U.S. patent application number 11/459845 was filed with the patent office on 2007-01-04 for human ccv polypeptides.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Ping Feng, Reiner Gentz, Geoffrey W. Krissansen, Jian Ni, Craig A. Rosen, Jeffrey Y. Su.
Application Number | 20070004008 11/459845 |
Document ID | / |
Family ID | 26710688 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070004008 |
Kind Code |
A1 |
Ni; Jian ; et al. |
January 4, 2007 |
HUMAN CCV POLYPEPTIDES
Abstract
The present invention relates to novel human proteins and
isolated nucleic acids containing the coding regions of the genes
encoding such proteins. Also provided are vectors, host cells and
recombinant methods for producing the proteins of the invention.
The invention further relates to diagnostic and therapeutic methods
useful for diagnosing and treating disorders related to these novel
human secreted proteins.
Inventors: |
Ni; Jian; (Germantown,
MD) ; Rosen; Craig A.; (Laytonsville, MD) ;
Gentz; Reiner; (Gauting, DE) ; Su; Jeffrey Y.;
(Clinton, NJ) ; Krissansen; Geoffrey W.;
(Auckland, NZ) ; Feng; Ping; (Germantown,
MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC.;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
Auckland Uniservices, Limited
Auckland
|
Family ID: |
26710688 |
Appl. No.: |
11/459845 |
Filed: |
July 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10314942 |
Dec 10, 2002 |
7098316 |
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11459845 |
Jul 25, 2006 |
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09010147 |
Jan 21, 1998 |
6653445 |
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10314942 |
Dec 10, 2002 |
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60034204 |
Jan 21, 1997 |
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60034205 |
Jan 21, 1997 |
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Current U.S.
Class: |
435/69.1 ;
435/226; 435/320.1; 435/325; 530/388.26; 536/23.2 |
Current CPC
Class: |
C07K 14/70535 20130101;
C12N 9/6475 20130101; C12N 9/6489 20130101; C12N 2799/026 20130101;
C07K 14/70503 20130101; A61K 38/00 20130101; C07K 14/47 20130101;
C12N 9/6491 20130101; A61P 43/00 20180101; A61P 35/00 20180101;
C12N 9/6421 20130101; C07K 14/705 20130101 |
Class at
Publication: |
435/069.1 ;
435/226; 435/320.1; 435/325; 530/388.26; 536/023.2 |
International
Class: |
C07K 16/40 20060101
C07K016/40; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 9/64 20060101 C12N009/64 |
Claims
1. An isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a nucleotide sequence
encoding a polypeptide selected from the group consisting of: (a)
the polypeptide shown in SEQ ID NO:2; (b) the polypeptide shown in
SEQ ID NO:4; (c) the mature polypeptide shown as residues 16-172 in
SEQ ID NO:4; (d) the polypeptide shown in SEQ ID NO:6; (e) the
mature polypeptide shown as residues 16-88 in SEQ ID NO:6; (f) the
mature polypeptide shown as residues 23-88 in SEQ ID NO:6; (g) the
polypeptide shown in SEQ ID NO:8; (h) the polypeptide shown in SEQ
ID NO:10; (i) the polypeptide shown in SEQ ID NO:12; (j) the
polypeptide shown in SEQ ID NO:14; (k) the polypeptide shown in SEQ
ID NO:16; (l) the polypeptide shown in SEQ ID NO:18; (m) the
polypeptide shown in SEQ ID NO:20; (n) the mature polypeptide shown
as residues 16-528 in SEQ ID NO:20; (o) the polypeptide shown in
SEQ ID NO:22; and (p) the polypeptide shown in SEQ ID NO:24.
2. The nucleic acid molecule of claim 1 comprising a nucleotide
sequence which is at least 95% identical to a nucleotide sequence
selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID
NO:23.
3. An isolated nucleic acid molecule of claim 3 comprising a
nucleotide sequence which is at least 95% identical to a sequence
of at least about 500 contiguous nucleotides in the nucleotide
sequence of SEQ ID NO:X.
4. An isolated nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule of
claim 1, wherein said nucleic acid molecule which hybridizes does
not hybridize under stringent hybridization conditions to a nucleic
acid molecule having a nucleotide sequence consisting of only A
residues or of only T residues.
5. An isolated nucleic acid molecule of claim 6 comprising a
nucleotide sequence which is at least 95% identical to sequence of
at least 500 contiguous nucleotides in the nucleotide sequence
encoded by said human cDNA clone.
6. An isolated polypeptide comprising an amino acid sequence which
is identical to a sequence of at least about 10 contiguous amino
acids in the amino acid sequence of SEQ ID NO:Y wherein Y is any
integer as defined in Table 1.
7. An isolated polypeptide of claim 6 comprising an amino acid
sequence at least 95% identical to the complete amino acid sequence
of SEQ ID NO:Y.
8. An isolated polypeptide comprising an amino acid sequence
identical to a sequence of at least about 10 contiguous amino acids
in the complete amino acid sequence of a secreted protein encoded
by a human cDNA clone identified by a cDNA Clone Identifier in
Table 1 and contained in the deposit with the ATCC Deposit Number
shown for said cDNA clone in Table 1.
9. A method of making a recombinant vector comprising inserting an
isolated nucleic acid molecule of claim 1 into a vector.
10. A recombinant vector produced by the method of claim 9.
11. A method of making a recombinant host cell comprising
introducing a vector of claim 10 into a host cell.
12. A recombinant host cell produced by the method of claim 12.
13. A method of making an isolated polypeptide comprising culturing
a recombinant host cell of claim 12 under conditions such that said
polypeptide is expressed and recovering said polypeptide.
14. An isolated polypeptide produced by the method of claim 13.
15. An isolated antibody capable of specifically binding to a
polypeptide of claim 6.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 10/314,942, filed Dec. 10, 2002, which is a divisional of U.S.
application Ser. No. 09/010,147, filed Jan. 21, 1998 (now U.S. Pat.
No. 6,653,445, issued Nov. 25, 2003), which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional App. Nos. 60/034,204,
filed Jan. 21, 1997 and 60/034,205, filed Jan. 21, 1997. Each of
the above-identified patent applications is hereby incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to genes encoding novel human
proteins which exhibit a variety useful biological activities. More
specifically, isolated nucleic acid molecules are provided which
encode polypeptides comprising various forms of human proteins.
Human polypeptides are also provided, as are vectors, host cells
and recombinant methods for producing the same. Also provided are
methods for detecting nucleic acids or polypeptides related to
those of the invention, for example, to aid in identification of a
biological sample or diagnosis of disorders related to expression
of protein genes of this invention. The invention further relates
to methods for identifying agonists and antagonists of the proteins
of the invention, as well as to methods for treatment of disorders
related to protein gene expression using polypeptides, antagonists
and agonists of the invention.
BACKGROUND OF THE INVENTION
[0003] Identification and sequencing of human genes is a major goal
of modern scientific research. For example, by identifying genes
and determining their sequences, scientists have been able to make
large quantities of valuable human gene products. These include
human insulin, interferon, Factor VIII, human growth hormone,
tissue plasminogen activator, erythropoeitin and numerous other
proteins. Additionally, knowledge of gene sequences can provide
keys to diagnosis, treatment or cure of genetic diseases such as
muscular dystrophy and cystic fibrosis.
[0004] Despite the great progress that has been made in recent
years, only a small number of genes which encode the presumably
thousands of human proteins have been identified and sequenced.
Therefore, there is a need for identification and characterization
of novel human proteins and corresponding genes which can play a
role in detecting, preventing, ameliorating or correcting disorders
related to abnormal expression of and responses to such
proteins.
SUMMARY OF THE INVENTION
[0005] The present invention provides isolated nucleic acid
molecules comprising polynucleotide sequences which have been
identified as sequences encoding human proteins of the invention.
Each protein of the invention is identified in Table 1, below (see
Example 2) by a reference number designated as a "Protein ID
(Identifier)" (e.g., "PF353-01"). Each protein of the invention is
related to a human complementary DNA (cDNA) clone prepared from a
messenger RNA (mRNA) encoding the related protein. The cDNA clone
related to each protein of the invention is identified by a "cDNA
Clone ID (Identifier)" in Table 1 (e.g., "HABCE99"). DNA of each
cDNA clone in Table 1 is contained in the material deposited with
the American Type Culture Collection and given the ATCC Deposit
Number shown for each cDNA Clone ID in Table 1, as further
described below.
[0006] The invention provides a nucleotide sequence determined for
an mRNA molecule encoding each protein identified in Table 1, which
is designated in Table 1 as the "Total NT (Nucleotide) Sequence."
This determined nucleotide sequence has been assigned a SEQ ID
NO="X" in the Sequence Listing hereinbelow, where the value of X
for the determined nucleotide sequence of each protein is an
integer specified in Table 1. The determined nucleotide sequence
provided for each protein of the invention was determined by
applying conventional automated nucleotide sequencing methods to
DNA of the corresponding deposited cDNA clone cited in Table 1.
[0007] The determined nucleotide sequence for the mRNA encoding
each protein of the invention has been translated to provide a
determined amino acid sequence for each protein which is identified
in Table 1 by a SEQ ID NO="Y" where the value of Y for each protein
is an integer defined in Table 1. The determined amino acid
sequence for each protein represents the amino acid sequence
encoded by the determined nucleotide sequence, beginning at or near
the translation initiation ("start") codon of the protein and
continuing until the first translation termination ("stop") codon.
Due to possible errors inherent in determining nucleotide sequences
from any DNA molecule, particularly using the conventional
automated sequencing technology used to sequence the cDNA clones
described herein, occasional nucleotide sequence errors are
expected in the determined nucleotide sequences of the invention.
These errors may include insertions or deletions of one or a few
nucleotides in the determined nucleotide sequence as compared to
the actual nucleotide sequence of the deposited cDNA. As one of
ordinary skill would appreciate, incorrect insertions or deletions
of one or two nucleotides into a determined nucleotide sequence
leads to a shift in the translation reading frame compared to the
reading frame actually encoded by a cDNA clone. Further, such a
shift in frame within an actual open reading frame frequently leads
to the appearance of a translation termination (stop) codon within
the sequence encoding the polypeptide. Accordingly, due to
occasional errors in the nucleotide sequences determined from the
deposited cDNAs and any related DNA clones used to prepare the
determined sequence for the mRNA encoding each secreted protein of
the invention, the translations shown as determined amino acid
sequences in SEQ ID NO:Y may represent only a portion of the
complete amino acid sequence of the human secreted protein actually
encoded by the mRNA represented by the corresponding cDNA clone in
the ATCC deposit identified in Table 1. In any event, the
determined amino acid sequence for each protein in Table 1, which
is shown in SEQ ID NO:Y for each protein, comprises at least a
portion of the amino acid sequence determined for that protein.
[0008] More particularly, the determined amino acid sequence is the
amino acid sequence translated from the determined nucleotide
sequence in the open reading frame of the first amino acid of the
ORF to the last amino acid of that frame. In other words, the
determined amino acid sequence is translated from the determined
nucleotide sequence beginning at the codon having as its 5' end the
nucleotide in the position of SEQ ID NO:X identified in Table 1 as
the 5' nucleotide of the first amino acid (abbreviated in Table 1
as "5' NT of First AA"). Translation of the determined nucleotide
sequence is continued in the reading frame of that first amino acid
codon to the first stop codon in that same open reading frame,
i.e., to the position in SEQ ID NO:X which encodes the amino acid
at the position in SEQ ID NO:Y identified as the "last amino acid
of the open reading frame" (abbreviated as "Last AA of ORF").
[0009] For any determined amino acid sequence in which the first
amino acid is the methionine encoded by the translation initiation
codon for the protein, Table 1 also identifies the position in SEQ
ID NO:X of the 5' nucleotide of the start codon ("5' NT of Start
Codon") as the same position in SEQ ID NO:X as that of the 5'
nucleotide of the first amino acid ("First AA").
[0010] Table 1 also identifies the positions in SEQ ID NO:Y of the
last amino acid of the signal peptide ("Last AA of Sig Pep") and
the first amino acid of the secreted portion ("First AA of Secreted
Portion") of the protein, for those polypeptide having a secretory
leader sequence. The "secreted portion" of a secreted protein in
the present context indicates that portion of the complete
polypeptide translated from an mRNA which remains after cleavage of
the signal peptide by a signal peptidase. In this context the term
"mature" may also be used interchangeably with "secreted portion"
although it is recognized that in other contexts "mature" may
designate a portion of a "proprotein" which is produced by further
cleavage of the polypeptide after cleavage of the signal
peptide.
[0011] Accordingly, in one aspect the invention provides an
isolated nucleic acid molecule comprising a nucleotide sequence
which is identical to the nucleotide sequence of SEQ ID NO:X, where
X is any integer as defined in Table 1. The invention also provides
an isolated nucleic acid molecule comprising a nucleotide sequence
which is identical to a portion of the nucleotide sequence of SEQ
ID NO:X, for instance, a sequence of at least 50, 100 or 150
contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.
Such a portion of the nucleotide sequence of SEQ ID NO:X may be
described most generally as a sequence of at least C contiguous
nucleotides in the nucleotide sequence of SEQ ID NO:X where: (1)
the sequence of at least C contiguous nucleotides begins with the
nucleotide at position N of SEQ ID NO:X and ends with the
nucleotide at position M of SEQ ID NO:X; (2) C is any integer in
the range beginning with a convenient primer size, for instance,
about 20, to the total nucleotide sequence length ("Total NT Seq.")
as set forth for SEQ ID NO:X in Table 1; (3) N is any integer in
the range of 1 to the first position of the last C nucleotides in
SEQ ID NO:X, or more particularly, N is equal to the value of Total
NT Seq. minus the quantity C plus 1 (i.e., Total NT Seq.-(C+1));
and (4) M is any integer in the range of C to Total NT Seq.
[0012] Preferably, the sequence of contiguous nucleotides in the
nucleotide sequence of SEQ ID NO:X is included in SEQ ID NO:X in
the range of positions beginning with the nucleotide at about the
5' nucleotide of the clone sequence ("5' NT of Clone Seq." in Table
1) and ending with the nucleotide at about the 3' nucleotide of the
clone sequence ("3' NT of Clone Seq." in Table 1). More preferably,
the sequence of contiguous nucleotides is in the range of positions
beginning with the nucleotide at about the position of the 5'
Nucleotide of the Start Codon ("5' NT of Start Codon" in Table 1)
and ending with the nucleotide at about the position of the 3'
Nucleotide of the Clone Sequence as set forth for SEQ ID NO:X in
Table 1. For instance, one preferred embodiment of this aspect of
the invention is an isolated nucleic acid molecule which comprises
a sequence at least 95%, 96%, 97%, 98%, or 99% identical to a
sequence of about 500 contiguous nucleotides included in the
nucleotide sequence of SEQ ID NO:X beginning at about the 5' NT of
Start Codon position as set forth for SEQ ID NO:X in Table 1.
