U.S. patent application number 09/251263 was filed with the patent office on 2002-05-02 for fibroblast growth factor homologous factor-1 (fhf-1) and methods of use.
This patent application is currently assigned to The Johns Hopkins University School of Medicine. Invention is credited to MACKE, JENNIFER P., NATHANS, JEREMY, SMALLWOOD, PHILIP M..
Application Number | 20020052477 09/251263 |
Document ID | / |
Family ID | 23745874 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020052477 |
Kind Code |
A1 |
NATHANS, JEREMY ; et
al. |
May 2, 2002 |
FIBROBLAST GROWTH FACTOR HOMOLOGOUS FACTOR-1 (FHF-1) AND METHODS OF
USE
Abstract
A novel protein, fibroblast growth factor homologous factor-1
(FHF-1), the polynucleotide sequence encoding FHF-1, and the
deduced amino acid sequence are disclosed. Also disclosed are
diagnostic and therapeutic methods of using the FHF-1 polypeptide
and polynucleotide sequences and antibodies which specifically bind
to FHF-1.
Inventors: |
NATHANS, JEREMY; (BALTIMORE,
MD) ; SMALLWOOD, PHILIP M.; (WOODBINE, MD) ;
MACKE, JENNIFER P.; (COLUMBIA, MD) |
Correspondence
Address: |
GARY CARY WARE & FRIENDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1600
SAN DIEGO
CA
92121-2189
US
|
Assignee: |
The Johns Hopkins University School
of Medicine
|
Family ID: |
23745874 |
Appl. No.: |
09/251263 |
Filed: |
February 16, 1999 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09251263 |
Feb 16, 1999 |
|
|
|
08867471 |
Jun 2, 1997 |
|
|
|
5872226 |
|
|
|
|
08867471 |
Jun 2, 1997 |
|
|
|
08439725 |
May 12, 1995 |
|
|
|
5693775 |
|
|
|
|
Current U.S.
Class: |
530/387.1 ;
530/388.23; 530/389.2 |
Current CPC
Class: |
C07K 14/50 20130101;
A61P 35/00 20180101; C07K 16/22 20130101; C12Q 2600/158 20130101;
A61P 25/00 20180101; A61P 27/02 20180101; A61K 38/00 20130101; C12Q
1/6886 20130101 |
Class at
Publication: |
530/387.1 ;
530/389.2; 530/388.23 |
International
Class: |
C07K 016/00; C07K
016/18; C07K 016/22 |
Claims
1. An antibody that binds to FHF-1 polypeptide or immunoreactive
fragments thereof.
2. The antibody of claim 1, wherein the antibody is polyclonal.
3. The antibody of claim 1, wherein the antibody is monoclonal.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 08/867,471, filed Jun. 2, 1997, issued Feb. 16, 1999 as
U.S. Pat. No. 5,872,226, which is a divisional of U.S. patent
application Ser. No. 08/439,725, filed May 12, 1995, issued Dec. 2,
1997 as U.S. Pat. No. 5,693,775.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates generally to growth factors and
specifically to a novel member of the fibroblast growth factor
family, denoted fibroblast growth factor homologous factor-1
(FHF-1) and the polynucleotide encoding FHF-1.
[0004] 2. Description of Related Art
[0005] The fibroblast growth factor family encompasses a group of
structurally related proteins with a wide range of growth
promoting, survival, and/or differentiation activities in vivo and
in vitro (reviewed in Baird, A., and Gospodarowicz, D. Ann N.Y.
Acad. Sci. 638: 1, 1991; Eckenstein, F. P., J Neurobiology 25:
1467, 1994; Mason, I. J. Cell 78: 547, 1994). As of December 1994,
nine members of this family had been characterized by molecular
cloning. The first two members of the family to be characterized,
acidic fibroblast growth factor (aFGF/FGF-1) and basic fibroblast
growth factor (bFGF/FGF-2), have been found in numerous tissues,
including for example brain, eye, kidney, placenta, and adrenal
(Jaye et al., Science 233: 541, 1986; Abraham et al., Science 233:
545, 1986). These factors have been shown to be potent mitogens and
survival factors for a variety of mesoderm and neurectoderm-derived
tissues, including fibroblasts, endothelial cells, hippocampal and
cerebral cortical neurons, and astroglia (Burgess, W. H. and
Maciag, T., Ann. Rev. Biochemistry 58: 575, 1989). Additional
members of the FGF family include: i-nt-2/FGF-3, identified as one
of the frequent sites of integration of the mouse mammary tumor
virus, and therefore a presumptive oncogenic factor (Smith et al.,
EMBO J 7:1013, 1988); FGF-4 (Delli-Bovi et al., Cell 50: 729, 1987)
and FGF-5 (Zhan et al., Mol. Cell Biol.8: 3487,1988) as
transforming genes in the NIH 3T3 transfection assay; FGF-6,
isolated by molecular cloning based on its homology to FGF-4
(Marics et al., Oncogene 4: 335 (1989); keratinocyte growth factor/
FGF-7, identified as a mitogen for keratinocytes (Finch et al.,
Science 245: 752, 1989); FGF-8 as an androgen-induced mitogen for
mammary carcinoma cells (Tanaka et al., Proc. Natl. Acad. Sci. USA
89: 8928, 1992); and FGF-9 as a mitogen for primary astrocytes
(Miyamoto et al., Mol. Cell Biol. 13: 4251, 1993). Several of the
FGFs, including aFGF and bFGF, lack a classical signal sequence;
the mechanism by which they are secreted is not known.
[0006] All members of the FGF family share approximately 25% or
more amino acid sequence identity, a degree of homology indicating
that they are likely to share nearly identical three-dimensional
structures. Support for this inference comes from a comparison of
the three-dimensional structures of bFGF and interleukin 1-beta
determined by x-ray diffraction (Eriksson et al., Proc. Natl. Acad.
Sci USA 88: 3441, 1991; Zhang et al., Proc. Natl. Acad. Sci USA 88:
3446, 1991; Ago et al., J Biochem. 110: 360, 1991). Although these
proteins share only 10% amino acid identity, the alpha carbon
backbones of the two crystal structures can be superimposed with a
root-mean square deviation of less than 2 angstroms (Zhang et al.,
Proc. Natl. Acad. Sci USA 88: 3446, 1991). Both proteins consist
almost entirely of beta-sheets, which form a barrel composed of
three copies of a four-stranded beta-meander motif. The likely
heparin- and receptor-binding regions are located on nearby regions
on one face of the protein.
[0007] aFGF, bFGF, and FGF-7/KGF have been shown to exert some or
all of their biological activity through high affinity binding to
cell surface tyrosine kinase receptors (e.g., Lee, P. L., et al.,
Science 245: 57, 1989; reviewed in Johnson, D. E. and Williams, L.
T., Adv. Cancer Res. 60: 1, 1993). Many members of the FGF family
also bind tightly to heparin, and a terniary complex of heparin,
FGF, and transmembrane receptor may be the biologically relevant
signalling species. Thus far four different genes have been
identified that encode receptors for FGF family members. Recent
work has shown that receptor diversity is increased by differential
mRNA splicing within the extracellular ligand binding domain, with
the result that multiple receptor isoforms with different ligand
binding properties can be encoded by the same gene (Johnson, D. E.
and Williams, L. T., Adv. Cancer Res. 60: 1, 1993). In tissue
culture systems, the binding of aFGF or bFGF to its cell surface
receptor activates phospholipase C-gamma (Burgess, W. H. et al.,
Mol. Cell Biol. 10: 4770, 1990), a pathway known to integrate a
variety of mitogenic signals.
[0008] Identification and characterization of new members of the
FGF family will provide insights into the mechanisms by which cells
and organs control their growth, survival, senescence,
differentiation, and recovery from injury.
SUMMARY OF THE INVENTION
[0009] The present invention provides a cell growth, survival or
differentiation factor, FHF-1 and a polynucleotide sequence which
encodes the factor. This factor is involved in the growth,
survival, and or differentiation of cells within the central
nervous system (CNS) as well as in peripheral tissues.
[0010] The invention provides a method for detecting alterations in
FHF-1 gene expression which are diagnostic of neurodegenerative or
neoplastic disorders. In another embodiment, the invention provides
a method for treating a neurodegenerative or neoplastic disorder by
modulating the expression or activity of FHF-1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the nucleotide and predicted amino acid
sequence of human FHF-1.
