clone R3-OF (2.9 kb) is indicated
by @. Consensus proteoglycan attachment site is indicated by +++.
Other potential glycosaminoglycan attachment sites are indicated by
+. The upstream in-frame stop codon (-42 to -44) is indicated by a
wavy line. Signal peptide cleavage site predicted by von Heijne's
algorithm (von Heijne, G., Nucl. Acid. Res. 14:4683-4690 (1986) is
indicated by an arrow.
[0012] FIGS. 2A-2B are the nucleotide sequence (SEQ ID NO: 7) of
the full-length type II TGF-.beta. receptor cDNA clone 3FF isolated
from a human HepG2 cell cDNA library (full insert size 5 kb). The
cDNA has an open reading frame encoding a 567 amino acid residue
protein.
[0013] FIG. 3 is the amino acid sequence (SEQ ID NO: 8) of the
full-length type II TGF-.beta. receptor.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The subject invention is based on the isolation and
sequencing of DNA of vertebrate, particularly mammalian, origin
which encodes TGF-.beta. type III receptor and DNA of mammalian
origin which encodes TGF-.beta. type II receptor, expression of the
encoded products and characterization of the expressed products. As
described, a full-length cDNA which encodes TGF-.beta. receptor
type III has been isolated from a cDNA library constructed from a
rat vascular smooth muscle cell line and a full-length cDNA which
encodes TGF-.beta. type II receptor has been isolated from a human
cDNA library. The human homologue of the type III gene has also
been cloned. A deposit of human TGF-.beta. type III cDNA in the
plasmid pBSK has been made under the terms of the Budapest Treaty
at the American Type Culture Collection (10/21/912) under Accession
Number 75127. All restrictions upon the availability of the
deposited material will be irrevocably removed upon granting of a
U.S. patent based on the subject application.
Isolation and Characterization of TGF-.beta. Type III Receptor
[0015] As described herein, two separate strategies were pursued
for the isolation of the TGF-.beta. type III receptor cDNA. In one
approach, monoclonal antibodies were generated against the type III
receptor protein and used to purify the receptor, which was then
subjected to microsequencing. (See Example 1.) Microsequencing of
several peptides resulting from partial proteolysis of the purified
receptor produced four oligopeptide sequences, which were used to
construct degenerate oligonucleotides. The degenerate
oligonucleotides were used either as primers in a cloning strategy
using the polymerase chain reaction (PCR) or as probes in screening
cDNA libraries. Although this strategy did not prove to be
productive, the oligopeptide sequences were useful in verifying the
identity of the receptor clones isolated by the second
strategy.
[0016] In the second approach to isolating TGF-.beta.
receptor-encoding clones, an expression cloning strategy was used
in COS cells; direct visualization of receptor positive cells was
used to isolate receptor cDNAs. (See Example 2). In this approach,
a cDNA library was constructed from A-10 cells, a rat vascular
smooth muscle cell line which expresses all three TGF-.beta.
receptors (Type I, II and III). COOS cells transfected with cDNA
components of this library in a vector carrying the cytomegalovirus
(CMV) transcriptional promoter and the SV40 origin of replication
were screened to identify cells expressing substantially higher
than normal levels of TGF-.beta. receptor. One transfectant
expressing such high levels of a TGF-.beta. binding protein was
identified and the original pool of expression constructs from
which it was derived was split into subpools, which were subjected
to a second round of screening. Two further rounds of sib-selection
resulted in isolation of one cDNA clone (R3-OF) with a 2.9 kb
insert which induced high levels of TGF-.beta. binding proteins in
approximable 10% of cells into which it was introduced. The
specificity of the TGF-.beta. binding was validated by showing that
addition of a 200-fold excess unlabeled competitor TGF-.beta.1
strongly reduced binding of 125 I-TGF-.beta. to transfected
cells.
[0017] The R3-OF cDNA encoded an open reading frame of 817 amino
acid residues, but did not contain a stop codon. R3-OF was used as
a probe to isolate a full-length cDNA from a rat 208F library. The
resulting clone, R3-OFF, is 6 kb in length and encodes a protein of
853 amino acids, which is colinear with clone R3-OF. The nucleotide
sequence of R3-OFF is shown in FIGS. 1A-1C, along with the
translated amino acid sequence.
[0018] Characterization of the receptor encoded by R3-OFF was
carried out, as described in Example 3. Results showed three
distinct TGF-.beta. binding protein species of TGF-.beta. on the
surface of mock-transfected COS cells, which is in accord with
results reported by others (Massague, J. et al., Ann. NY Acad. Sci.
593:59-72 (1990)). These included the two lower molecular weight
type I and II receptors (65 and 85 kD) and the higher molecular
weight type III proteoglycan, which migrates as a diffuse band of
280-330 kd. Enzymatic removal of the proteoglycan yielded a core
protein of approximately 100 kd. Binding to all three receptor
types is specific in that it was competed by 200-fold excess of
unlabeled TGF-.beta.1.
[0019] Transfecting the isolated cDNA caused a two-fold increase in
expression of the type III receptor. When a cell lysate derived
from COS cells transfected with clone R3-OFF was treated with
deglycosylating enzymes, the heterogeneous 280-330 kd band was
converted to a protein core which co-migrates with the type III
protein core seen in parental A10 cells. Importantly the
recombinant protein core migrated differently from the endogenous
COS cell type III protein core.
[0020] These observations were confirmed and extended using stably
transfected cells expressing the type III cDNA. L6 rat skeleton
muscle myoblasts do not express any detectable type III mRNA and no
endogenous surface type III receptor (Massague et al., 1986;
Segarini et al., (1989). These cells were transfected with the
isolated cDNA in the vector pcDNA-neo. Cell clones stably
expressing this clone in both the forward and reverse orientations
with respect to the CMV promoter were isolated and analyzed by
ligand binding assay.
[0021] Introduction of either the full-length clone R3-OFF or the
partial clone R3-OF in the forward orientation resulted in
expression of type III receptor. L6 cells transfected with the cDNA
clones in the reverse orientation did not express this protein.
Importantly, the apparent size of the protein core of the type III
receptor in cells transformed with the R3-OF clone is smaller than
that from R3-OFF transformed cells, consistent with the difference
in the sizes of the protein cores predicted from their nucleic acid
sequences.
[0022] Surprisingly, binding of radio-labeled ligand to the type II
receptor was increased by 2.5 fold in cells expressing the type III
cDNA. Binding to the type I receptor was unchanged. This apparently
specific up-regulation of ligand-binding to the type II receptor
was evident in all of the 15 stably transfected L6 cell lines
analyzed to date. Furthermore, this effect seems to be mediated
equally well by the full-length clone or a truncated clone (R3-OF)
that lacks the cytoplasmic domain of TGF-.beta. type III receptor
was expressed.
[0023] Expression of type III receptor mRNA was assessed by
Northern blot analysis and RNA blot analysis. Northern gel analysis
showed that the type III receptor mRNA is expressed as a single 6
kb message in several rat tissues. RNA dot blot analysis of several
different tissue culture cell lines was also carried out. Cells of
mouse origin (MEL and YH16) appear to express a smaller (.about.5.5
kb) message for the type III mRNA than those of pig, rat and human
origin. In all of these cells, expression or absence of the type
III mRNA is consistent with the expression or absence of detectable
cell surface type III receptors, with the notable exception of the
retinoblastoma cell lines (Y79, Weri-1, Weri-24, and Weri-27).
These cells lack detectable surface expression of type III
receptor, which confirms in earlier report. (Kimchi, A. et al.,
Science 240:196-198 (1988)). It is striking that the type III
receptor mRNA is expressed in these cells at a level comparable to
that of other cells that do indeed express type III receptor
proteins at readily detectable levels. It appears that TGF-.beta.
receptor III expression, which is substantial in normal
retinoblasts (AD12), has been down-regulated in these
retinoblastoma tumor cells, perhaps through post-transcriptional
mechanisms.
[0024] The nucleotide sequence full reading frame along with
flanking sequences of the full-length cDNA clone R3-OFF was
determined and is presented in FIGS. 1A-1C. The reading frame
encodes a protein of 853 amino acid residues, which is compatible
with the 100 kD size observed for the fully deglycosylated
TGF-.beta.1 type III receptor. The identity of the receptor as
TGF-.beta. type III was verified by searching for segments of the
putative transcription product which included the peptide sequences
determined by microsequencing of the isolated type III receptor.
(See Example 1). As indicated in FIGS. 1A-1C, two segments of
derived protein (underlined and italicized, residues 378-388 and
427-434) precisely match with the amino acid sequences of two
peptides (I and III) determined from direct biochemical analysis of
the purified type III receptor.
[0025] Further analysis showed that TGF-.beta. type III binding
protein has an unusual structure for a cytokine receptor.
Hydropathy analysis indicates that the protein includes a
N-terminal signal sequence, followed by a long, hydrophilic
N-terminal region. A 27 residue region of strong hydrophobicity
(underlined in FIGS. 1A-1C, residues 786-812) toward the C-terminus
represents the single putative transmembrane domain. This suggests
that nearly all of the receptor which is an N-terminal
extracellular domain is anchored to the plasma membrane near its
C-terminus. A relatively small C-terminal tail of 41 residues
represents the cytoplasmic domain.
[0026] Analysis of related sequences provides few clues to function
of TGF-.beta. type III protein. Only one other gene described to
date, a glycoprotein expressed in high quantities by endothelial
cells and termed endoglin, contains a related amino acid sequence.