Another preferred embodiment of this aspect of the invention is a
nucleic acid molecule comprising a nucleotide sequence which is at
least 95% identical to the nucleotide sequence of SEQ ID NO:X
beginning with the nucleotide at about the position of the 5'
Nucleotide of the First Amino Acid of the Signal Peptide and ending
with the nucleotide at about the position of the 3' Nucleotide of
the Clone Sequence as defined for SEQ ID NO:X in Table 1.
[0013] Further embodiments of the invention include isolated
nucleic acid molecules which comprise a nucleotide sequence at
least 90% identical, and more preferably at least 95%, 96%, 97%,
98%, 99% or 99.9% identical, to any of the determined nucleotide
sequences above. For instance, one such embodiment is an isolated
nucleic acid molecule comprising a nucleotide sequence which is at
least 95% identical to a sequence of at least 50 contiguous
nucleotides in the nucleotide sequence of SEQ ID NO:X wherein X is
any integer as defined in Table 1. Another embodiment of this
aspect of the invention is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
the complete nucleotide sequence of SEQ ID NO:X.
[0014] Isolated nucleic acid molecules which hybridize under
stringent hybridization conditions to a nucleic acid molecule
described above also are provided. Such a nucleic acid molecule
which hybridizes does not hybridize under stringent hybridization
conditions to a nucleic acid molecule having a nucleotide sequence
consisting of only A residues or of only T residues.
[0015] The invention further provides a composition of matter
comprising a nucleic acid molecule which comprises a human cDNA
clone identified by a cDNA Clone ID (Identifier) in Table 1, which
DNA molecule is contained in the material deposited with the
American Type Culture Collection and given the ATCC Deposit Number
shown in Table 1 for that cDNA clone. As described further in
Example 1, this deposited material comprises a mixture of plasmid
DNA molecules containing cloned cDNAs of the invention. Further,
the invention provides an isolated nucleic acid molecule comprising
a nucleotide sequence which is, for instance, at least 95%
identical to a sequence of at least 50, 150 or 500 contiguous
nucleotides in the nucleotide sequence encoded by a human cDNA
clone contained in the deposit given the ATCC Deposit Number shown
in Table 1. One preferred embodiment of this aspect is an isolated
nucleic acid molecule comprising a nucleotide sequence which is at
least 95% identical to the complete nucleotide sequence encoded by
a human cDNA clone identified in Table 1 and as contained in the
deposit with the ATCC Deposit Number shown in Table 1. Also
provided are isolated nucleic acid molecules which hybridize under
stringent hybridization conditions to a nucleic acid molecule
comprising a nucleotide sequence encoded by a human cDNA clone
identified in Table 1 and contained in the cited deposit.
[0016] These nucleic acid molecules of the invention may be used
for a variety of identification and diagnostic purposes. For
instance, the invention provides a method for detecting in a
biological sample a nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least
50 contiguous nucleotides in a nucleotide sequence of the
invention. The sequence of the nucleic acid molecule used in this
method is selected from the group consisting of: a nucleotide
sequence of SEQ ID NO:X wherein X is any integer as defined in
Table 1; and a nucleotide sequence encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC Deposit Number shown for said cDNA clone
in Table 1. This method of the invention comprises a step of
comparing a nucleotide sequence of at least one nucleic acid
molecule in the biological sample with a sequence selected from the
group above, and determining whether the sequence of the nucleic
acid molecule in the sample is at least 95% identical to the
selected sequence. The step of comparing sequences may comprise
determining the extent of nucleic acid hybridization between
nucleic acid molecules in the sample and a nucleic acid molecule
comprising the sequence selected from the above group.
Alternatively, this step may be performed by comparing the
nucleotide sequence determined from a nucleic acid molecule in the
sample, for instance by automated DNA sequence methods, with the
sequence selected from the above group.
[0017] In another aspect, the invention provides methods for
identifying the species, tissue or cell type of a biological sample
based on detecting nucleic acid molecules in the sample which
comprise a nucleotide sequence of a nucleic acid molecule of the
invention (for instance, a nucleic acid molecule comprising a
nucleotide sequence that is at least 95% identical to at least a
portion of a nucleotide sequence of SEQ ID NO:X or a nucleotide
sequence encoded by a human cDNA clone identified in Table 1 as
contained in the deposit with the ATCC Deposit Number shown
therein. This method may be conducted by detecting a nucleotide
sequence of an individual cDNA of the invention or using panel of
nucleotide sequences of the invention. Thus, this method may
comprise a step of detecting nucleic acid molecules comprising a
nucleotide sequence in a panel of at least two nucleotide
sequences, where at least one sequence in the panel is at least 95%
identical to at least a portion of a nucleotide sequence of SEQ ID
NO:X or a nucleotide sequence encoded by a human cDNA clone
contained in the ATCC deposit. In this method for identifying the
species, tissue or cell type of a biological sample, the detection
of nucleic acid molecules comprising nucleotide sequences of the
invention may be conducted by various techniques known in the art
including, for instance, hybridization of either DNA or RNA probes
to either DNA or RNA molecules obtained from the biological sample,
as well as computational comparisons of nucleotide sequences
determined from nucleic acids in a biological sample with
nucleotide sequences of the invention.
[0018] Similarly, nucleic acid molecules of the invention may be
used in a method for diagnosing in a subject a pathological
condition associated with abnormal structure or expression of a
gene encoding a protein identified in Table 1. This method may
comprise a step of detecting in a biological sample obtained from
the subject nucleic acid molecules comprising a nucleotide sequence
that is at least 95% identical to at least a portion of a
nucleotide sequence of SEQ ID NO:X or a nucleotide sequence encoded
by a human cDNA clone identified in Table 1 as contained in the
deposit with the given ATCC Deposit Number. Again, this diagnostic
method may involve analysis of individual nucleotide sequences or
panels of several nucleotide sequences, and the analysis of either
DNA or RNA species using either DNA or RNA probes.
[0019] For use in identification or diagnostic methods such as
those described above, therefore, the invention also provides a
composition of matter comprising isolated nucleic acid molecules in
which the nucleotide sequences of the nucleic acid molecules
comprise a panel of sequences, at least one of which is at least
95% identical to a sequence, either a nucleotide sequence of SEQ ID
NO:X or a nucleotide sequence encoded by a human cDNA clone
contained in the ATCC deposit in Table 1. In this composition, the
nucleic acid molecules may comprise DNA molecules or RNA molecules
or both, as well as polynucleotide equivalents of DNA and RNA which
are not naturally occurring but are known in the art as such.
[0020] Another aspect of the invention relates to polypeptides
comprising amino acid sequences encoded by nucleotide sequences of
the invention. For identification and diagnostic purposes, these
polypeptides need not include the amino acid sequence of a complete
secreted protein or even of the secreted form of such a protein,
since, for instance, antibodies may bind specifically to a linear
epitope of a polypeptide which comprises as few as 6 to 8 amino
acids. Accordingly, the invention also provides an isolated
polypeptide comprising an amino acid sequence at least 90%,
preferably 95%, 96%, 97%, 98%, or 99% identical to a sequence of at
least about 10, 30 or 100 contiguous amino acids in the amino acid
sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1. Preferably, the sequence of contiguous amino acids is
included in the amino acid sequence of SEQ ID NO:Y beginning with
the residue at about the position of the First Amino Acid of the
Secreted Portion where one exists or the first amino acid of the
open reading frame if the protein is not indicated as having a
signal peptide and ending with the residue at about the Last Amino
Acid of the Open Reading Frame as set forth for SEQ ID NO:Y in
Table 1. A preferred embodiment of this aspect relates to an
isolated polypeptide comprising an amino acid sequence at least 95%
identical to the complete amino acid sequence of SEQ ID NO:Y.
[0021] As noted above, however, the determined amino acid sequence
of SEQ ID NO:Y may not include the complete amino acid sequence of
the protein encoded by each cDNA in the ATCC deposit identified in
Table 1. Accordingly, the invention further provides an isolated
polypeptide comprising an amino acid sequence at least 90%
identical, preferably at least 95%, 96%, 97%, 98% or 99% identical
to a sequence of at least about 10, 300 or 100 contiguous amino
acids in the complete amino acid sequence of a secreted protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier
in Table 1 and contained in the deposit with the ATCC Deposit
Number shown for that cDNA clone in Table 1. A particularly
preferred embodiment of this aspect is a polypeptide in which the
sequence of contiguous amino acids is included in the amino acid
sequence of a secreted ("mature") portion of the protein encoded by
a human cDNA clone contained in the deposit, particularly a
polypeptide comprising the entire amino acid sequence of the
secreted portion of the secreted protein encoded by a human cDNA
clone of the invention.
[0022] For purposes such as tissue identification and diagnosis of
pathological conditions, the invention also provides an isolated
antibody which binds specifically to a polypeptide comprising an
amino acid sequence of the invention, (for instance, a sequence
that is identical to a sequence of at least 6, preferably at least
7, 8, 9 or 10, contiguous amino acids in an amino acid sequence of
SEQ ID NO:Y or in a complete amino acid sequence of a protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier
in Table 1 and contained in the deposit cited therein. Further in
the same vein, the invention provides a method for detecting in a
biological sample a polypeptide comprising an amino acid sequence
which is identical to a sequence of at least 6, preferably at least
7, 8, 9 or 10 contiguous amino acids in a sequence selected from
the group consisting of an amino acid sequence of SEQ ID NO:Y and a
complete amino acid sequence of a protein encoded by a human cDNA
clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC Deposit Number shown for
that cDNA clone in Table 1. This method comprises a step of
comparing an amino acid sequence of at least one polypeptide
molecule in said sample with a sequence selected from the above
group and determining whether the sequence of that polypeptide
molecule in the sample is identical to the selected sequence of at
least 6-10 contiguous amino acids. This step of comparing an amino
acid sequence of at least one polypeptide molecule in the sample
with a sequence selected from the above group may comprise
determining the extent of specific binding of polypeptides in the
sample to an antibody which binds specifically to a polypeptide
comprising an amino acid sequence of the invention. Alternatively,
this comparison step may be performed by comparing the amino acid
sequence determined from a polypeptide molecule in the sample with
the sequence selected from the above group, for instance, using
computational methods.
[0023] The invention further provides methods for identifying the
species, tissue or cell type of a biological sample comprising a
step of detecting polypeptide molecules in the sample which include
an amino acid sequence that is identical to a sequence of at least
6-10 contiguous amino acids an amino acid sequence of SEQ ID NO:Y
or of a cDNA identified in Table 1 and contained in the cited
deposit. This method may involve analyses of polypeptides for the
presence of individual amino acid sequences of the invention or of
panels of such sequences. Similarly provided are methods for
diagnosing in a subject a pathological condition associated with
abnormal structure or expression of a gene encoding a protein
identified in Table 1. In preferred embodiments of these methods of
the invention for identification or diagnosis, an antibody which
binds specifically to a polypeptide comprising an amino acid
sequence of the invention is used to analyze amino acid sequences
of polypeptides in a biological sample.
[0024] In yet another aspect, the invention provides recombinant
means for making a polypeptide comprising all or a portion of an
amino acid sequence of the invention. For this purpose, an isolated
nucleic acid molecule comprising a nucleotide sequence which is,
for instance, at least 95% identical to a nucleotide sequence
encoding a polypeptide which comprises an amino acid sequence of
the invention (for instance, one that is at least 90% identical to
SEQ ID NO:Y.
[0025] It will be readily appreciated by one of ordinary skill
that, due to the degeneracy of the genetic code, any nucleotide
sequence encoding the amino acid sequence of a given protein needs
to share only a low level of identity with the nucleotide sequence
of a human cDNA clone which encodes the identical amino acid
sequence of that protein. It will be further appreciated that the
nucleotide of the deposited cDNAs presumably all comprise codons
optimized for expression by human cells from which the cDNAs
originated. Therefore, for improved expression in recombinant
prokaryotic host cells, for instance, it may be desirable to alter
the codon usage in a nucleic acid molecule encoding an amino acid
sequence of the invention, selecting codons in accordance with the
redundancy of the genetic code, which provide optimal codon usage
in the selected host. Preferred nucleic acid molecules of this
aspect of the invention are those which encode a polypeptide which
comprises an complete amino acid sequence of SEQ ID NO:Y or a
complete amino acid sequence of a protein encoded by a human cDNA
clone identified in Table 1 and contained in the deposit cited
therein.
[0026] Using such nucleic acid molecules encoding polypeptides of
the invention, the invention further provides recombinant means for
making the polypeptides. Thus, included is a method of making a
recombinant vector comprising inserting an isolated nucleic acid
molecule of the invention into a vector, as well as a recombinant
vector produced by this method. Also included is a method of making
a recombinant host cell comprising introducing a vector of the
invention into a host cell, and a recombinant host so made. Such
cells are useful, for instance, in a method of making an isolated
polypeptide of the invention which comprises culturing a
recombinant host cell under conditions such that the polypeptide is
expressed and recovering the polypeptide.
[0027] In a preferred embodiment of this method, the recombinant
host cell is a eukaryotic cell and the polypeptide encoded by the
nucleic acid of the invention encodes the complete amino acid
sequence of a protein encoded by a cDNA identified in Table 1, so
that the polypeptide produced by this method is a secreted
("mature") portion of a human secreted protein of the invention
(i.e., one comprising an amino acid sequence of SEQ ID NO:Y
beginning with the residue at the position identified in Table 1 as
the First AA of Secreted Portion of SEQ ID NO:Y or an amino acid
sequence of a secreted portion of a secreted protein encoded by a
human cDNA clone identified in Table 1 and contained in the deposit
with the ATCC Deposit Number shown in Table 1. The invention
further provides an isolated polypeptide which is a secreted
portion of a human secreted protein produced by the above method.
Where the polypeptide shown in Table 1 does not have a leader
sequence one may be provided by the vector. Such vectors are known
in the art and are discussed below.