[0012] FIG. 2 shows the alignment of the amino acid sequence of
human FKF-1 and each of the other nine members of the FGF family.
Conserved residues are highlighted. The FGF family members are:
aFGF/FGF-1 (Jaye et al., Science 233: 541, 1986), bFGF/FGF-2
(Abraham et al., Science 233: 545, 1986), int-2/FGF-3 (Smith et
al., EMBO J 7: 1013, 1988), FGF-4 (Delli-Bovi et al., Cell 50: 729,
1987), FGF-5 (Zhan et al., Mol. Cell Biol. 8: 3487, 1988), FGF-6
(Marics et al., Oncogene 4: 335, 1989); keratinocyte growth factor/
FGF-7 (Finch et al., Science 245: 752, 1989), FGF-8 (Tanaka et al.,
Proc. Natl. Acad Sci. USA 89: 8928, 1992), and FGF-9 (Miyamoto et
al., Mol. Cell Biol. 13: 4251, 1993).
[0013] FIG. 3 shows a dendrogram in which the length of each path
connecting any pair of FGF family members is proportional to the
degree of amino acid sequence divergence of that pair.
[0014] FIG. 4 shows that the gene encoding FHF-1 is located on
human chromosome 3. The human specific hybridization is found on
chromosome 3.
[0015] FIG. 5 shows the production of FHF-1 in transfected human
embryonic kidney cells. Lanes 1, 3, and 5, total cell protein;
lanes 2, 4, and 6 protein present in the medium (secreted protein).
Lanes 1 and 2, transfection with cDNA encoding human growth
hormone; lanes 3 and 4, transfection with cDNA encoding FHF-1;
lanes 5 and 6, transfection with cDNA encoding a novel surface
receptor fused to an immunoglobulin constant region. Protein
standards are shown to the left; from top to bottom their molecular
masses are 220, 97, 66, 46, 30, 21.5, and 14.3 kD.
[0016] FIG. 6 shows the tissue specificity of FBF-1 expression. Ten
micrograms of total RNA from the indicated mouse tissues was
prepared (Chomczinski & Sacchi. Anal. Biochem. 162: 156, 1987)
and used for RNAse protection (Ausabel et al., Current Protocols in
Molecular Biology; New York: Wiley Interscience, 1987) with a mouse
FHF-1 antisense probe that spanned 212 bases of exon 1 and the
adjacent 100 bases of intron 1. RNAse protection at the size
expected for the 212 base exon 1 region of the probe (arrowhead)
was observed with RNA from brain, eye, and testis.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides a growth factor, FHF-1, and a
polynucleotide sequence encoding FHF-1. FHF-1 is expressed at high
levels in brain, eye and testes tissues. In one embodiment, the
invention provides a method for detection of a cell proliferative
disorder of central nervous system or testes origin which is
associated with FHF-1 expression or function. In another
embodiment, the invention provides a method for treating a cell
proliferative or immunologic disorder by using an agent which
suppresses or enhances FHF-1 expression or activity.
[0018] The structural homology between the FHF-1 protein of this
invention and the members of the FGF family, indicates that FHF-1
is a new member of the family of growth factors. Based on the known
activities of many of the other members it can be expected that
FHF-1 will also possess biological activities that will make it
useful as a diagnostic and therapeutic reagent.
[0019] Many growth factors have expression patterns or possess
activities that relate to the function of the nervous system. For
example, one growth factor in the TGF family, namely GDNF, has been
shown to be a potent neurotrophic factor that can promote the
survival of dopaminergic neurons (Lin, et al., Science, 260:1130).
Another family member, namely dorsalin-1, is capable of promoting
the differentiation of neural crest cells (Basler, et al., Cell,
73:687, 1993). The inhibins and activins have been shown to be
expressed in the brain (Meunier, et al., Proc. Nat'l. Acad. Sci.,
USA, 85:247, 1988; Sawchenkco, et al., Nature, 334:615, 1988), and
activin has been shown to be capable of functioning as a nerve cell
survival molecule (Schubert, et al., Nature, 344:868, 1990).
Another TGF family member, namely GDF-1, is nervous system-specific
in its expression pattern (Lee, Proc. Nat'l. Acad. Sci., USA,
8:4250, 1991), and certain other family members, such as Vgr-1
(Lyons, et al., Proc. Nat'l. Acad. Sci., USA, 86:4554, 1989; Jones,
et al., Development, 111:581, 1991), OP-1 (Ozkaynak, et al., J.
Biol. Chem., 267:25220, 1992), and BMP-4 (Jones, et al.,
Development, 111:531, 1991), are also known to be expressed in the
nervous system.
[0020] The expression of FHF-1 in brain and eye suggests that FHF-1
may also possess activities that relate to the function of the
nervous system. FHF-1 may have neurotrophic activities for various
neuronal populations. Hence, FHF-1 may have in vitro and in vivo
applications in the treatment of neurodegenerative diseases, such
as amyotrophic lateral sclerosis, or in maintaining cells or
tissues in culture prior to transplantation.
[0021] In a first embodiment, the present invention provides a
sbstantially pure fibroblast growth factor homologous factor-1
(FHF-1) characterized by having a molecular weight of about 30 kD
as determined by reducing SDS-PAGE and having essentially the amino
acid sequence of SEQ ID NO:2. The term "substantially pure" as used
herein refers to FHF-1 which is substantially free of other
proteins, lipids, carbohydrates or other materials with which it is
naturally associated. One skilled in the art can purify FHF-1 using
standard techniques for protein purification. The substantially
pure polypeptide will yield a single major band on a non-reducing
polyacrylamide gel. The purity of the FHF-1 polypeptide can also be
determined by amino-terminal amino acid sequence analysis. FHF-1
polypeptide includes functional fragments of the polypeptide, as
long as the activity of FHF-1 remains. Smaller peptides containing
the biological activity of FIF-1 are included in the invention.
[0022] The invention provides polynucleotides encoding the FHF-1
potypeptide. These polynucleotides include DNA, cDNA and RNA
sequences which encode FHF-1. It is understood that all
polynucleotides encoding all or a portion of FHF-1 are also
included herein, as long as they encode a polypeptide with FHF-1
activity. Such polynucleotides include naturally occurring,
synthetic, and intentionally manipulated polynucleotides. For
example, FHF-1 polynucleotide may be subjected to site-directed
mutagenesis. The polynucleotide sequence for FHF-1 also includes
antisense sequences. The polynucleotides of the invention include
sequences that are degenerate as a result of the genetic code.
There are 20 natural amino acids, most of which are specified by
more than one codon. Therefore, all degenerate nucleotide sequences
are included in the invention as long as the amino acid sequence of
FHF-1 polypeptide encoded by the nucleotide sequence is
functionally unchanged.
[0023] Specifically disclosed herein is a DNA sequence encoding the
human FHF-1 gene. The sequence contains an open reading frame
encoding a polypeptide 244 amino acids in length. The human FHF-1
inititiator methionine codon shown in FIG. 1 at position 332-334 is
the first ATG codon following the in-frame stop codon at
nucleotides 323-325; a good consensus ribosome binding site
(TGGCCATGG; Kozak, Nucleic Acids Res., 15:8125, 1987) is found at
this position. The next methionine codon within the open reading
frame is encountered 86 codons 3' of the putative initiator
methionine codon. As observed for aFGF and bFGF, the amino-terminus
of the primary translation product of FHF-1 does not conform to the
consensus sequence for a signal peptide to direct cotranslatioal
insertion across the endoplasmic reticulum membrane. The FHF-1
sequence lacks potential asn-X-ser/thr site for asparagine-linked
glycosylation. Preferably, the human FHF-1 nucleotide sequence is
SEQ ID NO:1 and the deduced amino acid sequence is preferably SEQ
ID NO:2.
[0024] The polynucleotide encoding FHF-1 includes SEQ ID NO:1 as
well as nucleic acid sequences complementary to SEQ ID NO: 1. A
complementary sequence may include an antisense nucleotide. When
the sequence is RNA, the deoxynucleotides A, G, C, and T of SEQ ID
NO:1 is replaced by ribonucleotides A, G, C, and U, respectively.
