Two Kinds of Protein Glycosylation in a Cell-Free Preparation from Developing Cotyledons of Phaseolus vulgaris

Abstract

Membrane preparations from developing cotyledons of red kidney bean (Phaseolas vulgaris L) transferred radioactive mannose from GDP-mannose (U-I'4Clmannose) to endogenous acceptor proteins. The transfer was inhibited by the antibiotic tunicamycin, suggesting the involvement of lipidoLigosaccharide intermediates typical of the pathway for glycosylation of asparagine residues. This was supported by the similarity of the linkage types of radioactive mannose in lipid-oligosaccharide and glycoprotein products; both contained labeled 2-linked mannose, 3,6-inked and terminal mannose typical of glycoprotein "core" oligosaccharides. As expected for "core" glycosylation, the transfer of labeled N-acetylgucosamine (GlcNAc) from UDP-GlcNAc (613HIGLcNAc) to 4-linkage in endogenous glycoproteins could also be demonstrated. However, most of the radioactive GlcNAc was incorporated into terminal linkage, in a reaction insensitive to tunicamycin. The proteins receiving "core" oligosaccharide in vitro were heterogeneous in size, in contrast to those receiving most of the GkcNAc (which chiefly comprised the seed reserve-proteins phaseolin and phytohemagglutinin). It is suggested that following "core" glycosylation, single GkcNAc residues are attached terminally to the oligosaccharides of these seed proteins, without the involvement of lipid-linked intermediates. Phaseolin from mature seeds does not possess a significant amount of terminal GIcNAc and so it is possible that these residues are subsequentiy removed in a processing event.cotyledons, is the identity of the endogenous glycoprotein products. Previous studies from our laboratory showed that crude cellfree extracts from this tissue synthesized lipid-linked GlcNAc4, dolichol monophosphoryl mannose, and lipid-oligosaccharide when supplied with UDP-GlcNAc and GDP-mannose, and also transferred GlcNAc and mannose to endogenous acceptor proteins (5, 9, 10). The lipid-linked sugars showed turnover, consistent with their roles as intermediates in protein glycosylation (9, 10). Recently we obtained evidence that the endogenous acceptor proteins for the in vitro transfer of GlcNAc are chiefly the principal reserve proteins of this seed, i.e. phaseolin5 and phytohemagglutinin (4). Both of these proteins contain typical asparagine-linked, "high mannose" type, oligosaccharide groups containing GlcNAc and mannose as sole constituent sugars (10, 17). We therefore expected concomitant incorporation of GlcNAc and mannose, via transfer of "core" oligosaccharides from lipid carriers in the usual pathway (7,22). However, in contrast to the results with GlcNAc, only a trace of incorporation of mannose into phaseolin could be shown (4). We suggested, therefore, the bulk of the transfer of GlcNAc to phaseolin and PHA did not represent "core" glycosylation. In this paper we present confirmatory evidence ofthis, demonstrating that two apparently independent kinds of glycosylation occur in this in vitro system, i.e. the attachment of GlcNAc residues to the identifiable (glyco)proteins phaseol...

UDP-GlcNAc:Glycoprotein GlcNAc-Transferase is Located in the Golgi Apparatus of Developing Bean Cotyledons

“…Most of the radioactivity was in the polypeptides of phaseolin and PHA, whether the organelle fraction was labeled in vitro with UDP-GlcNAc, or the cotyledons were labeled in vivo with GlcNAc. That the in vitro incorporation was mostly in PHA and phaseolin was confirmed by immunoaffinity chromatography using antibodies to PHA and phaseolin (data not shown) as was demonstrated by Davies and Delmer (3). It is obvious from Figure 1 that there is differential labeling of the individual polypeptides.…”

Transient N-acetylglucosamine in the biosynthesis of phytohemagglutinin: attachment in the Golgi apparatus and removal in protein bodies.

“…9. A glycosyltransferase that transfers G1cNAc residues from UDP G1cNAc to mannosyl residues of PHA and phaseolin has been shown to be associated with the membranous organelles of P. vulgaris cotyledons (24). When membranous organelle fractions were incubated with labeled UDP-G1cNAc it was found that most of the GIcNAc that became incorporated into phaseolin and PHA was terminal G1cNAc.…”

Contribution of processing events to the molecular heterogeneity of four banding types of phaseolin, the major storage protein of Phaseolus vulgaris L.

“…Phaseolin is a glycoprotein with high-mannose oligosaccaride sidechains which are susceptible to removal by endo-fl-N-acetyl glucosaminidase H (6,18). Some phaseolin polypeptides have only one high-mannose sidechain, while others have two (8).…”