U.S. patent application number 11/514821 was filed with the patent office on 2007-03-22 for fibroblast growth factor homologous factor-2 and methods of use.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE. Invention is credited to Jennifer P. Macke, Jeremy Nathans, Philip M. Smallwood.
Application Number | 20070066812 11/514821 |
Document ID | / |
Family ID | 23740672 |
Filed Date | 2007-03-22 |
United States Patent
Application |
20070066812 |
Kind Code |
A1 |
Nathans; Jeremy ; et
al. |
March 22, 2007 |
Fibroblast growth factor homologous factor-2 and methods of use
Abstract
A novel growth factor, fibroblast growth factor homologous
factor-2 (FHF-2) polypeptide, the polynucleotide sequence encoding
FHF-2 and the deduced amino acid sequence are disclosed. Also
disclosed are diagnostic and therapeutic methods of using the FHF-2
polypeptide and polynucleotide sequences and antibodies which
specifically bind to FHF-2.
Inventors: |
Nathans; Jeremy; (Baltimore,
MD) ; Smallwood; Philip M.; (Woodbine, MD) ;
Macke; Jennifer P.; (Columbia, MD) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.;DLA PIPER RUDNICK GRAY GARY US LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
THE JOHNS HOPKINS UNIVERSITY SCHOOL
OF MEDICINE
|
Family ID: |
23740672 |
Appl. No.: |
11/514821 |
Filed: |
September 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10192988 |
Jul 10, 2002 |
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11514821 |
Sep 1, 2006 |
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09261007 |
Mar 2, 1999 |
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10192988 |
Jul 10, 2002 |
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08438439 |
May 12, 1995 |
5876967 |
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09261007 |
Mar 2, 1999 |
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Current U.S.
Class: |
530/388.25 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 38/00 20130101; C07K 16/22 20130101; C07K 14/50 20130101 |
Class at
Publication: |
530/388.25 |
International
Class: |
C07K 16/22 20060101
C07K016/22 |
Claims
1. An isolated antibody that binds to an FHF-2 polypeptide
comprising the amino acid sequence set forth in SEQ ID NO.:2 or
immunoreactive fragments of the FHF-2 polypeptide.
2. The isolated antibody of claim 1, wherein the antibody is
polyclonal.
3. The isolated antibody of claim 2, wherein the antibody is
monoclonal.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] Under 35 USC .sctn. 120, this application is a continuation
application of U.S. application Ser. No. 10/192,988 filed Jul. 10,
2002; which is a continuation of U.S. application Ser. No.
09/261,007 filed Mar. 2, 1999; which is a divisional of U.S.
application Ser. No. 08/438,439 filed May 12, 1995 and issued as
U.S. Pat. No. 5,876,967. This disclosure of the prior applications
is considered part of and is incorporated by reference in the
disclosure of this application.
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-2
(FHF-2) and the polynucleotide encoding FHF-2.
[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: int-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 and
differentiation factor, FHF-2, 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) and in the heart.
[0010] The invention provides a method for detecting alterations in
FHF-2 gene expression which are diagnostic of neurodegenerative,
neoplastic, or cardiac disorders. In another embodiment, the
invention provides a method for treating a neurodegenerative,
neoplastic or cardiac disorder by enhancing or suppressing the
expression or activity of FHF-2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the nucleotide and predicted amino acid
sequence of human FHF-2 (SEQ ID NO:1 and SEQ ID NO:2).
[0012] FIGS. 2A and B show an alignment of the amino acid sequence
of human FHF-2 and each of the published nine members of the FGF
family. Conserved residues are highlighted. The FGF family members
are: aFGF/FGF-1 (SEQ ID NO:22) (Jaye et al., Science 233: 541,
1986), bFGF/FGF-2 (SEQ ID NO:24) (Abraham et al., Science 233: 545,
1986), int-2/FGF-3 (SEQ ID NO: 17) (Smith et al., EMBO J. 7: 1013,
1988), FGF-4 (SEQ ID NO:19) (Delli-Bovi et al., Cell 50: 729,
1987), FGF-5 (SEQ ID NO: 18) (Zhan et al., Mol. Cell Biol. 8, 3487,
1988), FGF-6 (SEQ ID NO:20) (Marics et al., Oncogene 4: 335, 1989);
keratinocyte growth factor/FGF-7 (SEQ ID NO:23) (Finch et al..
