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.
Highlights
The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans
We have identified two enzymes of the human glycosyltransferase 8 family, named GXYLT1 and GXYLT2, as UDP-D-xylose:D-glucoside ␣1,3-D-xylosyltransferases adding the first xylose
More than half of the 29 –36 epidermal growth factor (EGF)2 repeats of the mammalian Notch receptors are modified with O-fucose (Fuc), O-glucose (Glc), or O-N-acetylglucosamine (GlcNAc) glycans
Summary
Identification of Glycosyltransferase 8 Family Members as Xylosyltransferases Acting on O-Glucosylated Notch Epidermal Growth Factor Repeats*□S. More than half of the 29 –36 epidermal growth factor (EGF) repeats of the mammalian Notch receptors are modified with O-fucose (Fuc), O-glucose (Glc), or O-N-acetylglucosamine (GlcNAc) glycans. These O-glycans are linked to specific serine or threonine residues in distinct consensus sequences [1, 2], which are found on EGF repeats of various other proteins that include blood coagulation factor VII and Notch ligands Delta and Serrate/Jagged [3]. We describe the identification of two genes, named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), encoding enzymes able to transfer xylose to the O-Glc residue bound to Notch EGF repeats both in vitro and in vivo
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