Characterization of the Oligosaccharides from Lipid-Linked Oligosaccharides of Mung Bean Seedlings

Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseolus aureus) seedlings in the presence of GDP-[(14)C] mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P-4 columns. Five different oligosaccharides were purified in this way. Based on their relative elution constants (K(d)) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man(7)GlcNAc(2), Man(5)GlcNAc(2), Man(3)GlcNAc(2), Man(2)GlcNAc(2), and ManGlcNAc(2). These oligosaccharides were treated with endoglucosaminidase H and alpha- and beta-mannosidase, and the products were examined on Biogel P-4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB(3)H(4) reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as follows: E, Manbeta-GlcNAc-GlcNAc; D, Manalpha1-->3Manbeta-GlcNAc-GlcNAc; C, Manalpha1-->2Manalpha1-->3Manbeta-GlcNAc-GlcNAc; B, Manalpha1-->2Manalpha1-->2Manalpha1--> 3(Manalpha1-->6)Manbeta-GlcNAc-GlcNAc; and A, Manalpha1-->2Manalpha1--> 2Manalpha1-->3(Manalpha1--> [Manalpha1-->6]Manalpha1-->6) Manbeta-GlcNAc-GlcNAc. The synthesis of the Man(7)GlcNAc(2) was greatly diminished when tunicamycin (10 mug/ml) was added to the incubation mixtures.