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 capability to bind different ligands, e.g. glycoproteins from zona pellucida (ZP), soybean trypsin inhibitor and heparin, and is involved in capacitation and binding of spermatozoa to the homologous zona pellucida. Here, we report the characterization of N- and O-glycosylated isoforms of AWN-1. Non-glycosylated AWN-1 is present in seminal plasma and on epididymal and ejaculated spermatozoa whereas its N- and O-glycosylated isoforms are only secretory products of the seminal vesicles. Lectin mapping indicated the presence of the glycosylated AWN-1 isoform mixture of both fucosylated and non-fucosylated N-glycans, and of two different classes of O-linked carbohydrate chains. These N- and O-linked oligosaccharide chains are neither sialylated nor contain terminal Gal beta (1-4)-GlcNAc sequences. Noteworthy, N- and O-glycosylation (either class) are mutually exclusive on the same protein molecule, indicating that each glycosylated AWN-1 molecule contains a single oligosaccharide chain. Peptide mapping was used to locate the N- and the O-glycosylation sites. Glycosylation of AWN-1 with either of the carbohydrate chain types greatly impaired the ability of the spermadhesin to bind biotinylated zona pellucida glycoproteins and soybean trypsin inhibitor, suggesting that the blocking effect may be due to steric hindrance of the ligand-binding pocket.
Calvete
J J
JJ
Institut für Reproduktionsmedizin, Tierärztiche Hochschule, Hannover, Germany.
Solís
D
D
Sanz
L
L
Díaz-Mauriño
T
T
Töpfer-Petersen
E
E
D000176:Acrosin / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D007536:Isomerism; D037102:Lectins; D008297:Male; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000302:isolation & purification; D011485:Protein Binding; D029589:Seminal Plasma Proteins; D013094:Spermatozoa / Q000737:chemistry; D013552:Swine; D014357:Trypsin
D002352:Carrier Proteins; D037102:Lectins; D010455:Peptides; D029589:Seminal Plasma Proteins; C425042:spermadhesin; D006026:Glycoside Hydrolases; C020668:glycanase; D000176:Acrosin; D014357:Trypsin