Molecular cloning and characterization of functional domains of a human testis-specific isoform of calpastatin.
Human serum containing sperm-agglutinating antibodies was used to screen a testis cDNA expression library to identify the cognate antigens that may be responsible for this biological effect. The longest positive phage clone (1.9 kb) was sequenced and found to be a testis-specific isoform of calpastatin (tCAST). The testis-specific segment of tCAST is encoded by a single exon within intron 14 of the calpastatin gene. A unique protein isoform is produced that differs in domain structure from the somatic calpastatins (sCAST). Human sCAST most commonly has an N-terminal domain L plus the four functional calpain inhibitory domains. Human tCAST consists of a 40-amino-acid N-terminal T domain plus a part of domain II and all of domains III and IV from the somatic isoform. Our data show that the T domain can target cytosolic localization and membrane association of tCAST, whereas domain I of sCAST exhibits a nuclear localization function. Calpastatin is the endogenous inhibitor of calpain. The calpain/calpastatin system is involved in membrane fusion events for several cell types, and calpain has been localized to the sperm acrosome. We detected tCAST in human sperm and testes extracts by Western blotting with specific antisera. These observations suggest that tCAST may modulate calpain in the calcium-mediated acrosome reaction that is required for fertilization.
Li
S
S
Center for Recombinant Gamete Contraceptive Vaccinogens and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Liang
Z G
ZG
Wang
G Y
GY
Yavetz
B
B
Kim
E D
ED
Goldberg
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D013737:Testis / Q000502:physiology*
D002135:Calcium-Binding Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C033668:calpastatin