Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid |
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Authors: | Duffin Kevin L; Lange Gary W; Welply Joseph K; Florman Richard; O'Brien Paul J; Dell Anne; Reason Andrew J; Morris Howard R; Fliesler Steven J |
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Institution: | 1Monsanto Corporate Research St Louis, MO 63198
2The National Eye Institute, National Institutes of Health Bethesda, MD 20892
3Department of Biochemistry, Imperial College of Science, Technology and Medicine London SW7 2AZ
4The Eye Institute and the E.A.Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine St Louis, MO 63104, USA |
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Abstract: | The N-linked oligosaccharides of frog (Rana pipiens) rhodopsinwere analysed by sequential exoglycosidase digestion and gelfiltration chromatography, following reductive tritiation. Inaddition, selected tryptic glycopeptides obtained from frogretinal rod outer segment membranes were examined by electrospraymass spectrometry (ES-MS), fast atom bombardment mass spectrometry(FAB-MS), amino acid sequence and composition analysis, andcarbohydrate composition analysis. The amino acid sequence datademonstrated that the glycopeptides were derived from rhodopsinand confirmed the presence of twoN-glycosylation sites, at residuesAsn2 and Asn15. The predominant glycan ( 60% of total) had thestructure GlcNAcß12Man 13(Man 16)Manß14GlcNAcß14GlcNAc-(Asn),with the remaining structures containing 13 additionalhexose residues, as reported previously for bovine rhodopsin.Unlike bovine rhodopsin, however, a sizable fraction of thetotal giycans of frog rhodopsin also contained sialic acid (NeuAc),with the sialylated oligosaccharides being present exclusivelyat the Asn2 site. FAB-MS analysis of oligosaccharides releasedfrom the Asn2 site gave, among other signals, an abundant quasimolecularion corresponding to a glycan of composition NeuAc1Hex6HexNAc3(where Hex is hexose and HexNAc is N-acetylhexosamine), consistentwith a hybrid structure. The potential biological implicationsof these results are discussed in the context of rod outer segmentmembrane renewal. glycoforms oligosaccharide structure rhodopsin |
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