Isolation of glycopeptides containing O-linked oligosaccharides by lectin affinity chromatography on jacalin-agarose.
Jacalin is a lectin which has high specificity and affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides. Here, it is shown that this lectin can be used for isolation of glycopeptides bearing O-linked oligosaccharides. Peptides produced by digestion of reduced and carboxamidomethylated human plasminogen or of bovine protein Z were chromatographed on a column of jacalin-agarose. Reverse-phase high-performance liquid chromatography revealed that two peptides from plasminogen and one from protein Z were eluted from the jacalin-agarose column by alpha-methylgalactopyranoside. Amino acid sequence and compositional analysis showed that both of the peptides from plasminogen consisted of residues 330-357 and that the single peptide from protein Z represented residues 385-396. These sequences contain the single known site of attachment of O-linked oligosaccharides to these proteins. The present analysis suggested that there may be a fraction of plasminogen with two sites of O-linked glycosylation. The two tryptic peptides isolated from plasminogen represented the same segment of the protein but sequence analysis showed that one peptide was modified only at Thr346, the known site of glycosylation, and the other peptide contained a modification of Ser339 as well. Results of the present study indicate that lectin affinity chromatography using jacalin-agarose can be a useful technique for isolating glycopeptides containing O-linked oligosaccharides and thereby localizing sites of attachment of these oligosaccharides.
Hortin
G L
GL
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000302:isolation & purification*; D006031:Glycosylation; D006801:Humans; D037102:Lectins; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010958:Plasminogen / Q000737:chemistry
D000596:Amino Acids; D001798:Blood Proteins; D006020:Glycopeptides; D037102:Lectins; D037121:Plant Lectins; C043696:jacalin; C026080:plasma protein Z; D010958:Plasminogen