Characterization of AWN-1 glycosylated isoforms helps define the zona pellucida and serine proteinase inhibitor-binding region on boar spermadhesins.

Spermadhesin AWN-1 (14 kDa) belongs to a recently described family of boar sperm surface-associated proteins. AWN-1 is a multifunctional protein which possesses heparin-, serine proteinase inhibitor-, and zona pellucida glycoprotein-binding capability. Therefore it has been implicated in sperm capacitation and sperm-oocyte attachment. Here, we report the characterization of 22-25 kDa isoforms of AWN-1 isolated by heparin-affinity chromatography, which fail to bind to zona pellucida glycoproteins or serine proteinase inhibitors. Our results show that the structure of the high and low molecular mass AWN-1 forms differ in that the former is N-glycosylated at Asp50 and truncated at the C-terminus. The inability of the glycosylated AWN-1 molecules to bind ligands is due solely to the presence of the oligosaccharide moieties, however. This indicates that glycosylation of AWN-1 may modulate its ligand-binding capabilities. On the other hand, the effect of glycosylation on ligand-binding suggests that both the zona pellucida- and the serine proteinase inhibitor binding domain(s) may be located around the glycosylation point.