Glycosyltransferases from pea membranes : glucose and fucose incorporation into cell wall polysaccharides

Abstract

Synthesis from UDP-($sp{14}$C) glucose of charged lipid-linked glucosyl compounds by pea membranes was short-lived, and of very limited magnitude compared to the synthesis of 1,4- and 1,3-linked B-glucans. Lipid-linked monophosphoryl glucose was the only charged lipid formed at initial stages, and had properties similar to that of dolichol-monophosphoryl glucose. It exhibited no turnover during pulse-chase experiments. Lipid-linked pyrophosphoryl-glucose or -oligosaccharides were not detected. Coumarin inhibited the synthesis of SDS-soluble products and glucans, but not of the lipid-P-glucose. Transfer of the label from endogeneous lipid-P-($sp{14}$C) glucose or from dolichol-P-($sp3$H) glucose into non-lipid products was minimal. It was concluded that the lipid-linked phosphoryl saccharide formed from UDP-glucose was not an obligate intermediate in the formation of B-glucans in pea membranes.Fucose-containing lipid-linked intermediates were not involved in the biosynthesis of xyloglucans. However, pea microsomal membranes catalysed the transfer of $lbrack sp{14}{ rm C} rbrack$-fucose from GDP-$lbrack sp{14}$C) fucose, with or without added unlabelled UDP-glucose, UDP-xylose or UDP-galactose, to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, $lbrack sp{14}$C) fucose residues occurred exclusively in a fragment identified as the xyloglucan nonasaccharide, Glc$sb4$ Xyl$sb3$ Gal Fuc. By comparison, in incubations with UDP-$lbrack sp3$H) xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products was an octasaccharide. In the presence of both GDP-$lbrack sp{14}$C) -fucose and UDP-$lbrack sp3$H) xylose, a nonasaccharide containing both labels was produced. Fucose and xylose residues were transferred rapidly to acceptor molecules of MW up to 300,000. Such products did not elongate detectably over 60 min of incubation. We concluded that the nonasaccharide subunit of xyloglucan was generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis was dependent on exogenous GDP-fucose.Microsomal membranes were separated by rate-zonal centrifugation on renografin gradients. Transfer to xyloglucan of labelled fucose and xylose from GDP- ($sp{14}$C) fucose and UDP- ($sp{14}$C) xylose occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with ($sp3$H) fucose suggested that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles