Incorporation of fucose into the carbohydrate moiety of phytohemagglutinin in developing Phaseolus vulgaris cotyledons

Abstract

Incubation of developing cotyledons of P. vulgaris with [(3)H]fucose resulted in the incorporation of radioactivity into the cell wall, membranous organelles and soluble macromolecules. Fractionation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluorography, showed that phytohemagglutinin (PHA) was the major fucosylated protein synthesized in the cotyledons. Incorporation of fucose into PHA occurred in the membranous organelle fraction, and the radioactive fucose remained associated with the PHA during a 20-h chase of the radioactivity. Tunicamycin inhibited the incorporation of glucosamine and fucose into PHA to the same extent (65%), indicating the involvement of a lipid intermediate in the incorporation of fucose, or the attachment of fucose to the high-mannose oligosaccharide moiety of newly synthesized PHA. Digestion with proteinase K of [(3)H]fucose- or [(3)H]glucosamine-labeled PHA resulted in the formation of glycopeptides of similar size. These glycopeptides were partially resistant to digestion with endo-β-N-acetylglucosaminidase H, even after the removal of fucose by mild acid hydrolysis. We postulate, on the basis of these experiments, that the transport of PHA from the endoplasmic reticulum to the protein bodies is accompanied by the modification of its oligosaccharide side-chain. This modification involves inter alia the attachment of fucose, and renders the oligosaccharide side-chain resistant to digestion with endo-β-N-acetylglucosaminidase H. Analogy with animal glycoproteins indicates that this modification probably occurs in the Golgi apparatus.