The most homologous regions between the sequences of the type III
receptor and endoglin (74%) falls primarily in the putative
transmembrane and cytoplasmic domains. Similar to the general
structure of type III receptor, endoglin is a glycoprotein which
contains a large hydrophilic N-terminal domain which is presumably
extracellular, followed by a putative transmembrane domain and a
short cytoplasmic tail of 47 amino acid residues. The biological
role of endoglin is still unclear at present, although it has been
suggested that it may be involved in cell-cell recognition through
interactions of an "RGD" sequence on its ectodomain with other
adhesion molecules. Unlike the TGF-.beta. type III receptor,
endoglin does not carry GAG groups.
Isolation of TGF-.beta. Type II Receptor
[0027] The cDNA encoding the type II TGF-.beta. receptor was also
isolated, using expression cloning in COS cells. A full-length cDNA
(designated clone 3FF) was isolated by high stringency
hybridization from a human HepG2 cell cDNA library. Analysis showed
that the corresponding message is a 5 kb message which is expressed
in different cell lines and tissues. Sequence analysis indicated
that the cDNA has an open reading frame encoding a core 567 amino
acid residue protein. The nucleotide sequence of the full-length
type II TGF-.beta. receptor cDNA clone 3FF is shown in FIGS. 2A-2B;
the amino acid sequence is represented in FIG. 3.
[0028] The 567 amino acid residue protein has a single putative
transmembrane domain, several consensus glycosylation sites, and a
putative intracellular serine/threonine kinase domain. The
predicted size of the encoded protein core is .about.60 kd, which
is too large for a type I TGF-.beta. receptor. Instead, cross
linking experiments using iodinated TGF-.beta. and COS cells
transiently transfected with clone 3FF shows over-expression of a
protein approximately 70-80 kd which corresponds to the size of
type II TGF-.beta. receptors. Thus, clone 3FF encodes a protein
that specifically binds TGF-.beta. and has an expressed protein
size of 70-80 kd, both characteristic of the type II TGF-.beta.
receptor.
[0029] Plasmid 3FF was deposited in accordance with the provisions
of the Budapest Treaty at the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va., 20110-2209,
U.S.A. on Nov. 12, 1997, and assigned Accession Number ATCC
209455.
Uses of the Cloned TGF-.beta. Receptors and Related Products
[0030] For the first time, as a result of the work described
herein, DNAs encoding two of the three high affinity cell-surface
TGF-.beta. receptors have been isolated, their sequences and
expression patterns determined and the encoded proteins
characterized. Expression of the TGF-.beta. type III receptor in
cells which do not normally express the receptor, followed by
ligand binding assay, verified that the cloned type III
receptor-encoding DNA (i.e., either the full-length clone R3-OFF or
the partial clone R3-OF) encoded the receptor. In addition, the
work described herein resulted in the surprising finding that
binding of TGF-.beta. to type II receptors in cells expressing the
type III DNA was increased by 2.5 fold.
[0031] Additional insight into the role of the TGF-.beta. type III
receptor and its interaction with TGF-.beta. type II receptor is a
result of the work described. For example, the role of TGF-.beta.
type III receptor is unclear, but it has been proposed that it
serves a most unusual function of attracting and concentrating
TGF-.beta.s for eventual transfer to closely situated
signal-transducing receptors. While most cytokines bind to a single
cell surface receptor, members of the TGF-.beta. family bind with
greater or lesser affinity to three distinct cell surface proteins.
This has raised the question of why these three receptors are
displayed by most cell types and whether they subserve distinct
functions. Evidence obtained to date suggests that the type III
receptor may perform functions quite different form those of types
I and II. Thus, type III is substantially modified by GAGs while
types I and II appear to carry primarily the N-linked (and perhaps
O-linked) side chains that are characteristic of most growth factor
receptors. In addition, variant cells that have been selected for
their ability to resist TGF-.beta.-induced growth inhibition show
the absence of Type I or Type II receptors while continuing to
display Type III receptors. Together, these data have caused some
to propose that the Type I and II receptors represent bona fide
signal-transducing receptors while the type III receptor, described
here, plays another distinct role in the cell.
[0032] It remains possible that the type III receptor serves a most
unusual function of attracting and concentrating TGF-.beta.s on the
cell surface for eventual transfer to closely situated
signal-transducing receptors. Such a function would be
unprecedented for a proteinaceous receptor, although heparin
sulfate has been shown to activate basic FGF by binding to this
growth factor prior to FGF association with its receptor (Yayon, A.
et al., Cell 64:841-848 (1991). Parenthetically, since the type III
receptor also contains large quantities of heparin sulfate
side-chains, it may also bind and present basic FGF to its
receptor.
[0033] Evidence that is consistent with the role for the type III
receptor comes from the work with L6 rat myoblast cells which is
described herein. As described above, in L6 cells overexpressing
type III receptor, the binding of radiolabelled TGF-.beta. to the
type II receptor is increased several fold when compared with that
seen with parental cells. Further assessment of TGF-.beta. type III
function and interaction with type II and type I receptors will be
needed to answer these questions and can be carried out using the
materials and methods described here.
[0034] TGF-.beta. receptors, both type III and type II, can be
identified in other species, using all or a portion of the DNA
encoding the receptor to be identified as a probe and methods
described herein. For example, all or a portion of the DNA sequence
encoding TGF-.beta. type III receptor (shown in FIGS. 1A-1C) or all
or a portion of the DNA sequence encoding TGF-.beta. type II
receptor (shown in FIGS. 2A-2B) can be used to identify equivalent
sequences in other animals. Stringency conditions used can be
varied, as needed, to identify equivalent sequences in other
species. Once a putative TGF-.beta. receptor type III or type
II-encoding sequence has been identified, whether it encodes the
respective receptor type can be determined using known methods,
such as described herein for verification that the cDNA insert of
full-length clone R3-OFF and the cDNA insert of partial clone R3-OF
encode the type III receptor. For example, DNA isolated in this
manner can be expressed in an appropriate host cell which does not
express the receptor mRNA or the surface receptor (e.g., L6 rat
skeleton muscle myoblasts) and analyzed by ligand binding
(TGF-.beta. binding) assay, as described herein.
[0035] Also as a result of the work described herein, antibodies
(polyclonal or monoclonal) specific for the cloned TGF-.beta. type
III or the clones TGF-.beta. type II receptor can be produced,
using known methods. Such antibodies and host cells (e.g.,
hybridoma cells) producing the antibodies are also the subject of
the present invention. Antibodies specific for the cloned
TGF-.beta. receptor can be used to identify host cells expressing
isolated DNA thought to encode a TGF-.beta. receptor. In addition,
antibodies can be used to block or inhibit TGF-.beta. activity. For
example, antibodies specific for the cloned TGF-.beta. type III
receptor can be used to block binding of TGF-.beta. to the
receptor. They can be administered to an individual for whom
reduction of TGF-.beta. binding is desirable, such as in some
fibrotic disease (e.g., of skin, kidney and lung).
[0036] DNA and RNA encoding TGF-.beta. type III receptor and DNA or
RNA encoding TGF-.beta. type II receptor are now available. As used
herein, the term DNA or RNA encoding the respective TGF-.beta.
receptor includes any oligodeoxynucleotide or
oligodeoxyribonucleotide sequence which, upon expression, results
in production of a TGF-.beta. receptor having the functional
characteristics of the TGF-.beta. receptor. That is, the present
invention includes DNA and RNA which, upon expression in an
appropriate host cell, produces a TGF-.beta. type III receptor
which has an affinity for TGF-.beta. similar to that of the
TGF-.beta. type III receptor on naturally occurring cell surfaces
(e.g., it shows comparable affinities for all TGF-.beta. isotypes).
Similarly, the present invention includes DNA and RNA which, upon
expression in an appropriate host cell, produces a TGF-.beta. type
II receptor which has an affinity for TGF-.beta. similar to that of
TGF-.beta. type II receptor on naturally occurring cell surfaces
(e.g., it has a distinctive affinity for each member of the
TGF-.beta. family of ligands similar to that of the naturally
occurring TGF-.beta. type II receptor). The DNA or RNA can be
produced in an appropriate host cell or can be produced
synthetically (e.g., by an amplification technique such as PCR) or
chemically.
[0037] The present invention also includes the isolated TGF-.beta.
type III receptor encoded by the nucleotide sequence of full-length
R3-OFF, the isolated TGF-.beta. type III receptor encoded by the
nucleotide sequence of partial clone R3-OF, the isolated TGF-.beta.
type II receptor encoded by the nucleotide sequence of full-length
clone 3FF and TGF-.beta. type III and type II receptors which bind
TGF-.beta. isotypes with substantially the same affinity. The
isolated TGF-.beta. type III and type II receptors can be produced
by recombinant techniques, as described herein, or can be isolated
from sources in which they occur naturally or synthesized
chemically. As used herein, the terms cloned TGF-.beta. type III
and cloned TGF-.beta. type II receptors include the respective
receptors identified as described herein, and TGF-.beta. type III
and type II receptors (e.g., from other species) which exhibit
substantially the same affinity for the TGF-.beta. isotypes as the
respective receptors.
[0038] As described previously, cells in which the cloned
TGF-.beta. type III receptor is expressed bind TGF-.beta. in
essentially the same manner as do cells on which the type III
receptor occurs naturally. Further analysis of ligand interactions
with the cloned TGF-.beta. type III receptor, based upon
site-directed mutagenesis of both TGF-.beta. and the receptor, can
be carried out to identify residues important for binding. For
example, DNA having the sequence of FIGS. 1A-1C can be altered by
adding, deleting or substituting at least one nucleotide, in order
to produce a modified DNA sequence which encodes a modified cloned
TGF-.beta. type III receptor. The functional characteristics of the
modified receptor (e.g., its TGF-.beta.-binding ability and
association of the binding with effects normally resulting from
binding) can be assessed, using the methods described herein.