Science 245: 752. 1989), FGF-8 (SEQ ID NO: 16) (Tanaka et al.,
Proc. Nati. Acad. Sci. USA 89: 8928, 1992), and FGF-9 (SEQ ID
NO:21) (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 the location of the gene encoding FHF-2 on the
human X-chromosome. A Southern blot was prepared from DNA derived
from mouse-human or hamster-human hybrid cell lines, each of which
contains a single human chromosome, indicated above each lane. The
human specific hybridization is found on the X-chromosome.
[0015] FIG. 5 shows the production of FHF-2 in transfected human
embryonic kidney cells. Proteins were labeled biosynthetically with
.sup.35S-methionine and resolved by SDS-polyacrylamide gel
electrophoresis. Lanes 1, 3, 5, and 7: total cell protein; lanes 2,
4, 6, and 8: protein present in the medium (secreted protein).
Lanes 1 and 2, mock transfected cells; lanes 3 and 4, transfection
with cDNA encoding FHF-1, a closely related member of the FGF
family; lanes 5 and 6, transfection with cDNA encoding FHF-2; lanes
7 and 8, transfection with cDNA encoding human growth hormone.
Arrows indicate the FHF-1 and FHF-2 protein bands. 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 FHF-2 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-2 antisense probe that spanned 197 bases of the most 3' coding
region exon and the adjacent upstream 335 bases of intron sequence.
RNAse protection at the size expected for the 197 base exon region
of the probe (arrowheads) was observed with RNA from brain, eye,
and heart. A longer exposure reveals barely visible bands in all of
the other tissues but not in the tRNA control sample.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides a growth factor, FHF-2, and a
polynucleotide sequence encoding FHF-2. FHF-2 is expressed at high
levels in brain and heart tissues. In one embodiment, the invention
provides a method for detection of a cell proliferative or
immunologic disorder of central nervous system or cardiac tissue
origin which is associated with FHF-2 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-2 expression or activity.
[0018] The structural homology between the FHF-2 protein of this
invention and the members of the FGF family, indicates that FHF-2
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-2 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; Sawchenko, 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,
88:4250, 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-2 in brain and eye suggests that FHF-2
may also possess activities that relate to the function of the
nervous system. The known neurotrophic activities of other members
of this family and the expression of FHF-2 in muscle suggest that
one activity of FHF-2 may be as a trophic factor for motor neurons.
Alternatively, FHF-2 may have neurotrophic activities for other
neuronal populations. Hence, FHF-2 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] Growth factors have also been shown to inhibit the
differentiation of myoblasts in culture (Massague, et al., Proc.
Natl. Acad Sci., USA 83:8206, 1986). Moreover, because myoblast
cells may be used as a vehicle for delivering genes to muscle for
gene therapy, the properties of FHF-2, namely the elevated
expression in heart tissue (i.e., muscle), could be exploited for
maintaining cells prior to transplantation or for enhancing the
efficiency of the fusion process.
[0022] In a first embodiment, the invention provides substantially
pure fibroblast growth factor homologous factor-2 (FHF-2)
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-2 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-2 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-2 polypeptide can also be
determined by amino-terminal amino acid sequence analysis. FHF-2
polypeptide includes functional fragments of the polypeptide, as
long as the activity of FHF-2 remains. Smaller peptides containing
the biological activity of FHF-2 are included in the invention.
[0023] The invention provides polynucleotides encoding the FHF-2
protein. These polynucleotides include DNA, cDNA and RNA sequences
which encode FHF-2. It is understood that all polynucleotides
encoding all or a portion of FHF-2 are also included herein, as
long as they encode a polypeptide with FHF-2 activity. Such
polynucleotides include naturally occurring, synthetic, and
intentionally manipulated polynucleotides. For example, FHF-2
polynucleotide may be subjected to site-directed mutagenesis. The
polynucleotide sequence for FHF-2 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-2
polypeptide encoded by the nucleotide sequence is functionally
unchanged.
