Molecular cloning and characterization of P47, a novel boar sperm- associated zona pellucida-binding protein homologous to a family of mammalian secretory proteins

Abstract

P47, a peripherally associated 47-kDa protein of porcine spermatozoa, was identified by affinity chromatography in the fraction of solubilized plasma membrane proteins bound to immobilized porcine zona pellucida glycoproteins. N-terminal and internal amino acid sequences revealed structural similarity between P47 and rat O-acetyl ganglioside synthase, 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 against bovine MGP57/53 displayed cross-reactivity with P47. Indirect immunofluorescence analysis located porcine P47 on the acrosomal cap of testicular sperm and on sperm recovered along different sections of the ductus epididymidis, as well as on swim-up and in vitro-capacitated sperm. 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-associated protein, porcine milk P47 possesses affinity for isolated, biotinylated sow oocyte zona pellucida glycoproteins. Reverse transcription-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 sequences showed 60-100% amino acid sequence identity. These proteins display a mosaic structure organized into two N-terminal, tandemly arranged epidermal growth factor (EGF)-like domains followed by a region with similarity to C1 and C2 domains found in blood clotting factors V and VIII. The second EGF-like domain contains an arginine-glycine-aspartic acid sequence, a motif often found in integrin receptor ligands. P47-like proteins are not expressed solely in testicular and mammary gland tissues. Northern blot analysis showed that P47 mRNA is transcribed in several porcine and bovine tissues. These data indicate a potential role for boar sperm-associated P47 in membrane remodeling and/or as a zona pellucida binding protein.