Characterization of the carbohydrate moieties of the functional unit RvH1-a of Rapana venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion.
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:[structure: see text]
Institute of Organic Chemistry, Bulgarian Academy of Sciences, G. Bonchev 9, Sofia 1113, Bulgaria. pda54@yahoo.com
Pavlina
P
Dolashka-Angelova
Alexander
A
Beck
Alexandar
A
Dolashki
Mariano
M
Beltramini
Stefan
S
Stevanovic
Benedetto
B
Salvato
Wolfgang
W
Voelter
D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006433:Hemocyanins / Q000737:chemistry*; D008969:Molecular Sequence Data; D008974:Mollusca; D009844:Oligosaccharides / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D006020:Glycopeptides; D009844:Oligosaccharides; D006433:Hemocyanins