SITE-DIRECTED MUTAGENESIS OF BOAR PROACROSIN REVEALS RESIDUES INVOLVED IN BINDING OF ZONA-PELLUCIDA GLYCOPROTEINS

Proacrosin, the zymogen form of the serine protease p-acrosin, is thou ght to function as a secondary binding molecule between mammalian game tes during fertilization (Jansen et al., 1995: Int J Dev Biol 39, 501- 510). The interaction involves strong ionic bonds between positively c harged amino acids on proacrosin and negatively charged polysulphate g roups on zona pellucida glycoproteins. In this investigation, we ident ified the basic residues on proacrosin that are important for this bin ding. Site-directed mutagenesis shows that two groups of amino acids c omprising His47, Arg50, and Arg51 together with Arg250, Lys252, and Ar g253 are crucial because their deletion or replacement severely reduce s affinity for zona glycoproteins. Molecular models of proacrosin reve al that these residues are located along one face of the protein on tw o exposed surface loops that project over and around the catalytic sit e. These findings support the hypothesis that polysulphate binding sit es on proacrosin are formed by a restricted number of basic amino acid s on the surface of the protein, presenting a specific orientation tha t is complementary to negatively charged sulphate groups on zona glyco proteins. Identification and elucidation of the stereochemistry of the se charged moieties will aid design of new kinds of nonsteroidal antif ertility agents. (C) 1998 Wiley-Liss, Inc.