Characterization of the functional domains of boar acrosin involved in nonenzymatic binding to homologous zona pellucida glycoproteins.
During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida. To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs). The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody. The recombinant truncated beta-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation. Three of the five recombinant forms (truncated beta-acrosin, Ser/Ala222-truncated beta-acrosin, and truncated beta-acrosin "heavy chain") had the ability to bind ZPGPs. The two shorter forms (the amino and carboxy termini of truncated beta-acrosin) failed to bind. The catalytic site mutant (Ser/Ala222) of truncated beta-acrosin does not differ from the recombinant truncated beta-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process. Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site.
Crosby
J A
JA
Department of Biochemistry, Faculty of Medicine, University of Chile, Santiago. jcrosby@genes.bio.puc.cl
Jones
R
R
Barros
C
C
Carvallo
P
P
D000176:Acrosin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D016264:Dextran Sulfate / Q000378:metabolism / Q000494:pharmacology; D004527:Egg Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007457:Iodine Radioisotopes / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000072216:Zona Pellucida Glycoproteins; D016264:Dextran Sulfate; C007789:fucoidan; D000176:Acrosin