The primary structures of two biantennary N-glycans of the glycoprotein Rapana venosa (marine snail) haemocyaninwere 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.

Characterization of the carbohydrate moieties of the functional unit RvH(1)-a of Rapana venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion

BELTRAMINI, MARIANO;SALVATO, BENEDETTO;
2003

Abstract

The primary structures of two biantennary N-glycans of the glycoprotein Rapana venosa (marine snail) haemocyaninwere 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.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/2473773
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