title: Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of notch.
abstract: The extracellular domain of Notch contains epidermal growth factor (EGF) repeats that are extensively modified with different O-linked glycans. O-Fucosylation is essential for receptor function, and elongation with N-acetylglucosamine, catalyzed by members of the Fringe family, modulates Notch activity. Only recently, genes encoding enzymes involved in the O-glucosylation pathway have been cloned. In the Drosophila mutant rumi, characterized by a mutation in the protein O-glucosyltransferase, Notch signaling is impaired in a temperature-dependent manner, and a mouse knock-out leads to embryonic lethality. We have previously identified two human genes, GXYLT1 and GXYLT2, encoding glucoside xylosyltransferases responsible for the transfer of xylose to O-linked glucose. The identity of the enzyme further elongating the glycan to generate the final trisaccharide xylose-xylose-glucose, however, remained unknown. Here, we describe that the human gene C3ORF21 encodes a UDP-xylose:α-xyloside α1,3-xylosyltransferase, acting on xylose-α1,3-glucoseβ1-containing acceptor structures. We have, therefore, renamed it XXYLT1 (xyloside xylosyltransferase 1). XXYLT1 cannot act on a synthetic acceptor containing an α-linked xylose alone, but requires the presence of the underlying glucose. Activity on Notch EGF repeats was proven by in vitro xylosylation of a mouse Notch1 fragment recombinantly produced in Sf9 insect cells, a bacterially expressed EGF repeat from mouse Notch2 modified in vitro by Rumi and Gxylt2 and in vivo by co-expression of the enzyme with the Notch1 fragment. The enzyme was shown to be a typical type II membrane-bound glycosyltransferase localized in the endoplasmic reticulum.
authors: Department of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors: Maya K
authors: MK
authors: Sethi
authors: Falk F R
authors: FF
authors: Buettner
authors: Angel
authors: A
authors: Ashikov
authors: Vadim B
authors: VB
authors: Krylov
authors: Hideyuki
authors: H
authors: Takeuchi
authors: Nikolay E
authors: NE
authors: Nifantiev
authors: Robert S
authors: RS
authors: Haltiwanger
authors: Rita
authors: R
authors: Gerardy-Schahn
authors: Hans
authors: H
authors: Bakker
mesh_terms: D000818:Animals; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D051379:Mice; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D051883:Receptor, Notch2 / Q000235:genetics / Q000378:metabolism*; D000097608:UDP Xylose-Protein Xylosyltransferase
chemical_list: D029721:Drosophila Proteins; D005964:Glucosyltransferases; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010430:Pentosyltransferases; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D000097608:UDP Xylose-Protein Xylosyltransferase; C497761:NOTCH1 protein, human; C497764:NOTCH2 protein, human; C497762:Notch1 protein, mouse; C497765:Notch2 protein, mouse; C421265:TIMMDC1 protein, human; C527374:Rumi protein, Drosophila