Mapping the heparin-binding domain of boar spermadhesins

Boar spermadhesins are a group of seminal plasma, heparin-binding proteins which appear to be involved in sperm capacitation and gamete interaction. Using a proteolytic protection assay we have identified regions of AQN-1, AQN-3, PSP-I and AWN which remain attached to a heparin-Sepharose column following in-column digestion of bound spermadhesins with chymotrypsin and elastase. In addition, the complete amino acid sequence of spermadhesin AWN was synthesized as overlapping peptides, and their ability to bind to a heparin-Sepharose column and to inhibit the interaction of soluble heparin with purified ELISA plate-coated AWN was tested. Both approaches gave similar results and as a whole showed that different regions of AWN may converge in its tertiary structure to form a composite heparin-binding site. The conformational heparin-binding surface resides on the GFCC'C'' face of the proposed structural model for AWN and is in an opposite location to the carbohydrate-binding region of the spermadhesin.