Site-directed mutagenesis of boar proacrosin reveals residues involved in binding of zona pellucida glycoproteins.
Proacrosin, the zymogen form of the serine protease beta-acrosin, is thought to function as a secondary binding molecule between mammalian gametes during fertilization (Jansen et al., 1995: Int J Dev Biol 39, 501-510). The interaction involves strong ionic bonds between positively charged amino acids on proacrosin and negatively charged polysulphate groups on zona pellucida glycoproteins. In this investigation, we identified the basic residues on proacrosin that are important for this binding. Site-directed mutagenesis shows that two groups of amino acids comprising His47, Arg50, and Arg51 together with Arg250, Lys252, and Arg253 are crucial because their deletion or replacement severely reduces affinity for zona glycoproteins. Molecular models of proacrosin reveal that these residues are located along one face of the protein on two exposed surface loops that project over and around the catalytic site. These findings support the hypothesis that polysulphate binding sites on proacrosin are formed by a restricted number of basic amino acids on the surface of the protein, presenting a specific orientation that is complementary to negatively charged sulphate groups on zona glycoproteins. Identification and elucidation of the stereochemistry of these charged moieties will aid design of new kinds of nonsteroidal antifertility agents.
Jansen
S
S
Veterinary Institute of Göttingen, Germany. sjansen@gwdg.de
Jones
R
R
Jenneckens
I
I
Marschall
B
B
Kriegesmann
B
B
Coadwell
J
J
Brenig
B
B
D000176:Acrosin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004527:Egg Proteins / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D004792:Enzyme Precursors; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D000072216:Zona Pellucida Glycoproteins; C022438:proacrosin; D000176:Acrosin