Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein.
A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-(lambda)gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility.
Santhanam
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Division of Research, Department of Obstetrics and Gynecology, Medical College of Ohio, Toledo, Ohio 43614-5806, USA.
Naz
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D020101:Acrosome Reaction; D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D000954:Antigens, Surface / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003269:Contraception, Immunologic / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D058851:GPI-Linked Proteins; D006801:Humans; D008297:Male; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D013094:Spermatozoa / Q000276:immunology / Q000502:physiology*; D013737:Testis / Q000166:cytology / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000954:Antigens, Surface; D018076:DNA, Complementary; D058851:GPI-Linked Proteins; C439870:LY6E protein, human; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins