Characterization of N-linked carbohydrate chains of the crayfish, Astacus leptodactylus hemocyanin

The primary structure of the carbohydrate chains of hemocyanin from the crayfish Astacus leptodactylus were investigated. The carbohydrate content is 0.2% (w/w) as referred to total hemocyanin content, resp. 1.8% as referred only to the one subunit which is glycosylated. Mannose and N-acetylglucosamine are present in a molar ratio of 6:2. The carbohydrate chains are N-glycosidically linked as revealed by dot blot analysis using various lectins and enzymatic deglycosylation. Furthermore, they are part of only one hemocyanin subunit of A. leptodactylus. After enzymatic deglycosylation with PNGase F, the oligosaccharide pool was separated by FPLC on Mono Q and subsequent HPLC on Lichrosorb-NH2, the subfractions were characterized by 1H NMR spectroscopy. A total of six oligosaccharides, ranging from Man4GlcNAc2 to Man9GlcNAc2 is present, Man6GlcNAc2 representing the most abundant one with 57% of all oligosaccharides.