Modification of the cloned TGF-.beta. type III receptor can be
carried out to produce, for example, a form of the TGF-.beta. type
III receptor, referred to herein as soluble TGF-.beta. receptor,
which is not membrane bound and retains the ability to bind the
TGF-.beta. isotypes with an affinity substantially the same as the
naturally-occurring receptor. Such a TGF-.beta. type III receptor
could be produced, using known genetic engineering or synthetic
techniques; it could include none of the transmembrane region
present in the naturally-occurring TGF-.beta. type III receptor or
only a small portion of that region (i.e., small enough not to
interfere with its soluble nature). For example, it can include
amino acids 1 through 785 of the TGF-.beta. type III sequence of
FIGS. 1A-1C or a portion of that sequence sufficient to retain
TGF-.beta. binding ability (e.g., amino acids 24-785, which does
not include the signal peptide cleavage site present in the first
23 amino acids). A soluble TGF-.beta. type II receptor (e.g., one
which does not include the transmembrane and cytoplasmic domains)
can also be produced. For example, it can include amino acids 1
through 166, inclusive, of FIG. 3 or a sufficient portion thereof
to retain TGF-.beta. binding ability substantially the same as that
of TGF-.beta. type II receptor.
[0039] The TGF-.beta. type III receptor and/or type II receptor can
be used for therapeutic purposes. As described above, the
TGF-.beta. family of proteins mediate a wide variety of cellular
activities, including regulation of cell growth, regulation of cell
differentiation and control of cell metabolism. TGF-.beta. may be
essential to cell function and most cells synthesize TGF-.beta. and
have TGF-.beta. cell surface receptors. Depending on cell type and
environment, the effects of TGF-.beta. vary: proliferation can be
stimulated or inhibited, differentiation can be induced or
interrupted and cell functions can be stimulated or suppressed.
TGF-.beta. is present from embryonic stages through adult life and,
thus, can affect these key processes throughout life. The
similarities of a particular TGF-.beta. (e.g., TGF-.beta.1) across
species and from cell to cell are considerable. For example, the
amino acid sequence of a particular TGF-.beta. and the nucleotide
sequence of the gene which encodes it regardless of source, are
essentially identical across species. This further suggests that
TGF-.beta. has a critical role in essential processes.
[0040] Specifically, TGF-.beta. has been shown to have
anti-inflammatory and immune suppression capabilities, to play an
important role in bone formation (by increasing osteoblast
activity), inhibit cancer cell proliferation in culture, and
control proliferation of glandular cells of the prostate. As a
result, it has potential therapeutic applications in altering
certain immune system responses (and possibly in modifying
immune-mediated diseases); in treating systemic bone disease (e.g.,
osteoporosis) and conditions in which bone growth is to be enhanced
(e.g., repair of broken bones) and in controlling growth and
metastasis of cancer cells. In addition, TGF-.beta. appears to play
a role in determining whether some cell types undergo or do not
undergo mitosis. In this respect, TGF-.beta. may play an important
role in tissue repair. Some diseases or conditions appear to
involve low production or chronic overproduction of TGF-.beta..
(For example, results of animal studies suggest that there is a
correlation between the over production of TGF-.beta. and diseases
characterized by fibrosis in the lung, kidney, liver or in viral
mediated immune expression).
[0041] Clearly, TGF-.beta. has key roles in body processes and
numerous related potential clinical or therapeutic applications in
would healing, cancer, immune therapy and bone therapy.
Availability of TGF-.beta. receptor genes, the encoded products and
methods of using them in vitro and in vivo provides an additional
ability to control or regulate TGF-.beta. activity and effect in
the body. For example, the TGF-.beta. type II or type III receptor
encoded by the type II or the type III receptor genes of the
subject invention can be used, as appropriate, to alter the effects
of TGF-.beta. (e.g., to enhance the effect of TGF-.beta. in the
body or to inhibit or reduce (totally or partially) its effects).
It is also possible to administer to an individual in whom
TGF-.beta. bound to TGF-.beta. type III receptor, such as soluble
TGF-.beta. type III receptor. The present invention provides both a
TGF-.beta. agonist and a TGF-.beta. antagonist. For these purposes,
DNA gene encoding the entire TGF-.beta. type II or type III
receptor, the encoded type II or type III receptor or a soluble
form of either receptor can be used. Alternatively, antibodies or
other ligands designed based upon these sequences or specific for
them can be used for this purpose.
[0042] Knowledge of the amino acid sequences of TGF-.beta. type III
and type II receptors makes it possible to better understand their
structure and to design compounds which interfere with binding of
the receptor with TGF-.beta.. It makes possible identification of
existing compounds and design of new compounds which are type III
and/or type II receptor antagonists.
[0043] Cells expressing the type III and/or type II receptors of
the present invention can be used to screen compounds for their
ability to interfere with (block totally or partially) TGF binding
to the receptors. For example, cells which do not express
TGF-.beta. type III receptor (e.g., L6 rat skeleton muscle
myoblasts) but have been modified to do so by incorporation of the
type III cDNA in an appropriate vector can be used for this
purpose. A compound to be assessed is added, for example, to tissue
culture dishes containing type III expressing cells, along with
labeled TGF-.beta.. As a control, the same concentration of labeled
TGF-.beta. is added to tissue culture dishes containing the same
type of cells. After sufficient time for binding of TGF-.beta. to
the receptor to occur, binding of labeled TGF-.beta. to the cells
is assessed, using known methods (e.g., by means of a gamma
counter) and the extent to which it occurred in the presence and in
the absence of the compound to be assessed is determined.
Comparison of the two values show whether the test compound blocked
TGF-.beta. binding to the receptor (i.e., less binding in the
presence of the compound than in its absence is evidence that the
test compound has blocked binding of TGF-.beta. to the TGF-.beta.
type III receptor).
[0044] Alternatively, a cell line expressing the TGF-.beta.
receptor or cells expressing microinjected TGF-.beta. receptor RNA,
can be used to assess compounds for their ability to block
TGF-.beta. binding to the receptor. In this embodiment, a compound
to be assessed is added to tissue culture dishes containing the
cell line cells expressing microinjected TGF-.beta. receptor RNA,
along with TGF-.beta.. As a control, TGF-.beta. alone is added to
the same type of cells expressing microinjected endothelin receptor
RNA. After sufficient time for binding of TGF-.beta. to the
receptor to occur, the extent to which binding occurred is
measured, both in the presence and in the absence of the compound
to be assessed. Comparison of the two values shows whether the
compound blocked TGF-.beta. binding to the receptor. The TGF-.beta.
type III and type II receptors can also be used to identify
TGF-.beta.-like substances, to purify TGF-.beta. and to identify
TGF-.beta. regions which are responsible for binding to the
respective receptors. For example, the type III receptor can be
used in an affinity-based method to identify substances which bind
the receptor in a manner similar to TGF-.beta..
[0045] The invention will now be illustrated by the following
examples, which are not intended to be limiting in any way.
EXAMPLES
[0046] Materials and methods used in Examples 1-5 are described
below:
Materials
[0047] The following is a description of materials used in the work
described herein.
[0048] Recombinant human TGF-.beta.1 was provided by Rik Derynck of
Genetech. COS-M6 cells were provided by Brain Seed of the
Massachusetts General Hospital and Alejandro Aruffo of
Bristol-Myers-Squibb. Heparitinase was provided by Tetsuhito Kojima
and Robert Rosenberg of MIT. LLC-PK.sub.1 cells were a gift of
Dennis Ausiello of the Massachusetts General Hospital. YH-16 cell
were a gift of Edward Yeh of the Massachusetts General Hospital.
3-4 cells were a gift of Eugene Kaji of the Whitehead Institute for
Biomedical Research. All other cell lines were purchased from ATCC
and grown as specified by the supplier, except as noted.
Expression Cloning
[0049] Construction of cDNA Library and Generation of Plasmid
Pools
[0050] 10 .mu.g polyadenylated mRNA was prepared form A 10 cells by
the proteinase-K/SDS method (Gonda et. al., Molec. Cell. Biol.
2:617-624 (1982)). Double stranded cDNA was synthesized and linked
to nonpalindromic BstX1 adaptors as described by Seed, B. and A.
Aruffo, Proc. Natl. Acad. Sci. USA 84:3365-3369 (1987). Acaptored
cDNA was size-fractionated on a 5 to 20% potassium acetate
gradient, and inserts greater than 1 kb were ligated to the plasmid
vector pcDNA-1, and electroporated in the E. coli MC1061/P3,
yielding a primary library with a titer of >10.sup.7
recombinants. A portion of the cDNA was plated as pools of
.about.1.times.10.sup.4 recombinant bacteria colonies grown on 15
cm petri dishes with Luria-broth agar containing 7.5 mg/ml
tetracycline and 12.5 mg/ml ampicillin. Cells were scraped off the
plates in 10 mls of Luria-broth, and glycerol stocks of pooled
bacteria were stored at -70.degree. C. The remaining bacteria was
used to purify plasmid DNA using the alkaline lysis mini-prep
method (Sambrook, J. et al., Molecular Cloning: A Laboratory
Manual, 2d Ed. (Cold Spring Harbor, N.Y., Cold Spring Harbor
Laboratory Press (1989)).
COS Cell Transfections and Binding Assay
[0051] Plasmid pools (each representing .about.1.times.10.sup.4
clones) were transfected into COS-M6 (subclone of COS-7 cells) by
the DEAE-dextran/chloroquine method described by Seed, B. and A.
Aruffo, Proc. Natl. Acad. Sci. USA 84:3365-3369 (1987). Forty-eight
hours after transfection, cells were incubated with 50 pM
.sup.125I-TGF-.beta.1 (100 to 200 Ci/mmol) for 4 hours at 4.degree.
