Molecular Genetics of Non-processive Glycosyltransferases

Although Arabidopsis has become the model for plant genomic and developmental research, it remains behind the rest of the field with respect to biochemical and structural analyses. However, the availability of the genome sequence provides an alternative and probable first approach towards identification, by homology, of potentially interesting genes. Subsequently, such a genetic approach has been useful in identifying a class of enzymes involved in cell wall biosynthesis, the polysaccharide glycosyltransferases. Most enzymes that are involved in glycan biosynthesis are glycosyltransferases. These enzymes act mainly by adding monosaccharides (donor molecules) one at a time to specific positions on acceptor molecules and thus assemble monosaccharides into linear and branched sugar chains. It has been estimated that more than 100 distinct glycosidic linkages are present in the glycoconjugate repertoire of any given multicellular organism. Because each linkage is usually the product of a different glycosyltransferase, it appears that a large number of distinct glycosyltransferases is present in higher eukaryotes. Many, but not all, of these enzymes are found within the ER-Golgi pathway where the synthesis of many glycoconjugates occurs. Glycosyltransferases may act as either processive or non-processive enzymes. Processive glycosyltransferases (often referred to as polysaccharide synthases) add sugar moieties to the growing end of a linear polysaccharide without releasing the acceptor substrate. One classic example is cellulose synthase. Non-processive glycosyltransferases add single monosaccharides to specific positions of an acceptor molecule, then move on to other acceptor sites. These enzymes are usually type II membrane proteins possessing a single hydrophobic segment spanning the Golgi membrane, which functions as a signal-anchor sequence. A short amino-terminal portion faces the cytosol and the carboxy-terminal catalytic domain is positioned within the lumen of the Golgi apparatus (Figure 1). Figure 1. Schematic diagram of most Golgi-resident glycosyltransferases. They are characterized by a short NH2-terminus oriented toward the cytoplasmic side of the Golgi membrane, a single membrane spanning region, and a variable length stem connecting the globular ... Many glycosyltransferases have been identified and extensively studied in animal, fungal, and bacterial systems; these are classified into different families, based on the type of sugar that they transfer and the type of linkage that they establish. For instance, an enzyme transferring a galactose residue from UDP-D-galactose onto the 2-hydroxyl group of another sugar moiety would be considered a (1,2)-galactosyltransferase. The sugar moiety may be added in either alpha or beta configuration adding further complexity to the types of linkages that can be formed. If in the above example the newly attached galactose residue is in the beta configuration, the enzyme would be classified as a *b(1,2)-galactosyltransferase. Since the sugar moiety in UDP-D-galactose exists in the alpha configuration, b-galactosyltransferases act with inversion of configuration whereas a-galactosyltransferases act with retention of configuration. Like other eukaryotic organisms, plants have glycoproteins and glycolipids; however, a major feature distinguishing plants from animals is the presence of a complex wall surrounding every cell. These walls play a crucial role in development, signal transduction, and response to environmental factors, including microbial pathogens and insects. Plant cell walls are mainly composed of cellulose microfibrils and matrix polysaccharides that encompass hemicelluloses and pectins. The synthesis of polysaccharides can be divided into two main stages: synthesis of the backbone, performed by polysaccharide synthases, and addition of side-chain residues mediated by glycosyltransferases. The structural complexity of many of these polysaccharides is attributed to numerous modifications that involve additions of various sugars by glycosyltransferases followed by acetylations, methylations, and addition of phenolics. Although biochemical analyses have demonstrated the existence of many glycosyltransferases in plants (White et al., 1993; Keegstra & Raikhel, 2001 and references therein), to date only three genes encoding non-processive glycosyltransferases in cell wall polysaccharide biosynthesis have been cloned, and several genes that are involved in glycoprotein biosynthesis have been identified as well. Low amino acid sequence similarity between families of glycosyltransferases and the difficulties of obtaining acceptor substrates contribute to the lack of success in identifying plant cell wall glycosyltransferases. In this chapter we will highlights some of the advances made in this area over the past few years.