Also included in the invention are fragments of the above-described
nucleic acid sequences that are at least 15 bases in length, which
is sufficient to permit the fragment to selectively hybridize to
DNA that encodes the protein of SEQ ID NO:2 under physiological
conditions. Specifically, the fragments should hybridize to DNA
encoding FHF-1 protein under stringent conditions.
[0025] The FGF family member most homologous to FHF-1 is FGF-9,
which shares 27% amino acid identity when aligned with 10 gaps.
Minor modifications of the FHF-1 primary amino acid sequence may
result in proteins which have substantially equivalent activity as
compared to the FHF-1 polypeptide described herein. Such proteins
include those as defined by the term "having essentially the amino
acid sequence of SEQ ID NO:2". Such modifications may be
deliberate, as by site-directed mutagenesis, or may be spontaneous.
All of the polypeptides produced by these modifications are
included herein as long as the biological activity of FHF-1 still
exists. Further, deletion of one or more amino acids can also
result in a modification of the structure of the resultant molecule
without significantly altering its biological activity. This can
lead to the development of a smaller active molecule which would
have broader utility. For example, one can remove amino or carboxy
terminal amino acids which are not required for FHF-1 biological
activity.
[0026] The FHF-1 polypeptide of the invention encoded by the
polynucleotide of the invention includes the disclosed sequence
(SEQ ID NO:2) and conservative variations thereof. The term
"conservative variation" as used herein denotes the replacement of
an amino acid residue by another, biologically similar residue.
Examples of conservative variations include the substitution of one
hydrophobic residue such as isoleucine, valine, leucine or
methionine for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acid, or glutamine for asparagine, and the
like. The term "conservative variation" also includes the use of a
substituted amino acid in place of an unsubstituted parent amino
acid provided that antibodies raised to the substituted polypeptide
also immunoreact with the unsubstituted polypeptide.
[0027] DNA sequences of the invention can be obtained by several
methods. For example, the DNA can be isolated using hybridization
techniques which are well known in the art. These include, but are
not limited to: 1) hybridization of genomic or cDNA libraries with
probes to detect homologous nucleotide sequences, 2) polymerase
chain reaction (PCR) on genomic DNA or cDNA using primers capable
of annealing to the DNA sequence of interest, and 3) antibody
screening of expression libraries to detect cloned DNA fragments
with shared structural features.
[0028] Preferably the FHF-1 polynucleotide of the invention is
derived from a mammalian organism, and most preferably from human.
Screening procedures which rely on nucleic acid hybridization make
it possible to isolate any gene sequence from any organism,
provided the appropriate probe is available. Oligonucleotide
probes, which correspond to a part of the sequence encoding the
protein in question, can be synthesized chemically. This requires
that short, oligopeptide stretches of amino acid sequence must be
known. The DNA sequence encoding the protein can be deduced from
the genetic code, however, the degeneracy of the code must be taken
into account. It is possible to perform a mixed addition reaction
when the sequence is degenerate. This includes a heterogeneous
mixture of denatured double-stranded DNA. For such screening,
hybridization is preferably performed on either single-stranded DNA
or denatured double-stranded DNA. Hybridization is particularly
useful in the detection of cDNA clones derived from sources where
an extremely low amount of mRNA sequences relating to the
polypeptide of interest are present. In other words, by using
stringent hybridization conditions directed to avoid non-specific
binding, it is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement (Wallace, et al., Nucl. Acid Res., 9:879, 1981;
Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y. 1989).
[0029] The development of specific DNA sequences encoding FHF-1 can
also be obtained by: 1) isolation of double-stranded DNA sequences
from the genomic DNA; 2) chemical manufacture of a DNA sequence to
provide the necessary codons for the polypeptide of interest; and
3) in vitro synthesis of a double-stranded DNA sequence by reverse
transcription of mRNA isolated from a eukaryotic donor cell. In the
latter case, a double-stranded DNA complement of mRNA is eventually
formed which is generally referred to as cDNA.
[0030] Of the three above-noted methods for developing specific DNA
sequences for use in recombinant procedures, the isolation of
genomic DNA isolates is the least common. This is especially true
when it is desirable to obtain the microbial expression of
mammalian polypeptides due to the presence of introns.
[0031] The synthesis of DNA sequences is frequently the method of
choice when the entire sequence of amino acid residues of the
desired polypeptide product is known. When the entire sequence of
amino acid residues of the desired polypeptide is not known, the
direct synthesis of DNA sequences is not possible and the method of
choice is the synthesis of cDNA sequences. Among the standard
procedures for isolating cDNA sequences of interest is the
formation of plasmid- or phage-carrying cDNA libraries which are
derived from reverse transcription of mRNA which is abundant in
donor cells that have a high level of genetic expression. When used
in combination with polymerase chain reaction technology, even rare
expression products can be cloned. In those cases where significant
portions of the amino acid sequence of the polypeptide are known,
the production of labeled single or double-stranded DNA or RNA
probe sequences duplicating a sequence putatively present in the
target cDNA may be employed in DNA/DNA hybridization procedures
which are carried out on cloned copies of the cDNA which have been
denatured into a single-stranded form (Jay, et al., Nucl. Acid
Res., 11:2325, 1983).
[0032] A cDNA expression library, such as lambda gt11, can be
screened indirectly for FHF-1 peptides having at least one epitope,
using antibodies specific for FHF-1. Such antibodies can be either
polyclonally or monoclonally derived and used to detect expression
product indicative of the presence of FHF-1 cDNA.
[0033] DNA sequences encoding FHF-1 can be expressed in vitro by
DNA transfer into a suitable host cell. "Host cells" are cells in
which a vector can be propagated and its DNA expressed. The term
also includes any progeny of the subject host cell. It is
understood that all progeny may not be identical to the parental
cell since there may be mutations that occur during replication.
However, such progeny are included when the term "host cell" is
used. Methods of stable transfer, meaning that the foreign DNA is
continuously maintained in the host, are known in the art.
[0034] In the present invention, the FHF-1 polynucleotide sequences
may be inserted into a recombinant expression vector. The term
"recombinant expression" vector refers to a plasmid, virus or other
vehicle known in the art that has been manipulated by insertion or
incorporation of the FHF-1 genetic sequences. Such expression
vectors contain a promoter sequence which facilitates the efficient
transcription of the inserted genetic sequence of the host. The
expression vector typically contains an origin of replication, a
promoter, as well as specific genes which allow phenotypic
selection of the transformed cells. Vectors suitable for use in the
present invention include, but are limited to the T7-based
expression vector for expression in bacteria (Rosenberg, et al.,
Gene, 56:125, 1987), the pMSXND expression vector for expression in
mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988)
and baculovirus-derived vectors for expression in insect cells. The
DNA segment can be present in the vector operably linked to
regulatory elements, for example, a promoter (e.g., T7,
metallothionein I, or polyhedrin promoters).
[0035] Polynucleotide sequences encoding FHF-1 can be expressed in
either prokaryotes or eukaryotes. Hosts can include microbial,
yeast, insect and mammalian organisms. Methods of expressing DNA
sequences having eukaryotic or viral sequences in prokaryotes are
well known in the art. Biologically functional viral and plasmid
DNA vectors capable of expression and replication in a host are
known in the art. Such vectors are used to incorporate DNA
sequences of the invention.
[0036] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method using procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired.
[0037] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used. Eukaryotic cells can also be cotransformed with DNA
sequences encoding the FHF-1 of the invention, and a second foreign
DNA molecule encoding a selectable phenotype, such as the herpes
simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein. (see for example, Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
[0038] Isolation and purification of microbial expressed
polypeptide, or fragments thereof, provided by the invention, may
be carried out by conventional means including preparative
chromatography and immunological separations involving monoclonal
or polyclonal antibodies.
[0039] The FHF-1 polypeptides of the invention can also be used to
produce antibodies which are immunoreactive or bind to epitopes of
the FHF-1 polypeptides. Antibody which consists essentially of
pooled monoclonal antibodies with different epitopic specificities,
as well as distinct monoclonal antibody preparations are provided.
Monoclonal antibodies are made from antigen containing fragments of
the protein by methods well known in the art (Kohler, et al.,
Nature, 256:495, 1975; Current Protocols in Molecular Biology,
Ausubel, et al., ed., 1989).
