Glycoprotein synthesis in plants: I. Role of lipid intermediates

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 vicilin. Radioactivity from GDP-[(14)C]mannose can be incorporated by crude extracts into a single chloroform-methanol-soluble product as well as into insoluble product(s). Mannose is the sole (14)C-labeled constituent of the lipid. The kinetics of incorporation of (14)C, as determined by pulse and pulse-chase experiments using GDP-[(14)C]mannose, as well as direct incorporation from added [(14)C]mannolipid, shows that the mannolipid is an intermediate in the synthesis of the insoluble product(s). The characteristics of the mannolipid are consistent with it being a mannosyl phosphoryl polyprenol. The mannose is apparently attached to the lipid via a monophosphate linkage. Of the radioactivity in the insoluble product(s), about 20% is pronase-digestible during a "pulse experiment." After a chase with unlabeled GDP-mannose, about 40% is pronase-digestible; the other 60% is as yet uncharacterized. A radioactive product soluble in a mixture of chloroform-methanol-H(2)O can be extracted from the insoluble residue obtained during a pulse, but is no longer present after a chase. This product may be a lipid oligosaccharide, the final intermediate in glycoprotein synthesis. Data are presented on incorporation from UDP-N-[(14)C]acetylglucosamine into both chloroform-methanol-soluble and -insoluble product(s). The results are consistent with an involvement of lipid intermediates in the glycosylation of protein in this system, and support the concept that the mechanisms of glycoprotein synthesis in higher plants are similar to those which have been reported for mammalian systems.