IDENTIFICATION AND OLIGOSACCHARIDE STRUCTURE-ANALYSIS OF RHODOPSIN GLYCOFORMS CONTAINING GALACTOSE AND SIALIC-ACID

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were an alysed by sequential exoglycosidase digestion and gel filtration chrom atography, following reductive tritiation. In addition, selected trypt ic glycopeptides obtained from frog retinal rod outer segment membrane s were examined by electrospray mass spectrometry (ES-MS), fast atom b ombardment mass spectrometry (FAB-MS), amino acid sequence and composi tion analysis, and carbohydrate composition analysis. The amino acid s equence data demonstrated that the glycopeptides were derived from rho dopsin and confirmed the presence of two N-glycosylation sites, at res idues Asn2 and Asn15. The predominant glycan (approximately 60% of tot al) had the structure GlcNAcbeta1-2Manalpha1-3(Manalpha1-6) Manbeta1-4 GlcNAcbeta1-4GlcNAc-(Asn), with the remaining structures containing 1- 3 additional hexose residues, as reported previously for bovine rhodop sin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with th e sialylated oligosaccharides being present exclusively at the Asn2 si te. FAB-MS analysis of oligosaccharides released from the Asn2 site ga ve, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in t he context of rod outer segment membrane renewal.