The primary structures of two biantennary N-glycans of the glycoprotein Rapana venosa (marine snail) haemocyanin were determined. Two different structural subunits have been found in R. venosa haemocyanin: RvH1 and RvH2. The carbohydrate content of the N-terminal functional unit RvH1-a of RvH1 was studied and compared with the N-terminal functional unit RvH2-a of RvH2. Oligosaccharide fragments were released from the glycoprotein by Smith degradation of a haemocyanin pronase digest and separated on a Superdex 300 column. The glycopeptide fragments, giving a positive reaction for the orcinol/H2SO4 method, were separated by HPLC. In order to determine the linked sugar chains to the hinge glycopeptides isolated from the functional unit RvH1-a, several techniques were applied, including capillary electrophoresis, matrix-assisted laser desorption ionization-MS and electrospray ionization-MS in combination with glycosidase digestion. On the basis of these results and amino acid sequence analysis, we concluded that the functional unit RvH1-a contains 7% oligosaccharides N-glycosidically attached to Asn262 and Asn401, and the following structures were suggested:
Abbreviations used: ESI-MS, electrospray ionization MS; Fuc, fucose; GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; Glp1, glycoprotein 1; Glp2, glycoprotein 2; Hc, haemocyanin; MALDI-MS, matrix-assisted laser-desorption ionization MS; Man, d-mannose; 3MeGal, 3-O-methyl-d-galactose; PNGase F, peptide N-glycosidase F; RvH1 and RvH2, structural subunits of Rapana venosa Hc.