SITE-DIRECTED MUTAGENESIS OF RABBIT PROACROSIN - IDENTIFICATION OF RESIDUES INVOLVED IN ZONA-PELLUCIDA BINDING

The mammalian acrosomal sperm protease proacrosin plays a role in fert ilization by proteolysis of the oocyte's outer investments, In additio n to its serine protease activity, acrosin from several species is kno wn to have binding activity for the zona pellucida, and this action ma y serve to anchor sperm during zona penetration. In this study, proacr osin was purified from acid extracts of rabbit sperm and shown to bind to homologous zona pellucida using an in vitro assay. Measurement of this binding activity indicated a high affinity saturable interaction with a K-D = 1.4 x 10(-8) M. Using cDNAs obtained from previously clon ed and sequenced rabbit proacrosin and a splice variant that encodes a shorter form of acrosin (Richardson, R. T., and O'Rand, M. G. (1994) Biochim. Biophys. Acta 1219, 215-218), constructs of various sizes wer e produced using polymerase chain reaction and expressed as recombinan t proteins. In the same in vitro zona binding assay, a construct repre senting residues 1-279 of rabbit proacrosin was found to bind to zona with a high affinity similar to that of native proacrosin, K-D = 2.1 x 10(-8) M. By making smaller recombinant fragments and assaying them f or zona binding activity, the location of the binding site was mapped to residues 47-94. Protein modeling of rabbit proacrosin using chymotr ypsinogen A as a three-dimensional model indicated that an exposed loo p Asp(35) to His(40) in chymotrypsinogen A is extended with an additio nal five amino acid residues in rabbit proacrosin from Ile(43) to His( 53) containing arginine residues Arg(47), Arg(50) and Arg(51). Site-di rected mutagenesis of arginine residues Arg(50) and Arg(51) to alanine produced a recombinant without significant zona binding activity. The se results are consistent with the hypothesis that rabbit proacrosin c ontains a specific zona pellucida binding site and that the loop conta ining arginine residues 50 and 51 is critical for zona binding activit y.