[0040] The term "antibody" as used in this invention includes
intact molecules as well as fragments thereof, such as Fab,
F(ab').sub.2, and Fv which are capable of binding the epitopic
determinant. These antibody fragments retain some ability to
selectively bind with its antigen or receptor and are defined as
follows:
[0041] (1) Fab, the fragment which contains a monovalent
antigen-binding fragment of an antibody molecule can be produced by
digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain;
[0042] (2) Fab', the fragment of an antibody molecule can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody
molecule;
[0043] (3) (Fab').sub.2, the fragment of the antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds;
[0044] (4) Fv, defined as a genetically engineered fragment
containing the variable region of the light chain and the variable
region of the heavy chain expressed as two chains; and
[0045] (5) Single chain antibody ("SCA"), defined as a genetically
engineered molecule containing the variable region of the light
chain, the variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain
molecule.
[0046] Methods of making these fragments are known in the art. (See
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York (1988), incorporated herein by
reference).
[0047] As used in this invention, the term "epitope" means any
antigenic determinant on an antigen to which the paratope of an
antibody binds. Epitopic determinants usually consist of chemically
active surface groupings of molecules such as amino acids or sugar
side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
[0048] Antibodies which bind to the FHF-1 polypeptide of the
invention can be prepared using an intact polypeptide or fragments
containing small peptides of interest as the immunizing antigen.
The polypeptide or a peptide used to immunize an animal can be
derived from translated cDNA (see for example, EXAMPLES 4 and 6) or
chemical synthesis which can be conjugated to a carrier protein, if
desired. Such commonly used carriers which are chemically coupled
to the peptide include keyhole limpet hemocyanin (KLH),
thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The
coupled peptide is then used to immunize the animal (e.g., a mouse,
a rat, or a rabbit).
[0049] If desired, polyclonal or monoclonal antibodies can be
further purified, for example, by binding to and elution from a
matrix to which the polypeptide or a peptide to which the
antibodies were raised is bound. Those of skill in the art will
know of various techniques common in the immunology arts for
purification and/or concentration of polyclonal antibodies, as well
as monoclonal antibodies (See for example, Coligan, et al., Unit 9,
Current Protocols in Immunology, Wiley Interscience, 1994,
incorporated by reference).
[0050] It is also possible to use the anti-idiotype technology to
produce monoclonal antibodies which mimic an epitope. For example,
an anti-idiotypic monoclonal antibody made to a first monoclonal
antibody will have a binding domain in the hypervariable region
which is the "image" of the epitope bound by the first monoclonal
antibody.
[0051] The term "cell-proliferative disorder" denotes malignant as
well as non-malignant cell populations which often appear to differ
from the surrounding tissue both morphologically and genotypically.
Malignant cells (i.e. cancer) develop as a result of a multistep
process. The FHF-1 polynucleotide that is an antisense molecule is
useful in treating malignancies of the various organ systems,
particularly, for example, cells in the central nervous system,
including neural tissue, testes, and cells of the eye. Essentially,
any disorder which is etiologically linked to altered expression of
FET-1 could be considered susceptible to treatment with a FEF-1
suppressing reagent. One such disorder is a malignant cell
proliferative disorder, for example.
[0052] For purposes of the invention, an antibody or nucleic acid
probe specific for FHF-1 may be used to detect FHF-1 polypeptide
(using antibody) or polynucleotide (using nucleic acid probe) in
biological fluids or tissues. The invention provides a method for
detecting a cell proliferative disorder of neural tissue or testes,
for example, which comprises contacting an anti-FHF-1 antibody or
nucleic acid probe with a cell suspected of having a FHF-1
associated disorder and detecting binding to the antibody or
nucleic acid probe. The antibody reactive with FHF-1 or the nucleic
acid probe is preferably labeled with a compound which allows
detection of binding to FHF-1. Any specimen containing a detectable
amount of antigen or polynucleotide can be used. A preferred sample
of this invention is CNS, e.g., neural tissue or cerebrospinal
fluid, testes, or eye tissue. The level of FHF-1 in the suspect
cell can be compared with the level in a normal cell to determine
whether the subject has a FHF-1-associated cell proliferative
disorder. Preferably the subject is human.
[0053] When the cell component is nucleic acid, it may be necessary
to amplify the nucleic acid prior to binding with an FHF-1 specific
probe. Preferably, polymerase chain reaction (PCR) is used,
however, other nucleic acid amplification procedures such as ligase
chain reaction (LCR), ligated activated transcription (LAT) and
nucleic acid sequence-based amplification (NASBA) may be used.
[0054] The antibodies of the invention can be used in any subject
in which it is desirable to administer in vitro or in vivo
immunodiagnosis or immunotherapy. The antibodies of the invention
are suited for use, for example, in immunoassays in which they can
be utilized in liquid phase or bound to a solid phase carrier. In
addition, the antibodies in these immunoassays can be detectably
labeled in various ways. Examples of types of immunoassays which
can utilize antibodies of the invention are competitive and
non-competitive immunoassays in either a direct or indirect format.
Examples of such immnunooassays are the radioimmunoassay (RIA) and
the sandwich (immunometric) assay. Detection of the antigens using
the antibodies of the invention can be done utilizing immunoassays
which are run in either the forward, reverse, or simultaneous
modes, including immunohistochemical assays on physiological
samples. Those of skill in the art will know, or can readily
discern, other immunoassay formats without undue
experimentation.
[0055] The antibodies of the invention can be bound to many
different carriers and used to detect the presence of an antigen
comprising the polypeptide of the invention. Examples of well-known
carriers include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon, amylases, natural and modified celluloses,
polyacrylamnides, agaroses and magnetite. The nature of the carrier
can be either soluble or insoluble for purposes of the invention.
Those skilled in the art will know of other suitable carriers for
binding antibodies, or will be able to ascertain such, using
routine experimentation.
[0056] There are many different labels and methods of labeling
known to those of ordinary skill in the art. Examples of the types
of labels which can be used in the present invention include
enzymes, radioisotopes, fluorescent compounds, colloidal metals,
chemiluminescent compounds, phosphorescent compounds, and
bioluminescent compounds. Those of ordinary skill in the art will
know of other suitable labels for binding to the antibody, or will
be able to ascertain such, using routine experimentation.
[0057] Another technique which may also result in greater
sensitivity consists of coupling the antibodies to low molecular
weight haptens. These haptens can then be specifically detected by
means of a second reaction. For example, it is common to use such
haptens as biotin, which reacts with avidin, or dinitrophenyl,
puridoxal, and fluorescein, which can react with specific
antihapten antibodies.
[0058] In using the monoclonal antibodies of the invention for the
in vivo detection of antigen, the detectably labeled antibody is
given a dose which is diagnostically effective. The term
"diagnostically effective" means that the amount of detectably
labeled monoclonal antibody is administered in sufficient quantity
to enable detection of the site having the antigen comprising a
polypeptide of the invention for which the monoclonal antibodies
are specific.
[0059] The concentration of detectably labeled monoclonal antibody
which is administered should be sufficient such that the binding to
those cells having the polypeptide is detectable compared to the
background. Further, it is desirable that the detectably labeled
monoclonal antibody be rapidly cleared from the circulatory system
in order to give the best target-to-background signal ratio.
[0060] As a rule, the dosage of detectably labeled monoclonal
antibody for in vivo diagnosis will vary depending on such factors
as age, sex, and extent of disease of the individual. Such dosages
may vary, for example, depending on whether multiple injections are
given, antigenic burden, and other factors known to those of skill
in the art.
[0061] For in vivo diagnostic imaging, the type of detection
instrument available is a major factor in selecting a given
radioisotope. The radioisotope chosen must have a type of decay
which is detectable for a given type of instrument. Still another
important factor in selecting a radioisotope for in vivo diagnosis
is that deleterious radiation with respect to the host is
minimized. Ideally, a radioisotope used for in vivo imaging will
lack a particle emission, but produce a large number of photons in
the 140-250 keV range, which may readily be detected by
conventional gamma cameras.
[0062] For in vivo diagnosis radioisotopes may be bound to
immunoglobulin either directly or indirectly by using an
intermediate functional group. Intermediate functional groups which
often are used to bind radioisotopes which exist as metallic ions
to immunoglobulins are the bifunctional chelating agents such as
diethylenetriaminepentacetic acid (DTPA) and
ethylenediaminetetraacetic acid (EDTA) and similar molecules.