C.), autoradiographic analysis of transfected cells was performed
using NT-B2 photographic emulsion (Kodak) essentially as described
(Gearing, D. P. et al., EMBO J. 8:3667-3676 (1989)). After
development of slides, cells were air-dried and mounted with
mounting media and a glass coverslip. Slides were analyzed under an
Olympus OM-T1 inverted phase-contrast microscope using a dissection
trans-illuminator for darkfield illumination.
Subdivision of Positive Pool
[0052] Of 86 pools screened, one pool (#13) was identified as
positive and a glycerol stock of bacteria corresponding to this
pool was titered and 25 pools of 1000 clones were generated and
miniprep plasmid from these pools were transfected into COS cells
as described above. Several positive pools of 1000 were identified,
and one was replated as 25 plates of 100 colonies. A replica was
made of this positive plate and harvested. Once a positive pool was
identified, individual colonies were picked from the corresponding
master plate and grown overnight in 3 ml liquid culture. A
2-dimensional grid representing the 100 clones was generated and a
single clone, R3-OF, was isolated.
Cloning of R3-OFF
[0053] A 208F rat fibroblast library in lambda ZAP II (Stratagene)
was screened at high stringency with clone R3-OF insert, and
several clones with .about.6 kb inserts were isolated, one of which
is referred to as R3-OFF.
DNA Sequencing and Sequence Analysis
[0054] Double-stranded DNA was sequenced by the dideoxy chain
termination method using Sequenase reagents (United States
Biochemicals). Comparison of the sequence to the data bases was
performed using BLAST (Altschul, S. F. et al., J. Mol. Biol.
215:403-410 (1990)).
Iodination of TGF-.beta.
[0055] TGF-.beta.1 was iodinated using the chloramine-T method as
described (Cheifetz, S. and J. L. Andres, J. Biol. Chem.
263:16984-16991 (1988)).
Chemical Cross-Linking
[0056] Transfected COS cells grown on 10 cm dishes or subconfluent
L6 and A-10 cells grown on 3.5 cm dishes were incubated with
.sup.125I-TGF-.beta.1 in binding buffer (Frebs-Ringer buffered with
20 mM Hepes, pH 7.5, 5 mM MgSO.sub.4, 0.5% BSA), washed 4 times
with ice-cold binding buffer without BSA, and incubated for 15
minutes with binding buffer without BSA containing 60 ng/ml
disucinimidyl suberate at 4.degree. C. under constant rotation.
Cross linking was terminated by addition of 7% sucrose in binding
buffer. Cells were scraped, collected and pelleted by
centrifugation, then resuspended in lysis buffer (10 mM Tris, pH
7.4, 1 mM EDTA, pH 8.0, 1% Triton-X 100, 10 .mu.g/ml of pepstatin,
10 .mu.g/ml leupeptin, 10 .mu.g/ml antipain, 100 .mu.g/m;
benzamidine hydrochloride, 100 .mu.g/ml soybean trypsin inhibitor,
50 .mu.g/ml aprotonin, and 1 mM phenylmethylsulfonyl fluoride).
Solubilized material was analyzed by 7% SDS-PAGE and subjected to
autoradiographic analysis by exposure to X-AR film (Kodak) at
-70.degree. C.
Enzymatic Digestion
[0057] Digestion of solubilized TGF-.beta. receptors with
chondroitinase and heparitinase was performed as described
(Cheifetz, S. and J. L. Andres, J. Biol. Chem. 263:16984-16991
(1988); Segarini, P. R. and S. M. Seyedin, J. Biol. Chem.,
263:8366-8370 (1988)).
Generation of Stable Cell Lines
[0058] L6 myoblasts were split 1:10 into 10 cm dishes and
transfected the following day by the calcium phosphate method
(Chen, C. and H. Okayama, Molec. Cell. Biol. 7:2745-2752 (1987))
with clones R3-OF or R3-OFF in the forward and reverse orientations
in the vector pcDNA-neo (InVitrogen). Cells were subjected to
selection in the presence of G418 (Geneticin, GIBCO) for several
weeks until individual colonies were visible by the naked eye.
These clones were isolated and amplified.
RNA Blot Analyses
[0059] Rat tissue polyadenylated mRNA was prepared by the lithium
chloride/urea method (Auffrey, C. and F. Raugeon, Eur. J.
Biochemistry 107:303-313 (1980), followed by oligo-dT cellulose
chromatography (Aviv and Leder, 1972). Polyadenylated mRNA from
cell lines was prepared by the proteinase K/SDS method (Gonda, T.
J. et. al., Molec. cell. Biol. 2:617-624 (1982)). Samples of mRNA
were resolved by electrophoresis on 1% agarose-2.2M formaldehyde
gels, blotted onto nylon membranes (Biotrns, ICN) and incubated
with the 2.9 kb insert of clone R3-OF labeled with .sup.32P by
random priming as probe (Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual, 2d Ed., Cold Spring Harbor, N.Y., Cold Spring
Harbor Laboratory Press, (1989). Hybridizations were performed at
42.degree. C. in hybridization buffer containing 50% formamide
overnight, and blots were washed at 55.degree. C. in 0.2.times.SSC,
0.1% SDS, before exposure to X-AR film at -70.degree. C.
Example 1
Production of Anti-Type III Receptor Protein Antibodies and
Microsequencing of Peptides Resulting from Partial Proteolysis of
Purified Type III Receptor
[0060] Initially cellular proteoglycans were purified from human
placenta and then subjected to enzymatic deglycosylation with
heparitinase and chondroitinase. Protein cores in the molecular
weight range of 100-130 kilodaltons were further purified by
preparative gel electrophoresis; these should include the type III
receptor. This partially purified material was used as an immunogen
in mice. After screening 850 hybridoma lines developed from
immunized mice, three lines were found to produce antibodies that
specifically recognized and immunoprecipitated a deglycosylated
polypeptide species of 100-120 kD. This species could be
radiolabelled by incubation of whole cells with 125I-TGF-.beta.
followed by covalent cross-linking. Its size is consistent with
that of the protein core previously reported for the type III
receptor. (Massague, J., Annu. Rev. Cell. Biol. 6:597-641
(1990)).
[0061] Monoclonal antibody 94 was used to purify the type III
receptor from rat liver by affinity-chromatography. The purified
receptor was subjected to partial proteolysis and the resulting
peptides were resolved by high pressure liquid chromatography.
Several peptides were subjected to microsequencing and yielded the
following oligopeptide sequences:
TABLE-US-00001 Peptide I: ILLDPDHPPAL (SEQ ID NO: 1) Peptide II:
QAPFPINFMIA (SEQ ID NO: 2) Peptide III: QPIVPSVQ (SEQ ID NO: 3)
Peptide IV: FYVEQGYGR (SEQ ID NO: 4)
[0062] These peptide sequences were used to construct degenerate
oligonucleotides that served either as primers in a cloning
strategy using the spolymerase chain reaction (PCR) or as probes in
screening cDNA libraries. While this strategy was not productive,
the oligopeptide sequences proved useful in verifying the receptor
clones isolated by the second, alternative strategy (see Example
2).
Example 2
Expression Cloning of the Type III Receptor cDNA
[0063] An expression cloning strategy in COS cells, a procedure
which takes advantage of the considerable amplification of
individual cDNAs in transfected COS cells was used as an
alternative means to isolate TGF-.beta. receptor clones. Such
amplification is mediated by SV40 large T antigen expressed by the
COS cells interacting with a SV40 origin of replication in the cDNA
vector. Gearing, D. et. al., EMBO J. 8:3667-3676 (1989); Lin, H.
Y., et al., Proc. Natl. Acad. Sci. 88:3185-3189 (1991a); Lin, H. Y.
et. al., Science, in press (1991); Mathews, L. S. and Vale, W. W.,
Cell 65:973-982 (1991).
[0064] The strategy involved the construction of a cDNA library
from A-10 cells, a rat vascular smooth muscle cell line that
expresses all three high-affinity TGF-.beta. receptors. The
resulting cDNAs were inserted into the vector pcDNA-1, which
carries the CMV transcriptional promoter and the SV40 origin of
replication. The resulting library was then divided into pools of
10,000 independent recombinants each and DNA from each pool was
transfected into 1.5.times.10.sup.6 COS-7 cells grown on glass
flaskettes by means of DEAE-dextran transfection procedure. Aruffo,
A. and Seed, B., Proc. Natl. Acad. Sci. U.S.A. 84:8573-8577 (1987);
Gearing, D. et al, EMBO J. 8:3667-3676 (1989); Mathews, L. S. and
Vale, W. W., Cell 65:973-982 (1991). The transfected cells were
cultured for 48-60 hours and then exposed to radiolabelled
TGF-.beta.1 for four hours. Following this treatment, the glass
slides carrying these cells were washed extensively and fixed.
These slides were dipped in liquid photographic emulsion and
examined by darkfield microscopy. While all of the receptor genes
cloned to date by this procedure have undetectable or low
constitutive levels of expression in COS cells, we were hindered by
the fact that untransfected COS cells already express substantial
amounts of type III TGF-.beta. receptor. Such expression, estimated
to be approximately 2.times.10.sup.5 receptor molecules per cell,
might well have generated an unacceptably high level of background
binding. However, since the detection procedure involves
visualizing radiolabelled ligand-binding on individual cells, it
was hoped that identifying occasional cells expressing
substantially higher levels of vector-encoded receptor would be
possible. This hope was vindicated in the initial experiments.
[0065] After screening nearly one million cDNA clones in this
manner, a glass slide containing 20 positive transfectants was
identified. The original pool of expression constructs from which
one such transfectant was derived was split into 25 subpools of
1000 clones each and these were subjected to a second round of
screening. Two further rounds of sib-selection resulted in the
isolation of a cDNA clone (R3-OF) with a 2.9 kb insert that induced
high levels of TGF-.beta.-binding proteins in approximately 10% of
COS cells into which it was transfected.
