title: Structural relationship of sperm soluble hyaluronidase to the sperm membrane protein PH-20.
abstract: The sperm plasma membrane protein PH-20 has a hyaluronidase activity that enables acrosome-intact sperm to pass through the cumulus cell layer of the egg. In this study we analyzed the relationship of guinea pig PH-20 and the "classical" soluble hyaluronidase released at the time of the acrosome reaction of guinea pig sperm. PH-20 is a membrane protein, anchored in the plasma and inner acrosomal membranes by a glycosyl phosphatidyl inositol anchor. Several types of experiments indicate a structural relationship of PH-20 and the soluble hyaluronidase released during the acrosome reaction. First, an antiserum raised against purified PH-20 is positive in an immunoblot of the soluble protein fraction released during the acrosome reaction. In the released, soluble protein fraction, the anti-PH-20 antiserum recognizes a protein of approximately 64 kDa, i.e., identical in molecular mass to PH-20 (approximately 64 kDa). Second, the enzymatic activity of the released hyaluronidase is completely inhibited (100%) by the anti-PH-20 antiserum. Third, almost all (97%) of the soluble hyaluronidase is removed from the released protein fraction by a single pass through an affinity column made with an anti-PH-20 monoclonal antibody. These findings suggest that the released, soluble hyaluronidase is a soluble form of PH-20 (sPH-20). During the acrosome reaction, PH-20 undergoes endoproteolytic cleavage into two disulfide-linked fragments whereas the released sPH-20 is not cleaved, suggesting the possible activity of a membrane-bound endoprotease on PH-20. We searched for a cDNA encoding sPH-20 but none was found. This result suggests that sPH-20 may arise from the enzymatic release of PH-20 from its membrane anchor, possibly at the time of acrosome reaction.
authors.affiliation: Department of Physiology, University of Connecticut Health Center, Farmington 06030, USA.
authors.forename: G R
authors.initials: GR
authors.lastname: Hunnicutt
authors.forename: K
authors.initials: K
authors.lastname: Mahan
authors.forename: W F
authors.initials: WF
authors.lastname: Lathrop
authors.forename: C S
authors.initials: CS
authors.lastname: Ramarao
authors.forename: D G
authors.initials: DG
authors.lastname: Myles
authors.forename: P
authors.initials: P
authors.lastname: Primakoff
mesh_terms: D000177:Acrosome / Q000737:chemistry / Q000201:enzymology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D018076:DNA, Complementary / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006821:Hyaluronoglucosaminidase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008297:Male; D013094:Spermatozoa / Q000737:chemistry* / Q000201:enzymology* / Q000648:ultrastructure
chemical_list: D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20