Analysis of polysulfate-binding domains in porcine proacrosin, a putative zona adhesion protein from mammalian spermatozoa.

Proacrosin is one of the major proteins found within the acrosomal vesicle of mammalian spermatozoa. Previous work has shown that it binds non-enzymatically and with high affinity to polysulfate groups on zona pellucida glycoproteins (ZPGPs) thereby leading to the hypothesis that at fertilization it functions as a secondary ligand molecule to retain acrosome-reacted spermatozoa on the surface of the egg. In the present work we have investigated the nature and extent of the polysulfate binding domain on boar sperm proacrosin using a combination of group-specific modifying reagents, fragmentation analysis, peptide synthesis and expression of deletion recombinants in E. coli bacteria. Taken overall, our results show that arginine, lysine and histidine residues located between Gly 93 and Ala 275, together with the participation of His 47 and Arg 50, are necessary for maximum polysulfate binding activity. The secondary and tertiary structure of this central peptide domain is also important to ensure correct alignment of basic residues with complementary sulfate groups on ZPGPs. Proacrosin, therefore, has many properties in common with other polysulfate binding proteins, such as antithrombin III and sea urchin sperm binding, in having a conformation-dependent domain containing basic amino acids that mediates specific protein-protein interactions. These observations strengthen the hypothesis that proacrosin is a multifunctional protein with a major role as a ligand molecule at fertilization.