[0066] The specificity of this binding was validated by showing
that addition of a 200-fold excess of unlabeled TGF-.beta.
competitor strongly reduced binding of .sup.125I-TGF-.beta. to
transfected cells. By taking into account a transfection efficiency
of 10% and the high background of endogenous receptor expression,
we calculated that the level of total .sup.125I-TGF-.beta. binding
to each glass slide of cells transfected with the cDNA clone was
only 2-fold above the level seen with mock transfectants (data not
shown). Nonetheless, this marginal increase in ligand-binding was
sufficient to identify rare transfectants amidst a large field of
cells expressing this background level of receptor.
[0067] The R3-OF cDNA encoded an open reading frame of 836 amino
acid residues of which the 3' most 18 were encoded by vector
sequence, clearly indicating that clone R3-OF was an incomplete
cDNA insert which ended prematurely at the codon specifying alanine
818. R3-OF was used as a probe to isolate a full-length cDNA from a
rat 208F lambda phage library. This clone, termed R3-OFF, was 6 kb
in length and encoded a protein of 853 amino acids; its sequence
was co-linear with that of clone R3-OF.
Example 3
Characterization of the Product of the Full Length Clone R3-OFF
[0068] Characterization of the product of the full length clone
R3-OFF was undertaken in order to determine which of the three
TGF-.beta. receptors it specified. To do so, COS transfectants were
incubated with radioiodinated TGF-.beta., chemical crosslinker was
added and the labeled receptors were resolved by polyacrylamide gel
electrophoresis.
[0069] Labeling of cell surface TGF-.beta. receptors in this way
resulted in the detection of three distinct species on the surface
of COS cells, as extensively by others (Massague, J. et al., Ann.
NY Acad. Sci. 593:59-72 (1990). These included the two lower
molecular weight type I and II receptors (65 and 85 kD) and the
higher molecular weight type III proteoglycan, which migrated as a
diffuse band of 280-330 kd. Enzymatic treatment of the proteoglycan
with chondroitinase and heparitinase yielded a core protein of
approximately 100 kd. Binding to all three receptor types was
specific, in that it was completed by 200-fold excess of unlabeled
TGF-.beta.1.
[0070] Transfecting the R3-OFF cDNA caused a two-fold increase in
expression of the type III receptor. When a cell lysate derived
form COS cells transfected with clone R3-OFF was treated with
deglycosylating enzymes, the heterogenous 280-330 kb band was
converted to a protein core which co-migrated with the type III
protein core seen in untransfected A10 cells. Importantly, the
recombinant protein core migrates differently from the endogenous
COS cell type III protein core.
[0071] These observations were confirmed and extended in
experiments using stably transfected cells expressing the R3-OFF
cDNA. L6 rat skeleton muscle myoblasts normally do not express
detectable type III mRNA or endogenous type III receptor
(determined by radiolabelled ligand-binding assay). Such cells were
transfected with the isolated cDNA in the vector pcDNA-neo. Cell
clones stably expressing this clone in both the forward and reverse
orientations with respect to the CMV promoter were isolated and
analyzed by ligand-binding assay.
[0072] Introduction of either the full length clone R3-OFF or the
partial clone R3-OF in the forward orientation led to the de novo
expression of the type III receptor. L6 cells transfected with the
cDNA in reversed orientation did not express this protein. The
apparent size of the protein core of the type III receptor in cells
transfected with the R3-OF clone is smaller than that expressed by
R3-OFF transfected cells, consistent with the difference in the
sizes of the protein cores predicted form the respective nucleic
acid sequences (FIGS. 1A-1C).
[0073] Unexpectedly, the amount of radio-labeled ligand
cross-linked to the type II receptor is increased by 2.5 fold in
cells expressing the type III cDNA, while the amount cross-linked
to the type I receptor remained unchanged. The apparent specific
up-regulation of ligand-binding to the type II receptor could be
detected with all of the stably transfected L6 cell lines analyzed
so far. This effect seems to be mediated by the truncated clone
R3-OF which lacks the cytoplasmic domain as well as by the
full-length clone R3-OFF.
Example 4
Expression of Type III Receptor
[0074] Northern blot analysis demonstrated that the type III
receptor mRNA is expressed as a single 6 kb message in several rat
tissues. The level of mRNA expression in the liver was less than in
other tissues, a result expected from earlier surveys of various
tissues using radioiodinated TGF-.beta.1. Based on this
information, it appears that clone R3-OFF, with a .about.6 kb cDNA
insert, represents a full length rat type III cDNA clone.
[0075] Cells of mouse origin (MEL and YH16) appear to express a
smaller (.about.5.5 kb) message for the type III mRNA than those of
pig, rat and human origin. In all of these cells, expression or
absence of the type III mRNA is consistent with the expression or
absence of detectable cell surface type III receptors with the
notable exception of the retinoblastoma cell lines (Y79, Weri-1,
Weri-24, and Weri-27). These cells have previously been shown to
lack detectable surface expression of type III receptor, a result
confirmed by our own unpublished work. It is striking that the type
III receptor mRNA is expressed in these cells at a level comparable
to that of other cells that do indeed express type III receptor
proteins at readily detectable levels. At this moment, we can only
conclude that TGF-.beta. receptor III expression, which is
substantial in normal retinoblasts (AD12), has been down-regulated
in these retinoblastoma tumor cells, perhaps through
post-transcriptional mechanisms.
Example 5
Sequence Analysis of the Full-Length Type III cDNA
[0076] The full-length cDNA clone (R3-OFF), described in Example 4,
was subjected to sequence analysis. The full reading frame along
with flanking sequences is presented in FIGS. 1A-1C. This reading
frame encodes a protein of 853 amino acid residues, which is
compatible with the 100 kD observed for the fully deglycosylated
TGF-.beta. type III receptor.
[0077] Two segments of derived protein sequence (underlined and
italicized, residues 378-388 and 427-434) precisely match those
determined earlier form direct biochemical analysis of the purified
receptor protein. This further secured the identity of this
isolated cDNA clone as encoding the rat type III receptor.
[0078] This TGF-.beta. binding protein has an unusual structure for
a cytokine receptor. Hydropathy analysis indicates a N-terminal
signal sequence, followed by a long, hydrophilic N-terminal region
(Kyte, J. and R. F. Doolittle, J. Mol. Biol. 157:105-132 (1982)). A
27 residue region of strong hydrophobicity (underlined, residues
786-812) toward the C-terminus represents the single putative
transmembrane domain. This suggests that nearly all of the receptor
is composed of an N-terminal extracellular domain that is anchored
to the plasma membrane near its C-terminus. A relatively short
C-terminal tail of 41 residues represents the putative cytoplasmic
domain.
[0079] The clone R3-OF was also analyzed and found to be a
truncated version of R3-OFF, with an identical open reading frame
but whose last encoded residue is alanine 818 (FIGS. 1A-1C).
[0080] In R3-OFF there are six consensus N-linked glycosylation
sites and 15 cysteines (indicated in FIGS. 1A-1C). There is at
least one consensus glycosaminoglycan addition site at serine 535
(Bernfield, M. and K. C. Hooper, Ann. N.Y. Acad. Sci. in press
(1991), and numerous Ser-Gly residues that are potential sites for
GAG conjugation. A consensus protein kinase C site is also present
at residue 817.
[0081] Only one other gene described to date, a glycoprotein
expressed in high quantities by endothelial cells and termed
endoglin (Gougos and Letarte, 1990), contains a related amino acid
sequence. Overall, there is .about.30% identity with the type III
receptor over the entire 645 amino acid residue length of endoglin.
The most homologous regions between the sequences of the type III
receptor and endoglin (74% identity) falls primarily in the
putative transmembrane and cytoplasmic domains. Similar to the
general structure of type III receptor, endoglin is a glycoprotein
which contains a large hydrophilic and presumably extracellular
N-terminal domain followed by a putative transmembrane domain and a
short cytoplasmic tail of 47 amino acid residues. The biological
role of endoglin is unclear, though it has been suggested that it
may involve cell-cell recognition through interactions of an "RGD"
sequence on its ectodomain with other adhesion molecules. Unlike
the TGF-.beta. type III receptor, endoglin does not carry GAG
groups.
