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

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.

FIGURE 1.

Sequence alignment. The complete amino acid sequence of four human GLT8 family members is aligned with the N-terminal catalytic domain of UGGT starting at amino acid 1243. Sequence conservation is shown when three out of five amino acids are identical. Putative transmembrane domains are shown shaded, and the conserved DXD motif is underlined.

FIGURE 2.

In vitro enzymatic activity. A, activity of GLT8D1, GLT8D2, GXYLT1, and GXYLT2 with acceptors mimicking the two naturally occurring xylosyltransferase substrates on EGF domains using UDP-Xyl as donor substrate. no Acc., no acceptor. B, donor substrate specificity using Glc-R as substrate. C, acceptor substrate specificity of GXYLT1 and GXYLT2 using para-nitrophenol (pNP)-linked monosaccharides as acceptor substrates. D, glucosylated EGF1 of factor VII when compared with unglucosylated EGF1 and Glc-R as acceptor, now measured at 2 μm acceptor concentration (when compared with 100 μm in A–C) to adapt to the low availability of factor VII EGF1. E, quantification of enzyme bound to 0.5 μl of IgG beads using a protein A standard and Coomassie Blue staining, of which 10 μl was used per assay in A–D.

FIGURE 3.

Xylosyltransferase activity using Notch EGF1–5 as acceptor. EGF1–5 produced in UDP-Xyl synthase-negative cells (A) was incubated with GXYLT1 or GXYLT2 (B and C), and tryptic peptides were analyzed by liquid chromatography-electrospray ionization-TOF mass spectrometry. Shown is the O-glucosylated peptide of EGF4. GXYLT1 and GXYLT2 were able to extend the O-glucosylated peptide by one xylose but not to produce the full trisaccharide that is produced in wild type CHO cells (D). To confirm that the m/z values shown represent the differentially glycosylated peptides of EGF4, sequencing was done by tandem mass spectrometry and shown in supplemental Fig. S2. MW, molecular weight.

FIGURE 4.

In vivo xylosyltransferase activity. Shown are the MALDI-mass spectra of insect cell-produced EGF1–5 that was co-infected with GLT8D1 as a negative control (A), GXYLT1 (B), or GXYLT2 (C). The glucosylated peptide with the calculated nominal mass of 3498 Da is most dominant in A but is reduced in favor of the Xyl-Glc-peptide (3630 Da) after incubation with GXYLT1 or GXYLT2. As for Fig. 3, peptide sequencing was performed and is presented in supplemental Fig. S2.