Processing of N-linked oligosaccharides in soybean cultured cells

Abstract

Evidence, based on both in vivo and in vitro studies with suspension-cultured soybean cells, is presented to demonstrate the processing of the oligosaccharide chain of plant N-linked glycoproteins. Following a 1-h incubation of soybean cells with [2- 3H]mannose, the predominant glycopeptide obtained by pronase digestion of the membrane fraction was a Man 7- or Man 8GlcNAc 2-Asn (GlcNAc, N-acetylglucosamine). However, the major oligosaccharide isolated from the lipid-linked oligosaccharides of these cells was a Glc 2- or Glc 3Man 9GlcNAc 2. Soybean cells were incubated with [2- 3H]mannose and the incorporation of mannose into Pronase-released glycopeptides was followed during a 2-h chase. During the first 10 min of labeling, the radioactivity was mostly in a large-sized glycopeptide that appeared to be a Glc 1Man 9GlcNAc 2-peptide. During the next 60 to 90 min of chase, this radioactivity was shifted to smaller and smaller-sized glycopeptides indicating that removal of sugars (i.e., processing) had occurred. Both glucosidase and mannosidase activity was detected in membrane preparations of soybean cells. Nine different glycopeptides were isolated from Pronase digests of soybean cell membrane fractions. These glycopeptides were purified by repeated gel filtration on columns of Bio-Gel P-4. Partial characterization of these glycopeptides by endoglucosaminidase H and α-mannosidase digestion, and by analysis of the products, suggested the following glycopeptides: Glc 1Man 9GlcNAc 2-Asn, Man 8GlcNAc 2-Asn, Man 7GlcNAc 2-Asn, Man 6GlcNAc 2-Asn, and Man 5GlcNAc 2-Asn.