[0024] Specifically disclosed herein is a DNA sequence encoding the
human FHF-2 gene. The sequence contains an open reading frame
encoding a polypeptide 245 amino acids in length. The human FHF-2
inititiator methionine codon shown in FIG. 1 at position 352-354
corresponds to the location of the initiator methionine codon of
another FGF family member, FHF-1, when the two sequences are
aligned; 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
124 codons 3' of the putative initiator methionine codon. As
observed for aFGF and bFGF, the amino-terminus of the primary
translation product of FHF-2 does not conform to the consensus
sequence for a signal peptide to direct cotranslational insertion
across the endoplasmic reticulum membrane. The FHF-2 sequence has
one potential asn-X-ser/thr site for asparagine-linked
glycosylation four amino acids from the carboxy-terminus.
Preferably, the human FHF-2 nucleotide sequence is SEQ ID NO:1 and
the deduced amino acid sequence is probably SEQ ID NO:2.
[0025] The polynucleotide encoding FHF-2 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-2 protein under stringent conditions.
[0026] The most homologous FGF family member is FGF-9, which shares
28% amino acid identity with FHF-2, when aligned with 6 gaps. Minor
modifications of the FHF-2 primary amino acid sequence may result
in proteins which have substantially equivalent activity as
compared to the FHF-2 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-2 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-2 biological
activity.
[0027] The nucleotide sequence encoding the FHF-2 polypeptide 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.
[0028] 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.
[0029] Preferably the FHF-2 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).
[0030] The development of specific DNA sequences encoding FHF-2 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.
[0031] 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.
[0032] 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).
[0033] A cDNA expression library, such as lambda gt11, can be
screened indirectly for FHF-2 peptides having at least one epitope,
using antibodies specific for FHF-2. Such antibodies can be either
polyclonally or monoclonally derived and used to detect expression
product indicative of the presence of FHF-2 cDNA.
[0034] DNA sequences encoding FHF-2 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.
[0035] In the present invention, the FHF-2 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-2 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 not 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 1, or polyhedrin promoters).
[0036] Polynucleotide sequences encoding FHF-2 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.
[0037] 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.
[0038] 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-2 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).
[0039] 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.
[0040] The FHF-2 polypeptides of the invention can also be used to
produce antibodies which are immunoreactive or bind to epitopes of
the FHF-2 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).
[0041] 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: [0042] (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; [0043] (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; [0044] (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; [0045] (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 [0046] (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.
[0047] 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).
[0048] 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.
[0049] Antibodies which bind to the FHF-2 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, EXAMPLE 4) 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).
[0050] 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).
[0051] 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.
[0052] 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-2 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, heart, and cells of the eye. Essentially,
any disorder which is etiologically linked to altered expression of
FHF-2 could be considered susceptible to treatment with a FHF-2
suppressing reagent. One such disorder is a malignant cell
proliferative disorder, for example.
[0053] For purposes of the invention, an antibody or nucleic acid
probe specific for FHF-2 may be used to detect FHF-2 polypeptide
(using antibody) or polynucleotide (using nucleic acid probe) in
biological tissues or fluids. The invention provides a method for
detecting a cell proliferative disorder of cardiac tissue or neural
tissue, for example, which comprises contacting an anti-FHF-2
antibody or nucleic acid probe with a cell suspected of having a
FHF-2 associated disorder and detecting binding of FHF-2 antigen or
mRNA to the antibody or nucleic acid probe, respectively. The
antibody or nucleic acid probe reactive with FHF-2 is preferably
labeled with a compound which allows detection of binding to FHF-2.
Any specimen containing a detectable amount of antigen can be used.
A preferred sample of this invention is neural tissue or heart
tissue. The level of FHF-2 in the suspect cell can be compared with
the level in a normal cell to determine whether the subject has a
FHF-2-associated cell proliferative disorder. Preferably the
subject is human.
