Identification by affinity chromatography of boar sperm membrane-associated proteins bound to immobilized porcine zona pellucida. Mapping of the phosphorylethanolamine-binding region of spermadhesin AWN.

Abstract

We have identified boar sperm membrane components recovered by affinity chromatography on a porcine zona pellucida affinity column. The major zona pellucida-bound proteins were spermadhesins AWN and AQN-3, the heparin-binding protein pAIF, and a homolog of the mouse milk fat globule membrane protein. All these proteins are phospholipid-binding proteins peripherally associated with the plasma membrane. Our data suggest that coating proteins tightly bound to the external lipid bilayer may act as major zona pellucida-binding molecules. Using a synthetic peptide approach we show that the regions of spermadhesin AWN comprising residues 6-12 and 104-108 possess affinity for phosphorylethanolamine. These two amino acid sequences are in close proximity in the predicted structural model for AWN, and in opposite location to its carbohydrate-recognition domain. Taken together, our data provide further evidence for the possible involvement of members of the porcine spermadhesin protein family in gamete interaction and suggest a model for the ultrastructural disposition of functional domains of spermadhesin AWN bound to the sperm surface.