Structural characterization of the N-glycans of gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus

Gp273, a glycoprotein of the egg extracellular coats of the mollusc bivalve Unio elongatulus, is the ligand molecule for sperm-egg interaction during fertilization. In this study we have analyzed the N-glycans from gp273. N-glycans were enzymatically released by PNGase F digestion and their structures were elucidated by normal phase HPLC profiling of the 2-aminobenzamide-labeled N-glycans, MALDI-TOF mass spectrometry and ¹H NMR spectroscopy. The combined data revealed that the N-glycans of gp273 consist of Glc₁Man₉GlcNAc₂ and Man₉GlcNAc₂. In Unio, the presence of noncomplex-type N-glycans parallels the inefficacy of these glycans in the ligand function. Their role in the protection of the polypeptide chain from proteolytic attack is suggested by the electrophoretic patterns obtained after enzymatic digestion of the native and the N-deglycosylated protein. These results are discussed in the light of the evolution of the recognition and adhesion properties of oligosaccharide chains in the fertilization process.     

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. Mol. Reprod. Dev. 54:203–207, 1999. © 1999 Wiley‐Liss, Inc.