[0054] When the cell component is nucleic acid, it may be necessary
to amplify the nucleic acid prior to binding with an FHF-2 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.
[0055] 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 immunoassays 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.
[0056] 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,
polyacrylamides, 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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 .sup.201Tl.
[0064] 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.
[0065] 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-2-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-2-associated
disease is effective. The term "ameliorate" denotes a lessening of
the detrimental effect of the FHF-2-associated disease in the
subject receiving therapy.
[0066] 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-2 expression is accomplished by
hybridization of nucleic acids isolated from a cell suspected of
having an FHF-2 associated proliferative disorder with an FHF-2
polynucleotide of the invention. Analyses, such as Northern Blot
analysis, are utilized to quantitate expression of FHF-2. Other
standard nucleic acid detection techniques will be known to those
of skill in the art.
[0067] Treatment of an FHF-2 associated cell proliferative disorder
include modulation of FHF-2 gene expression and FHF-2 activity. The
term "modulate" envisions the suppression of expression of FHF-2
when it is over-expressed, or augmentation of FHF-2 expression when
it is under-expressed. Where a cell-proliferative disorder is
associated with the expression of FHF-2, nucleic acid sequences
that interfere with FHF-2 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-2 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.
[0068] 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-2-producing cell. The
use of antisense methods to inhibit the in vitro translation of
genes is well known in the art (Marcus-Sakura, AnalBiochem.,
172:289, 1988).
[0069] 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).
[0070] 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.
[0071] 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.
[0072] The present invention also provides gene therapy for the
treatment of cell proliferative or immunologic disorders which are
mediated by FHF-2 protein. Such therapy would achieve its
therapeutic effect by introduction of the FHF-2 antisense
polynucleotide into cells having the proliferative disorder.
Delivery of antisense FHF-2 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.
[0073] 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 can be identified and
generated. By inserting a FHF-2 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-2 antisense
polynucleotide.
[0074] 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
e-ncapsidation. Helper cell lines which have deletions of the
packaging signal include, but are not limited to .PSI.2, PA317 and
PA 12, 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.
[0075] 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.
[0076] Another targeted delivery system for FHF-2 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 macro-molecules. 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).
[0077] 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.
[0078] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are d-iacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0079] 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.
[0080] 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.
[0081] Due to the expression of FHF-2 in heart, 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-2 may be useful in various gene
therapy procedures.
[0082] The identification of a novel member of the FGF family
provides a useful tool for diagnosis, prognosis and therapeutic
strategies associated with FHF-2 mediated disorders. Measurement of
FHF-2 levels using anti-FHF-2 antibodies is 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 p-igmentosa, or viral encephalitis. The presence
of high levels of FHF-2 in the central nervous system suggests that
the observed low level of FHF-2 in a number of peripheral tissues
could reflect FHF-2 in peripheral nerve, and therefore measurement
of FHF-2 levels using anti-FHF-2 antibodies could be diagnostic for
peripheral neuropathy. The presence of high levels of FHF-2 in the
heart suggests that measurement of FHF-2 levels using anti-FHF-2
antibodies is useful as a diagnostic for myocardial infarction,
viral endocarditis, or other cardiac disorders.
[0083] Like other members of the FGF family, FHF-2 likely has
mitogenic and/or cell survival activity, therefore FHF-2 or an
analogue that mimics FHF-2 action could be used to promote tissue
repair or replacement. The presence of FHF-2 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. The presence of high levels of FHF-2 in
the heart suggests that FHF-2 or an analogue of FHF-2 could be used
to accelerate recovery from myocardial infarction or could promote
increased cardiac output by increasing heart muscle.
[0084] Conversely, blocking FHF-2 action either with anti-FHF-2
antibodies or with an FHF-2 antagonist might slow or ameliorate
diseases in which excess cell growth is pathological, most
obviously cancer.
[0085] 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
Isolation of FHF-2, a Novel Member of the FGF Family
[0086] 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.
[0087] 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 arrayed
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.
