Abstract
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.
Highlights
Vious work in our laboratorythat threedistinct, differentially glycosylated I-Aka chains (a1,a2,and a3)exist and to compare the carbohydrate of the a chain with that of the p chain
Gly~opeptides-[~H]mannose-labeled I-Ak a1, 02, a3, and /3 chains were isolated as previously described (31),and lectin affinity profiies wergeenerated for glycopeptides derived from each
In order to localize each of the different oligosaccharide structures from intact chains to a particular tryptic-chymotryptic fragment, we examined the lectin affinity profiles of glycopeptides derived from pronase digests of each of the az and a3 HPLC-isolated tryptic-chymotryptic fragments
Summary
Lectin affinity analysis is a recently developed technique which shouldprove to be invaluable in the analysis of N linked oligosaccharides of glycoproteins present in limited quantity This technique, described by Cummings and Kornfeld (7), is applicable to small quantities (2000-5000 cpm) of [3H]mannose-labeled glycopeptides. The binding pattern of a given glycopeptide to a series of lectins suggests a minimal structure for the oligosaccharide which it bears This technique permits a rapid and efficient way to screenisolated glycoproteins for nantly high mannose or hybrid-type N-linked oligosac- glycosylation differences and, in addition, to understand the charides. We have recently described the isolation of three distinct I-Aka chain subpopulations (ala,z,and a3)which possess the same polypeptide backbone but are differentially glycosylated (31).The a,and a2chains appear to bear complex-type N-linked oligosaccharides since (i) they radiolabel with ["Hlmannose and [3H] fucose, and(ii) their electrophoretic mobility is altered by treatment with neuraminidase, suggestingthat theybear sialic acid residues. We have employed the technique of lectin affinity analysis to extend our examination of the nature of the glycosylation differences between aI, a2,and 1x3 and to investigate the glycosylation pattern of the /3 chain
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