[0028] In yet another aspect, the invention provides a method of
treatment of an individual in need of an increased level of a
secreted protein activity. As described herein, diagnostic methods
of the invention enable the identification of such individuals,
that is, individuals with a pathological condition involving a
particular organ, tissue or cell type, exhibiting lower levels of
expression product (e.g., mRNA or antigen) of a given secreted
protein in that organ, tissue or cell type, or those with mutant
expression products, compared with normal individuals not suffering
from the pathology. The method of the invention for treatment of an
individual with such a pathological condition comprises
administering to such an individual a pharmaceutical composition
comprising an amount of an isolated polypeptide of a secreted
protein of the invention effective to increase the level of
activity of that secreted protein in the individual.
[0029] Agonists and antagonists of the polypeptides of the
invention and methods for using these also are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIGS. 1A-C show the nucleotide sequence and deduced amino
acid sequence of CCV (HEMFI85), SEQ ID NOS:1 and 2,
respectively.
[0031] FIGS. 2A-B show the nucleotide sequence and deduced amino
acid sequence of CAT-1 (HTXET53), SEQ ID NOS:3 and 4,
respectively.
[0032] FIGS. 3A-B show the nucleotide sequence and deduced amino
acid sequence of CAT-2 (HT3SG28), SEQ ID NOS:5 and 6,
respectively.
[0033] FIG. 4 shows the nucleotide sequence and deduced amino acid
sequence of MIA-2 (HBXAK03), SEQ ID NOS:7 and 8, respectively.
[0034] FIGS. 5A-C show the nucleotide sequence and deduced amino
acid sequence of MIA-3 (HLFBD44), SEQ ID NOS:9 and 10,
respectively.
[0035] FIGS. 6A-B show the nucleotide sequence and deduced amino
acid sequence of AIF-2 (HEBGM49), SEQ ID NOS:11 and 12,
respectively.
[0036] FIGS. 7A-B show the nucleotide sequence and deduced amino
acid sequence of AIF-3 (HNGBH45), SEQ ID NOS:13 and 14,
respectively.
[0037] FIGS. 8A-C show the nucleotide sequence and deduced amino
acid sequence of Annexin HSAAL25, SEQ ID NOS:15 and 16,
respectively.
[0038] FIGS. 9A-K show the nucleotide sequence and deduced amino
acid sequence of ES/130-like I, SEQ ID NOS:17 and 18,
respectively.
[0039] FIGS. 10A-F show the nucleotide sequence and deduced amino
acid sequence of BEF, SEQ ID NOS:19 and 20, respectively.
[0040] FIGS. 11A-E show the nucleotide sequence and deduced amino
acid sequence of ADF, SEQ ID NOS:21 and 22, respectively.
[0041] FIGS. 12A-D show the nucleotide sequence and deduced amino
acid sequence of Bcl-like, SEQ ID NOS:23 and 24, respectively.
DETAILED DESCRIPTION
Nucleic Acid Molecules
[0042] Nucleotide Sequences and ATCC Deposits of cDNA Clones
Encoding Human Proteins
[0043] The present invention provides isolated nucleic acid
molecules comprising polynucleotide sequences which have been
identified as sequences encoding human proteins. The invention
further provides a nucleotide sequence determined from an mRNA
molecule encoding each human protein identified in Table 1, which
comprises all or a substantial portion of the complete nucleotide
sequence of the mRNA encoding each protein of the invention and has
been assigned a SEQ ID NO="X" in the Sequence Listing and Figures
hereinbelow,
[0044] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring nucleic
acid molecule or polynucleotide present in a living organism is not
isolated, but the same nucleic acid molecule or polynucleotide,
separated from some or all of the coexisting materials in the
natural environment, is isolated. Such nucleic acid molecule could
be part of a vector and/or such polynucleotide could be part of a
composition, and still be isolated in that such vector or
composition is not part of the natural environment of the nucleic
acid molecule or polynucleotide.
[0045] By "nucleotide sequence" of a nucleic acid molecule or
polynucleotide is intended, for a DNA molecule or polynucleotide, a
sequence of deoxyribonucleotides, and for an RNA molecule or
polynucleotide, the corresponding sequence of ribonucleotides (A,
G, C and U), where each thymidine deoxyribonucleotide (T) in the
specified deoxyribonucleotide sequence is replaced by the
ribonucleotide uridine (U).
[0046] Using the information provided herein, such as a nucleotide
sequence shown in the sequence listing, a nucleic acid molecule of
the present invention encoding a polypeptide may be obtained using
standard cloning and screening procedures, such as those for
cloning cDNAs using mRNA as starting material. The present
invention provides not only the determined nucleotide sequences of
the mRNA encoding each human secreted protein of the invention, as
set forth in SEQ ID NO:X for each protein, but also a sample of
plasmid DNA containing a cDNA of the invention deposited with the
American Type Culture Collection (Rockville, Md.), as set forth in
Table 1. These deposits enable recovery of each cDNA clone and
recombinant production of each secreted protein of the invention
actually encoded by a cDNA clone identified in Table 1, as further
described hereinbelow.
[0047] Nucleic acid molecules of the present invention may be in
the form of RNA, such as mRNA, or in the form of DNA, including,
for instance, cDNA and genomic DNA obtained by cloning or produced
synthetically. The DNA may be double-stranded or single-stranded.
Single-stranded DNA or RNA may be the coding strand, also known as
the sense strand, or it may be the non-coding strand, also referred
to as the anti-sense strand.
[0048] In addition to nucleic acid molecules comprising a
determined nucleotide sequence in SEQ ID NO:X or the nucleotide
sequence of a deposited human cDNA clone, isolated nucleic acid
molecules of the invention include DNA molecules which comprise a
sequence substantially different from those described above but
which, due to the degeneracy of the genetic code, still encode the
proteins shown in the sequence listing or those encoded by the
clones contained in the deposited plasmids. Of course, the genetic
code and species-specific codon preferences are well known in the
art. Thus, it would be routine for one skilled in the art to
generate the degenerate variants described above, for instance, to
optimize codon expression for a particular host (e.g., change
codons in the human mRNA to those preferred by a bacterial host
such as E. coli). Preferably, this nucleic acid molecule will
encode a secreted portion (mature polypeptide) encoded by the
deposited cDNA.
[0049] The invention further provides a nucleic acid molecule
having a sequence complementary to one of the above sequences. Such
isolated molecules, particularly DNA molecules, are useful as
probes for gene mapping, by in situ hybridization with chromosomes,
and for detecting expression of the corresponding gene(s) in human
tissue, for instance, by Northern blot analysis.
[0050] The present invention is further directed to nucleic acid
molecules encoding portions of the nucleotide sequences described
herein as well as to fragments of the isolated nucleic acid
molecules described herein. By a "fragment" of an isolated nucleic
acid molecule having the nucleotide sequence of the deposited cDNA
or the nucleotide sequence shown in the sequence listing is
intended fragments at least about 15 nt, and more preferably at
least about 20 nt, still more preferably at least about 30 nt, and
even more preferably, at least about 40 nt in length which are
useful as diagnostic probes and primers as discussed herein. Of
course, larger fragments 50-500 nt in length are also useful
according to the present invention as are fragments corresponding
to most, if not all, of the nucleotide sequence of the deposited
cDNA or as shown in the sequence listing. By a fragment "at least
20 nt in length," for example, is intended fragments which include
20 or more contiguous bases from the nucleotide sequence of the
deposited cDNA or the determined nucleotide sequence shown in SEQ
ID NO:X. Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding epitope-bearing portions of
the polypeptides of the present invention, as described further
below.
[0051] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of a nucleic
acid molecule of the invention described above, for instance, a
cDNA contained in the plasmid sample deposited with the ATCC. By
"stringent hybridization conditions" is intended overnight
incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu./ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0052] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of
the reference polynucleotide. These are useful as diagnostic probes
and primers as discussed above and in more detail below. For
certain applications, such as the FISH technique for gene mapping
on chromosomes, probes of 500 nucleotides up to 2000 nucleotides
may be preferred.
[0053] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in SEQ ID
NO:X). Of course, a polynucleotide which hybridizes only to a poly
A sequence (such as any 3' terminal poly(A) tract of a cDNA shown
in the sequence listing), or to a complementary stretch of T (or U)
residues, would not be included in a polynucleotide of the
invention used to hybridize to a portion of a nucleic acid of the
invention, since such a polynucleotide would hybridize to any
nucleic acid molecule containing a poly (A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA
clone).
[0054] Also encoded by nucleic acids of the invention are the amino
acid sequences of the invention together with additional,
non-coding sequences, including for example, but not limited to
introns and non-coding 5' and 3' sequences, such as the
transcribed, non-translated sequences that play a role in
transcription, mRNA processing, including splicing and
polyadenylation signals, for example--ribosome binding and
stability of mRNA; and additional coding sequence which codes for
additional amino acids, such as those which provide additional
functionalities.
[0055] Thus, the sequence encoding the polypeptide may be fused to
a marker sequence, such as a sequence encoding a peptide which
facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for convenient purification of the fusion protein. The
"HA" tag is another peptide useful for purification which
corresponds to an epitope derived from the influenza hemagglutinin
protein, which has been described by Wilson et al., Cell 37: 767
(1984). As discussed below, other such fusion proteins include
those fused to Fc at the N- or C-terminus.
[0056] Sequences Encoding Signal Peptide and Secreted Portions
[0057] According to the signal hypothesis, proteins secreted by
eukaryotic cells have a signal peptide (or secretory leader
sequence) which is cleaved from the complete polypeptide to produce
a secreted portion or "mature" form of the protein. Methods for
predicting whether a protein has a signal peptide (or "secretory
leader") as well as the cleavage point for that leader sequence are
well known in the art. See, for instance, von Heinje, supra. The
determined amino acid sequence of several proteins of the
invention, determined by translation of the determined nucleotide
sequence identified in Table 1, have been found to comprise an
amino acid sequence of a secretory signal peptide. The sequence and
cleavage site of that signal peptide are described in Table 1 and
in the Examples and the signal sequence is underlined in the
Figures, to the extent that these have been determined for each
secreted protein of the invention.
[0058] More in particular, the present invention provides nucleic
acid molecules encoding a secreted portion (mature form) of each
secreted protein identified in Table 1. Most mammalian cells and
even insect cells cleave signal peptides from secreted proteins
with approximately the same specificity. However, in some cases,
cleavage of the signal peptide (as referred to herein as a "leader
sequence" or "leader") from a secreted protein is not entirely
uniform, which results in more than one secreted (also herein
"mature") for or species of the protein. Further, it has long been
known that the cleavage specificity of a secreted protein is
ultimately determined by the primary structure of the complete
protein, that is, it is inherent in the amino acid sequence of the
initial polypeptide translated from its mRNA. Therefore, the
present invention provides not only a determined nucleotide
sequence and translated amino acid sequence identifying a signal
peptide and secreted portion of each secreted protein of the
invention, but also a deposited sample of a cDNA clone encoding a
secreted (mature) form of each secreted protein of the
invention.
[0059] More particularly, the invention further provides an
isolated polypeptide comprising an amino acid sequence at least 90%
identical, preferably 95%, 96%, 97%, 98% or 99% identical, to a
sequence of at least about 25, 50 or 100 contiguous amino acids in
the complete amino acid sequence of a protein encoded by a human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC Deposit Number shown for
that cDNA clone in Table 1. A particularly preferred embodiment of
this aspect of the invention is a polypeptide in which the sequence
of contiguous amino acids is included in the amino acid sequence of
a secreted portion of a secreted protein encoded by a human cDNA
clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC Deposit Number shown for
said cDNA clone in Table 1. By the "secreted portion" or mature
form of a secreted protein encoded by a human cDNA clone identified
by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the ATCC Deposit Number shown for said cDNA clone in Table 1"
is meant the secreted portion(s) or mature form(s) of the protein
produced by expression in any eukaryotic cell (for instance, cells
of an established insect or mammalian cell line), preferably a
human cell (for instance, cells of the well known HeLa cell line),
of the complete open reading frame encoded by the human cDNA clone
identified in Table 1 and contained in the deposit cited in Table
1.
[0060] Variant and Mutant Polynucleotides
[0061] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the secreted proteins. Variants
may occur naturally, such as a natural allelic variant. By an
"allelic variant" is intended one of several alternate forms of a
gene occupying a given locus on a chromosome of an organism. Genes
II, Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0062] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants may
be altered in coding regions, non-coding regions, or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the secreted protein or portions thereof. Also
especially preferred in this regard are conservative
substitutions.
[0063] Most highly preferred are nucleic acid molecules encoding a
secreted portion (mature form) of a protein described in Table 1
and having the amino acid sequence shown in the sequence listing as
SEQ ID NO:X, or the amino acid sequence of the secreted portion
(mature form) of the protein encoded by a deposited cDNA clone.
Further embodiments include an isolated nucleic acid molecule
comprising a polynucleotide having a nucleotide sequence at least
85% identical, more preferably at least 90% identical, and most
preferably at least 95%, 96%, 97%, 98% or 99% identical to a
polynucleotide of the invention described in Table 1, or a
polynucleotide which hybridizes under stringent hybridization
conditions to such a polynucleotide. This polynucleotide which
hybridizes does not hybridize under stringent hybridization
conditions to a polynucleotide having a nucleotide sequence
consisting of only A residues or of only T residues. An additional
nucleic acid embodiment of the invention relates to an isolated
nucleic acid molecule comprising a polynucleotide which encodes the
amino acid sequence of an epitope-bearing portion of a secreted
polypeptide having an amino acid sequence of SEQ ID NO:Y or an
amino acid sequence of a secreted protein encoded by a cDNA clone
in the deposit identified in Table 1.
[0064] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a secreted polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the secreted polypeptide. In other
words, to obtain a polynucleotide having a nucleotide sequence at
least 95% identical to a reference nucleotide sequence, up to 5% of
the nucleotides in the reference sequence may be deleted or
substituted with another nucleotide, or a number of nucleotides up
to 5% of the total nucleotides in the reference sequence may be
inserted into the reference sequence. These mutations of the
reference sequence may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0065] As a practical matter, whether any particular nucleic acid
molecule is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical
to, for instance, the nucleotide sequence shown in SEQ ID NO:1, or
to the nucleotide sequence of a deposited cDNA can be determined
conventionally using known computer programs such as the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711). Bestfit uses the local homology
algorithm of Smith and Waterman, Advances in Applied Mathematics
2:482-489 (1981), to find the best segment of homology between two
sequences. When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for
instance, 95% identical to a reference sequence according to the
present invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0066] Uses for Nucleic Acid Molecules of the Invention
[0067] Each of the nucleic acid molecules identified herein can be
used in numerous ways as polynucleotide reagents. The
polynucleotides can be used as diagnostic probes for the presence
of a specific mRNA in a particular cell type. In addition, these
polynucleotides can be used as diagnostic probes suitable for use
in genetic linkage analysis (polymorphisms). Further, the
polynucleotides can be used as probes for locating gene regions
associated with genetic disease, as explained in more detail
below.