Typical examples of metallic ions which can be bound to the
monoclonal antibodies of the invention are .sup.111In, .sup.97Ru,
.sup.67Ga, .sup.68Ga, .sup.72As, .sup.89Zr, and and .sup.201TI.
[0063] The monoclonal antibodies of the invention can also be
labeled with a paramagnetic isotope for purposes of in vivo
diagnosis, as in magnetic resonance imaging (MRI) or electron spin
resonance (ESR). In general, any conventional method for
visualizing diagnostic imaging can be utilized. Usually gamma and
positron emitting radioisotopes are used for camera imaging and
paramagnetic isotopes for MRI. Elements which are particularly
useful in such techniques include .sup.157Gd, .sup.55Mn,
.sup.162Dy, .sup.52Cr, and .sup.56Fe.
[0064] The monoclonal antibodies or polynucleotides of the
invention can be used in vitro and in vivo to monitor the course of
amelioration of a FHF-1-associated disease in a subject. Thus, for
example, by measuring the increase or decrease in the number of
cells expressing antigen comprising a polypeptide of the invention
or changes in the concentration of such antigen present in various
body fluids, it would be possible to determine whether a particular
therapeutic regimen aimed at ameliorating the FHF-1-associated
disease is effective. The term "ameliorate" denotes a lessening of
the detrimental effect of the FHF-1-associated disease in the
subject receiving therapy.
[0065] The present invention identifies a nucleotide sequence that
can be expressed in an altered manner as compared to expression in
a normal cell, therefore it is possible to design appropriate
therapeutic or diagnostic techniques directed to this sequence.
Detection of elevated levels of FHF-1 expression is accomplished by
hybridization of nucleic acids isolated from a cell suspected of
having an FHF-1 associated proliferative disorder with an FHF-1
polynucleotide of the invention. Analysis, such as Northern Blot
analysis, are utilized to quantitate expression of FHF-1. Other
standard nucleic acid detection techniques will be known to those
of skill in the art.
[0066] Treatment of an FHF-1 associated cell proliferative disorder
include modulation of FHF-1 gene expression and FHF-1 activity. The
term "modulate" envisions the suppression of expression of FHF-1
when it is over-expressed, or augmentation of FHF-1 expression when
it is under-expressed. Where a cell-proliferative disorder is
associated with the expression of FHF-1, nucleic acid sequences
that interfere with FHF-1 expression at the translational level can
be used. This approach utilizes, for example, antisense nucleic
acid, ribozymes, or triplex agents to block transcription or
translation of a specific FHF-1 mRNA, either by masking that mRNA
with an antisense nucleic acid or triplex agent, or by cleaving it
with a ribozyme. Such disorders include neurodegenerative diseases,
for example.
[0067] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub, Scientific American, 262:40, 1990). In the cell, the
antisense nucleic acids hybridize to the corresponding mRNA,
forming a double-stranded molecule. The antisense nucleic acids
interfere with the translation of the mRNA, since the cell will not
translate a mRNA that is double-stranded. Antisense oligomers of
about 15 nucleotides are preferred, since they are easily
synthesized and are less likely to cause problems than larger
molecules when introduced into the target FHF-1-producing cell. The
use of antisense methods to inhibit the in vitro translation of
genes is well known in the art (Marcus-Sakura, Anal. Biochem.,
172:289, 1988).
[0068] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. and Dev., 1(3):227, 1991;
Helene, C., Anticancer Drug Design, 6(6):569, 1991).
[0069] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030,
1988). A major advantage of this approach is that, because they are
sequence-specific, only mRNAs with particular sequences are
inactivated.
[0070] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-based
recognition sequences are preferable to shorter recognition
sequences.
[0071] The present invention also provides gene therapy for the
treatment of cell proliferative or immunologic disorders which are
mediated by FHF-1 protein. Such therapy would achieve its
therapeutic effect by introduction of the FHF-1 antisense
polynucleotide into cells having the proliferative disorder.
Delivery of antisense FHF-1 polynucleotide can be achieved using a
recombinant expression vector such as a chimeric virus or a
colloidal dispersion system. Especially preferred for therapeutic
delivery of antisense sequences is the use of targeted
liposomes.
[0072] Various viral vectors which can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
Preferably, when the subject is a human, a vector such as the
gibbon ape leukemia virus (GaLV) is utilized. A number of
additional retroviral vectors can incorporate multiple genes. All
of these vectors can transfer or incorporate a gene for a
selectable marker so that transduced cells car, be identified and
generated. By inserting a FHF-1 sequence of interest into the viral
vector, along with another gene which encodes the ligand for a
receptor on a specific target cell, for example, the vector is now
target specific. Retroviral vectors can be made target specific by
attaching, for example, a sugar, a glycolipid, or a protein.
Preferred targeting is accomplished by using an antibody to target
the retroviral vector. Those of skill in the art will know of, or
can readily ascertain without undue experimentation, specific
polynucleotide sequences which can be inserted into the retroviral
genome or attached to a viral envelope to allow target specific
delivery of the retroviral vector containing the FHF-1 antisense
polynucleotide.
[0073] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence which enables
the packaging mechanism to recognize an RNA transcript for
encapsidation. Helper cell lines which have deletions of the
packaging signal include, but are not limited to .PSI.2, PA317 and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
cells in which the packaging signal is intact, but the structural
genes are replaced by other genes of interest, the vector can be
packaged and vector virion produced.
[0074] Alternatively, NIH 3T3 or other tissue culture cells can be
directly transfected with plasmids encoding the retroviral
structural genes gag, pol and env, by conventional calcium
phosphate transfection. These cells are then transfected with the
vector plasmid containing the genes of interest. The resulting
cells release the retroviral vector into the culture medium.
[0075] Another targeted delivery system for FHF-1 antisense
polynucleotides is a colloidal dispersion system. Colloidal
dispersion systems include macromolecule complexes, nanocapsules,
microspheres, beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, and liposomes. The preferred
colloidal system of this invention is a liposome. Liposomes are
artificial membrane vesicles which are useful as delivery vehicles
in vitro and in vivo. It has been shown that large unilamellar
vesicles (LUV), which range in size from 0.2-4.0 .mu.m can
encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules. RNA, DNA and intact virions can be
encapsulated within the aqueous interior and be delivered to cells
in a biologically active form (Fraley, et al., Trends Biochem.
Sci., 6:77, 1981). In addition to mammalian cells, liposomes have
been used for delivery of polynucleotides in plant, yeast and
bacterial cells. In order for a liposome to be an efficient gene
transfer vehicle, the following characteristics should be present:
(1) encapsulation of the genes of interest at high efficiency while
not compromising their biological activity; (2) preferential and
substantial binding to a target cell in comparison to non-target
cells; (3) delivery of the aqueous contents of the vesicle to the
target cell cytoplasm at high efficiency; and (4) accurate and
effective expression of genetic information (Mannino, et al.,
Biotechniques, 6:682. 1988).
[0076] The composition of the liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0077] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmltoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0078] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0079] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various linking groups can
be used for joining the lipid chains to the targeting ligand.
[0080] Due to the expression of FHF-1 in the testes, eye and brain,
or neural tissue, there are a variety of applications using the
polypeptide, polynucleotide, and antibodies of the invention,
related to these tissues. Such applications include treatment of
cell proliferative and immunologic disorders involving these and
other tissues. In addition, FHF-1 may be useful in various gene
therapy procedures.
[0081] Due to the high level of expression of FHF-1 in the testes,
there are a variety of applications using the polypeptide,
polynucleotide, and antibodies of the invention related to this
tissue. Such applications include treatment of cell proliferative
disorders associated with FHF-1 expression in the testes. Various
testicuiar developmental or acquired disorders can also be subject
to FHF-1 applications. These may include, but are not limited to
viral infection (e.g., viral orchitis), autoimmunity, sperm
production or dysfunction, trauma, and testicular tumors. The
presence of high levels of FHF-1 in the testis suggests that FHF-1
or an analogue of FHF-1 could be used to increase or decrease male
fertility.
