title: Biochemical characterization of Silene alba alpha4-fucosyltransferase and Lewis a products.
abstract: alpha1,4-Fucosylation has been recently detected in Arabidopsis thaliana [Leonard et al. (2002), Glycobiology 12: 299-306], and corresponding enzymes have also been characterized in Beta vulgaris [Bakker et al. (2001), FEBS Lett, 507: 307-312], and Lycopersicum aesculentum [Wilson (2001), Glycoconjugate J., 18: 439-447]. Here we demonstrated fucosyltransferase activity (FucT) in Silene alba cells and tissues. The Fuc linkage to GlcNAc residues of the lactosamine moiety of the Type I acceptor was confirmed by mass spectrometry experiments. Le(a)-glycoconjugates are found in the Golgi apparatus and plasma membrane of plant cells. In planta, the highest levels of activity were detected in seedlings, young roots and male flowers. The enzyme was stable up to 45( composite function)C and the optimum pH of reaction was 8.0. The enzyme required Mg(2+) or Mn(2+) for activity and was inhibited by Zn(2+) and ethylenediaminetetraacetic acid. Chemical modification of the enzyme with group-selective reagents revealed that selective modifications of arginine and lysine residues had no effect on enzyme activity. However the enzyme contains histidine and tryptophan residues that are essential for its activity. In contrast to human FUT3, the S. alba alpha4-FucT was insensitive to N-ethylmaleimide (NEM) treatment. Measurement of enzyme activity in S. alba cell fractions indicated that the enzyme is bound to microsomal membranes, furthermore a soluble isoform of the protein may be present.
authors.affiliation: Laboratoire de Chimie des Substances Naturelles, Groupe de Glycobiologie Forestière (EA1069), Faculté des Sciences et Techniques de Limoges, LIMOGES, France.
authors.forename: R
authors.initials: R
authors.lastname: Léonard
authors.forename: S
authors.initials: S
authors.lastname: Lhernould
authors.forename: M
authors.initials: M
authors.lastname: Carlué
authors.forename: P
authors.initials: P
authors.lastname: Fleurat
authors.forename: A
authors.initials: A
authors.lastname: Maftah
authors.forename: G
authors.initials: G
authors.lastname: Costa
mesh_terms: D002462:Cell Membrane / Q000201:enzymology; D004795:Enzyme Stability; D005647:Fucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D011134:Polysaccharides / Q000378:metabolism*; D029751:Silene / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D013379:Substrate Specificity
chemical_list: D011134:Polysaccharides; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase