Importance of Glycosylation and Disulfide Bonds in Hyaluronidase Activity of Macaque Sperm Surface PH‐20

Abstract

ABSTRACT: PH‐20 is a glycoprotein located on the surface of the sperm plasma membrane and on the inner acrosomal membrane. The best understood function of sperm surface PH‐20 is its hyaluronidase activity, which results in hydrolysis of the hyaluronic acid‐rich cumulus matrix during sperm penetration of this extracellular oocyte investment. In this study, we investigated whether alterations in the secondary and tertiary structures of sperm surface PH‐20 would affect its enzyme activity. Proteins were isolated from the sperm plasma membrane by treatment of living cells with phosphatidylinositol‐specific phospholipase C (PI‐PLC). PH‐20 was purified from the PI‐PLC released proteins by immunoaffinity chromatography. Two‐dimensional electrophoresis of purified PH‐20 revealed 6 isoforms with isoelectric points ranging from 5.1 to 6.0. Removal of the N‐linked glycans from PH‐20 with N‐glycosidase F shifted the molecular weight from 64 kd to approximately 54 kd, its deduced molecular weight based on sequence analysis, suggesting that most if not all, of the potential N‐glycosylation sites are linked to oligosaccharides. The lectins Con A and PSA recognized purified sperm surface PH‐20 after Western blotting, suggesting that mannose is a major sugar within or at the terminal end of the linked glycan. The lectins UEA and LPA did not recognize PH‐20 Western blot, suggesting that fucose and sialic acid are not terminal sugars of sperm surface PH‐20. Deglycosylation of sperm surface PH‐20 resulted in a complete loss of its hyaluronidase activity. The reduction of disulfide bonds with β‐mercaptoethanol or dithiothreitol also resulted in loss of enzyme activity. We conclude that the hyaluronidase activity of sperm surface PH‐20 is dependent on structural features established by sulfhydryl linkages, as well as glycosylation.