EQUIVALENTS
[0082] Those skilled in the art will recognize, or be able to
ascertain using not 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
8111PRTArtificial SequenceOligopeptide sequence 1Ile Leu Leu Asp
Pro Asp His Pro Pro Ala Leu 1 5 10211PRTArtificial
SequenceOligopeptide sequence 2Gln Ala Pro Phe Pro Ile Asn Phe Met
Ile Ala 1 5 1038PRTArtificial SequenceOligopeptide sequence 3Gln
Pro Ile Val Pro Ser Val Gln 1 549PRTArtificial SequenceOligopeptide
sequence 4Phe Tyr Val Glu Gln Gly Tyr Gly Arg 1
553237DNAgenomicCDS(241)...(2799) 5caggaggtga aagtccccgg cggtccggat
ggcgcagttg cactgcgctg ctgagctcgc 60ggccgcctgc gcacactggg gggactcgct
tcggctagta actcctccac ctcgcggcgg 120acgaccggtc ctggacacgc
tgcctgcgag gcaagttgaa cagtgcagag aaggatctta 180aagctacacc
cgacttgcca cgattgcctt caatctgaag aaccaaaggc tgttggagag 240atg gca
gtg aca tcc cac cac atg atc ccg gtg atg gtt gtc ctg atg 288Met Ala
Val Thr Ser His His Met Ile Pro Val Met Val Val Leu Met 1 5 10
15agc gcc tgc ctg gcc acc gcc ggt cca gag ccc agc acc cgg tgt gaa
336Ser Ala Cys Leu Ala Thr Ala Gly Pro Glu Pro Ser Thr Arg Cys Glu
20 25 30ctg tca cca atc aac gcc tct cac cca gtc cag gcc ttg atg gag
agc 384Leu Ser Pro Ile Asn Ala Ser His Pro Val Gln Ala Leu Met Glu
Ser 35 40 45ttc acc gtt ctg tct ggc tgt gcc agc aga ggc acc acc ggg
ctg cca 432Phe Thr Val Leu Ser Gly Cys Ala Ser Arg Gly Thr Thr Gly
Leu Pro 50 55 60agg gag gtc cat gtc cta aac ctc cga agt aca gat cag
gga cca ggc 480Arg Glu Val His Val Leu Asn Leu Arg Ser Thr Asp Gln
Gly Pro Gly 65 70 75 80cag cgg cag aga gag gtt acc ctg cac ctg aac
ccc att gcc tcg gtg 528Gln Arg Gln Arg Glu Val Thr Leu His Leu Asn
Pro Ile Ala Ser Val 85 90 95cac act cac cac aaa cct atc gtg ttc ctg
ctc aac tcc ccc cag ccc 576His Thr His His Lys Pro Ile Val Phe Leu
Leu Asn Ser Pro Gln Pro 100 105 110ctg gtg tgg cat ctg aag acg gag
aga ctg gcc gct ggt gtc ccc aga 624Leu Val Trp His Leu Lys Thr Glu
Arg Leu Ala Ala Gly Val Pro Arg 115 120 125ctc ttc ctg gtt tcg gag
ggt tct gtg gtc cag ttt cca tca gga aac 672Leu Phe Leu Val Ser Glu
Gly Ser Val Val Gln Phe Pro Ser Gly Asn 130 135 140ttc tcc ttg aca
gca gaa aca gag gaa agg aat ttc cct caa gaa aat 720Phe Ser Leu Thr
Ala Glu Thr Glu Glu Arg Asn Phe Pro Gln Glu Asn145 150 155 160gaa
cat ctc gtg cgc tgg gcc caa aag gaa tat gga gca gtg act tcg 768Glu
His Leu Val Arg Trp Ala Gln Lys Glu Tyr Gly Ala Val Thr Ser 165 170
175ttc act gaa ctc aag ata gca aga aac atc tat att aaa gtg gga gaa
816Phe Thr Glu Leu Lys Ile Ala Arg Asn Ile Tyr Ile Lys Val Gly Glu
180 185 190gat caa gtg ttt cct cct acg tgt aac ata ggg aag aat ttc
ctc tca 864Asp Gln Val Phe Pro Pro Thr Cys Asn Ile Gly Lys Asn Phe
Leu Ser 195 200 205ctc aat tac ctt gcc gag tac ctt caa ccc aaa gcc
gcc gaa ggt tgt 912Leu Asn Tyr Leu Ala Glu Tyr Leu Gln Pro Lys Ala
Ala Glu Gly Cys 210 215 220gtc ctg ccc agt cag ccc cat gaa aag gaa
gta cac atc atc gag tta 960Val Leu Pro Ser Gln Pro His Glu Lys Glu
Val His Ile Ile Glu Leu225 230 235 240att acc ccc agc tcg aac cct
tac agc gct ttc cag gtg gat ata ata 1008Ile Thr Pro Ser Ser Asn Pro
Tyr Ser Ala Phe Gln Val Asp Ile Ile 245 250 255gtt gac ata cga cct
gct caa gag gat ccc gag gtg gtc aaa aac ctt 1056Val Asp Ile Arg Pro
Ala Gln Glu Asp Pro Glu Val Val Lys Asn Leu 260 265 270gtc ctg atc
ttg aag tgc aaa aag tct gtc aac tgg gtg atc aag tct 1104Val Leu Ile
Leu Lys Cys Lys Lys Ser Val Asn Trp Val Ile Lys Ser 275 280 285ttt
gac gtc aag gga aac ttg aaa gtc att gct ccc aac agt atc ggc 1152Phe
Asp Val Lys Gly Asn Leu Lys Val Ile Ala Pro Asn Ser Ile Gly 290 295
300ttt gga aaa gag agt gaa cga tcc atg aca atg acc aaa ttg gta aga
1200Phe Gly Lys Glu Ser Glu Arg Ser Met Thr Met Thr Lys Leu Val
Arg305 310 315 320gat gac atc cct tcc acc caa gag aat ctg atg aag
tgg gca ctg gac 1248Asp Asp Ile Pro Ser Thr Gln Glu Asn Leu Met Lys
Trp Ala Leu Asp 325 330 335aat ggc tac agg cca gtg acg tca tac aca
atg gct ccc gtg gct aat 1296Asn Gly Tyr Arg Pro Val Thr Ser Tyr Thr
Met Ala Pro Val Ala Asn 340 345 350aga ttt cat ctt cgg ctt gag aac
aac gag gag atg aga gat gag gaa 1344Arg Phe His Leu Arg Leu Glu Asn
Asn Glu Glu Met Arg Asp Glu Glu 355 360 365gtc cac acc att cct cct
gag ctt cgt atc ctg ctg gac cct gac cac 1392Val His Thr Ile Pro Pro
Glu Leu Arg Ile Leu Leu Asp Pro Asp His 370 375 380ccg ccc gcc ctg
gac aac cca ctc ttc cca gga gag gga agc cca aat 1440Pro Pro Ala Leu
Asp Asn Pro Leu Phe Pro Gly Glu Gly Ser Pro Asn385 390 395 400ggt
ggt ctc ccc ttt cca ttc ccg gat atc ccc agg aga ggc tgg aag 1488Gly
Gly Leu Pro Phe Pro Phe Pro Asp Ile Pro Arg Arg Gly Trp Lys 405 410
415gag ggc gaa gat agg atc ccc cgg cca aag cag ccc atc gtt ccc agt
1536Glu Gly Glu Asp Arg Ile Pro Arg Pro Lys Gln Pro Ile Val Pro Ser
420 425 430gtt caa ctg ctt cct gac cac cga gaa cca gaa gaa gtg caa
ggg ggc 1584Val Gln Leu Leu Pro Asp His Arg Glu Pro Glu Glu Val Gln
Gly Gly 435 440 445gtg gac atc gcc ctg tca gtc aaa tgt gac cat gaa
aag atg gtc gtg 1632Val Asp Ile Ala Leu Ser Val Lys Cys Asp His Glu
Lys Met Val Val 450 455 460gct gta gac aaa gac tct ttc cag acc aat
ggc tac tca ggg atg gag 1680Ala Val Asp Lys Asp Ser Phe Gln Thr Asn
Gly Tyr Ser Gly Met Glu465 470 475 480ctc acc ctg ttg gat cct tcg
tgt aaa gcc aaa atg aat ggt act cac 1728Leu Thr Leu Leu Asp Pro Ser
Cys Lys Ala Lys Met Asn Gly Thr His 485 490 495ttt gtt ctc gag tct
ccc ctg aat ggc tgt ggt act cga cat cgg agg 1776Phe Val Leu Glu Ser
Pro Leu Asn Gly Cys Gly Thr Arg His Arg Arg 500 505 510tcg acc ccg
gat ggt gtg gtt tac tat aac tct att gtg gtg cag gct 1824Ser Thr Pro
Asp Gly Val Val Tyr Tyr Asn Ser Ile Val Val Gln Ala 515 520 525ccg
tcc cct ggg gat agc agt ggc tgg cct gat ggc tat gaa gac ttg 1872Pro
Ser Pro Gly Asp Ser Ser Gly Trp Pro Asp Gly Tyr Glu Asp Leu 530 535
540gag tca ggc gat aat gga ttt cct gga gac ggg gat gaa gga gaa act
1920Glu Ser Gly Asp Asn Gly Phe Pro Gly Asp Gly Asp Glu Gly Glu
Thr545 550 555 560gcc ccc ctg agc cga gct gga gtg gtg gtg ttt aac
tgc agc ttg cgg 1968Ala Pro Leu Ser Arg Ala Gly Val Val Val Phe Asn
Cys Ser Leu Arg 565 570 575cag ctg agg aat ccc agt ggc ttc cag ggc
cag ctc gat gga aat gct 2016Gln Leu Arg Asn Pro Ser Gly Phe Gln Gly
Gln Leu Asp Gly Asn Ala 580 585 590acc ttc aac atg gag ctg tat aac
aca gac ctc ttt ctg gtg ccc tcc 2064Thr Phe Asn Met Glu Leu Tyr Asn
Thr Asp Leu Phe Leu Val Pro Ser 595 600 605cca ggg gtc ttc tct gtg
gca gag aac gag cat gtt tat gtt gag gtg 2112Pro Gly Val Phe Ser Val
Ala Glu Asn Glu His Val Tyr Val Glu Val 610 615 620tct gtc acc aag
gct gac caa gat ctg gga ttc gcc atc caa acc tgc 2160Ser Val Thr Lys
Ala Asp Gln Asp Leu Gly Phe Ala Ile Gln Thr Cys625 630 635 640ttt
ctc tct cca tac tcc aac cca gac aga atg tct gat tac acc atc 2208Phe
Leu Ser Pro Tyr Ser Asn Pro Asp Arg Met Ser Asp Tyr Thr Ile 645 650
655atc gag aac atc tgt ccg aaa gac gac tct gtg aag ttc tac agc tcc
2256Ile Glu Asn Ile Cys Pro Lys Asp Asp Ser Val Lys Phe Tyr Ser Ser
660 665 670aag aga gtg cac ttt ccc atc ccg cat gct gag gtg gac aag
aag cgc 2304Lys Arg Val His Phe Pro Ile Pro His Ala Glu Val Asp Lys
Lys Arg 675 680 685ttc agc ttc ctg ttc aag tct gtg ttc aac acc tcc
ctg ctc ttc ctg 2352Phe Ser Phe Leu Phe Lys Ser Val Phe Asn Thr Ser
Leu Leu Phe Leu 690 695 700cac tgc gag ttg act ctg tgc tcc agg aag
