Site-directed mutagenesis of rabbit proacrosin. Identification of residues involved in zona pellucida binding.
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.
Richardson
R T
RT
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
O'Rand
M G
MG
D000176:Acrosin / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000201:enzymology*; D013329:Structure-Activity Relationship; D015044:Zona Pellucida / Q000378:metabolism*
D004792:Enzyme Precursors; D011994:Recombinant Proteins; C022438:proacrosin; D000176:Acrosin