Biosynthesis of the fucose-containing xyloglucan nonasaccharide by pea microsomal membranes

Abstract

Pea microsomal membranes catalyze the transfer of [(14)C]fucose (Fuc) from GDP-[U-(14)C]fucose, with or without added unlabeled UDP-glucose (Glc), UDP-xylose (Xyl) or UDP-galactose (Gal), to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, [(14)C] fucose residues occur exclusively in a fragment corresponding in size to the xyloglucan nonasaccharide, Glc(4) Xyl(3) Gal Fuc. This fragment contains a single labeled fucose residue per oligomer, alpha-linked in a terminal nonreducing position. By comparison, in incubations where GDP-[(14)C] fucose is absent and replaced by UDP-[(3)H]xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products is an octasaccharide. In the presence of both GDP-[(14)C]fucose and UDP-[(3)H]xylose, a nonasaccharide containing the two labels is produced. Fucose and xylose residues are transferred within a few minutes to acceptor molecules of molecular weight up to 300,000. Such products do not elongate detectably over 60 minutes of incubation. The data support the conclusion that the nonasaccharide subunit of xyloglucan may be generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis is dependent on the inclusion of exogenous GDP-fucose.