[0088] 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. This
sequence was named FHF-1 and is the subject of a pending patent
application (U.S. patent application Ser. No. 08/439,725,
"Fibroblast Growth Factor Homologous Factor-1 (FHF-1) and Methods
of Use", Nathans et al., filed May 12, 1995). A search of partial
cDNA sequences (`expressed sequence tags`, ESTs) in the public
databases revealed a human adult testis cDNA fragment (NCBI ID
28057, EST ID EST06895, Genbank ID T09003; Adams, et al., Nature
Genetics, 4: 373, 1993) with strong homology to FHF-1, but only
weak homology to other members of the FGF family. The homology
between this EST and other members of the FGF family is
sufficiently low that no indication of that homology was noted in
the description associated with the clone in Genbank or in the
publication describing the EST sequence (Adams, et al., supra,
1993). Based on the EST sequence, this fragment was amplified by
PCR from a human retina cDNA library and used as a probe to isolate
multiple independent cDNA clones from that library, including two
that encompass the entire open reading frame and from which
complete nucleotide sequences were determined. This sequence was
named FHF-2.
EXAMPLE 2
Deduced Primary Structure FHF-2
[0089] FIG. 1 shows the sequence of human FHF-2 deduced from the
nucleotide sequences of two independent human retina cDNA clones.
The primary translation product of human FHF-2 is predicted to be
245 amino acids in length. The human FHF-2 inititiator methionine
codon shown in FIG. 1 at position 352-354 corresponds to the
location of the initiator methionine codon of FHF-1 when the two
sequences are aligned; a good consensus ribosome binding site
(TGGCCATGG; Kozak, Nucleic Acids Res., 15: 8125, 1987) (SEQ ID
NO:5) is found at this position. The next methionine codon within
the open reading frame is encountered 124 codons 3' of the putative
initiator methionine codon. As observed for aFGF and bFGF, the
amino-terminus of the primary translation product of FHF-2 does not
conform to the consensus sequence for a signal peptide to direct
cotranslational insertion across the endoplasmic reticulum
membrane. The FHF-2 sequence has one potential asn-X-ser/thr site
for asparagine-linked glycosylation four amino acids from the
carboxy-terminus.
[0090] Alignment of FHF-2 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 28% amino acid identity with
FHF-2 when aligned with 6 gaps. Note that in the central region of
each polypeptide, all FGF family members, including FHF-2, share 11
invariant amino acids.
EXAMPLE 3
Chromosomal Localization of FHF-2
[0091] The chromosomal location of FHF-2 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-2 probe to human,
mouse, and hamster genomic DNA produces distinct hybridizing
fragment sizes. The human-specific hybridization pattern is seen
only in the lane corresponding to the hybrid cell line carrying the
human X-chromosome.
EXAMPLE 4
Production of FHF-2 in Tranfected Human Cells
[0092] To express FHF-2 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) (SEQ ID NO:5) by cutting the FHF-2 coding
region at the initiator methionine with Nco 1 (which recognizes
CCATGG) and ligating to the expression vector. 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 35S methionine for 6 hours in the
absence of serum. As shown in FIG. 5, cells transfected with FHF-2
synthesize a single polypeptide with an apparent molecular mass of
30 kD that is not produced by untransfected cells or by cells
transfected with an unrelated construct. This polypeptide
corresponds closely to the predicted molecular mass of the primary
translation product, 27.6 kD. FIG. 5 also shows that cells
transfected with a human growth hormone (hGH) expression plasmid
efficiently secrete hGH, whereas FHF-2 accumulates within the
transfected cells and fails to be secreted in detectable
quantities.
EXAMPLE 5
Tissue Distribution of FHF-2 mRNA
[0093] To determine the tissue distribution of FHF-2 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-2 gene isolated by hybridization with the
full-length human FHF-2 cDNA. As seen in FIG. 6, the highest levels
of FHF-2 expression are in the brain, eye, and heart. Very low
levels of FHF-2 expression were detected in all of the other
tisssues on a longer exposure of the autoradiogram.
