A 140-kDa glycopeptide from the sperm ligand of the vitelline coat of the freshwater bivalve Unio elongatulus, only contains O-linked oligosaccharide chains and mediates sperm-egg interaction.
In previous studies we found that sperm binding activity in the vitelline coat of the freshwater bivalve Unio elongatulus is located on the O-linked oligosaccharide chains of gp273, one of the two major components of the extracellular coat, and that fucose plays a key role in this interaction. In this paper we report the partial characterization of a large glycopeptide (about 140 kDa) obtained by cyanogen bromide fragmentation of gp273, that maintains sperm binding activity. Lectin blotting revealed that the glycopeptide reacted with lectins from Arachis hypogaea (PNA) and Lotus tetragonolobus (LTA) but not Canavalia ensiformis (ConA). No other PNA-positive fragments could be detected in the electrophoretic pattern of fragmented gp273 but several ConA-positive fragments of lower molecular weight were present indicating that all the O-linked chains are clustered together in this fragment. Two-dimensional gel electrophoresis of the fragment revealed it to be acidic in nature in contrast with the neutral character of the whole gp273 molecule. Competition binding assay showed that this fragment is a strong inhibitor of the interaction, whereas no effect was detected using the ConA-positive peptides. This confirms that the sperm receptor activity of gp273 is related to its O-linked chains. The immunodominance of this fragment is also discussed.
Capone
A
A
Department of Evolutionary Biology, University of Siena, Siena, Italy.
Rosati
F
F
Focarelli
R
R
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001667:Binding, Competitive; D003208:Concanavalin A / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D037102:Lectins / Q000494:pharmacology; D008297:Male; D008974:Mollusca / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D013084:Sperm-Ovum Interactions / Q000187:drug effects / Q000276:immunology*; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism*; D014817:Vitelline Membrane / Q000737:chemistry / Q000378:metabolism*
D000906:Antibodies; D006020:Glycopeptides; D037102:Lectins; D009844:Oligosaccharides; D003208:Concanavalin A