GLYCOSYLATED BOAR SPERMADHESIN AWN-1 ISOFORMS - BIOLOGICAL ORIGIN, STRUCTURAL CHARACTERIZATION BY LECTIN MAPPING, LOCALIZATION OF O-GLYCOSYLATION SITES, AND EFFECT OF GLYCOSYLATION ON LIGAND-BINDING

Spermadhesin AWN-1 is a 133-residues boar sperm surface lectin with ca pability to bind different ligands, e.g. glycoproteins from zona pellu cida (ZP), soybean trypsin inhibitor and heparin, and is involved in c apacitation and binding of spermatozoa to the homologous zona pellucid a. Here, we report the characterization of N- and O-glycosylated isofo rms of AWN-1. Non-glycosylated AWN-1 is present in seminal plasma and on epididymal and ejaculated spermatozoa whereas its N- and O-glycosyl ated isoforms are only secretory products of the seminal vesicles. Lec tin mapping indicated the presence of the glycosylated AWN-1 isoform m ixture of both fucosylated and non-fucosylated N-glycans, and of two d ifferent classes of O-linked carbohydrate chains. These N- and O-linke d oligosaccharide chains are neither sialylated nor contain terminal G al beta(1-4)-GlcNAc sequences. Noteworthy, N- and O-glycosylation (eit her class) are mutually exclusive on the same protein molecule, indica ting that each glycosylated AWN-1 molecule contains a single oligosacc haride chain. Peptide mapping was used to locate the N- and the O-glyc osylation sites. Glycosylation of AWN-1 with either of the carbohydrat e chain types greatly impaired the ability of the spermadhesin to bind biotinylated zona pellucida glycoproteins and soybean trypsin inhibit or, suggesting that the blocking effect may be due to steric hindrance of the ligand-binding pocket.