[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
25 1 1150 DNA Homo sapiens CDS (353)..(1087) 1 aattccgctt
gcacagtgtc cgccgggcgc aggggccgac cgcacgcagt cgcgcagttc 60
tgcctccgcc tgccagtctc gcccgcgatc ccggcccggg gctgtggcgt cgactccgac
120 ccaggcagcc agcagcccgc gcgggagccg gaccgccgcc ggaggagctc
ggacggcatg 180 ctgagccccc tccttggctg aagcccgagt gcggagaagc
ccgggcaaac gcaggctaag 240 gagaccaaag cggcgaagtc gcgagacagc
ggacaagcag cggaggagaa ggaggaggag 300 gcgaacccag agaggggcag
caaaagaagc ggtggtggtg ggcgtcgtgg cc atg gcg 358 Met Ala 1 gcg gct
atc gcc agc tcg ctc atc cgt cag aag agg caa gcc cgc gag 406 Ala Ala
Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala Arg Glu 5 10 15 cgc
gag aaa tcc aac gcc tgc aag tgt gtc agc agc ccc agc aaa ggc 454 Arg
Glu Lys Ser Asn Ala Cys Lys Cys Val Ser Ser Pro Ser Lys Gly 20 25
30 aag acc agc tgc gac aaa aac aag tta aat gtc ttt tcc cgg gtc aaa
502 Lys Thr Ser Cys Asp Lys Asn Lys Leu Asn Val Phe Ser Arg Val Lys
35 40 45 50 ctc ttc ggc tcc aag aag agg cgc aga aga aga cca gag cct
cag ctt 550 Leu Phe Gly Ser Lys Lys Arg Arg Arg Arg Arg Pro Glu Pro
Gln Leu 55 60 65 aag ggt ata gtt acc aag cta tac agc cga caa ggc
tac cac ttg cag 598 Lys Gly Ile Val Thr Lys Leu Tyr Ser Arg Gln Gly
Tyr His Leu Gln 70 75 80 ctg cag gcg gat gga acc att gat ggc acc
aaa gat gag gac agc act 646 Leu Gln Ala Asp Gly Thr Ile Asp Gly Thr
Lys Asp Glu Asp Ser Thr 85 90 95 tac act ctg ttt aac ctc atc cct
gtg ggt ctg cga gtg gtg gct atc 694 Tyr Thr Leu Phe Asn Leu Ile Pro
Val Gly Leu Arg Val Val Ala Ile 100 105 110 caa gga gtt caa acc aag
ctg tac ttg gca atg aac agt gag gga tac 742 Gln Gly Val Gln Thr Lys
Leu Tyr Leu Ala Met Asn Ser Glu Gly Tyr 115 120 125 130 ttg tac acc
tcg gaa ctt ttc aca cct gag tgc aaa ttc aaa gaa tca 790 Leu Tyr Thr
Ser Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys Glu Ser 135 140 145 gtg
ttt gaa aat tat tat gtg aca tat tca tca atg ata tac cgt cag 838 Val
Phe Glu Asn Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr Arg Gln 150 155
160 cag cag tca ggc cga ggg tgg tat ctg ggt ctg aac aaa gaa gga gag
886 Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu Gly Glu
165 170 175 atc atg aaa ggc aac cat gtg aag aag aac aag cct gca gct
cat ttt 934 Ile Met Lys Gly Asn His Val Lys Lys Asn Lys Pro Ala Ala
His Phe 180 185 190 ctg cct aaa cca ctg aaa gtg gcc atg tac aag gag
cca tca ctg cac 982 Leu Pro Lys Pro Leu Lys Val Ala Met Tyr Lys Glu
Pro Ser Leu His 195 200 205 210 gat ctc acg gag ttc tcc cga tct gga
agc ggg acc cca acc aag agc 1030 Asp Leu Thr Glu Phe Ser Arg Ser
Gly Ser Gly Thr Pro Thr Lys Ser 215 220 225 aga agt gtc tct ggc gtg
ctg aac gga ggc aaa tcc atg agc cac aat 1078 Arg Ser Val Ser Gly
Val Leu Asn Gly Gly Lys Ser Met Ser His Asn 230 235 240 gaa tca acg
tagccagtga gggcaaaaga agggctctgt aacagaacct 1127 Glu Ser Thr 245
tacctccagg tgctgttgaa ttc 1150 2 245 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 Arg Glu Lys Ser Asn Ala Cys Lys Cys Val Ser Ser Pro Ser 20 25
30 Lys Gly Lys Thr Ser Cys Asp Lys Asn Lys Leu Asn Val Phe Ser Arg
35 40 45 Val Lys Leu Phe Gly Ser Lys Lys Arg Arg Arg Arg Arg Pro
Glu Pro 50 55 60 Gln Leu Lys Gly Ile Val Thr Lys Leu Tyr Ser Arg
Gln Gly Tyr His 65 70 75 80 Leu Gln Leu Gln Ala Asp Gly Thr Ile Asp
Gly Thr Lys Asp Glu Asp 85 90 95 Ser Thr Tyr Thr Leu Phe Asn Leu
Ile Pro Val Gly Leu Arg Val Val 100 105 110 Ala Ile Gln Gly Val Gln
Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu 115 120 125 Gly Tyr Leu Tyr