aag ggc tcc ctg aag ctg 2400His Cys Glu Leu Thr Leu Cys Ser Arg Lys
Lys Gly Ser Leu Lys Leu705 710 715 720ccg agg tgt gtg act cct gac
gac gcc tgc act tct ctc gat gcc acc 2448Pro Arg Cys Val Thr Pro Asp
Asp Ala Cys Thr Ser Leu Asp Ala Thr 725 730 735atg atc tgg acc atg
atg cag aat aag aag aca ttc acc aag ccc ctg 2496Met Ile Trp Thr Met
Met Gln Asn Lys Lys Thr Phe Thr Lys Pro Leu 740 745 750gct gtg gtc
ctc cag gta gac tat aaa gaa aat gtt ccc agc act aag 2544Ala Val Val
Leu Gln Val Asp Tyr Lys Glu Asn Val Pro Ser Thr Lys 755 760 765gat
tcc agt cca att cct cct cct cct cca cag att ttc cat ggc ctg 2592Asp
Ser Ser Pro Ile Pro Pro Pro Pro Pro Gln Ile Phe His Gly Leu 770 775
780gac acg ctc acc gtg atg ggc att gca ttt gca gca ttt gtg atc gga
2640Asp Thr Leu Thr Val Met Gly Ile Ala Phe Ala Ala Phe Val Ile
Gly785 790 795 800gcg ctc ctg acg ggg gcc ttg tgg tac atc tac tcc
cac aca ggg gag 2688Ala Leu Leu Thr Gly Ala Leu Trp Tyr Ile Tyr Ser
His Thr Gly Glu 805 810 815aca gca cga agg cag caa gtc cct acc tcg
ccg cca gcc tcg gag aac 2736Thr Ala Arg Arg Gln Gln Val Pro Thr Ser
Pro Pro Ala Ser Glu Asn 820 825 830agc agc gcg gcc cac agc atc ggc
agc act cag agt acc ccc tgc tct 2784Ser Ser Ala Ala His Ser Ile Gly
Ser Thr Gln Ser Thr Pro Cys Ser 835 840 845agc agc agc aca gcc
taggtggaca gacagacgcc cgcccaccgc agccagggca 2839Ser Ser Ser Thr Ala
850gggcccgatg ccagtgctgc gtgtccacag tcagaagtct tgatctgggc
tccctgtaaa 2899gaaagagtga atttcagtat acagacagcc agttctaccc
accccttacc acggcccaca 2959taaatgtgac cctgggcatc tgtcacacga
aagctaagct ggtggccttc cccaccagcc 3019cctcgcagga tgggggtttc
aatgtgaaac atctgccagt tttgttttgt ttttttaatg 3079ctgctttgtc
caggtgtcca aacatccatc atttggggtg gtctgtttta cagagtaaag
3139gaggcggtga agggacgtca gctagtgtgt agagccaagg ggagacagct
aggattctcg 3199cctagctgaa ccaaggtgta aaatagaaga cacgctcc
32376853PRTgenomic 6Met Ala Val Thr Ser His His Met Ile Pro Val Met
Val Val Leu Met 1 5 10 15Ser Ala Cys Leu Ala Thr Ala Gly Pro Glu
Pro Ser Thr Arg Cys Glu 20 25 30Leu Ser Pro Ile Asn Ala Ser His Pro
Val Gln Ala Leu Met Glu Ser 35 40 45Phe Thr Val Leu Ser Gly Cys Ala
Ser Arg Gly Thr Thr Gly Leu Pro 50 55 60Arg Glu Val His Val Leu Asn
Leu Arg Ser Thr Asp Gln Gly Pro Gly65 70 75 80Gln Arg Gln Arg Glu
Val Thr Leu His Leu Asn Pro Ile Ala Ser Val 85 90 95His Thr His His
Lys Pro Ile Val Phe Leu Leu Asn Ser Pro Gln Pro 100 105 110Leu Val
Trp His Leu Lys Thr Glu Arg Leu Ala Ala Gly Val Pro Arg 115 120
125Leu Phe Leu Val Ser Glu Gly Ser Val Val Gln Phe Pro Ser Gly Asn
130 135 140Phe Ser Leu Thr Ala Glu Thr Glu Glu Arg Asn Phe Pro Gln
Glu Asn145 150 155 160Glu His Leu Val Arg Trp Ala Gln Lys Glu Tyr
Gly Ala Val Thr Ser 165 170 175Phe Thr Glu Leu Lys Ile Ala Arg Asn
Ile Tyr Ile Lys Val Gly Glu 180 185 190Asp Gln Val Phe Pro Pro Thr
Cys Asn Ile Gly Lys Asn Phe Leu Ser 195 200 205Leu Asn Tyr Leu Ala
Glu Tyr Leu Gln Pro Lys Ala Ala Glu Gly Cys 210 215 220Val Leu Pro
Ser Gln Pro His Glu Lys Glu Val His Ile Ile Glu Leu225 230 235
240Ile Thr Pro Ser Ser Asn Pro Tyr Ser Ala Phe Gln Val Asp Ile Ile
245 250 255Val Asp Ile Arg Pro Ala Gln Glu Asp Pro Glu Val Val Lys
Asn Leu 260 265 270Val Leu Ile Leu Lys Cys Lys Lys Ser Val Asn Trp
Val Ile Lys Ser 275 280 285Phe Asp Val Lys Gly Asn Leu Lys Val Ile
Ala Pro Asn Ser Ile Gly 290 295 300Phe Gly Lys Glu Ser Glu Arg Ser
Met Thr Met Thr Lys Leu Val Arg305 310 315 320Asp Asp Ile Pro Ser
Thr Gln Glu Asn Leu Met Lys Trp Ala Leu Asp 325 330 335Asn Gly Tyr
Arg Pro Val Thr Ser Tyr Thr Met Ala Pro Val Ala Asn 340 345 350Arg
Phe His Leu Arg Leu Glu Asn Asn Glu Glu Met Arg Asp Glu Glu 355 360
365Val His Thr Ile Pro Pro Glu Leu Arg Ile Leu Leu Asp Pro Asp His
370 375 380Pro Pro Ala Leu Asp Asn Pro Leu Phe Pro Gly Glu Gly Ser
Pro Asn385 390 395 400Gly Gly Leu Pro Phe Pro Phe Pro Asp Ile Pro
Arg Arg Gly Trp Lys 405 410 415Glu Gly Glu Asp Arg Ile Pro Arg Pro
Lys Gln Pro Ile Val Pro Ser 420 425 430Val Gln Leu Leu Pro Asp His
Arg Glu Pro Glu Glu Val Gln Gly Gly 435 440 445Val Asp Ile Ala Leu
Ser Val Lys Cys Asp His Glu Lys Met Val Val 450 455 460Ala Val Asp
Lys Asp Ser Phe Gln Thr Asn Gly Tyr Ser Gly Met Glu465 470 475
480Leu Thr Leu Leu Asp Pro Ser Cys Lys Ala Lys Met Asn Gly Thr His
485 490 495Phe Val Leu Glu Ser Pro Leu Asn Gly Cys Gly Thr Arg His
Arg Arg 500 505 510Ser Thr Pro Asp Gly Val Val Tyr Tyr Asn Ser Ile
Val Val Gln Ala 515 520 525Pro Ser Pro Gly Asp Ser Ser Gly Trp Pro
Asp Gly Tyr Glu Asp Leu 530 535 540Glu Ser Gly Asp Asn Gly Phe Pro
Gly Asp Gly Asp Glu Gly Glu Thr545 550 555 560Ala Pro Leu Ser Arg
Ala Gly Val Val Val Phe Asn Cys Ser Leu Arg 565 570 575Gln Leu Arg
Asn Pro Ser Gly Phe Gln Gly Gln Leu Asp Gly Asn Ala 580 585 590Thr
Phe Asn Met Glu Leu Tyr Asn Thr Asp Leu Phe Leu Val Pro Ser 595 600
605Pro Gly Val Phe Ser Val Ala Glu Asn Glu His Val Tyr Val Glu Val
610 615 620Ser Val Thr Lys Ala Asp Gln Asp Leu Gly Phe Ala Ile Gln
Thr Cys625 630 635 640Phe Leu Ser Pro Tyr Ser Asn Pro Asp Arg Met
Ser Asp Tyr Thr Ile 645 650 655Ile Glu Asn Ile Cys Pro Lys Asp Asp
Ser Val Lys Phe Tyr Ser Ser 660 665 670Lys Arg Val His Phe Pro Ile
Pro His Ala Glu Val Asp Lys Lys Arg 675 680 685Phe Ser Phe Leu Phe
Lys Ser Val Phe Asn Thr Ser Leu Leu Phe Leu 690 695 700His Cys Glu
Leu Thr Leu Cys Ser Arg Lys Lys Gly Ser Leu Lys Leu705 710 715
720Pro Arg Cys Val Thr Pro Asp Asp Ala Cys Thr Ser Leu Asp Ala Thr
725 730 735Met Ile Trp Thr Met Met Gln Asn Lys Lys Thr Phe Thr Lys
Pro Leu 740 745 750Ala Val Val Leu Gln Val Asp Tyr Lys Glu Asn Val
Pro Ser Thr Lys 755 760 765Asp Ser Ser Pro Ile Pro Pro Pro Pro Pro
Gln Ile Phe His Gly Leu 770 775 780Asp Thr Leu Thr Val Met Gly Ile
Ala Phe Ala Ala Phe Val Ile Gly785 790 795 800Ala Leu Leu Thr Gly
Ala Leu Trp Tyr Ile Tyr Ser His Thr Gly Glu 805 810 815Thr Ala Arg
Arg Gln Gln Val Pro Thr Ser Pro Pro Ala Ser Glu Asn 820 825 830Ser
Ser Ala Ala His Ser Ile Gly Ser Thr Gln Ser Thr Pro Cys Ser 835 840
845Ser Ser Ser Thr Ala 85072090DNAgenomicCDS(336)...(2038)
7gttggcgagg agtttcctgt ttcccccgca gcgctgagtt gaagttgagt gagtcactcg
60cgcgcacgga gcgacgacac ccccgcgcgt gcacccgctc gggacaggag ccggactcct
120gtgcagcttc cctcggccgc cgggggcctc cccgcgcctc gccggcctcc
aggcccctcc 180tggctggcga gcgggcgcca catctggccc gcacatctgc
gctgccggcc cggcgcgggg 240tccggagagg gcgcggcgcg gagcgcagcc
aggggtccgg gaaggcgccg tccgtgcgct 300gggggctcgg tctatgacga
gcagcggggt ctgcc atg ggt cgg ggg ctg ctc 353
Met Gly Arg Gly Leu Leu 1 5agg ggc ctg tgg ccg ctg cac atc gtc ctg
tgg acg cgt atc gcc agc 401Arg Gly Leu Trp Pro Leu His Ile Val Leu
Trp Thr Arg Ile Ala Ser 10 15 20acg atc cca ccg cac gtt cag aag tcg
gtt aat aac gac atg ata gtc 449Thr Ile Pro Pro His Val Gln Lys Ser
Val Asn Asn Asp Met Ile Val 25 30 35act gac aac aac ggt gca gtc aag
ttt cca caa ctg tgt aaa ttt tgt 497Thr Asp Asn Asn Gly Ala Val Lys
Phe Pro Gln Leu Cys Lys Phe Cys 40 45 50gat gtg aga ttt tcc acc tgt
gac aac cag aaa tcc tgc atg agc aac 545Asp Val Arg Phe Ser Thr Cys
Asp Asn Gln Lys Ser Cys Met Ser Asn 55 60 65 70tgc agc atc acc tcc
atc tgt gag aag cca cag gaa gtc tgt gtg gct 593Cys Ser Ile Thr Ser
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 75 80 85gta tgg aga aag
aat gac gag aac ata aca cta gag aca gtt tgc cat 641Val Trp Arg Lys
Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 90 95 100gac ccc
