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 Fragments1

Abstract

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.

A 70 kDa protein is transferred from the outer acrsomal to the plasma membrane during capacitation

“…Transfer of proteins from the acrosome to the cell surface has been shown to occur during spermatogenesis and epididymal maturation. PH-20 is a 60 kDa guinea pig sperm protein present on the acrosomal membrane of round spermatids which is subsequently transferred to the plasma membrane [14,15]. APZ is a 55 kDa boar sperm surface protein present in the acrosome of testicular sperm, which is observed in both the acrosome and plasma membrane of ejaculated sperm [10,27].…”

“…To date such proteins have been identified in several systems. Among these are the trypsin inhibitor-sensitive site on the surface of hamster and mouse sperm [4,5], galactosyl transferase in mouse [6,7], proacrosine [8,9] and APZ [10,11] in the pig, rabbit sperm autoantigen [ 12,13], and PH-20 in the guinea pig [14,15]. In ejaculated sperm, ZP-binding proteins may be masked by epididymal or seminal secretions until after capacitation.…”

Prospects for Sperm Immunocontraceptive Vaccines

“…PH-20 is a bi-functional protein since it has both hyaluronidase activity and a separate zona-binding moiety [34]. Fully effective and reversible contraception was obtained in male and female guinea-pigs immunized with even low doses of the antigen [35,36]. In another study, to analyze the immunopathological effects of PH-20 in the reproductive tract, male guinea pigs were immunized with PH-20.…”

New horizons in contraception

“…Subsequent characterization of the sperm surface antigen involved in mediating zona recognition in the guinea pig was achieved by Primakoff 15 using a monoclonal antibody directed against a guinea-pig sperm antigen, PH-20, associated with three proteins, a major DURING the time it takes to read this chances of a successful reversal ever component of relative molecular mass article, about 1,000 new individuals will being performed '. 64,000 (64K), a component of 56K and an have been added to the world's popula-…”