N-glycosylation site mapping of human serotransferrin by serial lectin affinity chromatography, fast atom bombardment-mass spectrometry, and 1H nuclear magnetic resonance spectroscopy.
This report describes the N-glycosylation site mapping of human serotransferrin (h-STF). Reduced and S-carboxymethylated h-STF was digested with trypsin or chymotrypsin. Glycopeptides in the proteolytic digests were isolated by serial concanavalin A (Con A), Sambucus nigra agglutinin (SNA), and Phaseolus vulgaris leukoagglutinin (LPHA) affinity chromatography and subjected to preliminary analysis by 1H NMR spectroscopy. The glycopeptide fractions were then individually digested with N-glycanase. One part of the digest of each fraction was analyzed by fast atom bombardment-mass spectrometry (FAB-MS) to identify the peptide sequences of the glycosylation sites. The other part was used to isolate the oligosaccharide by the corresponding lectin affinity chromatography and to characterize the structures of the isolated oligosaccharides by 1H NMR spectroscopy and FAB-MS. The oligosaccharides in the Con A-bound fraction were shown to have bi-alpha(2-->6)-sialyl, diantennary structures. The SNA-bound fraction was shown to contain trisialyl, triantennary structures. Di- and triantennary oligosaccharides were found to occur on each of the two N-glycosylation sites of h-STF (Asn413 and Asn611) in the ratio of approximately 85:15. The SNA-bound glycopeptides were further fractionated by LPHA affinity chromatography. Two different oligosaccharides were characterized, namely, a trisialyl 2,4-triantennary and a trisialyl 2,6-triantennary glycan. The ratio of 2,4-triantennary vs 2,6-triantennary oligosaccharides attached to glycosylation site Asn413 was found to be approximately 5:1, whereas the two isomeric triantennary oligosaccharides were found to be attached to glycosylation site Asn611 in the ratio approximately 1:1.
Fu
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Complex Carbohydrate Research Center, University of Georgia, Athens 30602-4712.
van Halbeek
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D037102:Lectins; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014168:Transferrin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D006020:Glycopeptides; D037102:Lectins; D009844:Oligosaccharides; D014168:Transferrin; D014357:Trypsin