[0068] The polynucleotides of the present invention are also
valuable for chromosome identification. Each polynucleotide is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of cDNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0069] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the sequences shown in the
sequence listing. Computer analysis of the sequences is used to
rapidly select primers that do not span more than one exon in the
genomic DNA, thus complicating the amplification process. These
primers are then used for PCR screening of somatic cell hybrids
containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the secreted protein
will yield an amplified fragment.
[0070] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular nucleic acid sequence to a particular
chromosome. Three or more clones can be assigned per day using a
single thermal cycler. Using the present invention with the same
oligonucleotide primers, sublocalization can be achieved with
panels of fragments from specific chromosomes or pools of large
genomic clones in an analogous manner. Other mapping strategies
that can similarly be used to map a gene to its chromosome include
in situ hybridization, prescreening with labeled flow-sorted
chromosomes and preselection by hybridization to construct
chromosome specific-cDNA libraries.
[0071] Fluorescence in situ hybridization (FISH) of a cDNA clone to
a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 500 or 600 bases; however, clones larger than
2,000 bp have a higher likelihood of binding to a unique
chromosomal location with sufficient signal intensity for simple
detection. For example, 2,000 bp is good, 4,000 is better, and more
than 4,000 is probably not necessary to get good results a
reasonable percentage of the time. For a review of this technique,
see Verma et al., Human Chromosomes: a Manual of Basic Techniques.
Pergamon Press, New York (1988).
[0072] Reagents for chromosome mapping can be used individually (to
mark a single chromosome or a single site on that chromosome) or as
panels of reagents (for marking multiple sites and/or multiple
chromosomes). Reagents corresponding to noncoding regions of the
genes actually are preferred for mapping purposes. Coding sequences
are more likely to be conserved within gene families, thus
increasing the chance of cross hybridizations during chromosomal
mapping.
[0073] Once a polynucleotide sequence has been mapped to a precise
chromosomal location, the physical position of the sequence on the
chromosome can be correlated with genetic map data. (Such data are
found, for example, in V. McKusick, Mendelian Inheritance in Man
(available on line through Johns Hopkins University Welch Medical
Library).) The relationship between genes and diseases that have
been mapped to the same chromosomal region are then identified
through linkage analysis (coinheritance of physically adjacent
genes).
[0074] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0075] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb.)
[0076] Comparison of affected and unaffected individuals generally
involves first looking for structural alterations in the
chromosomes, such as deletions or translocations that are visible
from chromosome spreads or detectable using PCR based on that cDNA
sequence. Ultimately, complete sequencing of genes from several
individuals is required to confirm the presence of a mutation and
to distinguish mutations from polymorphisms.
[0077] In addition to the foregoing, the polynucleotides of the
invention, as broadly described, can be used to control gene
expression through triple helix formation or antisense DNA or RNA,
both of which methods are based on binding of a polynucleotide
sequence to DNA or RNA. Polynucleotides suitable for use in these
methods are usually 20 to 40 bases in length and are designed to be
complementary to a region of the gene involved in transcription
(triple helix--see Lee et al, Nucl. Acids Res., 6:3073 (1979);
Cooney et al, Science, 241:456 (1988); and Dervan et al, Science,
251: 1360 (1991)) or to the mRNA itself (antisense--Okano, J.
Neurochem., 56:560 (1991) Oligodeoxy-nucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
Triple helix formation optimally results in a shut-off of RNA
transcription from DNA, while antisense RNA hybridization blocks
translation of an mRNA molecule into polypeptide. Both techniques
have been demonstrated to be effective in model systems.
Information contained in the sequences of the present invention is
necessary for the design of an antisense or triple helix
oligonucleotide.
[0078] Nucleic acid molecules of the present invention are also a
useful in gene therapy which requires isolation of the
disease-associated gene in question as a prerequisite to the
insertion of a normal gene into an organism to correct a genetic
defect. The high specificity of the cDNA probes according to this
invention offer means of targeting such gene locations in a highly
accurate manner.
[0079] The sequences of the present invention, as broadly defined,
are also useful for identification of individuals from minute
biological samples. The United States military, for example, is
considering the use of restriction fragment length polymorphism
(RFLP) for identification of its personnel. In this technique, an
individual's genomic DNA is digested with one or more restriction
enzymes, and probed on a Southern blot to yield unique bands for
identifying personnel. This method does not suffer from the current
limitations of "Dog Tags" which can be lost, switched, or stolen,
making positive identification difficult. The sequences of the
present invention are useful as additional DNA markers for
RFLP.
[0080] However, RFLP is a pattern based technique, which does not
require the DNA sequence of the individual to be sequenced. The
polynucleotides and sequences of the present invention can be used
to provide an alternative technique that determines the actual
base-by-base DNA sequence of selected portions of an individual's
genome. These sequences can be used to prepare PCR primers for
amplifying and isolating such selected DNA. One can, for example,
take a sequence of the invention and prepare two PCR primers. These
are used to amplify an individual's DNA, corresponding to the gene
or gene fragment. The amplified DNA is sequenced.
[0081] Panels of corresponding DNA sequences from individuals, made
this way, can provide unique individual identifications, as each
individual will have a unique set of such DNA sequences, due to
allelic differences. The sequences of the present invention can be
used to particular advantage to obtain such identification
sequences from individuals and from tissue, as further described in
the Examples. The polynucleotide sequences shown in the sequence
listing and the inserts contained in the deposited cDNAs uniquely
represent portions of the human genome. Allelic variation occurs to
some degree in the coding regions of these sequences, and to a
greater degree in the noncoding regions. It is estimated that
allelic variation between individual humans occurs with a frequency
of about once per each 500 bases. Each of the sequences comprising
a part of the present invention can, to some degree, be used as a
standard against which DNA from an individual can be compared for
identification purposes. Because greater numbers of polymorphisms
occur in the noncoding regions, fewer sequences are necessary to
differentiate individuals.
[0082] If a panel of reagents from sequences of this invention is
used to generate a unique ID database for an individual, those same
reagents can later be used to identify tissue from that individual.
Positive identification of that individual, living or dead can be
made from extremely small tissue samples.
[0083] Another use for DNA-based identification techniques is in
forensic biology. PCR technology can be used to amplify DNA
sequences taken from very small biological samples such as tissues,
e.g., hair or skin, or body fluids, e.g., blood, saliva, semen,
etc. In one prior art technique, gene sequences are amplified at
specific loci known to contain a large number of allelic
variations, for example the DQa class II HLA gene (Erlich, H., PCR
Technology, Freeman and Co. (1992)). Once this specific area of the
genome is amplified, it is digested with one or more restriction
enzymes to yield an identifying set of bands on a Southern blot
probed with DNA corresponding to the DQa class II HLA gene.
[0084] The sequences of the present invention can be used to
provide polynucleotide reagents specifically targeted to additional
loci in the human genome, and can enhance the reliability of
DNA-based forensic identifications. Those sequences targeted to
noncoding regions are particularly appropriate. As mentioned above,
actual base sequence information can be used for identification as
an accurate alternative to patterns formed by restriction enzyme
generated fragments. Reagents for obtaining such sequence
information are within the scope of the present invention. Such
reagents can comprise complete genes, ESTs or corresponding coding
regions, or fragments of either of at least 20 bp, preferably at
least 50 bp, most preferably at least 500 to 1,000 bp.
[0085] There is also a need for reagents capable of identifying the
source of a particular tissue. Such need arises, for example, in
forensics when presented with tissue of unknown origin. Appropriate
reagents can comprise, for example, DNA probes or primers specific
to particular tissue prepared from the sequences of the present
invention. Panels of such reagents can identify tissue by species
and/or by organ type. In a similar fashion, these reagents can be
used to screen tissue cultures for contamination.
[0086] The present application is directed to nucleic acid
molecules at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
a nucleic acid sequence referenced in Table 1 and shown in the
sequence listing or to the nucleic acid sequence of a deposited
cDNA, irrespective of whether they encode a polypeptide having
biological activity. This is because even where a particular
nucleic acid molecule does not encode a polypeptide having
biological activity, one of skill in the art would still know how
to use the nucleic acid molecule, for instance, for one of the uses
above.
[0087] Preferred, however, are nucleic acid molecules having
sequences at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
the nucleic acid sequence shown in FIGS. 1A-C (SEQ ID NO:1) or to
the nucleic acid sequence of the deposited cDNA which do, in fact,
encode a secreted polypeptide having biological activity. By "a
polypeptide having biological activity" is intended polypeptides
exhibiting activity similar, but not necessarily identical, to an
activity of the mature protein of the invention, as measured in a
particular biological assay. "A polypeptide having biological
activity" includes polypeptides that also exhibit any of the same
activities as a protein of the invention in an assay in a
dose-dependent manner. Although the degree of dose-dependent
activity need not be identical to that of the protein, preferably,
"a polypeptide having biological activity" will exhibit
substantially similar dose-dependence in a given activity as
compared to the protein (i.e., the candidate polypeptide will
exhibit greater activity or not more than about 25-fold less and,
preferably, not more than about tenfold less activity relative to
the reference protein).
[0088] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence of the deposited cDNA or the nucleic acid sequence shown
in the sequence listing will encode a polypeptide "having
biological activity." In fact, since degenerate variants of these
nucleotide sequences all encode the same polypeptide, this will be
clear to the skilled artisan even without performing the comparison
assay. It will be further recognized in the art that, for such
nucleic acid molecules that are not degenerate variants, a
reasonable number will also encode a polypeptide having biological
activity. This is because the skilled artisan is fully aware of
amino acid substitutions that are either less likely or not likely
to significantly affect protein function (e.g., replacing one
aliphatic amino acid with a second aliphatic amino acid), as
further described below.
[0089] Vectors, Host Cells and Protein Production
[0090] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of polypeptides or fragments thereof by recombinant
techniques. The vector may be, for example, a phage, plasmid, viral
or retroviral vector. Retroviral vectors may be replication
competent or replication defective. In the latter case, viral
propagation generally will occur only in complementing host
cells.
[0091] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0092] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the transcripts
expressed by the constructs will preferably include a translation
initiating codon at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0093] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and
Bowes melanoma cells; and plant cells. Appropriate culture mediums
and conditions for the above-described host cells are known in the
art.
[0094] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc., supra; pBluescript
vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from Stratagene Cloning Systems, Inc.; and ptrc99a,
pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech,
Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44,
pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and
pSVL available from Pharmacia. Other suitable vectors will be
readily apparent to the skilled artisan.
[0095] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0096] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to stabilize
and purify proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobulin molecules together
with another human protein or part thereof. In many cases, the Fc
part in a fusion protein is thoroughly advantageous for use in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as hIL-5, have been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5.
See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995) and
K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0097] A protein of this invention can be recovered and purified
from recombinant cell cultures by well-known methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include: products purified from natural sources, including bodily
fluids, tissues and cells, whether directly isolated or cultured;
products of chemical synthetic procedures; and products produced by
recombinant techniques from a prokaryotic or eukaryotic host,
including, for example, bacterial, yeast, higher plant, insect and
mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may
be glycosylated or may be non-glycosylated. In addition,
polypeptides of the invention may also include an initial modified
methionine residue, in some cases as a result of host-mediated
processes. Thus, it is well known in the art that the N-terminal
methionine encoded by the translation initiation codon generally is
removed with high efficiency from any protein after translation in
all eukaryotic cells. While the N-terminal methionine on most
proteins also is efficiently removed in most prokaryotes, for some
proteins this prokaryotic removal process is inefficient, depending
on the nature of the amino acid to which the N-terminal methionine
is covalently linked.
Polypeptides and Fragments
[0098] The invention further provides isolated polypeptides having
an amino acid sequence encoded by a deposited cDNA, or an amino
acid sequence in the sequence listing identified SEQ ID NO:Y as
defined in Table 1, or a peptide or polypeptide comprising a
portion of the above polypeptides. At the simplest level, the amino
acid sequence can be synthesized using commercially available
peptide synthesizers. This is particularly useful in producing
small peptides and fragments of larger polypeptides. Such fragments
are useful, for example, in generating antibodies against the
native polypeptide.
[0099] Variant and Mutant Polypeptides
[0100] To improve or alter the characteristics of the polypeptides
of the invention, protein engineering may be employed. Recombinant
DNA technology known to those skilled in the art can be used to
create novel mutant proteins or "muteins" including single or
multiple amino acid substitutions, deletions, additions or fusion
proteins. Such modified polypeptides can show, e.g., enhanced
activity or increased stability. In addition, they may be purified
in higher yields and show better solubility than the corresponding
natural polypeptide, at least under certain purification and
storage conditions.
[0101] For instance, for many proteins, including the mature
form(s) of a secreted protein, it is known in the art that one or
more amino acids may be deleted from the N-terminus or C-terminus
without substantial loss of biological function. For instance, Ron
et al., J. Biol. Chem., 268:2984-2988 (1993) reported modified KGF
proteins that had heparin binding activity even if 3, 8, or 27
amino-terminal amino acid residues were missing. Similarly, many
examples of biologically functional C-terminal deletion muteins are
known. For instance, Interferon gamma shows up to ten times higher
activities by deleting 8-10 amino acid residues from the carboxy
terminus of the protein (Dobeli et al., J Biotechnology 7:199-216
(1988). Furthermore, even if deletion of one or more amino acids
from the N-terminus or C-terminus of a protein results in
modification or loss of one or more biological functions of the
protein, other biological activities may still be retained. Thus,
the ability of the shortened protein to induce and/or bind to
antibodies which recognize the complete or mature form of the
protein generally will be retained when less than the majority of
the residues of the complete or mature form of the protein are
removed from the N-terminus or C-terminus. Whether a particular
polypeptide lacking N- or C-terminal residues of a complete protein
retains such immunologic activities can readily be determined by
routine methods described herein and otherwise known in the
art.