aag ctc ccc tac cat gac ttt att ctg gaa gat gct gct tct 689Asp Pro
Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 105 110
115cca aag tgc att atg aag gaa aaa aaa aag cct ggt gag act ttc ttc
737Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
120 125 130atg tgt tcc tgt agc tct gat gag tgc aat gac aac atc atc
ttc tca 785Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile
Phe Ser135 140 145 150gaa gaa tat aac acc agc aat cct gac ttg ttg
cta gtc ata ttt caa 833Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu
Leu Val Ile Phe Gln 155 160 165gtg aca ggc atc agc ctc ctg cca cca
ctg gga gtt gcc ata tct gtc 881Val Thr Gly Ile Ser Leu Leu Pro Pro
Leu Gly Val Ala Ile Ser Val 170 175 180atc atc atc ttc tac tgc tac
cgc gtt aac cgg cag cag aag ctg agt 929Ile Ile Ile Phe Tyr Cys Tyr
Arg Val Asn Arg Gln Gln Lys Leu Ser 185 190 195tca acc tgg gaa acc
ggc aag acg cgg aag ctc atg gag ttc agc gag 977Ser Thr Trp Glu Thr
Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu 200 205 210cac tgt gcc
atc atc ctg gaa gat gac cgc tct gac atc agc tcc acg 1025His Cys Ala
Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr215 220 225
230tgt gcc aac aac atc aac cac aac aca gag ctg ctg ccc att gag ctg
1073Cys Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu
235 240 245gac acc ctg gtg ggg aaa ggt cgc ttt gct gag gtc tat aag
gcc aag 1121Asp Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys
Ala Lys 250 255 260ctg aag cag aac act tca gag cag ttt gag aca gtg
gca gtc aag atc 1169Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val
Ala Val Lys Ile 265 270 275ttt ccc tat gag gag tat gcc tct tgg aag
aca gag aag gac atc ttc 1217Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys
Thr Glu Lys Asp Ile Phe 280 285 290tca gac atc aat ctg aag cat gag
aac ata ctc cag ttc ctg acg gct 1265Ser Asp Ile Asn Leu Lys His Glu
Asn Ile Leu Gln Phe Leu Thr Ala295 300 305 310gag gag cgg aag acg
gag ttg ggg aaa caa tac tgg ctg atc acc gcc 1313Glu Glu Arg Lys Thr
Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr Ala 315 320 325ttc cac gcc
aag ggc aac cta cag gag tac ctg acg cgg cat gtc atc 1361Phe His Ala
Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His Val Ile 330 335 340agc
tgg gag gac ctg cgc aag ctg ggc agc tcc ctc gcc cgg ggg att 1409Ser
Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile 345 350
355gct cac ctc cac agt gat cac act cca tgt ggg agg ccc aag atg ccc
1457Ala His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro
360 365 370atc gtg cac agg gac ctc aag agc tcc aat atc ctc gtg aag
aac gac 1505Ile Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys
Asn Asp375 380 385 390cta acc tgc tgc ctg tgt gac ttt ggg ctt tcc
ctg cgt ctg gac cct 1553Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser
Leu Arg Leu Asp Pro 395 400 405act ctg tct gtg gat gac ctg gct aac
agt ggg cag gtg gga act gca 1601Thr Leu Ser Val Asp Asp Leu Ala Asn
Ser Gly Gln Val Gly Thr Ala 410 415 420aga tac atg gct cca gaa gtc
cta gaa tcc agg atg aat ttg gag aat 1649Arg Tyr Met Ala Pro Glu Val
Leu Glu Ser Arg Met Asn Leu Glu Asn 425 430 435gct gag tcc ttc aag
cag acc gat gtc tac tcc atg gct ctg gtg ctc 1697Ala Glu Ser Phe Lys
Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu 440 445 450tgg gaa atg
aca tct cgc tgt aat gca gtg gga gaa gta aaa gat tat 1745Trp Glu Met
Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr455 460 465
470gag cct cca ttt ggt tcc aag gtg cgg gag cac ccc tgt gtc gaa agc
1793Glu Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser
475 480 485atg aag gac aac gtg ttg aga gat cga ggg cga cca gaa att
ccc agc 1841Met Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile
Pro Ser 490 495 500ttc tgg ctc aac cac cag ggc atc cag atg gtg tgt
gag acg ttg act 1889Phe Trp Leu Asn His Gln Gly Ile Gln Met Val Cys
Glu Thr Leu Thr 505 510 515gag tgc tgg gac cac gac cca gag gcc cgt
ctc aca gcc cag tgt gtg 1937Glu Cys Trp Asp His Asp Pro Glu Ala Arg
Leu Thr Ala Gln Cys Val 520 525 530gca gaa cgc ttc agt gag ctg gag
cat ctg gac agg ctc tcg ggg agg 1985Ala Glu Arg Phe Ser Glu Leu Glu
His Leu Asp Arg Leu Ser Gly Arg535 540 545 550agc tgc tcg gag gag
aag att cct gaa gac ggc tcc cta aac act acc 2033Ser Cys Ser Glu Glu
Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr Thr 555 560 565aaa ta
gctcttatgg ggcaggctgg gcatgtccaa agaggctgcc cctctcacca 2088Lysaa
20908567PRTgenomic 8Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu
His Ile Val Leu 1 5 10 15Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro
His Val Gln Lys Ser Val 20 25 30Asn Asn Asp Met Ile Val Thr Asp Asn
Asn Gly Ala Val Lys Phe Pro 35 40 45Gln Leu Cys Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60Lys Ser Cys Met Ser Asn Cys
Ser Ile Thr Ser Ile Cys Glu Lys Pro65 70 75 80Gln Glu Val Cys Val
Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95Leu Glu Thr Val
Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110Leu Glu
Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120
125Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
130 135 140Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp Leu145 150 155 160Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser
Leu Leu Pro Pro Leu 165 170 175Gly Val Ala Ile Ser Val Ile Ile Ile
Phe Tyr Cys Tyr Arg Val Asn 180 185 190Arg Gln Gln Lys Leu Ser Ser
Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205Leu Met Glu Phe Ser
Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220Ser Asp Ile
Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu225 230 235
240Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala
245 250 255Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln
Phe Glu 260 265 270Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr
Ala Ser Trp Lys 275 280 285Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn
Leu Lys His Glu Asn Ile 290 295 300Leu Gln Phe Leu Thr Ala Glu Glu
Arg Lys Thr Glu Leu Gly Lys Gln305 310 315 320Tyr Trp Leu Ile Thr
Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr 325 330 335Leu Thr Arg
His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350Ser
Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys 355 360
365Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn
370 375 380Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe
Gly Leu385 390 395 400Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp
Asp Leu Ala Asn Ser 405 410 415Gly Gln Val Gly Thr Ala Arg Tyr Met
Ala Pro Glu Val Leu Glu Ser 420 425 430Arg Met Asn Leu Glu Asn Ala
Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445Ser Met Ala Leu Val
Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450 455 460Gly Glu Val
Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu465 470 475
480His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly
485 490 495Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile
Gln Met 500 505 510Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp
Pro Glu Ala Arg 515 520 525Leu Thr Ala Gln Cys Val Ala Glu Arg Phe
Ser Glu Leu Glu His Leu 530 535 540Asp Arg Leu Ser Gly Arg Ser Cys
Ser Glu Glu Lys Ile Pro Glu Asp545 550 555 560Gly Ser Leu Asn Thr
Thr Lys 565
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