Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats

J Biol Chem. 2010 Jan 15;285(3):1582-6. doi: 10.1074/jbc.C109.065409. Epub 2009 Nov 25.

Abstract

The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous alpha1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:beta-d-glucoside alpha1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for beta-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the alpha1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Epidermal Growth Factor / chemistry*
  • Glucose / metabolism*
  • Glycosylation
  • Glycosyltransferases / chemistry
  • Glycosyltransferases / genetics
  • Glycosyltransferases / metabolism*
  • Humans
  • Molecular Sequence Data
  • Oxygen / metabolism*
  • Pentosyltransferases / metabolism*
  • Receptors, Notch / chemistry
  • Receptors, Notch / metabolism*
  • Repetitive Sequences, Amino Acid*
  • Xylose / metabolism

Substances

  • Receptors, Notch
  • Epidermal Growth Factor
  • Xylose
  • Glycosyltransferases
  • Pentosyltransferases
  • UDP xylose-protein xylosyltransferase
  • Glucose
  • Oxygen