[0082] The identification of a novel member of the FGF family
provides a useful tool for diagnosis, prognosis and therapeutic
strategies associated with FHF-1 mediated disorders. Measurement of
FHF-1 levels using anti-FHF-1 antibodies is a useful diagnostic for
following the progression or recovery from diseases of the nervous
system, including: cancer, stroke, neurodegenerative diseases such
as Parkinson's disease or Alzheimer's disease, retinal diseases
such as retinitis pigmentosa, or viral encephalitis. The presence
of high levels of FHF-1 in the central nervous system suggests that
the observed low level of FHF-1 in a number of peripheral tissues
could reflect FHF-1 in peripheral nerve, and therefore measurement
of FHF-1 levels using anti-FHF-1 antibodies could be diagnostic for
peripheral neuropathy. The presence of high levels of FHF-1 in the
testis suggests that measurement of FHF-1 levels using anti-FHF-1
antibodies could be diagnostic for testicular cancer.
[0083] Like other members of the FGF family, FHF-1 likely has
mitogenic and/or cell survival activity, therefore FHF-1 or an
analogue that mimics FHF-1 action could be used to promote tissue
repair or replacement. The presence of FHF-1 in the CNS suggests
such a therapeutic role in diseases of the nervous system,
including: stroke, neurodegenerative diseases such as Parkinson's
disease or Alzheimer's disease, or in retinal degenerative diseases
such as retinitis pigmentosa or macular degeneration, or in
peripheral neuropathies. Conversely, blocking FHF-1 action either
with anti-FHF-1 antibodies or with an FHF-1 antagonist might slow
or ameliorate diseases in which excess cell growth is pathological,
most obviously cancer.
[0084] The following examples are intended to illustrate but not
limit the invention. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
EXAMPLE 1
Identification of FHF-1, A Novel Member of the FGF Family
[0085] To identify novel gene products expressed in the human
retina, random segments of human retina cDNA clones were partially
sequenced, and the resulting partial sequences compared to the
sequences available in the public databases.
[0086] In detail, an adult human retina cDNA library constructed in
lambda gt10 (Nathans, et al., Science 232: 193, 1986) was
amplified, and the cDNA inserts were excised en mass by cleavage
with EcoR I and purified free of the vector by agarose gel
electrophoresis. Following heat denaturation of the purified cDNA
inserts, a synthetic oligonucleotide containing an EcoR I site at
its 5' end and six random nucleotides at its 3' end (5'
GACGAGATATTAGAATTCTACTCGNNNNNN) (SEQ ID NO:3) was used to prime two
sequential rounds of DNA synthesis in the presence of the Klenow
fragment of E. coli DNA polymerase. The resulting duplex molecules
were amplified using the polymerase chain reaction (PCR) with a
primer corresponding to the unique 5' flanking sequence (5'
CCCCCCCCCGACGAGATATTAGAATTCTACTCG) (SEQ ID NO:4). These PCR
products, representing a random sampling of the original cDNA
inserts, were cleaved with EcoR I, size fractionated by preparative
agarose gel electrophoresis to include only segments of
approximately 500 bp in length, and cloned into lambda gt10. Three
thousand single plaques from this derivative library were arranged
in 96-well trays and from these clones the inserts were amplified
by PCR using flanking vector primers and then sequenced using the
dideoxy method and automated fluorescent detection (Applied
Biosystems). A single sequencing run from one end of each insert
was conceptually translated on both strands in all three reading
frames and the six resulting amino acid sequences were used to
search for homology in the GenBank nonredundant protein database
using the BLASTX searching algorithm.
[0087] One partial cDNA sequence was found that showed
statistically significant homology to previously described members
of the FGF family. Using this partial cDNA as a probe, multiple
independent cDNA clones were isolated from the human retina cDNA
library, including two that encompass the entire open reading frame
and from which complete nucleotide sequences were determined.
EXAMPLE 2
Deduced Primary Structure of FHF-1
[0088] FIG. 1 shows the sequence of human FHF-1 deduced from the
nucleotide sequences of two independent human retina cDNA clones.
The primary translation product of human FHF-1 is predicted to be
244 amino acids in length. The human FEF-1 inititiator methionine
codon shown in FIG. 1 at position 332-334 is the first ATG codon
following the in-frame stop codon at nucleotides 323-325; a good
consensus ribosome binding site (CAGCTATGG (SEQ ID NO:5); Kozak,
Nucleic Acids Res. 15:8125 1987) is found at this position. The
next methionine codon within the open reading frame is encountered
86 codons 3' of the putative initiator methionine codon. As
observed for aFGF and bFGF, the amino-termninus of the primary
translation product of FHF-1 does not conform to the consensus
sequence for a signal peptide to direct cotranslational insertion
across the endoplasmic reticulum membrane. The FHF-1 sequence lacks
asn-X-ser/thr sites for asparagine-linked glycosylation.
[0089] Alignment of FHF-1 with the other known members of the FGF
family is shown in FIG. 2 and a dendrogram showing the degree of
amino acid similarity is shown in FIG. 3. The most homologous FGF
family member is FGF-9 which shows 27% amino acid identity with
FHF-1 when aligned with 10 gaps. Note that in the central region of
each polypeptide, all FGF family members, including FHF-1, share 11
invariant amino acids.
EXAMPLE 3
Chromosomal Localization of FHF-1
[0090] The chromosomal location of FHF-1 was determined by probing
a Southern blot containing restriction enzyme digested DNA derived
from a panel of 24 human-mouse and human-hamster cell lines, each
containing a different human chromosome (Oncor, Gaithersburg, Md.).
As seen in FIG. 4, hybridization of the human FHF-1 probe to human,
mouse, and hamster genomic DNA produces distinct hybridizing
fragment sizes. Among the hybrid panels, the human-specific
hybridization pattern is seen only in the lane corresponding to the
hybrid cell line carrying human chromosome 3.
EXAMPLE 4
Production of FHF-1 in Transfected Human Cells
[0091] To express FHF-1 in human cells, the complete open reading
frame was inserted into the eukaryotic expression vector pCIS
(Gorman, et al., DNA Protein Eng. Tech. 2: 3, 1990). To increase
the efficiency of translation, the region immediately 5' of the
initiator methionine coding was converted to an optimal ribosome
binding site (CCACCATGG) by PCR amplification with a primer that
carried the desired sequence. Following transient transfection of
human embryonic kidney cells with the expression construct and a
plasmid expressing the simian virus 40 (SV40) large T-antigen
(pRSV-TAg; Gorman, et al., supra), cells were metabolically labeled
with .sup.35S methionine for 6 hours in the absence of serum. As
shown in FIG. 5, cells transfected with FHF-1 synthesize a single
abundant polypeptide with an apparent molecular mass of 30 kD that
is not produced by cells transfected with either of two unrelated
constructs. This polypeptide corresponds closely to the predicted
molecular mass of the primary translation product, 27.4 kD. FIG. 5
also shows that cells transfected with a human growth hormone (hGH)
expression plasmid efficiently secrete hGH, whereas FHF-1
accumulates within the transfected cells and fails to be secreted
in detectable quantities.
EXAMPLE 5
Tissue Distribution of FHF-1 mRNA
[0092] To determine the tissue distribution of FHF-1 mRNA, RNase
protection analysis was performed on total RNA from mouse brain,
eye, heart, kidney, liver, lung, spleen, and testis, as well as a
yeast tRNA negative control. The probe used was derived from a
segment of the mouse FHF-1 gene isolated by hybridization with the
full-length human FHF-1 cDNA. As seen in FIG. 6, the highest levels
of FHF-1 expression are in the brain, eye, and testis. Very low
levels of FHF-1 expression were detected in kidney, liver, and lung
on a five-fold longer exposure of the autoradiogram.
EXAMPLE 6
Production of Antibodies Specific for FHF-1
[0093] To generate anti-FHF-1 antibodies, a DNA segment
encompassing the carboxy-terminal 190 amino acids of FHF-1 was
inserted into the E. coli expression vector pGEMEX (Studier, et
al., Meth. Enzymol. 185: 60, 1990). The recombinant fusion protein
between the T7 gene 10 protein and the carboxy-terminal 190 amino
acids of FHF-1 was produced in E. coli, purified by preparative
polyacrylamide gel electrophoresis, and used to immunize rabbits.
Anti-FHF-1 antibodies from immune serum were affinity purified
using the fusion protein immobilized onto nitrocellulose; those
antibodies directed against the pGEMEX T7 gene 10 protein fusion
partner were removed by absorption to the purified T7 gene 10
protein immobilized onto nitrocellulose. By Western blotting, the
affinity purified anti-FHF-1 antibodies were shown to recognize
recombinant FHF-1 produced either in E. coli or in human embryonic
kidney cells. By immunohistochemical staining the antibodies also
specifically recognized recombinant FHF-1 produced in human
embryonic kidney cells transfected with the FHF-1 expression
plasmid described above. Immuno-staining of neural tissues shows
anti-FHF-1 immunostaining in the ganglion cell layer and inner
nuclear layers of adult mouse and macaque monkey retinas and in a
large number of regions within the adult mouse brain.
