The mammalian acrosomal sperm
protease proacrosin plays a role in fertilization by proteolysis of the oocyte's outer investments. In addition to its
serine protease activity,
acrosin from several species is known to have binding activity for the zona pellucida, and this action may serve to anchor sperm during
zona penetration. In this study,
proacrosin 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 KD = 1.4 x 10(-8) M. Using cDNAs obtained from previously cloned 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 were produced using polymerase chain reaction and expressed as
recombinant proteins. In the same in vitro
zona binding assay, a construct representing residues 1-279 of rabbit
proacrosin was found to bind to
zona with a high affinity similar to that of native
proacrosin, KD = 2.1 x 10(-8) M. By making smaller recombinant fragments and assaying them for
zona binding activity, the location of the binding site was mapped to residues 47-94.
Protein modeling of rabbit
proacrosin using
chymotrypsinogen A as a three-dimensional model indicated that an exposed loop Asp35 to His40 in
chymotrypsinogen A is extended with an additional five
amino acid residues in rabbit
proacrosin from Ile43 to His53 containing
arginine residues Arg47, Arg50 and Arg51. Site-directed mutagenesis of
arginine residues Arg50 and Arg51 to
alanine produced a recombinant without significant
zona binding activity. These results are consistent with the hypothesis that rabbit
proacrosin contains a specific zona pellucida binding site and that the loop containing
arginine residues 50 and 51 is critical for
zona binding activity.