Covalently bonded and adventitious glycans in germin.

Germin was previously shown to contain covalently bonded and adventitious glycans. The object of the present investigation was to characterize the two types of glycan. The presence of N- but not O-glycans in germin is indicated by the biosynthesis of altered forms, including an unglycosylated form of germin when wheat embryos are germinated in the presence of tunicamycin. After treating the doublet of germin pentamers (G and G') from normally germinated embryos with beta-N-acetylglucosaminidase, G is converted to a form that co-migrates with G' during electrophoresis in sodium dodecyl sulfate-polyacrylamide, but G' is unaffected. This suggests that the N-glycans in G contain antennary N-acetylglucosamine but that those in G' do not. This conclusion has been confirmed and elaborated by doubly labeling G and G' in vivo with [3H]glucosamine and [35S]methionine, and by characterizing sugar-labeled glycopeptides from G and G' by gel filtration, before and after their degradation by exoglycosidases. In the context of proven structures for the complex N-glycans in other plant glycoproteins, the findings, when combined with monosaccharide analyses of G and G', permit plausible speculation about the structure of the single N-glycan that is likely present in each G monomer (GlcNAc2(Man)2(Man-Xyl)(GlcNAc)(GlcNAc-Fuc] and G' monomer ((Man)2(Man-Xyl)(GlcNAc)(GlcNAc-Fuc)). The adventitious glycans, which can be removed by phenolic extraction of germin, have a composition similar to that expected for the characteristic hemicelluloses and pectins in monocot cell walls. The possible significance of this finding is discussed in relation to our continuing efforts to define the biochemical involvements of germin. In allied studies, affinity of its N-linked glycans for concanavalin A has been used to concentrate small amounts of germin from large volumes of wheat extract and to fractionate germin from tunicamycin-treated and normally germinated wheat embryos.