[0102] In addition to terminal deletion forms of the protein
discussed above, it also will be recognized by one of ordinary
skill in the art that some amino acid sequences of a polypeptide
can be varied without significant effect of the structure or
function of the protein. If such differences in sequence are
contemplated, it should be remembered that there will be critical
areas on the protein which determine activity.
[0103] Thus, the invention further includes variants of a
polypeptide which show substantial biological activity or which
include regions of the protein such as the portions discussed
below. Such mutants include deletions, insertions, inversions,
repeats, and type substitutions selected according to general rules
known in the art so as have little effect on activity. For example,
guidance concerning how to make phenotypically silent amino acid
substitutions is provided in Bowie, J. U. et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid
Substitutions," Science 247:1306-1310 (1990), wherein the authors
indicate that there are two main approaches for studying the
tolerance of an amino acid sequence to change. The first method
relies on the process of evolution, in which mutations are either
accepted or rejected by natural selection. The second approach uses
genetic engineering to introduce amino acid changes at specific
positions of a cloned gene and selections or screens to identify
sequences that maintain functionality.
[0104] As the authors state, these studies have revealed that
proteins are surprisingly tolerant of amino acid substitutions. The
authors further indicate which amino acid changes are likely to be
permissive at a certain position of the protein. For example, most
buried amino acid residues require nonpolar side chains, whereas
few features of surface side chains are generally conserved. Other
such phenotypically silent substitutions are described in Bowie, J.
U. et al., supra, and the references cited therein. Typically seen
as conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the
acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0105] Thus, the fragment, derivative or analog of a polypeptide
shown in the figures (and sequence listing), or one encoded by the
deposited cDNA, may be (i) one in which one or more of the amino
acid residues are substituted with a conserved or non-conserved
amino acid residue (preferably a conserved amino acid residue) and
such substituted amino acid residue may or may not be one encoded
by the genetic code, or (ii) one in which one or more of the amino
acid residues includes a substituent group, or (iii) one in which
the mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the above form of the polypeptide, such as an
IgG Fc fusion region peptide or leader or secretory sequence or a
sequence which is employed for purification of the above form of
the polypeptide or a proprotein sequence. Such fragments,
derivatives and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein
[0106] Thus, the mature polypeptide of the present invention may
include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human manipulation. As
indicated, changes are preferably of a minor nature, such as
conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 2).
TABLE-US-00001 TABLE 2 CONSERVATIVE AMINO ACID SUBSTITUTIONS
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0107] Amino acids in the protein of the present invention that are
essential for function can be identified by methods known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as receptor binding or in vitro
or in vitro proliferative activity.
[0108] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids which may produce
proteins with highly desirable improved characteristics, such as
less aggregation. Aggregation may not only reduce activity but also
be problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic (Pinckard et al., Clin. Exp. Immunol.
2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987);
Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993).
[0109] Replacement of amino acids can also change the selectivity
of the binding of a ligand to cell surface receptors. For example,
Ostade et al., Nature 361:266-268 (1993) describes certain
mutations resulting in selective binding of TNF-.alpha. to only one
of the two known types of TNF receptors. Sites that are critical
for ligand-receptor binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904
(1992) and de Vos et al. Science 255:306-312 (1992)).
[0110] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of a polypeptide of the
invention can be substantially purified by the one-step method
described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides
of the invention also can be purified from natural or recombinant
sources using antibodies of the invention raised against the
protein in methods which are well known in the art of protein
purification.
[0111] Further polypeptides of the present invention include
polypeptides which have at least 90% similarity, more preferably at
least 95% similarity, and still more preferably at least 96%, 97%,
98% or 99% similarity to those described above. The polypeptides of
the invention also comprise those which are at least 80% identical,
more preferably at least 90% or 95% identical, still more
preferably at least 96%, 97%, 98% or 99% identical to a polypeptide
encoded by a deposited cDNA or to the polypeptide of SEQ ID NO:Y,
and also include portions of such polypeptides with at least 30
amino acids and more preferably at least 50 amino acids.
[0112] By "% similarity" for two polypeptides is intended a
similarity score produced by comparing the amino acid sequences of
the two polypeptides using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711)
and the default settings for determining similarity. Bestfit uses
the local homology algorithm of Smith and Waterman (Advances in
Applied Mathematics 2:482-489, 1981) to find the best segment of
similarity between two sequences.
[0113] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
polypeptide described herein is intended that the amino acid
sequence of the polypeptide is identical to the reference sequence
except that the polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the reference amino
acid of the polypeptide of the invention. In other words, to obtain
a polypeptide having an amino acid sequence at least 95% identical
to a reference amino acid sequence, up to 5% of the amino acid
residues in the reference sequence may be deleted or substituted
with another amino acid, or a number of amino acids up to 5% of the
total amino acid residues in the reference sequence may be inserted
into the reference sequence. These alterations of the reference
sequence may occur at the amino or carboxy terminal positions of
the reference amino acid sequence or anywhere between those
terminal positions, interspersed either individually among residues
in the reference sequence or in one or more contiguous groups
within the reference sequence.
[0114] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
an amino acid sequence shown in the sequence listing or to an amino
acid sequence encoded by the deposited cDNA can be determined
conventionally using known computer programs such the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711). When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0115] The polypeptide of the present invention could be used as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0116] As described in detail below, the polypeptides of the
present invention can also be used to raise polyclonal and
monoclonal antibodies, which are useful in assays for detecting the
corresponding protein expression as described below or as agonists
and antagonists capable of enhancing or inhibiting function of the
protein. Further, such polypeptides can be used in the yeast
two-hybrid system to "capture" receptors of secreted proteins which
are also candidate agonists and antagonists according to the
present invention. The yeast two hybrid system is described in
Fields and Song, Nature 340:245-246 (1989).
[0117] Epitope-Bearing Portions
[0118] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
[0119] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A.
(1983) "Antibodies that react with predetermined sites on
proteins," Science, 219:660-666. Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals. Antigenic epitope-bearing peptides and
polypeptides of the invention are therefore useful to raise
antibodies, including monoclonal antibodies, that bind specifically
to a polypeptide of the invention. See, for instance, Wilson et
al., Cell 37:767-778 (1984) at 777.
[0120] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention.
[0121] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. See, e.g.,
Houghten, R. A. (1985) "General method for the rapid solid-phase
synthesis of large numbers of peptides: specificity of
antigen-antibody interaction at the level of individual amino
acids." Proc. Natl. Acad. Sci. USA 82:5131-5135; this "Simultaneous
Multiple Peptide Synthesis (SMPS)" process is further described in
U.S. Pat. No. 4,631,211 to Houghten et al. (1986).
[0122] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, for instance, Sutcliffe et al., supra; Wilson et al.,
supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and
Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).
Immunogenic epitope-bearing peptides of the invention, i.e., those
parts of a protein that elicit an antibody response when the whole
protein is the immunogen, are identified according to methods known
in the art. See, for instance, Geysen et al., supra. Further still,
U.S. Pat. No. 5,194,392 to Geysen (1990) describes a general method
of detecting or determining the sequence of monomers (amino acids
or other compounds) which is a topological equivalent of the
epitope (i.e., a "mimotope") which is complementary to a particular
paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a
method of detecting or determining a sequence of monomers which is
a topographical equivalent of a ligand which is complementary to
the ligand binding site of a particular receptor of interest.
Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996)
on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl
peralkylated oligopeptides and sets and libraries of such peptides,
as well as methods for using such oligopeptide sets and libraries
for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
[0123] Fusion Proteins
[0124] As one of skill in the art will appreciate, polypeptides of
the present invention and the epitope-bearing fragments thereof
described above can be combined with parts of the constant domain
of immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins facilitate purification and show an increased
half-life in vivo. This has been shown, e.g., for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins (EP A 394,827; Traunecker et
al., Nature 331:84-86 (1988)). Fusion proteins that have a
disulfide-linked dimeric structure due to the IgG part can also be
more efficient in binding and neutralizing other molecules than the
monomeric secreted protein or protein fragment alone (Fountoulakis
et al., J. Biochem. 270:3958-3964 (1995)).
Antibodies
[0125] Protein-species specific antibodies for use in the present
invention can be raised against an intact protein or an antigenic
polypeptide fragment thereof, which may be presented together with
a carrier protein, such as an albumin, to an animal system (such as
rabbit or mouse) or, if it is long enough (at least about 25 amino
acids), without a carrier.
[0126] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody fragments (such as, for example, Fab and F(ab')2
fragments) which are capable of specifically binding to protein.
Fab and F(ab')2 fragments lack the Fe fragment of intact antibody,
clear more rapidly from the circulation, and may have less
non-specific tissue binding of an intact antibody (Wahl et al., J.
Nucl. Med. 24:316-325 (1983)). Thus, these fragments are
preferred.
[0127] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, cells expressing the
protein of the present invention or an antigenic fragment thereof
can be administered to an animal in order to induce the production
of sera containing polyclonal antibodies. In a preferred method, a
preparation of the secreted protein is prepared and purified to
render it substantially free of natural contaminants. Such a
preparation is then introduced into an animal in order to produce
polyclonal antisera of greater specific activity.
[0128] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or protein binding fragments
thereof). Such monoclonal antibodies can be prepared using
hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler
et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.
Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies
and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-681). In
general, such procedures involve immunizing an animal (preferably a
mouse) with a protein antigen of the invention or, more preferably,
with a protein-expressing cell. Such cells may be cultured in any
suitable tissue culture medium; however, it is preferable to
culture cells in Earle's modified Eagle's medium supplemented with
10% fetal bovine serum (inactivated at about 56.degree. C.), and
supplemented with about 10 g/l of nonessential amino acids, about
1,000 U/ml of penicillin, and about 100 .mu./ml of streptomycin.
The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP20), available
from the American Type Culture Collection, Rockville, Md. After
fusion, the resulting hybridoma cells are selectively maintained in
HAT medium, and then cloned by limiting dilution as described by
Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma
cells obtained through such a selection are then assayed to
identify clones which secrete antibodies capable of binding the
protein antigen.
[0129] Alternatively, additional antibodies capable of binding to
the protein antigen of the invention may be produced in a two-step
procedure through the use of anti-idiotypic antibodies. Such a
method makes use of the fact that antibodies are themselves
antigens, and that, therefore, it is possible to obtain an antibody
which binds to a second antibody. In accordance with this method,
protein specific antibodies are used to immunize an animal,
preferably a mouse. The splenocytes of such an animal are then used
to produce hybridoma cells, and the hybridoma cells are screened to
identify clones which produce an antibody whose ability to bind to
the protein-specific antibody can be blocked by the protein
antigen. Such antibodies comprise anti-idiotypic antibodies to the
protein-specific antibody and can be used to immunize an animal to
induce formation of further protein-specific antibodies.
[0130] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). Alternatively, protein-binding fragments can be
produced through the application of recombinant DNA technology or
through synthetic chemistry.
[0131] For in vivo use of antibodies in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric antibodies are known in the
art. See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).
[0132] Identification and Diagnostic Applications
[0133] Assaying protein levels in a biological sample can occur
using antibody-based techniques. For example, protein expression in
tissues can be studied with classical immunohistological methods
(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen,
M., et al., J. Cell. Biol. 105:3087-3096 (1987)). Other
antibody-based methods useful for detecting protein gene expression
include immunoassays, such as the enzyme linked immunosorbent assay
(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay
labels are known in the art and include enzyme labels, such as,
glucose oxidase, and radioisotopes, such as iodine (.sup.125I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.112In), and technetium (.sup.99mTc), and
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0134] In addition to assaying protein levels in a biological
sample obtained from an individual, protein can also be detected in
vivo by imaging. Antibody labels or markers for in vivo imaging of
protein include those detectable by X-radiography, NMR or ESR. For
X-radiography, suitable labels include radioisotopes such as barium
or cesium, which emit detectable radiation but are not overtly
harmful to the subject. Suitable markers for NMR and ESR include
those with a detectable characteristic spin, such as deuterium,
which may be incorporated into the antibody by labeling of
nutrients for the relevant hybridoma.
[0135] A protein-specific antibody or antibody fragment which has
been labeled with an appropriate detectable imaging moiety, such as
a radioisotope (for example, .sup.131I, .sup.112In, .sup.99mTc), a
radio-opaque substance, or a material detectable by nuclear
magnetic resonance, is introduced (for example, parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined
for immune system disorder. It will be understood in the art that
the size of the subject and the imaging system used will determine
the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the
quantity of radioactivity injected will normally range from about 5
to 20 millicuries of .sup.99mTc. The labeled antibody or antibody
fragment will then preferentially accumulate at the location of
cells which contain the specific protein. In vivo tumor imaging is
described in S. W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor
Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and
B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
Treatment of Conditions Related to Proteins of the Invention
[0136] It will be appreciated that conditions caused by a decrease
in the standard or normal expression level of a protein of the
invention, particularly a secreted protein, in an individual can be
treated by administration of the polypeptide (in the form of a
mature protein for secreted polypeptides). Thus, the invention also
provides a method of treatment of an individual in need of an
increased level of the protein of the present invention comprising
administering to such an individual a pharmaceutical composition
comprising an amount of the isolated polypeptide of the invention
effective to increase the activity level of the protein in such an
individual.
[0137] Formulations
[0138] Polypeptide composition will be formulated and dosed in a
fashion consistent with good medical practice, taking into account
the clinical condition of the individual patient (especially the
side effects of treatment with the polypeptide alone), the site of
delivery, the method of administration, the scheduling of
administration, and other factors known to practitioners. The
"effective amount" for purposes herein is thus determined by such
considerations.
[0139] As a general proposition, the total pharmaceutically
effective amount of a polypeptide administered parenterally per
dose will be in the range of about 1 .mu.g/kg/day to 10 mg/kg/day
of patient body weight, although, as noted above, this will be
subject to therapeutic discretion. More preferably, this dose is at
least 0.01 mg/kg/day, and most preferably for humans between about
0.01 and 1 mg/kg/day for the hormone. If given continuously, the
polypeptide is typically administered at a dose rate of about 1
.mu.g/kg/hour to about 50 .mu.g/kg/hour, either by 1-4 injections
per day or by continuous subcutaneous infusions, for example, using
a mini-pump. An intravenous bag solution may also be employed. The
length of treatment needed to observe changes and the interval
following treatment for responses to occur appears to vary
depending on the desired effect.
[0140] Pharmaceutical compositions containing the protein of the
invention may be administered orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as
by powders, ointments, drops or transdermal patch), bucally, or as
an oral or nasal spray. By "pharmaceutically acceptable carrier" is
meant a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. The
term "parenteral" as used herein refers to modes of administration
which include intravenous, intramuscular, intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and
infusion.
