Importance of glycosylation and disulfide bonds in hyaluronidase activity of macaque sperm surface PH-20.
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 beta-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.
Li
Ming-Wen
MW
Department of Obstetrics and Gynecology, University of California, Davis 95616, USA.
Yudin
Ashley I
AI
Robertson
Kathryn R
KR
Cherr
Gary N
GN
Overstreet
James W
JW
D000581:Amidohydrolases / Q000494:pharmacology; D000818:Animals; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006031:Glycosylation; D006821:Hyaluronoglucosaminidase / Q000737:chemistry* / Q000378:metabolism*; D008252:Macaca fascicularis; D008297:Male; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D010738:Type C Phospholipases / Q000494:pharmacology
D015815:Cell Adhesion Molecules; D004220:Disulfides; D020033:Protein Isoforms; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D043265:Phosphatidylinositol Diacylglycerol-Lyase