MOLECULAR-CLONING AND CHARACTERIZATION OF P47, A NOVEL BOAR SPERM-ASSOCIATED ZONA PELLUCIDA-BINDING PROTEIN HOMOLOGOUS TO A FAMILY OF MAMMALIAN SECRETORY PROTEINS

P47, a peripherally associated 47-kDa protein of porcine spermatozoa, was identified by affinity chromatography in the fraction of solubiliz ed plasma membrane proteins bound to immobilized porcine zona pellucid a glycoproteins. N-terminal and internal amino acid sequences revealed structural similarity between P47 and rat O-acetyl ganglioside syntha se, bovine mammary gland protein (MPG)57/53 and mouse milk fat globule protein E8-polypeptides of unknown function secreted by mammary gland epithelial cells in both species. A polyclonal antibody directed agai nst bovine MGP57/53 displayed crossreactivity with P47. Indirect immun ofluorescence analysis located porcine P47 on the acrosomal cap of tes ticular sperm and on sperm recovered along different sections of the d uctus epididymidis, as well as on swim-up and in vitro-capacitated spe rm. Porcine P47 was demonstrated on sperm bound to the zona pellucida of a homologous oocyte. Western blot analysis identified P47 (or MGP57 /53) homologous proteins in porcine and human milk. Like the sperm-ass ociated protein, porcine milk P47 possesses affinity for isolated, bio tinylated sow oocyte zona pellucida glycoproteins. Reverse transcripti on-polymerase chain reaction was used to isolate P47 homologous cDNAs from porcine testis and mammary gland tissues as well as from bovine, mouse, and human testis. P47 proteins deduced from these cDNA sequence s showed 60-100% amino acid sequence identity. These proteins display a mosaic structure organized into two N-terminal, tandemly arranged ep idermal growth factor (EGF)-like domains followed by a region with sim ilarity to C1 and C2 domains found in blood clotting factors V and VII I. The second EGF-like domain contains an arginine-glycine-aspartic ac id sequence, a motif often found in integrin receptor ligands. P47-lik e proteins are not expressed solely in testicular and mammary gland ti ssues. Northern blot analysis showed that P47 mRNA is transcribed in s everal porcine and bovine tissues. These data indicate a potential rol e for boar sperm-associated P47 in membrane remodeling and/or as a zon a pellucida binding protein.