Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats.
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.
Sethi
Maya K
MK
Department of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
Buettner
Falk F R
FF
Krylov
Vadim B
VB
Takeuchi
Hideyuki
H
Nifantiev
Nikolay E
NE
Haltiwanger
Robert S
RS
Gerardy-Schahn
Rita
R
Bakker
Hans
H
D000595:Amino Acid Sequence; D000818:Animals; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005947:Glucose / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D010430:Pentosyltransferases / Q000378:metabolism*; D051880:Receptors, Notch / Q000737:chemistry / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D014994:Xylose / Q000378:metabolism; D000097608:UDP Xylose-Protein Xylosyltransferase
D004815:Epidermal Growth Factor; D005947:Glucose; D016695:Glycosyltransferases; D010100:Oxygen; D010430:Pentosyltransferases; D051880:Receptors, Notch; D014994:Xylose; D000097608:UDP Xylose-Protein Xylosyltransferase