[0141] The polypeptide is also suitably administered by
sustained-release systems. Suitable examples of sustained-release
compositions include semi-permeable polymer matrices in the form of
shaped articles, e.g., films, or mirocapsules. Sustained-release
matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman,
U. et al., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethyl
methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277
(1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene
vinyl acetate (R. Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
compositions also include liposomally entrapped polypeptides.
Liposomes containing the polypeptide are prepared by methods known
per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA)
82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA)
77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949;
EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045
and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the
small (about 200-800 Angstroms) unilamellar type in which the lipid
content is greater than about 30 mol. percent cholesterol, the
selected proportion being adjusted for the optimal polypeptide
therapy.
[0142] For parenteral administration, in one embodiment, the
polypeptide is formulated generally by mixing it at the desired
degree of purity, in a unit dosage injectable form (solution,
suspension, or emulsion), with a pharmaceutically acceptable
carrier, i.e., one that is non-toxic to recipients at the dosages
and concentrations employed and is compatible with other
ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other compounds
that are known to be deleterious to polypeptides.
[0143] Generally, the formulations are prepared by contacting the
polypeptide uniformly and intimately with liquid carriers or finely
divided solid carriers or both. Then, if necessary, the product is
shaped into the desired formulation. Preferably the carrier is a
parenteral carrier, more preferably a solution that is isotonic
with the blood of the recipient. Examples of such carrier vehicles
include water, saline, Ringer's solution, and dextrose solution.
Non-aqueous vehicles such as fixed oils and ethyl oleate are also
useful herein, as well as liposomes.
[0144] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, mannose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0145] The polypeptide is typically formulated in such vehicles at
a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10
mg/ml, at a pH of about 3 to 8. It will be understood that the use
of certain of the foregoing excipients, carriers, or stabilizers
will result in the formation of polypeptide salts.
[0146] Any polypeptide to be used for therapeutic administration
must be sterile. Sterility is readily accomplished by filtration
through sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic polypeptide compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0147] Polypeptides ordinarily will be stored in unit or multi-dose
containers, for example, sealed ampoules or vials, as an aqueous
solution or as a lyophilized formulation for reconstitution. As an
example of a lyophilized formulation, 10-ml vials are filled with 5
ml of sterile-filtered 1% (w/v) aqueous polypeptide solution, and
the resulting mixture is lyophilized. The infusion solution is
prepared by reconstituting the lyophilized polypeptide using
bacteriostatic Water-for-Injection.
[0148] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the polypeptides of the present
invention may be employed in conjunction with other therapeutic
compounds.
[0149] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Isolation of A Selected cDNA Clone From the Deposited Sample
[0150] Each protein of the invention is related to a human
complementary DNA (cDNA) clone prepared from a messenger RNA (mRNA)
encoding the related protein. The cDNA clone related to each
protein of the invention is identified by a "cDNA Clone ID
(Identifier)" in Table 1, below (e.g., "HABCE99"). DNA of each cDNA
clone in Table 1 is contained in the material deposited with the
American Type Culture Collection and given the ATCC Deposit Number
shown for each cDNA Clone ID in Table 1. All deposits containing
such clones have been submitted to the American Type Culture
Collection (10801 University Blvd, Mannasas, Va. 20110-2209) on the
date indicated for each given accession number indicated in Table
1. All deposits have been made in accordance with the Budapest
Treaty, and in full compliance with 37 CFR .sctn.1.801 et seq.
[0151] The cDNA clones contained in the ATCC deposits cited in
Table 1 can be utilized by those of skill in the art by reference
to the information describing each clone, and by reference to SEQ
ID NO:X, provided in Table 1 for the determined nucleotide sequence
of each deposited clone. The following additional information is
provided for convenience. Each cDNA clone in a cited ATCC deposit
is contained in a plasmid vector. Table 1 identifies the vector
used to construct the cDNA library from which each clone was
isolated. In many cases the vector used to construct the library is
a phage vector from which a plasmid has been excised. The table
immediately below provides a correlation of the related plasmid for
each such phage vector used in construction of the cDNA library
from which each cDNA clone listed in Table 1 originally was
isolated. For example, where a particular clone is identified in
Table 1 as being isolated in the vector "Lambda Zap," it can be
seen from the following table that this cDNA clone contained in the
biological deposit in pBluescript. TABLE-US-00002 Vector Used to
Construct Library Corresponding Deposited Plasmid Lambda Zap
pBluescript (pBS) Uni-Zap XR pBluescript (pBS) Zap Express pBK
lafmid BA plafmid BA pSport1 pSport1 pCMVSport 2.0 pCMVSport 2.0
pCMVSport 3.0 pCMVSport 3.0 pCR .RTM.2.1 pCR .RTM.2.1
[0152] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636),
Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express
(U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short,
J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees,
M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK
(Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are
commercially available from Stratagene Cloning Systems, Inc., 11011
N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an
ampicillin resistance gene and pBK contains a neomycin resistance
gene. Both may be transformed into E. coli strain XL-1 Blue, also
available from Stratagene. pBS comes in 4 forms SK+, SK-, KS+ and
KS-. The S and K refer to the orientation of the polylinker to the
T7 and T3 primer sequences which flank the polylinker region ("S"
is for SacI and "K" is for KpnI which are the first restriction
enzyme sites on each respective end of the linker). "+" or "-"
refer to the orientation of the f1 origin of replication ("ori"),
such that in one orientation single stranded rescue initiated from
the f1 ori generates sense strand DNA and in the other,
antisense.
[0153] Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were
obtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg,
Md. 20897. All Sport vectors contain an ampicillin resistance gene
and may be transformed into E. coli strain DH10B, also available
from Life Technologies. See, for instance, Gruber, C. E., et al.,
Focus 15:59-(1993). Vector lafmid BA (Bento Soares, Columbia
University, NY) contains an ampicillin resistance gene and can be
transformed into E. coli strain XL-1 Blue. Vector pCR.RTM.2.1,
which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad,
Calif. 92008, contains an ampicillin resistance gene and may be
transformed into E. coli strain DH10B, available from Life
Technologies. See, for instance, Clark, J. M., Nuc. Acids Res.
16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9:
(1991).
[0154] The deposited material in the sample assigned the ATCC
Deposit Number cited in Table 1 for any given cDNA clone also may
contain one or more additional plasmids, each comprising a cDNA
clone different from that given clone. Thus, each cited deposit
contains at least a plasmid for each cDNA clone identified in Table
1 as sharing the same ATCC Deposit Number.
[0155] Two approaches are used herein to isolate a particular clone
from the deposited sample of plasmid DNAs cited for that clone in
Table 1, although others are known in art. In the first, a plasmid
is isolated directly by screening clones using an oligonucleotide
probe. To isolate a particular clone, a specific oligonucleotide
with 30-40 nucleotides is synthesized using an Applied Biosystems
DNA synthesizer according to the sequence reported. The
oligonucleotide is labeled, for instance, with .sup.32P-.gamma.-ATP
using T4 polynucleotide kinase and purified according to routine
methods (e.g., Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Press, Cold Spring, N.Y., 1982). The
plasmid mixture is transformed into a suitable host, as indicated
above (such as XL-1 Blue (Stratagene)) using techniques known to
those of skill in the art such as those provided by the vector
supplier or in related publications or patents cited above. The
transformants are plated on 1.5% agar plates (containing the
appropriate selection agent, e.g., ampicillin) to a density of
about 150 transformants (colonies) per plate. These plates are
screened using Nylon membranes according to routine methods for
bacterial colony screening (e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor
Laboratory Press, pages 1.93 to 1.104), or other technique known to
those of skill in the art.
[0156] An alternative approach to isolate any polynucleotide of
interest in the deposited library is to prepare two oligonucleotide
primers of 17-20 nucleotides derived from both ends of the
determined sequence for the selected clone (i.e., within the region
of SEQ ID NO:X bounded by the 5' NT of the clone and the 3' NT of
the clone defined in Table 1 for each cDNA clone identified
therein. These two oligonucleotide primers are used to amplify the
polynucleotide of interest using the deposited cDNA plasmid as a
template. The polymerase chain reaction is carried out under
routine conditions, for instance, in 25 .mu.l of reaction mixture
with 0.5 ug of the above cDNA template. A convenient reaction
mixture is 1.5-5 mM MgCl.sub.2, 0.01% (w/v) gelatin, 20 .mu.M each
of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of
Taq polymerase. Thirty five cycles of PCR (denaturation at
94.degree. C. for 1 min; annealing at 55.degree. C. for 1 min;
elongation at 72.degree. C. for 1 min) are performed with a
Perkin-Elmer Cetus automated thermal cycler. The amplified product
is analyzed by agarose gel electrophoresis and the DNA band with
expected molecular weight is excised and purified. The PCR product
is verified to be the selected sequence by subcloning and
sequencing the DNA product.
[0157] Several methods are available for the identification of the
5' or 3' non-coding portions of a gene which may not be present in
the deposited clone. These methods include but are not limited to
filter probing, clone enrichment using specific probes and
protocols similar or identical to 5' and 3' "RACE" protocols which
are well known in the art. For instance, a method similar to 5'
RACE is available for generating the missing 5' end of a desired
full-length transcript. (Fromont-Racine et al., Nucleic Acids Res.,
21(7):1683-1684 (1993). Briefly, a specific RNA oligonucleotide is
ligated to the 5' ends of a population of RNA presumably containing
full-length gene RNA transcript and a primer set containing a
primer specific to the ligated RNA oligonucleotide and a primer
specific to a known sequence of the gene of interest, is used to
PCR amplify the 5' portion of the desired full-length gene which
may then be sequenced and used to generate the full length gene.
This method starts with total RNA isolated from the desired source;
poly A RNA may be used but is not a prerequisite for this
procedure. The RNA preparation may then be treated with phosphatase
if necessary to eliminate 5' phosphate groups on degraded or
damaged RNA which may interfere with the later RN-A ligase step.
The phosphatase if used is then inactivated and the RNA is treated
with tobacco acid pyrophosphatase in order to remove the cap
structure present at the 5' ends of messenger RNAs. This reaction
leaves a 5' phosphate group at the 5' end of the cap cleaved RNA
which can then be ligated to an RNA oligonucleotide using T4 RNA
ligase. This modified RNA preparation can then be used as a
template for first strand cDNA synthesis using a gene specific
oligonucleotide. The first strand synthesis-reaction can then be
used as a template for PCR amplification of the desired 5' end
using a primer specific to the ligated RNA oligonucleotide and a
primer specific to the known sequence of the gene of interest. The
resultant product is then sequenced and analyzed to confirm that
the 5' end sequence belongs to the desired gene.
Example 2
Features of Proteins of the Invention
[0158] Table 1, below, describes particular features of the
proteins and related nucleotide and amino acid sequences of this
invention. TABLE-US-00003 TABLE 1 FEATURES OF PROTEINS OF THE
INVENTION ATCC NT SEQ Protein ID cDNA Deposit Nr ID NO: Total 5' NT
of 3' NT of (Group-Nr) Clone ID and Date Vector X NT Seq. Clone
Seq. Clone Seq. PF353-1 HEMFI85 209053 pBluescript SK- 1 1091 1
1091 May 16, 1997 PF353-2 HTXET53 209053 pBluescript SK- 3 887 1
887 May 16, 1997 PF353-3 HT3SG28 209053 pBluescript SK- 5 540 1 540
May 16, 1997 PF353-4 HBZAK03 209053 pSport 1.0 7 520 1 520 May 16,
1997 PF353-5 HDFUB43 209053 pBluescript SK- 9 1352 1 1352 May 16,
1997 PF353-6 HEBGM49 209054 pBluescript SK- 11 632 1 632 May 16,
1997 PF353-7 HNGBH54 209054 Uni-ZAP XR 13 582 1 582 May 16, 1997
PF353-8 HSAAL25 209054 pBluescript SK- 15 1356 1 1356 May 16, 1997
PF353-9 HUSAX55 209054 pBluescript SK- 17 2934 1 2934 May 16, 1997
PF353-10 HSXCK41 209054 pBluescript SK- 19 1587 1 1587 May 16, 1997
PF353-11 HFKFY79 209054 pBluescript SK- 21 1359 1 1359 May 16, 1997
PF353-12 HAICH28 209054 Uni-Zap XR 23 1098 1 1098 May 16, 1997 AA
SEQ Last First AA of Protein ID 5' NT of 5' NT of ID NO: First AA
of Secreted Last AA (Group-Nr) NOTE Start Codon First AA Y AA Sig
Pep Portion of ORF PF353-1 118 118 2 1 103 PF353-2 64 64 4 1 15 16
172 PF353-3 19 19 6 1 22 23 88 PF353-4 112 112 8 1 59 PF353-5 55 55
10 1 116 PF353-6 88 88 12 1 150 PF353-7 1 1 14 1 193 PF353-8 115
115 16 1 324 PF353-9 1 1 18 1 977 PF353-10 1 1 20 1 15 16 528
PF353-11 1 1 22 1 452 PF353-12 1 1 24 1 365
[0159] Features of the Protein Encoded by SEQ ID NO: 1
[0160] The novel full-length chemotactic cytokine V (CCV)
polypeptide exhibits significant sequence identity to a chemotactic
protein isolated from the murine S100 fraction designated CP-10
(chemotactic protein, 10 kD). The chemotactic cytokine V cDNA clone
contains an 1091 nucleotide insert (SEQ ID NO:1) which encodes a
103 amino acid polypeptide (SEQ ID NO:2), both shown in FIGS. 1A-C.
The clone was obtained from an induced endothelial cell cDNA
library. A sequence alignment analysis of the deduced amino acid
sequence of HEMFI85 shows that CCV shares approximately 24%
identity and 69% similarity to the amino acid sequence of the
murine CP-10 protein. In addition, it was determined by a BLAST
analysis that the amino acid sequence of chemotactic cytokine V
also exhibits approximately 31% identity and 67% similarity to the
previously described rat intracellular Ca2+-binding protein. An
examination of expression of chemotactic cytokine V in the HGS
database reveals a widespread cell and tissue distribution of this
gene. Expression of this clone was observed in a wide variety of
human cDNA libraries in the Human Genome Sciences, Inc. (HGS)
express sequence tag (EST) database including colon carcinoma (HCC)
cell line, smooth muscle, amygdala depression, keratinocytes,
uninduced endothelial cells, osteoblasts, and others.
