Fucosylation of Membrane Proteins in Soybean Cultured Cells

Abstract

Cultures of soybean cells incorporate [5,6-(3)H]-l-fucose into various cellular components including lipids and proteins. The membrane glyco-proteins were digested with pronase to produce glycopeptides, and the glycopeptides were isolated on columns of Biogel P-4. The major fucoselabeled glycopeptide sized as a Hexose(15-17)-N-acetylglucosamine(2) (GlcNAc(2)) on columns of Biogel P-4. Fucose incorporation was also examined in the presence of the processing inhibitor swainsonine, and the glycosylation inhibitor tunicamycin. In the presence of swainsonine, the incorporation of fucose was not reduced but the glycopeptides were smaller in size and migrated like Hexose(12-13)-GlcNAc(2) structures. On the other hand, tunicamycin inhibited the incorporation of fucose into the glycopeptides by 70 to 80%, indicating that the l-fucose was present in N-linked oligosaccharides.The membrane glycoproteins were doubly-labeled by incubating soybean cells in [(3)H]fucose and [(14)C]mannose. By repeated separation on Biogel P-4, six glycopeptides were purified that ranged in size from Hexose(8)GlcNAc(2) to Hexose(15-17)-GlcNAc(2). The three larger glycopeptides (I, II, III) were highly labeled with [(3)H]fucose and also contained [(14)C] mannose. Evidence that both isotopes were in the same glycopeptide was obtained by the finding that the mannose-labeled glycopeptides were shifted to smaller-sized structures when the [(3)H]fucose was removed by mild acid hydrolysis. Glycopeptide IV also contained [(3)H]fucose and [(14)C] mannose but only part of the [(3)H]fucose was released by mild hydrolysis. Glycopeptides V and VI contained only small amounts of tritium, but were labeled with [(14)C]mannose. None of the six glycopeptides was susceptible to the action of endo-beta-N-acetylglucosaminidase H, and none of these glycopeptides was bound to columns of concanavalin A-sepharose. The smaller glycopeptides (IV, V, VI) were partially susceptible to alpha-mannosidase digestion, but this enzyme did not release any radioactive mannose from the larger-sized glycopeptides. These data indicate that the fucosylated glycopeptides are N-linked structures containing fucose, mannose, GlcNAc, and probably other sugars, and that the mannose units are blocked by other sugars. Thus, these results indicate that plant membrane glycoproteins contain a significant amount of modified oligosaccharide side chains.