Thr Ser Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys 130 135 140 Glu Ser
Val Phe Glu Asn Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr 145 150 155
160 Arg Gln Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu
165 170 175 Gly Glu Ile Met Lys Gly Asn His Val Lys Lys Asn Lys Pro
Ala Ala 180 185 190 His Phe Leu Pro Lys Pro Leu Lys Val Ala Met Tyr
Lys Glu Pro Ser 195 200 205 Leu His Asp Leu Thr Glu Phe Ser Arg Ser
Gly Ser Gly Thr Pro Thr 210 215 220 Lys Ser Arg Ser Val Ser Gly Val
Leu Asn Gly Gly Lys Ser Met Ser 225 230 235 240 His Asn Glu Ser Thr
245 3 30 DNA Artificial sequence Synthetic oligonucleotide
containing an EcoR I site misc_feature (1)..(30) n is any
nucleotide 3 gacgagatat tagaattcta ctcgnnnnnn 30 4 33 DNA
Artificial sequence PCR primer 4 cccccccccg acgagatatt agaattctac
tcg 33 5 9 DNA Artificial sequence Consensus ribosome binding site
5 tggccatgg 9 6 215 PRT Unknown Mammalian 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 7 245 PRT
Unknown Mammalian 7 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 8 268 PRT Unknown Mammalian 8
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 9 206 PRT
Unknown Mammalian 9 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 10 198 PRT
Unknown Mammalian 10 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 11 246 PRT Unknown Mammalian 11 Met Ala Ala Ala Ile
Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala 1 5 10 15 Arg Glu Arg
Glu Lys Ser Asn Ala Cys Lys Cys Val Ser Ser Pro Ser 20 25 30 Lys
Gly Lys Thr Ser Cys Asp Lys Asn Lys Leu Asn Val Phe Ser Arg 35 40
45 Val Lys Leu Phe Gly Ser Lys Lys Arg Arg Arg Arg Arg Pro Glu Pro
50 55 60 Gln Leu Lys Gly Ile Val Thr Lys Leu Tyr Ser Arg Gln Gly
Tyr His 65 70 75 80 Leu Gln Leu Gln Ala Asp Gly Thr Ile Asp Gly Thr
Lys Asp Glu Asp 85 90 95 Ser Thr Tyr Thr Leu Phe Asn Leu Ile Pro
Val Gly Leu Arg Val Val 100 105 110 Ala Ile Gln Gly Val Gln Thr Lys
Leu Tyr Leu Ala Met Asn Ser Glu 115 120 125 Gly Tyr Leu Tyr Thr Ser
Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys 130 135 140 Glu Ser Val Phe
Glu Asn Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr 145 150 155 160 Arg
Gln Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu 165 170
175 Gly Glu Ile Met Lys Gly Asn His Val Lys Lys Asn Lys Pro Ala Ala
180 185 190 His Phe Leu Pro Lys Pro Leu Lys Val Ala Met Tyr Lys Glu
Pro Ser 195 200 205 Leu His Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser
Gly Thr Pro Thr 210 215 220 Lys Ser Arg Ser Val Ser Gly Val Leu Asn
Gly Gly Lys Ser Met Ser 225 230 235 240 His Asn Glu Ser Thr Thr 245
12 208 PRT Unknown Mammalian 12 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 13 155 PRT Unknown Mammalian 13 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 Ile 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 14 155 PRT
Unknown Mammalian 14 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 15 194 PRT Unknown Mammalian 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 16 215 PRT Unknown Mammalian 16 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 Glu 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 Glu Gly 65 70 75 80 Glu Pro Phe Ala Lys Leu Ile Val Glu Thr Glu
Thr Phe Gly Ser Arg 85 90 95 Val Arg Val Arg Gly Ala Glu Thr Gly
Leu Tyr Ile Gln Met Asn Lys 100 105 110 Lys Gly Lys Leu Ile Ala Lys
Ser Asn Gly Lys Gly Lys Glu Gln 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 17 245