[0161] CP-10 is a potent factor capable of extravascular
recruitment of polymorphonuclear cells (PMN) and monocytes from
circulation. Optimal chemotactic activity of CP-10 for murine PMN
and neutrophils is in the range of 10-11 and 10-13 M, making this
factor one of the most potent chemotactic factors reported to date.
CP-10 is the murine homologue of a human S100 protein designated
migration inhibition factor-related protein 8 (MRP8). MRP 8 can
occur as a complex with an additional human S100 protein termed
MRP14 (the complex has previously been reported as the cystic
fibrosis antigen, calgranulin A and B, or L1 antigen). This complex
can comprise as much as 10-20% of the total cytoplasmic protein
content of resting neutrophils and, although a significantly lower
percentage of total cytoplasmic protein content, MRP8/14 complexes
can also be found in resting monocytes. There is also evidence that
suggests that MRP8/14 may be released from myeloid cells, although
it is not clear whether the complex is actively released as part of
a response to inflammation or passively as a part of the demise of
such cells during the inflammatory process.
[0162] The function(s) of MRP8/14 complexes, CP-10, and related
S100 fraction Ca2+-binding proteins are not entirely clear.
However, it is thought that a major functional role of such
proteins is in the recruitment of certain populations of immune
cells to areas of inflammation. Devery and coworkers (J. Immunol.
152, 1888-1897; 1994) have demonstrated that expression of cell
surface molecules such as Mac-1, which is involved in the process
of cell adhesion as well as several additional cellular processes,
may be influenced by prior interaction of the cell with chemotactic
factors such as CP-10. These studies have also been performed in
vivo where it was observed that CP-10 protein accumulated on the
endothelial lining of small blood vessels in LPS-inflamed footpads.
Furthermore, increased levels of MRP8/14 have been observed in the
sera of patients afflicted with several inflammatory diseases
including rheumatoid arthritis. It has also been suggested that
chemotactic cytokine molecules such as CP-10 or MRP8/14 may
function as a type of "calcium sink" during times of elevated
intracellular levels of calcium for sustained periods of time.
Alternatively, it has been suggested that MRP8/14 may function as a
specific inhibitor of casein kinase II activity. Although the
precise functional role(s) of many of the currently defined
chemotactic cytokine-like proteins containing significant regions
of sequence identity to HEMFI85 are not known in any detail, a
number of studies with these proteins strongly suggest one or more
roles for these proteins in a variety of human disease states
including rheumatoid arthritis, sarcoidosis, tuberculosis,
onchocerciasis, and other chronic inflammatory disease states. As a
result, the discovery of a novel chemotactic cytokine-like molecule
is believed to be of value in a variety of therapeutic and
diagnostic capacities.
[0163] Owing to the homology to CP-10 and other calcium binding
proteins it is expected that the CCV polypeptide shares possess
common bioactivities. The activity of CCV may be assayed by any of
several biological assays known in the art, preferably calcium
binding assays. The homology to CP-10 and other calcium binding
proteins indicates that the CCV polypeptide is useful in the
detection and treatment of chronic inflammatory diseases such as
rheumatoid arthritis, sarcoidosis, tuberculosis and
onchocerciasis.
Features of the Proteins Encoded by SEQ ID NOS: 3 and 5
[0164] The full-length nucleotide sequences of two novel human cDNA
clones (HTXET53 and HT3SG28) which encode splice variants of the
previously reported and highly related chemokines LAG-2, NKG5, and
519 have recently been identified. See for example, Hercend and
Triebel (WPI Acc. No. 90-132241/17). These two clones have been
designated Chemokine from Activated T-Cells-1 (CAT-1) (HTXET53),
and Chemokine from Activated T-Cells-2 (CAT-2) (HT3SG28).
[0165] The HTXET53 clone was obtained from a human activated (12
hour) T-cell cDNA library and contains a 887 nucleotide insert (SEQ
ID NO:3) which encodes a 172 amino acid polypeptide (SEQ ID NO:4),
shown in FIGS. 2A-B. The HT3SG28 clone was obtained from a human
activated (8 hour) T-cell cDNA library and contains a 550
nucleotide insert (SEQ ID NO:5) which encodes an 88 amino acid
polypeptide (SEQ ID NO:6), shown in FIGS. 3A-B. The predicted amino
acid sequences of the novel full-length CAT splice variants contain
several regions of nearly perfect sequence identity to the
previously reported human LAG-2, NKG5, and 519 lymphokines.
Alignment of the amino acid sequences shows perfect identity
between the two novel molecules with LAG-2 and NKG5, with the
exception of a 27 amino acid insertion near the amino terminus of
HTXET53, and a 57 amino acid deletion very near the carboxy
terminus of HT3SG28. The 519 amino acid sequence differs from each
of the novel clones and from LAG-2 and NKG5 by an 18 amino acid
deletion of the hydrophobic amino terminus.
[0166] The HTXET53 polypeptide is predicted to have a 15 amino acid
secretory leader sequence. The HT3SG28 polypeptide is predicted by
the computer program PSORT to have either a 15 or a 22 amino acid
leader sequence. The leader sequences are underlined in FIGS. 2A-B
and 3A-B. Applicants believe that both the shorter and longer form
of the HT3SG28 polypeptides (i.e., beginning at either residue 16
or residue 23) are active.
[0167] Expression profiles of the two novel genes are qualitatively
identical in the HGS database. Additional HGS human cDNA libraries
which contain the two novel CAT clones are resting T-cells,
apoptotic T-cells, activated T-cells, spleen (chronic lymphocytic
leukemia), activated monocytes, pituitary, and 9 week early stage
human. The mRNA expression patterns of these novel genes have not
been examined by Northern blot analysis.
[0168] The original molecule cloned from this group the
T-cell-specific clone 519. NKG5 was a term used to describe a group
of identical clones isolated from a human natural killer (NK) cell
cDNA library. These genes are highly related and are thought to be
expressed only in NK and T-cells. A genomic clone of the gene which
encodes both 519 and NKG5 consists of at least five exons and four
introns which are likely responsible for the generation of the
related, but unique gene products. The genomic clone also reveals a
number of T-cell-specific and activation state-specific regulatory
sequences indicating that expession of the gene is highly
restricted to certain functions of a small subset of cell
types.
[0169] The novel and previously described molecules discussed
herein also contain approximately 33% identity with a recently
reported clone designated NK-lysin. NK-lysin has been found to
exhibit a potent anti-bacterial activity against such organisms as
Escherichia coli, Bacillus megaterium, Acinetobacter calcoaceticus,
and Streptococcus pyogenes. In addition, NK-lysin was also observed
to possess a marked lytic activity against an NK-cell-sensitive
mouse tumor cell line (YAC-1), but had no such activity against
erythrocytes. As a result, there are a number of potential
therapeutic and/or diagnostic applications for a factor such as
those encoded by HTXET53 and HT3SG28. Applications may include the
detection and treatment of such clinical presentations as various
bacterial infections, a number of lymphomas, immunological
disorders, autoimmune diseases, inflammatory diseases, various
allergies, and possibly as anti-infectious agents.
Features of the Proteins Encoded by SEQ ID NOS: 7 and 9
[0170] The novel Melanoma Inhibitory Activity Protein (MIA)-2 and
-3 cDNA clones presented herein are shown in FIGS. 4 and 5A-C. The
cDNA clone HBZAK03 contains a 520 nucleotide insert (SEQ ID NO:7)
which encodes a 59 amino acid polypeptide (SEQ ID NO:8), as shown
in FIG. 4. A BLAST analysis of the predicted amino acid sequence of
HBZAK03 demonstrates that this novel clone appears to be a splice
variant of another cDNA clone designated HLFBD44. The nucleotide
sequence of HLFBD44 (SEQ ID NO:9) and deduced amino acid sequence
(SEQ ID NO:10) are shown in FIGS. 5A-C. Both of these HGS clones
exhibit significant sequence identity to a human gene termed
melanoma inhibitory activity (MIA) protein. BestFit analysis
demonstrates that the HBZAK03 protein exhibits approximately 20%
identity and 58% similarity to the MIA protein over a region of
roughly 60 amino acids. The expression profile of the HBZAK03 cDNA
in the HGS database reveals that it appears in a number of HGS
human cDNA libraries in addition to the prostate cDNA library from
which it was cloned. Some of the cDNA libraries in which this clone
appears include fetal lung, the bone marrow cell line (RS4;11),
macrophage, serum-treated smooth muscle, epileptic frontal cortex,
subtracted fetal brain, HSA 172 cell line, induced endothelial
cells, and others.
[0171] The highest sequence identity of the novel cDNA clones
presented herein suggests that they may possess a function involved
in the regulation of melanoma progression. The previously described
MIA protein functions as a component of a highly complex and only
partially characterized system of stimulatory and inhibitory
factors which together dictate the progression of a melanoma. MIA
is secreted by malignant melanoma cells and has the capacity to
inhibit the growth of melanoma cells in culture. Investigators have
examined the expression profile of the MIA gene by Northern blot
and RT-PCR analysis and have determined that it is expressed in all
melanoma cell lines, a few glioma cell lines, approximately half of
the benign melanomas, all malignant melanomas, and from all lymph
node metastases of malignant melanomas examined (Bosserhoff et al.,
J. Biol. Chem. 271, 490-495; 1996). In contrast, no MIA expression
was detected by these methods in samples obtained from any other
skin-derived cells including normal fibroblasts, HaCaT
keratinocytes, COS cells, HeLa cells, HepG2 cells, DU 145 (human
prostate carcinoma) cells, and J82 (human bladder carcinoma)
cells.
[0172] Based on the sequence similarity between these polypeptides
MIA-2 and -3 are predicted to be useful in the detection and
regulation of malignant melanoma, in immune system modulation, and
in the treatment of cardiac arrest and stroke. Other activities of
MIA-1 as well as assays for detecting MIA-1 activity are outlined
in WO 95/03328, hereby incorporated herein by reference in its
entirety. MIA-2 and -3 activity can be assayed accordingly.
Features of the Proteins Encoded by SEQ ID NOS: 11 and 13
[0173] A macrophage-specific protein, termed AIF-1, has only very
recently been molecularly cloned. AIF-1 appears to function in
macrophage activation in the pathogenesis of chronic cardiac
rejection following transplantation. A characteristic manifestation
of cardiac tissue rejection following transplantation is an
immune-mediated arteriosclerosis which ultimately results in graft
failure and creates the need for retransplantation during the first
postoperative year. It is thought that the arteriosclerotic state
results from an alloimmune response involving activated immune
cells, particularly macrophages, which stimulate smooth muscle-cell
migration and proliferation into the area of the transplant leading
to lesions in donor vessels. AIF-1 was identified by Utans and
coworkers (J. Clin. Invest. 95, 2954-2962; 1995) in ongoing studies
of inducible gene expression patterns in macrophage cells in a
chronic rejecting rat heart allograft model. AIF-1 was expressed in
response to INF-g in the chronic cardiac rejection model referenced
above. Expression of AIF-1 was seen selectively in activated
macrophages, neutrophils, and the macrophage-like cell lines THP-1,
U937, and HL60, but not in several other human cells and tissues
examined. Furthermore, low levels of AIF-1 expression can be
observed in endomyocardial biopsy samples obtained from human heart
transplant patients.
[0174] The cDNA clone designated HEBGM49 or "AIF-2" contains a 632
nucleotide cDNA insert (SEQ ID NO:11) encoding a 150 amino acid
polypeptide (SEQ ID NO:12), as shown in FIGS. 6A-B. The cDNA clone
was isolated from a human early stage brain cDNA library. This
clone also appears in several other cDNA libraries constructed from
a variety of human cell and tissue types including fetal
epithelium, fetal kidney, hippocampus, tongue, and osteoblastoma
HOS cells. A BLAST analysis of the amino acid sequence of HEBGM49
demonstrated that this clone exhibits approximately 65% identity
and 80% similarity with AIF-1 over its entire length.
[0175] The cDNA clone HNGBH45 or "AIF-3" contains a 757 nucleotide
cDNA insert (SEQ ID NO:13) encoding a 193 amino acid polypeptide
(SEQ ID NO:14), as shown in FIGS. 7A-B. The cDNA clone was isolated
from a human neutrophil cDNA library. This clone appears in a
number of additional cDNA libraries including aortic endothelium,
cerebellum, corpus collosum, CD34-depleted buffy coat, activated
neutrophil, colon cancer, resting T-cells, tonsils, and others. A
BLAST analysis of the amino acid sequence of HNGBH45 demonstrated
that this clone exhibits approximately 25% identity and 47%
similarity over approximately 70 amino acids of the AIF-1
molecule.
[0176] AIF-2 and AIF-3 are believed to be valuable clinical markers
for assessing varying degrees of acute and chronic rejection of
transplanted cardiac tissue. In addition, monitoring the level of
AIF-2 and/or AIF-3 expression may also be useful in determining the
level of macrophage or neutrophil infiltration into area of the
transplanted tissue. In addition, AIF-2 and -3 may be used as
targets in assays for the identification of antagonists such as
small organic molecules which act to block AIF activity. Such
assays are known in the art.
Features of Protein Encoded by SEQ ID NO: 15
[0177] The full-length nucleotide sequence of a novel human cDNA
clone (HSAAL25) has been isolated which is believed to encode a new
member of the annexin/lipocortin supergene family. The novel
polypeptide is termed herein "Annexin HSAAL25". The
annexin/lipocortin supergene family is composed of at least ten
calcium-binding proteins proposed to function in a variety of
cellular roles including phospholipase A2 and protein kinase C
inhibition, anti-coagulation, endo- and exo-cytosis, inositol
phosphate metabolism, and as calcium channel proteins. Eukaryotic
calcium-binding proteins are typically classified as proteins which
bind calcium by a mechanism which either includes or does not
include an E-F hand motif. The annexin/lipocortin superfamily is
the largest group of calcium-binding proteins whose interaction
with calcium is not mediated by an E-F hand motif. Structurally,
all known annexins may be characterized by a common carboxy
terminal region consisting of four similar amino acid sequences, of
approximately seventy amino acids each, termed the "annexin
repeats". Conversely, the amino termini of annexin/lipocortin
proteins vary widely in both length and amino acid composition
between member protein sequences. Typical expression patterns of
annexin/lipocortin proteins include a wide variety of cells and
tissues including lung, kidney, bone marrow, spleen, thymus, brain,
macrophage, placenta, ovary, uterus, skeletal muscle, and
others.
