Glycoprotein Synthesis in Plants

Abstract

The enzymic processes involved in glycoprotein synthesis have been studied using crude extracts obtained from developing cotyledons of Phaseolus vulgaris harvested at the time of active deposition of vidin. Radioactivity from GDP-['4CJmannose can be incorporated by crude extracts into a single chloroform-methanol-soluble product as well as into insoluble product(s). Mannose is the sole '4C-labeled constituent of the lipid. The kinetics of incorporation of 14C, as determined by pulse and pulse-chase experiments using GDP-['4Cjmannose, as weU as direct incorporation from added ['4CImannolipid, shows In those systems where glycoprotein synthesis has been studied in more detail, the mechanism of lipid transfer of sugar to protein seems to depend upon the type of carbohydrate-peptide linkage that the finished glycoprotein will have. In the case of the formation of O-glycosidic linkages between carbohydrate and serine or threonine, such as those studied in yeast (3), the first sugar is added via a polyprenol monophosphate (in this case dolichol monophosphate) and all subsequent sugars seem to be added directly to the final acceptor from nucleoside diphosphate sugars. However, in glycoproteins which have the carbohydrate attached by an N-glycosidic linkage between N-acetyl-D-glucosamine and asparagine, the mechanism of attachment is more complex. The N-acetyl-D-glucosamine residue which will be directly attached to the protein is transferred from UDP-N-acetylo-glucosamine to form a polyprenol pyrophosphate derivative.The rest of the sugars that go to make up the carbohydrate moiety are transferred from nucleoside diphosphate sugars to polyprenol monophosphate lipids and from these lipids to the polyprenol pyrophosphate-N-acetyl-u-glucosamine. In this way, a lipid-bound oligosaccharide is made that is transferred, in its entirety, to the protein acceptor. In the animal systems where this mechanism has been demonstrated, the polyprenol monophosphate is dolichol monophosphate and the polyprenol pyrophosphate is dolichol pyrophosphate (12).Vicilin, the major storage protein of Phaseolus vulgaris seeds, is a glycoprotein containing mannose and glucosamine (29). Because this glycoprotein is made in large quantity at a specific time, developing seeds of this plant provide an excellent system in which to study glycoprotein synthesis. Here, we show that enzyme preparations from the developing seeds of P. vulgaris (kidney bean)

Heterogeneous Distribution of Glycosyltransferases in the Endoplasmic Reticulum of Castor Bean Endosperm

“…4) were predominantly located in the upper ER subtraction. The monosaccharide lipid intermediates have previously been identified as dolicholpyrophosphate N-acetylglucosamine and the dolicholmonophosphate mannose in castor bean endosperm (25) as they have been in other plant tissues (2,13,14). Both sugars are probably incorporated into the same dolichollinked oligosaccharide (14,15 (Figs.…”

Glycoprotein Synthesis in Plants

“…Such acid lability is characteristic of glycosyl-phosphoryl-polyprenols (11,18). Similarly, the previously reported chromatographic properties of this mannolipid (5) in three separate solvent systems are indicative of such compounds.…”

“…The Figure 4. monophosphate linkage is our previous result (5) showing that synthesis of the mannolipid is reversed by addition of excess GDP but not GMP to the reaction. The stability of the mannolipid to the mild alkaline conditions of deacylation is also consistent with the properties of a mannosyl-phosphoryl-polyprenol (11,18), and was used to advantage in purification as a means to eliminate contamination by other phospholipids susceptible to degradation by deacylation.…”

“…However, recent evidence from Elbein's laboratory (8,9) and our own (5) has provided strong support for the concept that such lipid intermediates do function in the glycosylation of plant proteins.…”

Profile of Deborah P. Delmer

“…''But the kinds of things we did do while at Michigan State were still valuable,'' she adds. From the work of postdoctoral researcher Mary Ericson, Delmer's laboratory was the first to show that plants, like animals, used a lipid intermediate in the process of protein glycosylation (7,8). Graduate student Maureen Meinert carried out initial studies on the structure and development of cotton fiber cell walls (9), and a group led by postdoctoral researcher Nick Carpita performed some of the first measurements of the porosity of plant cell walls (10).…”