Carbohydrate structures of the glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen

The glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen was treated with pepsin and glycopeptidase A to release asparagine-linked oligosaccharides. The reducing ends of the oligosaccharides were aminated with the fluorescent reagent 2-aminopyridine. The oligosaccharide derivatives were purified by gel permeation chromatography and reversed-phase HPLC. Their structures were determined by sequential exoglycosidase digestion and 500 MHz 1H-NMR spectroscopy. Four oligosaccharide structures, A, B, C, and D, were identified as the xylose-containing complex-type. They were present at a molar ratio of 8:1:6:1. By amino acid sequence analyses of the tryptic peptides, Asn-170 and Asn-333 of Cry j I were found to carry asparagine-linked oligosaccharides.