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178. Saxena, I.M. and R. M. Brown, Jr. 1999 Are the reversibly glycosylated polypeptides implicated in plant cell wall biosynthesis non-processive b-glycosyltransferases. Trends in Plant Science 4: 6-7.
Introduction: The biosynthesis of plant cell wall polysaccharides takes place on the plasma membrane, where enzymes for cellulose biosynthesis are localized, and in the Golgi apparatus, where the non-cellulosic polysaccharides are synthesized. Studies aimed at identifying the proteins responsible for biosynthesis of the cell wall polysaccharides have revealed that specific polypeptides are reversibly glycosylated by UDP-glucose1. The cloning of the gene for a reversibly glycosylated polypeptide (RGP) from pea2, led to the identification of similar genes in other plants2,3. The RGPs are soluble proteins found in association with the Golgi, and sequence analysis revealed that the proteins lack any signal sequences that might lead to their insertion into membranes. The RGPs bind UDP-glucose, UDP-xylose, and UDP-galactose in a reversible manner: the bound sugar nucleotides can be exchanged with UDP or any of these sugar nucleotides, but not with glucose alone. The binding of UDP-glucose is inhibited by UDP-xylose and UDPgalactose, but not by UDP-mannose, GDPglucose, ADP-glucose, or TDP-glucose – suggesting both nucleotide and sugar specificity. The binding takes place in the presence of Mg2+ or Mn2+, but experiments on a GST fusion of RGP1p from Arabidopsis3 have shown that this can also occur in the absence of metal ions. Because the binding is resistant to boiling in SDS, it has been suggested that a glycosidic bond links the protein and the nucleotide-sugar. Attempts have been made to identify the UDP-glucose-binding region in these proteins by comparing their sequences with cellulose synthase and other glycosyltransferases2,3. However, these alignments failed to reveal a consensus UDP-glucose-binding region. In our analysis, we have identified a region in the RGPs that contains two conserved aspartic acid residues at positions that are characteristic of domain A of cellulose synthases and other b-glycosyltransferases4 (Fig. 1). In addition, hydrophobic cluster analysis of this region predicts a secondary structure that is similar to that observed for domain A of the b-glycosyltransferases4. However, the residues that are characteristic of domain B, which are present in the processive b-glycosyltransferases, were not observed in these proteins. This suggests that RGPs are non-processive b-glycosyltransferases, which transfer a single sugar residue from a nucleotide-sugar to an acceptor molecule that may be an intermediate in the synthesis of polysaccharides. The intermediate may be either the RGP and/or another molecule, such as a membrane-linked oligosaccharide subunit involved in the synthesis of polysaccharides. To date, no genes that encode nucleotide sugar transporters in higher plant Golgi membranes have been identified, and the suggestion that RGPs may represent nucleotide sugar transporters does not concur with the known properties of these transporters in mammalian cells, yeasts and other organisms5. Instead, the RGPs may function in binding tightly to nucleotide sugars in the cytosol and delivering them to the Golgi surface where they transfer the sugar to an acceptor molecule for polysaccharide biosynthesis. Although the mechanism of reversible glycosylation is not fully understood, the sequence analysis shown in Fig. 1 indicates that RGPs have a nucleotide sugar-binding region that is conserved with the b-glycosyltransferases. In site-directed mutagenesis experiments using the Acetobacter xylinum cellulose synthase, substitution of the conserved aspartic acid residues (shown in Fig. 1) led to a loss of cellulose synthase activity6. The precise function of these conserved aspartic acid residues has yet to be determined, but the conserved aspartic acid residues present in the DXD motif is observed in a large number of glycosyltransferases including the bglycosyltransferases7. These residues might function in coordinating the metal ion (Mg2+ or Mn2+) with the phosphates of the UDP moiety of UDP-sugars, or as a base in the catalytic reaction.
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