Molecular identity of a sperm acrosome antigen recognized by HS-63 monoclonal antibody.
The molecular identity of mouse sperm acrosome antigen recognized by HS-63 monoclonal antibody was analyzed by various biochemical, immunological and molecular biological methods. When its cognate antigen, MSA-63 was isolated from mouse testis by immunoaffinity chromatography, a group of protein spots with wide range of molecular sizes and isoelectric points were identified. Through previous studies, it was established that most of these protein spots were actin-like molecules co-purified with MSA-63 protein from mouse testis. To analyze the molecular size heterogeneity of the isolated MSA-63 proteins, rabbit antisera against a computer-predicted antigenic synthetic peptide (amino acid residue No. 160-171) and a recombinant glutathione S-transferase (GST) fusion protein (GST-63) were raised. These two antisera and those raised against the isolated MSA-63 protein were used as the probes in comparative Western blot assay, indirect immunofluorescent assay and enzyme-linked immunosorbent assay (ELISA). Using ELISA, antisera against GST-63 and computer-predicted antigenic synthetic peptides were shown to cross-react with affinity-isolated MSA-63 protein coated on microwells. However, little immunological cross-reactivity was observed between GST-63 fusion protein and the synthetic peptide. By using a Western blot assay, two major protein bands of 22 and 32 kDa, respectively were commonly detected on mouse testis homogenate strips by both anti-MSA-63 and anti-GST-63. In addition, anti-MSA-63 also recognized several protein bands with molecular masses greater than 35 kDa. The results of this study suggested that the molecular heterogeneity of MSA-63 protein isolated from mouse testis and sperm, is due to a series of post-translational modifications on a single gene product. These modifications may include glycosylations, proteolytic digestions and tight non-covalent associations with other testicular cytoskeletal proteins, such as actins.
Lee
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Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada.
Wong
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Hsu
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Kowalik
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Huang
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D000177:Acrosome / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000941:Antigens / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D001483:Base Sequence; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D012739:Gonadal Steroid Hormones / Q000032:analysis; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011817:Rabbits
C080034:Acrv1 protein, mouse; D000911:Antibodies, Monoclonal; D000941:Antigens; D012739:Gonadal Steroid Hormones; D008565:Membrane Proteins