[0094] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
15 1 1422 DNA Homo sapiens CDS (332)...(1060) 1 gaattccgca
cactgcgttc ggggtaccaa gtggaagggg aagaacgatg cccaaaataa 60
caagacgtgc ctgggaccgc cccgccccgc cccccggccg ccagaggttg gggaagttta
120 catctggatt ttcacacatt ttgtcgccac tgcccagact ttgactaacc
ttgtgagcgc 180 cgggttttcg atactgcagc ctcctcaaat tttagcactg
cctccccgcg actgcccttt 240 ccctggccgc ccaggtcctg ccctcgcccc
ggcggagcgc aagccggagg gcgcagtaga 300 ggctggggcc tgaggccctc
gctgagcagc t atg gct gcg gcg ata gcc agc 352 Met Ala Ala Ala Ile
Ala Ser 1 5 tcc ttg atc cgg cag aag cgg cag gcg agg gag tcc aac agc
gac cga 400 Ser Leu Ile Arg Gln Lys Arg Gln Ala Arg Glu Ser Asn Ser
Asp Arg 10 15 20 gtg tcg gcc tcc aag cgc cgc tcc agc ccc agc aaa
gac ggg cgc tcc 448 Val Ser Ala Ser Lys Arg Arg Ser Ser Pro Ser Lys
Asp Gly Arg Ser 25 30 35 ctg tgc gag agg cac gtc ctc ggg gtg ttc
agc aaa gtg cgc ttc tgc 496 Leu Cys Glu Arg His Val Leu Gly Val Phe
Ser Lys Val Arg Phe Cys 40 45 50 55 agc ggc cgc aag agg ccg gtg agg
cgg aga cca gaa ccc cag ctc aaa 544 Ser Gly Arg Lys Arg Pro Val Arg
Arg Arg Pro Glu Pro Gln Leu Lys 60 65 70 ggg att gtg aca agg tta
ttc agc cag cag gga tac ttc ctg cag atg 592 Gly Ile Val Thr Arg Leu
Phe Ser Gln Gln Gly Tyr Phe Leu Gln Met 75 80 85 cac cca gat ggt
acc att gat ggg acc aag gac gaa aac agc gac tac 640 His Pro Asp Gly
Thr Ile Asp Gly Thr Lys Asp Glu Asn Ser Asp Tyr 90 95 100 act ctc
ttc aat cta att ccc gtg ggc ctg cgt gta gtg gcc atc caa 688 Thr Leu
Phe Asn Leu Ile Pro Val Gly Leu Arg Val Val Ala Ile Gln 105 110 115
gga gtg aag gct agc ctc tat gtg gcc atg aat ggt gaa ggc tat ctc 736
Gly Val Lys Ala Ser Leu Tyr Val Ala Met Asn Gly Glu Gly Tyr Leu 120
125 130 135 tac agt tca gat gtt ttc act cca gaa tgc aaa ttc aag gaa
tct gtg 784 Tyr Ser Ser Asp Val Phe Thr Pro Glu Cys Lys Phe Lys Glu
Ser Val 140 145 150 ttt gaa aac tac tat gtg atc tat tct tcc aca ctg
tac cgc cag caa 832 Phe Glu Asn Tyr Tyr Val Ile Tyr Ser Ser Thr Leu
Tyr Arg Gln Gln 155 160 165 gaa tca ggc cga gct tgg ttt ctg gga ctc
aat aaa gaa ggt caa att 880 Glu Ser Gly Arg Ala Trp Phe Leu Gly Leu
Asn Lys Glu Gly Gln Ile 170 175 180 atg aag ggg aac aga gtg aag aaa
acc aag ccc tca tca cat ttt gta 928 Met Lys Gly Asn Arg Val Lys Lys
Thr Lys Pro Ser Ser His Phe Val 185 190 195 ccg aaa cct att gaa gtg
tgt atg tac aga gaa cca tcg cta cat gaa 976 Pro Lys Pro Ile Glu Val
Cys Met Tyr Arg Glu Pro Ser Leu His Glu 200 205 210 215 att gga gaa
aaa caa ggg cgt tca agg aaa agt tct gga aca cca acc 1024 Ile Gly
Glu Lys Gln Gly Arg Ser Arg Lys Ser Ser Gly Thr Pro Thr 220 225 230
atg aat gga ggc aaa gtt gtg aat caa gat tca aca tagctgagaa 1070 Met
Asn Gly Gly Lys Val Val Asn Gln Asp Ser Thr 235 240 ctctcccctt
cttccctctc tcatcccttc cccttccctt ccttcccatt tacccatttc 1130
cttccagtaa atccacccaa ggagaggaaa ataaaatgac aacgcaagac ctagtggcta
1190 agattctgca ctcaaaatct tcctttgtgt aggacaagaa aattgaacca
aagcttgctt 1250 gttgcaatgt ggtagaaaat tcacgtgcac aaagattagc
acacttaaaa gcaaaggaaa 1310 aaataaatca gaactccata aatattaaac
taaactgtat tgttattagt agaaggctaa 1370 ttgtaatgaa gacattaata
aagatgaaat aaacttatta ctttcggaat tc 1422 2 243 PRT Homo sapiens 2
Met Ala Ala Ala Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala 1 5
10 15 Arg Glu Ser Asn Ser Asp Arg Val Ser Ala Ser Lys Arg Arg Ser
Ser 20 25 30 Pro Ser Lys Asp Gly Arg Ser Leu Cys Glu Arg His Val
Leu Gly Val 35 40 45 Phe Ser Lys Val Arg Phe Cys Ser Gly Arg Lys
Arg Pro Val Arg Arg 50 55 60 Arg Pro Glu Pro Gln Leu Lys Gly Ile
Val Thr Arg Leu Phe Ser Gln 65 70 75 80 Gln Gly Tyr Phe Leu Gln Met
His Pro Asp Gly Thr Ile Asp Gly Thr 85 90 95 Lys Asp Glu Asn Ser
Asp Tyr Thr Leu Phe Asn Leu Ile Pro Val Gly 100 105 110 Leu Arg Val
Val Ala Ile Gln Gly Val Lys Ala Ser Leu Tyr Val Ala 115 120 125 Met
Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr Pro Glu 130 135
140 Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser
145 150 155 160 Ser Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp
Phe Leu Gly 165 170 175 Leu Asn Lys Glu Gly Gln Ile Met Lys Gly Asn
Arg Val Lys Lys Thr 180 185 190 Lys Pro Ser Ser His Phe Val Pro Lys
Pro Ile Glu Val Cys Met Tyr 195 200 205 Arg Glu Pro Ser Leu His Glu
Ile Gly Glu Lys Gln Gly Arg Ser Arg 210 215 220 Lys Ser Ser Gly Thr
Pro Thr Met Asn Gly Gly Lys Val Val Asn Gln 225 230 235 240 Asp Ser
Thr 3 30 DNA Artificial Sequence primer for DNA synthesis 3
gacgagatat tagaattcta ctcgnnnnnn 30 4 33 DNA Artificial Sequence
oligonucleotide for PCR 4 cccccccccg acgagatatt agaattctac tcg 33 5
9 DNA Artificial Sequence good consensus ribosome binding site 5
cagctatgg 9 6 215 PRT Homo sapiens 6 Met Gly Ser Pro Arg Ser Ala
Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15 Val Leu Cys Leu Gln
Ala Gln Val Thr Val Gln Ser Ser Pro Asn Phe 20 25 30 Thr Gln His
Val Arg Glu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg 35 40 45 Arg
Leu Ile Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His 50 55
60 Val Gln Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu Asp Gly
65 70 75 80 Asp Pro Phe Ala Lys Leu Ile Val Glu Thr Asp Thr Phe Gly
Ser Arg 85 90 95 Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile
Cys Met Asn Lys 100 105 110 Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly
Lys Gly Lys Asp Cys Val 115 120 125 Phe Ile Glu Ile Val Leu Glu Asn
Asn Tyr Thr Ala Leu Gln Asn Ala 130 135 140 Lys Tyr Glu Gly Trp Tyr
Met Ala Phe Thr Arg Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Ser
Lys Thr Arg Gln His Gln Arg Glu Val His Phe Met Lys 165 170 175 Arg
Leu Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg Phe Glu 180 185
190 Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg
195 200 205 Thr Trp Ala Pro Glu Pro Arg 210 215 7 208 PRT Homo
sapiens 7 Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln
Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp
Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu Ala
Gly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr Asp Leu Asp His
Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr
Gly Phe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80 Thr Ile Gln Gly
Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85 90 95 Phe Ile
Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser 100 105 110
Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115
120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn
Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp
Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly
Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln Lys Phe
Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys Val Pro
Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205 8 208 PRT Homo
sapiens 8 Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln
Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp
Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu Ala
Gly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr Asp Leu Asp His
Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr
Gly Phe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80 Thr Ile Gln Gly
Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85 90 95 Phe Ile
Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser 100 105 110
Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115