PRT Unknown Mammalian 17 Met Gly Leu Ile Tyr Leu Leu Leu Leu Ser
Leu Leu Glu Pro Ser Tyr 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 Gly 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 Tyr
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 18 269 PRT Unknown
Mammalian 18 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 Arg Lys Gly Cys Ser Pro Arg Val Lys Pro 195 200 205 Gln His Ile
Ser Thr His Phe Leu Pro Arg Glu Phe Lys Gln Ser Glu 210 215 220 Gln
Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro 225 230
235 240 Pro Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys
Asn 245 250 255 Thr Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly
260 265 19 206 PRT Unknown Mammalian 19 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 Trp Gly Gly Ala Ala Ala Pro 20 25 30 Thr Ala Pro
Asn Gly Thr Leu Glu Ala Glu Leu Glu Trp Trp Trp Glu 35 40 45 Ser
Leu Val Ala Leu Ser Leu Ala Trp 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 Trp Leu Trp Trp Leu Tyr Cys Asn Val Gly Ile Gly Phe
His Leu 85 90 95 Gln Ala Leu Pro Asp Gly Trp Ile Gly Gly Ala His
Ala Asp Thr Trp 100 105 110 Asp Ser Leu Leu Glu Leu Ser Pro Val Glu
Trp Gly Val Val Ser Ile 115 120 125 Phe Gly Val Ala Ser Trp 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 Trp 180 185
190 Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Trp Leu 195 200
205 20 198 PRT Unknown Mammalian 20 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 21 193 PRT Unknown Mammalian 21 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 Glu Ser 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 22 191 PRT Unknown Mammalian 22 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 Ile Leu
Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95 Glu Cys Ile Leu Phe Leu
Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 100 105 110 Tyr Ile Ser Lys
Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 115 120 125 Lys Asn
Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 130 135 140
Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Met Tyr Arg Glu Pro Ser 145
150 155 160 Leu His Glu Ile Gly Glu Lys Gln Gly Arg Ser Arg Lys Ser
Ser Gly 165 170 175 Thr Pro Thr Met Asn Gly Gly Lys Val Val Asn Gln
Asp Ser Thr 180 185 190 23 194 PRT Unknown Mammalian 23 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 Arg 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 24 154 PRT Unknown Mammalian 24 Ala Ala
Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly Gly 1 5 10 15
Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr 20
25 30 Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg
Val 35 40 45 Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu
Gln Leu Gln 50 55 60 Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly
Val Cys Ala Asn Arg 65 70 75 80 Tyr Leu Ala Met Lys Glu Asp Gly Arg
Leu Leu Ala Ser Lys Cys Val 85 90 95 Thr Asp Glu Cys Phe Phe Phe
Glu Arg Leu Glu Ser Asn Asn Tyr Asn 100 105 110 Thr Tyr Arg Ser Arg
Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg 115 120 125 Thr Gly Gln
Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys Ala 130 135 140 Ile
Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 25 243 PRT Unknown
Mammalian 25 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 Gly 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
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