Structural relationship of sperm soluble hyaluronidase to the sperm membrane protein PH 20 (PDF)
Hunnicutt,Gary R.; Mahan,Kimberly; Lathrop,William F.; Ramarao,Chodavarapu S.; Myles,Diane G.; Primakoff,Paul
Biology of Reproduction 54(6): 1343-1349
Publication date: 1996
The sperm plasma membrane protein PH-20 has a hyaluronidase activity thatenables acrosome-intact sperm to pass through the cumulus cell layer of theegg. In this study we analyzed the relationship of guinea pig PH-20 and the"classical" soluble hyaluronidase released at the time of the acrosomereaction of guinea pig sperm. PH-20 is a membrane protein, anchored in theplasma and inner acrosomal membranes by a glycosyl phosphatidyl inositolanchor. Several types of experiments indicate a structural relationship ofPH-20 and the soluble hyaluronidase released during the acrosome reaction.First, an antiserum raised against purified PH-20 is positive in animmunoblot of the soluble protein fraction released during the acrosomereaction. In the released, soluble protein fraction, the anti-PH-20antiserum recognizes a protein of approximately 64 kDa, i.e., identical inmolecular mass to PH-20 (approximately 64 kDa). Second, the enzymaticactivity of the released hyaluronidase is completely inhibited (100%) bythe anti-PH-20 antiserum. Third, almost all (97%) of the solublehyaluronidase is removed from the released protein fraction by a singlepass through an affinity column made with an anti-PH-20 monoclonalantibody. These findings suggest that the released, soluble hyaluronidaseis a soluble form of PH-20 (sPH-20). During the acrosome reaction, PH-20undergoes endoproteolytic cleavage into two disulfide- linked fragmentswhereas the released sPH-20 is not cleaved, suggesting the possibleactivity of a membrane-bound endoprotease on PH-20. We searched for a cDNAencoding sPH-20 but none was found. This result suggests that sPH-20 mayarise from the enzymatic release of PH-20 from its membrane anchor,possibly at the time of acrosome reaction.
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