Studies on N-glycosylation by elongating tissues and membranes from pea stems.

Glucosamine and mannose were incorporated into oligosaccharides linked to either polar membrane-lipids or to asparagine residues of endogenous proteins in apical growing tissues of the etiolated pea stem. The glycolipids were subject to turnover in pulse-chase tests and protein-linked oligosaccharides accumulated with time, as expected for a precursor-product relationship. The newly formed glycoproteins were hydrolyzed by endo-beta-N-acetylglucosaminidase H to oligosaccharides in the same size range as those released by dilute acid from the lipid-linked oligosaccharides formed during the pulse. The glycoproteins were also partly degraded to free N-acetylglucosamine by beta-N-acetylhexosaminidase. Affinity of the carbohydrate moiety of the protein for concanavalin A increased between the beginning and the end of the chase, indicating processing following core glycosylation.The addition of UDP-N-acetyl-[(14)C]glucosamine plus external peptide acceptors (derived from carboxymethylated alpha-lactalbumin) to membrane preparations from the pea stem resulted in peptide glycosylation at the expense of lipid-linked oligosaccharide. Glycosylation of endogenous protein acceptors did not take place via lipid intermediates but directly from the sugar nucleotide substrate. Tunicamycin inhibited glycosyltransfer to both glycolipids and added peptides, but not to endogenous protein. It is concluded that limiting factors for N-glycosylation by pea membranes in vitro could include the unavailability of endogenous acceptors or the inability to fully elongate and internalize lipid precursors, but is not due to any limitation in capacity for N-glycosylation.