Testis-specific cystatin-like protein cystatin T

Details Testis-specific cystatin-like protein cystatin T Testis-specific cystatin-like protein cystatin T

2000-07-17

2001-06-12

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S252330, C435S320100, C435S069100, C536S023400, C536S023500, C530S350000

Reexamination Certificate

active

06245529

ABSTRACT:

BACKGROUND OF THE INVENTION
The cystatin superfamily is an evolutionarily related group of proteins consisting of at least three families, i.e., stefins (type 1), cystatins (type 2), and kininogens (type 3). See, for example, Barrett,
TIBS
12: 193-196, 1987. Generally, stefin family members are unglycosylated proteins consisting of about 100 amino acids that are devoid of disulfide bonds. In contrast, cystatin family members are proteins consisting of about 115 amino acids and characterized by two disulfide bonds in the carboxy-terminal region of the protein. Finally, kininogens contain three regions containing two disulfide loops, similar to the carboxy terminal domain found in members of the cystatin family. The cystatin superfamily are inhibitors of cysteine proteinases (also referred to as cysteine proteases) and are believed to function in a protective role with regard to pathological action of endogenous or exogenous cysteine proteinases. It is believe that cystatins form equimolar reversible complexes with cysteine proteinases.
Cystatin-like proteins have also been identified. One such protein, cystatin-related epididymal specific gene (CRES) does not contain the conserved sequence motifs necessary for cysteine proteinase inhibitory activity (Cornwell et al.,
Mol. Endocrinol.
6:1653-64, 1992 and Cornwell and Hann,
Mol. Reprod. Dev.
41:37-46, 1995). Also, unlike the ubiquitous expression of many of the cystatins, CRES is restricted to the proximal caput epididymal epithelium and testis. CRES expression is stage specific during spermatogenesis and CRES is found in both round and elongating spermatids suggesting a specialized role during spermatogenesis. Cystatins are also found with male reproductive tissues and secretions. Cystatin C for example is found in highest abundance in human semen and participates in spermatogenesis and spermiogenesis and is associated with the sperm throughout its time in the male genital tract (Esnard wt al.,
FEBS Lett.
300:131-5, 1992). Testatin is believed to be involved in early testis development. Expression is restricted to fetal gonads and adult testis and it is expressed during testis cord formation in pre-Sertoli cells (Töhönen et al.,
Proc. Natl. Acad. Sci. USA
95:14208-13, 1998).
Proteins capable of modulating spermatogenesis are sought for the study, diagnosis and treatment of conditions associated with reproductive disorders. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
SUMMARY OF THE INVENTION
Within one aspect the invention provides an isolated polypeptide comprising 10 or more contiguous amino acid residues of SEQ ID NO:2, wherein the polypeptide comprises SEQ ID NO:14 and specifically binds to an antibody to which a polypeptide of SEQ ID NO:2 specifically binds. Within one embodiment the polypeptide comprises SEQ ID NO:13. Within another embodiment the polypeptide comprises amino acid residues 76-138 of SEQ ID NO:2. Within yet another embodiment the polypeptide further comprises an affinity tag or binding domain.
The invention also provides an isolated polypeptide comprising a sequence of amino acid residues that is selected from the group consisting of: a) a sequence of amino acid residues that is 80% identical to the amino acid sequence of SEQ ID NO:2, wherein the sequence comprises cysteine residues corresponding to amino acid residues 94, 104, 118 and 138 of SEQ ID NO:2 and wherein the polypeptide comprising the amino acid sequence specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO:2; and b) a sequence of amino acid residues encoded by a polynucleotide sequence which hybridizes under stringent conditions to a similarly sized polynucleotide sequence of SEQ ID NO:1. Within one embodiment any difference between the amino acid sequence of the isolated polypeptide and the corresponding amino acid sequence of SEQ ID NO:2 is due to a conservative amino acid substitution. Within another embodiment amino acid percent identity is determined using a FASTA program with ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=blosum62, with other parameters set as default.
The invention also provides an isolated polypeptide consisting of amino acid residues 76-138 of SEQ ID NO:2. Additionally the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2.
Within another aspect of the invention is provided a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-20 of SEQ ID NO:2, wherein the secretory signal sequence is operably linked to an additional polypeptide. In one embodiment the first portion comprising a polypeptide as described above; and the second portion comprising another polypeptide.
Within yet another aspect of the invention is provided an isolated polynucleotide encoding a polypeptide as described above. Within one embodiment the polypeptide comprises SEQ ID NO:13. Within another embodiment the polypeptide comprises amino acid residues 76-138 of SEQ ID NO:2. Within another embodiment the polynucleotide encodes a polypeptide further comprising an affinity tag or binding domain.
The invention also provides an isolated polynucleotide molecule that encodes a polypeptide, wherein the polypeptide comprises a sequence of amino acid residues that is selected from the group consisting of: a) a sequence of amino acid residues that is 80% identical to the amino acid sequence of SEQ ID NO:2, wherein the sequence comprises cysteine residues corresponding to amino acid residues 94, 104, 118 and 138 of SEQ ID NO:2 and wherein the polypeptide comprising the amino acid sequence specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO:2; b) degenerate nucleotide sequence of a); c) nucleotide sequences complementary to a) or b); and d) a sequence of amino acid residues encoded by a polynucleotide sequence which hybridizes under stringent conditions to a similarly sized polynucleotide sequence of SEQ ID NO:1. Within one embodiment any difference between the amino acid sequence encoded by the polynucleotide molecule and the corresponding amino acid sequence of SEQ ID NO:2 is due to one or more conservative amino acid substitutions. Within another embodiment the amino acid percent identity is determined using a FASTA program with ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=blosum62, with other parameters set as default.
The invention also provides an isolated polynucleotide molecule consisting of nucleotides 271-459 of SEQ ID NO:1. The invention further provides an isolated polynucleotide molecule comprising the nucleotide sequence of nucleotides 46 to 468 of SEQ ID NO:1.
The invention provides a polynucleotide encoding a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-20 of SEQ ID NO:2, wherein the secretory signal sequence is operably linked to an additional polypeptide. Within one embodiment the first portion comprises a polypeptide as described above; and the second portion comprising another polypeptide.
Within another aspect the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator. Within one embodiment the expression vector further comprises a secretory signal sequence operably linked to the polypeptide encoded by the DNA segment. Within another aspect the DNA segment encodes a polypeptide covalently linked amino terminally or carboxy terminally to an affinity tag. Within yet another embodiment is provided a cultured cell into which has been introduced an expression vector as described above, wherein the cultured cell expresses the polypeptide encoded by the DNA segment.

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