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

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Abstract
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.