[0178] Annexin/lipocortin proteins are involved in a wide variety
of physiologically important cellular processes. For example,
lipocortin-1 (LC-1; also known as annexin-I) appears to function as
a second messenger in the anti-inflammatory glucocorticoid signal
transduction cascade. Most LC-1 molecules are cell
surface-associated and attached to the plasma membrane by a
Ca2+-dependent interaction with unrelated plasma membrane binding
molecules. The process of extravasation, in which polymorphonuclear
leukocytes (PMNs) migrate into an area of inflammation, adhere to
the vascular wall, and eventually pass through the vascular wall
into the surrounding tissue, may be delayed by glucocorticoids,
and, as a result of LC-1 function, the overall process of
inflammation may be delayed. As an example of the diversity of
LC-1, and other annexin/lipocortin superfamily member, function,
LC-1 has also been shown to play a major regulatory role in a
number of possibly unrelated cellular systems such as cell growth
regulation and differentiation, response of the CNS to cytokines,
neuroendocrine secretion, anti-coagulation, and
neurodegeneration.
[0179] Annexin HSAAL25 contains a 1356 nucleotide cDNA insert (SEQ
ID NO:15) encoding a 324 amino acid polypeptide (SEQ ID NO:16), as
is shown in FIGS. 8A-C. HSAAL25 was isolated from a cDNA library
made from the HSA 172 cell line. Although previously described
annexin/lipocortin proteins are widely expressed, this clone also
appears only once in the HSA 172 cell line cDNA library and does
not appear in any other tissue type assayed for. A BLAST analysis
of the amino acid sequence of HSAAL25 demonstrated that this clone
exhibits at least 30% identity and 55% similarity over the entire
length of a molecule designated human annexin-III, a member of the
annexin/lipocortin supergene family.
[0180] There is clearly a need for identifying and exploiting novel
members of the annexin/lipocortin superfamily such as the cDNA
clone described herein. Plasma membrane-associated molecules, such
as the novel potential members of the annexin/lipocortin
superfamily detailed here, should prove useful in target based
screens for small molecules and other such pharmacologically
valuable factors that may be useful for regulating the complex
processes of inflammation. Furthermore, Annexin HSAAL25 is believed
to be useful as a regulator of coagulation (anti-coagulant) by
affecting Ca2+-dependent cell to cell aggregation. In addition,
this annexin-like clone may prove valuable in a number of other
therapeutically useful roles as an anti-inflammatory agent
including regulation of ischemia, tumor metastasis, rheumatoid
arthritis, other inflammatory diseases, wound healing,
arteriosclerosis, and other heart diseases.
Features of Protein Encoded by SEQ ID NO: 17
[0181] The full-length nucleotide sequence of a novel human cDNA
(HUSAX55) which encodes a previously unidentified "ES/130-like I"
protein has been identified. The translation product of the novel
full-length ES/130-like I cDNA clone exhibits significant sequence
identity to the chicken EDTA-soluble/130 kDa protein (ES/130) gene.
The ES/130-like I cDNA clone contains an 3036 nucleotide insert
(SEQ ID NO:17) which encodes a 977 amino acid polypeptide (SEQ ID
NO:18), as shown in FIGS. 9A-K. The clone was obtained from an
umbilical vein endothelial cell cDNA library. A BLAST analysis of
the deduced amino acid sequence of HUSAX55 exhibits approximately
66% identity and 83% similarity to the amino acid sequence of the
chicken ES/130 gene over a 573 amino acid stretch. Expression of
ES/130-like I is detected in a wide collection of HGS human cDNA
libraries including amygdala depression, thymus, smooth muscle,
endometrial tumor, synovial sarcoma, macrophage, fetal heart, and a
number of others. Northern blot analyses performed on expression of
the ES/130-like I gene indicates a high level of expression in
pancreas and liver and moderate to low expression elsewhere.
[0182] The in vitro process of endothelial cell transformation to
mesenchymal tissue models a similar in vivo process in the
developing heart where closely associated epithelial cells undergo
a transformation to cardiac mesenchyme tissue. This transformation
is a required event for the development of a multichambered heart
from the primative, single chambered heart tube. ES/130 was
originally identified as a 130 kD antigen isolated from the
100,000.times.g pellet fraction of non-cytolytic EDTA extracts of
developing chicken cardiac tissue. Inclusion of this fraction in
cardiac endothelial cell cultures results in formation of
mesenchymal tissue. ES/130 is an extracellular, secreted protein
which, in addition to endothelial cell transformation, has been
proposed to function in the regulation of adhesion molecule
expression and limb bud ectoderm, neural tube, and notocord
development. Potential therapeutic and/or diagnostic applications
for the ES130-like I protein include such clinical presentations as
atherosclerosis, restenosis, or as a general factor following a
number of types of surgery.
Features of the Protein Encoded by SEQ ID NO: 19
[0183] The full-length nucleotide sequence of a human cDNA clone
(HSXCK41) which encodes a novel brain-enriched hyaluronan-binding
factor ("BEF") has been determined. The novel BEF cDNA clone
presented herein was discovered in a human substantia nigra cDNA
library. The clone contains a 1757 nucleotide insert (SEQ ID NO:19)
which is predicted to encode a 528 amino acid polypeptide (SEQ ID
NO:20). A BLAST analysis of the predicted amino acid sequence of
HSXCK41 demonstrates significant sequence identity to the bovine
brevican mRNA (GenBank entry X75887), a member of the
aggrecan/versican family of cell surface proteoglycans. The HSXCK41
amino acid sequence exhibits approximately 92% identity and 95%
similarity over an approximately 400 amino acid stretch of the
brevican sequence. This clone has been identified in a number
additional HGS human cDNA libraries, many of which originate from
neural tissues. These include epileptic frontal cortex, early stage
brain, skin tumor, hippocampus, cerebellum, hemangiopericytoma,
infant brain, fetal brain, and fetal bone.
[0184] The aggrecan/versican family of cell surface proteoglycans
may be characterized by the presence of chondroitin sulfate side
chains, a hyaluronic acid (HA)-binding motif in the amino terminal
domain, and at least one epidermal growth factor (EGF)-like repeat,
a lectin-like motif, and one or more complement regulatory protein
(CRP)-like motifs in the carboxy terminal domain. The
aggrecan/versican family includes a number of members such as
brevican, aggrecan, decorin, versican, and neurocan. Brevican is
expressed predominantly in the brain and in primary cerebellar
astrocytes, but not in neurons. Meanwhile, both aggrecan and
versican are expressed in chondrocytes in human articular cartilage
obtained from subjects of a wide range of ages. Aggrecan messenger
RNAs undergo alternative splicing events which vary the inclusion
or exclusion of the single EGF-like motif in the carboxy terminal
domain. Alternatively, versican contains two EGF-like motifs and a
single CRP-like motif, all of which are present in all expression
patterns examined. Finally, the expression of two recently
described members of the aggrecan/versican family isolated from the
human sciatic nerve is significantly increased following lesioning
of the nerve.
[0185] The functional roles of members of the aggrecan/versican
family are rather varied. Aggrecan itself aggregates with HA to
function as a major space-filling component of cartilage. Brevican,
an aggrecan/versican family member which is a conditional
chondroitan sulfate proteoglycan, appears in a secreted, soluble
form as well as in a GPI-anchored form. Both brevican isoforms have
been implicated as functional components of the terminally
differentiating and adult nervous systems. It will likely be
determined that molecules such as these and the novel BEF cDNA
clone HSXCK41 may play a role in one or more of a variety of
cellular processes which typically involve intercellular contact
and communication mediated through cell surface and/or secreted
glycoprotein factors. Such cellular processes might include cell
adhesion, proliferation, tumor metastasis, and lymphocyte migration
into areas of inflammation. Related polypeptides are believed to be
expressed at a higher level in tumors such as gliomas. Thus, BEF
polynucleotides and polypeptides are useful as diagnostic markers
and reagents for detection of tumors such as gliomas.
FEATURES OF THE PROTEIN ENCODED BY SEQ ID NO: 21
[0186] The full-length nucleotide sequence of a human cDNA clone
(HFKFY79) which encodes a novel adipose differentiation factor
("ADF") has recently been determined. The novel ADF cDNA clone
presented herein was originally isolated from a human fetal kidney
cDNA library. The clone contains a 1550 nucleotide insert (SEQ ID
NO:21) which encodes a 452 amino acid polypeptide (SEQ ID NO:22),
as shown in FIGS. 11A-E. A BLAST analysis of the predicted amino
acid sequence of HFKFY79 demonstrates that this clone exhibits its
highest degree of sequence relatedness in the GenBank public
database to the murine ADF protein (GenBank accession number
M93275). Based on its homology to murine ADF, human ADF is believed
to share common biological activities. A BestFit analysis of the
predicted amino acid sequence of HFKFY79 versus the murine ADF
amino acid sequence demonstrates that the two protein sequences
exhibit approximately 39% identity and 79% similarity. The
expression profile of the HFKFY79 clone suggests a widely
distributed expression pattern. In addition to the human fetal
kidney library from which this clone was obtained, it also appears
in a large number of human cDNA libraries including ulcerative
colitis, adult testis, hypothalamus, induced endothelial cells,
Jurkat T-cell line in S-phase, serum-treated and control smooth
muscle, adipocytes, adult small intestine, lymph node breast
cancer, infant brain, and many others.
[0187] The murine ADF gene was cloned by Jiang & Serrero (Proc.
Natl. Acad. Sci. USA 89, 7856-7860; 1992, incorporated herein by
reference) in an effort to identify genes whose expression profiles
change significantly during the process of 1246 adipocyte cell and
primary adipocyte differentiation. The murine ADF gene product
identified by Jiang & Serrero is a 50 kD, membrane-bound
protein expressed abundantly in mouse fat pads. The novel cDNA
presented herein also exhibits sequence identity to several
additional lipid-specific proteins. The first of the putative
homologues is the major substrate for cAMP-dependent protein kinase
A (PKA) in adipocytes and is termed perilipin. Perilipin is
expressed in two alternatively spliced forms designated perilipins
A and B. Both forms of perilipins are expressed exclusively at the
surface of lipid storage droplets. It is thought that perilipids
may function as a barrier to deny access of lipase to lipid
reservoir of unstimulated cells. This event may be regulated by
PKA-dependent phosphorylation of perilipin which allows exposure of
lipid molecules to lipase. In addition, ADF is also related by
sequence identity to a gene cloned from a human bone marrow-derived
stromal cell line (KM-102) designated adipogenesis inhibitory
factor (AGIF). AGIF has been shown to inhibit the process of
adipogenesis in the mouse preadipocyte cell line 3T3-L1. Thus,
human ADF may be useful among other things as a therapeutic
modulator of lipid metabolism in the human body.
Features of the Protein Encoded by SEQ ID NO: 23
[0188] The novel "Bcl-like" cDNA clone (HAICH28) presented herein
was originally identified in a TNF-a/IFN-induced endothelial cell
cDNA library. The clone contains a 1211 nucleotide insert (SEQ ID
NO:23) which encodes a 365 amino acid polypeptide (SEQ ID NO:24). A
BLAST analysis of the predicted amino acid sequence of HAICH28
demonstrates that this clone exhibits strong sequence similarity to
two previously reported genes termed bovine polyA binding protein
II and human Bcl-w (GenBank accession numbers X89969 and U59747,
respectively). The expression profile of the HAICH28 clone suggests
a widely distributed expression pattern. In addition to the
TNF-a/IFN-induced endothelial cell library from which this clone
was obtained, it also appears in a large number of human cDNA
libraries including PHA-stimulated T-cells, osteoblasts,
schizophrenic hypothalamus, activated monocytes, adrenal gland
tumor, primary dendritic cells, and a number of others.
[0189] The protein product of the related Bcl-w gene has been
determined to function as a key player in the cellular apoptosis or
cell death pathway. Apoptosis is a term which describes the process
of programmed cell death in vertebrates. During the process of
apoptosis, the cell membrane shrinks and blebs resulting in a loss
of membrane integrity and intercellular contact. In addition, the
chromatin is condensed and cleaved into a characteristic
ladder-like organization and, finally, vesicular remnants of the
cell are quickly engulfed and destroyed by neighboring cells. The
signal for the cell to enter the apoptotic pathway likely begins
with the binding of Fas ligand or tumor necrosis factor (TNF), or
the recently discovered TRAIL ligand, to the Fas/CD95/APO-1 or TNF
(p55), or DR4 or DR5 receptors, respectively. These ligand/receptor
interactions recruit a cellular protein designated FLICE to the
cell membrane to act as a physical link between the Fas/CD95/APO-1
and TNF receptor complexes, also termed death receptors, and the
cysteine proteases belonging to the interleukin-1b (IL-1b)
converting enzyme (ICE)/CED-3 family to carry out the process of
apoptosis.
[0190] The t(14:18) chromosomal translocation is often associated
with human follicular lymphoma. In this chromosomal abnormality,
the immunoglobulin heavy chain locus becomes translocated adjacent
to the Bcl-2 gene, resulting in a drastic overexpression of the
Bcl-2 gene. Bcl-2 blocks the process of apoptosis by an unknown
mechanism. It has been proposed that Bcl-2 controls the process of
apoptosis by regulating endoplasmic reticulum-associated Ca2+
fluxes. Several other genes have been identified which have
significant regions of sequence identity with Bcl-2, including
Ced-9, BHRF 1, Bax, Bcl-xS, Bcl-xL, Bcl-w, Bak, Mcl-1, and GRS. The
protein product of each of these genes can affect the process of
apoptosis in either a positive (for example, Bax or Bcl-xS) or
negative (for example Bcl-2, BHRF 1, Ced-9, or Bcl-xL) fashion.
[0191] A large number of cells fall victim to the apoptotic process
throughout development and during the lifetime of the organism.
Clearly, strict regulation of the functional molecules comprising
such a potentially dangerous process is an extremely necessary and
valuable facet of the repertoire of cellular regulatory pathways.
As a result, the identification of novel molecules related to Bcl-2
or Bcl-w, such as that encoded by the novel cDNA clone described
herein, represents a major step in understanding, and, in turn,
exploiting the complex process of controlled cell death.
Accordingly, the Bcl-like polypeptide of the present invention is
thought to be useful as a therapeutic in an anti-viral or
anti-tumor capacity or, alternatively, in a diagnostic capacity.
Sequence CWU 1
1
* * * * *