Analysis of murine Ia antigen glycosylation by lectin affinity chromatography. I-Ak alpha chain subspecies and beta chains are each glycosylated differently.
Murine Ia antigens consist of two glycosylated polypeptide chains, the alpha chain and the beta chain. We have used lectin affinity chromatography to confirm previous work in our laboratory that three distinct, differentially glycosylated I-Ak alpha chains (alpha 1, alpha 2, and alpha 3) exist and to compare the carbohydrate of the alpha chain with that of the beta chain. Glycopeptides derived from pronase digestion of [3H]mannose-labeled I-Ak alpha 1, alpha 2, alpha 3, and beta chains were sequentially passed over columns of immobilized concanavalin A, Lens culinaris lectin, phytohemagglutinin-E, and phytohemagglutinin-L in a prescribed manner to generate a lectin affinity profile, which, in turn, allowed assignment of a minimal oligosaccharide structure for each glycopeptide studied. The lectin affinity profile for each chain was unique. The alpha 1, alpha 2, and beta chains each possess complex-type N-linked oligosaccharides, although the branching pattern and specific sugar residues found on each differ. The alpha 3 chain, on the other hand, possesses predominantly high mannose or hybrid-type N-linked oligosaccharides. Lectin affinity analysis of glycopeptides derived from pronase digestion of high pressure liquid chromatography-isolated tryptic-chymotryptic fragments from alpha 2 and alpha 3 and tryptic fragments from beta revealed that specific minimal oligosaccharide structures were associated with particular fragments. In addition, although tryptic-chymotryptic peptide maps of alpha 2 and alpha 3 were similar, alpha 2 fragments bear predominantly complex-type N-linked oligosaccharides, whereas homologous alpha 3 fragments bear high mannose or hybrid-type N-linked oligosaccharides. Possible explanations of the oligosaccharide heterogeneity are discussed.
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006020:Glycopeptides / Q000032:analysis; D006027:Glycosides / Q000032:analysis; D000949:Histocompatibility Antigens Class II; D037102:Lectins; D046911:Macromolecular Substances; D051379:Mice; D009844:Oligosaccharides / Q000032:analysis
D006020:Glycopeptides; D006027:Glycosides; D000949:Histocompatibility Antigens Class II; D037102:Lectins; D046911:Macromolecular Substances; D009844:Oligosaccharides