120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn
Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp
Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly
Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln Lys Phe
Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys Val Pro
Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205 9 155 PRT Homo
sapiens 9 Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu
Lys Phe 1 5 10 15 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu
Leu Tyr Cys Ser 20 25 30 Asn Gly Gly His Phe Leu Arg Ile Leu Pro
Asp Gly Thr Val Asp Gly 35 40 45 Thr Arg Asp Arg Ser Asp Gln His
Ile Gln Leu Gln Leu Ser Ala Glu 50 55 60 Ser Val Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80 Ala Met Asp Thr
Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95 Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115
120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
Ala 130 135 140 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150
155 10 155 PRT Homo sapiens 10 Met Ala Ala Gly Ser Ile Thr Thr Leu
Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro
Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly
Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly
Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln
Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70
75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys
Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser
Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp
Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser
Lys Thr Gly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met
Ser Ala Lys Ser 145 150 155 11 245 PRT Homo sapiens 11 Met Gly Leu
Ile Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Ser Trp 1 5 10 15 Pro
Thr Thr Gly Pro Gly Thr Arg Leu Arg Arg Asp Ala Gly Gly Arg 20 25
30 Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu
35 40 45 Tyr Cys Ala Thr Lys Tyr His Leu Gln Leu His Pro Ser Gly
Arg Val 50 55 60 Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu
Glu Ile Thr Ala 65 70 75 80 Val Glu Val Gly Val Val Ala Ile Lys Gly
Leu Phe Ser Gly Arg Tyr 85 90 95 Leu Ala Met Asn Lys Arg Gly Arg
Leu Tyr Ala Ser Asp His Tyr Asn 100 105 110 Ala Glu Cys Glu Phe Val
Glu Arg Ile His Glu Leu Gly Tyr Asn Thr 115 120 125 Tyr Ala Ser Arg
Leu Tyr Arg Thr Gly Ser Ser Gly Pro Gly Ala Gln 130 135 140 Arg Gln
Pro Gly Ala Gln Arg Pro Trp Tyr Val Ser Val Asn Gly Lys 145 150 155
160 Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser
165 170 175 Leu Phe Leu Pro Arg Val Leu Gly His Lys Asp His Glu Met
Val Arg 180 185 190 Leu Leu Gln Ser Ser Gln Pro Arg Ala Pro Gly Glu
Gly Ser Gln Pro 195 200 205 Arg Gln Arg Arg Gln Lys Lys Gln Ser Pro
Gly Asp His Gly Lys Met 210 215 220 Glu Thr Leu Ser Thr Arg Ala Thr
Pro Ser Thr Gln Leu His Thr Gly 225 230 235 240 Gly Leu Ala Val Ala
245 12 268 PRT Homo sapiens 12 Met Ser Leu Ser Phe Leu Leu Leu Leu
Phe Phe Ser His Leu Ile Leu 1 5 10 15 Ser Ala Trp Ala His Gly Glu
Lys Arg Leu Ala Pro Lys Gly Gln Pro 20 25 30 Gly Pro Ala Ala Thr
Asp Arg Asn Pro Ile Gly Ser Ser Ser Arg Gln 35 40 45 Ser Ser Ser
Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala 50 55 60 Ala
Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln 65 70
75 80 Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val
Gly 85 90 95 Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly Lys Val
Asn Gly Ser 100 105 110 His Glu Ala Asn Met Leu Ser Val Leu Glu Ile
Phe Ala Val Ser Gln 115 120 125 Gly Ile Val Gly Ile Arg Gly Val Phe
Ser Asn Lys Phe Leu Ala Met 130 135 140 Ser Lys Lys Gly Lys Leu His
Ala Ser Ala Lys Phe Thr Asp Asp Cys 145 150 155 160 Lys Phe Arg Glu
Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser 165 170 175 Ala Ile
His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu 180 185 190
Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro 195
200 205 Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu
Gln 210 215 220 Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys
Asn Pro Pro 225 230 235 240 Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser
Ala Pro Arg Lys Asn Thr 245 250 255 Asn Ser Val Lys Tyr Arg Leu Lys
Phe Arg Phe Gly 260 265 13 206 PRT Homo sapiens 13 Met Ser Gly Pro
Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val Leu 1 5 10 15 Leu Ala
Leu Leu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro 20 25 30
Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 35
40 45 Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln
Pro 50 55 60 Lys Glu Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu
Leu Gly Ile 65 70 75 80 Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly
Ile Gly Phe His Leu 85 90 95 Gln Ala Leu Pro Asp Gly Arg Ile Gly
Gly Ala His Ala Asp Thr Arg 100 105 110 Asp Ser Leu Leu Glu Leu Ser
Pro Val Glu Arg Gly Val Val Ser Ile 115 120 125 Phe Gly Val Ala Ser
Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys 130 135 140 Leu Tyr Gly
Ser Pro Phe Phe Thr Asp Glu Cys Ile Phe Lys Glu Ile
145 150 155 160 Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys
Tyr Pro Gly 165 170 175 Met Phe Ile Ala Leu Ser Lys Asn Gly Lys Thr
Lys Lys Gly Asn Arg 180 185 190 Val Ser Pro Thr Met Lys Val Thr His
Phe Leu Pro Arg Leu 195 200 205 14 198 PRT Homo sapiens 14 Met Ser
Arg Gly Ala Gly Arg Leu Gln Gly Thr Leu Trp Ala Leu Val 1 5 10 15
Phe Leu Gly Ile Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Thr 20
25 30 Arg Ala Asn Asn Thr Leu Leu Asp Ser Arg Gly Trp Gly Thr Leu
Leu 35 40 45 Ser Arg Ser Arg Ala Gly Leu Ala Gly Glu Ile Ala Gly
Val Asn Trp 50 55 60 Glu Ser Gly Tyr Leu Val Gly Ile Lys Arg Gln
Arg Arg Leu Tyr Cys 65 70 75 80 Asn Val Gly Ile Gly Phe His Leu Gln
Val Leu Pro Asp Gly Arg Ile 85 90 95 Ser Gly Thr His Glu Glu Asn
Pro Tyr Ser Leu Leu Glu Ile Ser Thr 100 105 110 Val Glu Arg Gly Val
Val Ser Leu Phe Gly Val Arg Ser Ala Leu Phe 115 120 125 Val Ala Met
Asn Ser Lys Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gln 130 135 140 Glu
Glu Cys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala 145 150
155 160 Tyr Glu Ser Asp Leu Tyr Gln Gly Thr Tyr Ile Ala Leu Ser Lys
Tyr 165 170 175 Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro Ile Met
Thr Val Thr 180 185 190 His Phe Leu Pro Arg Ile 195 15 194 PRT Homo
sapiens 15 Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu
Tyr Arg 1 5 10 15 Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile
Ser Leu Ala Cys 20 25 30 Asn Asp Met Thr Pro Glu Gln Met Ala Thr
Asn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg His Thr Arg Ser Tyr
Asp Tyr Met Glu Gly Gly Asp Ile 50 55 60 Arg Val Arg Arg Leu Phe
Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp 65 70 75 80 Lys Arg Gly Lys
Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 85 90 95 Ile Met
Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly 100 105 110
Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115
120 125 Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile
Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr
His Asn Gly 145 150 155 160 Gly Glu Met Phe Val Ala Leu Asn Gln Lys
Gly Ile Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gln Lys
Thr Ala His Phe Leu Pro Met Ala 180 185 190 Ile Thr
* * * * *