Purification of the guinea pig sperm PH-20 antigen and detection of a site-specific endoproteolytic activity in sperm preparations that cleaves PH-20 into two disulfide-linked fragments.
Previous work has indicated that the guinea pig sperm membrane protein, PH-20, functions in sperm-egg adhesion and that its surface expression is regulated by the acrosome reaction. The PH-20 protein was purified by monoclonal antibody affinity chromatography. Sixty-seven to one hundred percent of the PH-20 antigenic activity present in an octylglucoside (OG) extract of sperm was recovered in the purified protein. From 10(10) sperm, approximately 0.4 mg of PH-20 protein was obtained, which was about 0.24% of the total protein in the OG extract. The purified protein retained the ability to bind the three anti-PH-20 monoclonal antibodies we have isolated. Silver staining of purified PH-20 on overloaded sodium dodecyl sulfate (SDS) gels allowed the estimate that silver-stainable contaminants were present at a level of one part in 2000. The purified PH-20 protein exists in three forms separable on SDS-polyacrylamide gel electrophoresis: a major form with a molecular mass of 64 kDa, a minor form of 56 kDa, and an endoproteolytically cleaved form composed of two disulfide-linked fragments of 41-48 kDa and 27 kDa. Cleveland digests of the 64 kDa and 56 kDa polypeptides indicated that they were structurally related. A proportion of the 64 kDa polypeptide in each purified preparation had undergone endoproteolysis at a specific site, so that it was cleaved into the two disulfide-linked fragments, 41-48 kDa and 27 kDa. It is speculated that the site-specific endoproteolysis of PH-20 may occur during the acrosome reaction and have biological significance.
D000177:Acrosome / Q000276:immunology; D000818:Animals; D015815:Cell Adhesion Molecules / Q000302:isolation & purification*; D002462:Cell Membrane / Q000276:immunology; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism; D006168:Guinea Pigs; D006821:Hyaluronoglucosaminidase; D008297:Male; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification; D008970:Molecular Weight; D013094:Spermatozoa / Q000201:enzymology / Q000276:immunology*
D015815:Cell Adhesion Molecules; D008565:Membrane Proteins; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20; D010450:Endopeptidases