In Vitro Assays of Orphan Glycosyltransferases and Their Application to Identify Notch Xylosyltransferases

Abstract

Here we describe a systematic approach to determine the activity of putative glycosyltransferases with a focus on orphan members of the glycosyltransferase 8 family. An assay that measures the hydrolysis activity of glycoslytransferases can indicate the donor nucleotide sugar specificity without previous knowledge about the acceptor. Knowing the donor specificity, the acceptor specificity can subsequently be determined using synthetic acceptors. Three putative glycosyltransferases, now renamed GXYLT1, GXYLT2, and XXYLT1, have been identified this way as xylosyltransferases and in addition have been shown to act on O-glucosylated EGF repeats of Notch.

The Capsule Polymerase CslB of Neisseria meningitidis Serogroup L Catalyzes the Synthesis of a Complex Trimeric Repeating Unit Comprising Glycosidic and Phosphodiester Linkages

“…As discussed before, the recombinant enzyme facilitates the hydrolysis of UDP-GlcNAc to GlcNAc 1-phosphate and UMP. Because this behavior suggests a functional hexose-1-phosphate transferase (31,49), one may speculate that CslA functions as an initiating glycosyltransferase that transfers the first residue of the CPSL repeating unit onto the universal ␤-Kdo acceptor that was recently described, thus priming it for elongation by CslB (48,65). In line with the variety of CPs, no or little conservation was expected to exist between these initiating glycosyltransferases, but they are hypothesized to be encoded in region A of the capsule gene locus (6,48).…”

C3orf21 and Notch Signaling in Cancer：A Potential Biomarker

“…Notch activation is modulated by the differential O-linked glycosylation of its extracellular domain (NECD) [20], whereby O-linked glucose is added to a subset of EGF repeats in the NECD domain by Protein O-glucosyltransferase 1 (POGLUT1) [5,27,28]. This can be subsequently extended to xylose α1-3 glucose trisaccharide by the sequential action of Glucoside α1-3 xylosyl transferase (GXYLT1/2) [17] and xyloside α1-3 xylosyl transferase (XXYLT1) [29]. GXYLT1 and GXYLT2 (glucoside-xylosyltransferase 1/2) transfer the first 1,3-linked xylose to O-glucosylated mammalian Notch EGF repeats [29], while XXYLT1 can transfer the second xylose to o-glucosylated EGF repeats of Notch [7].…”

“…The XXYLT1 played an important role in the trisaccharide synthesis, which catalyzes the transfer of xylose to Xylα1-3Glc, and form D-Xylpα1-3-D-Xylpαl-3-D-Glc, X 2 G. XXYLT1 elongates the O-linked xylose-glucose disaccharide attached to the EGF-like repeats in the extracellular domain of target proteins by catalyzing the addition of the second xylose [7,15,16]. XXYLT1 participated in the biosynthesis of Glc-O-type sugar chains and encodes xylosyltransferase activity [17]. XXYLT1 was active only when linked with an acceptor containing Xylα1, 3Glcβ1, while no activity was observed when linked with xylose linked to pNP or with Xyl-O-EGF16 [18].…”

XXYLT1 Methylation Contributes to the Occurrence of Lung Adenocarcinoma

“…XXYLT1 also plays a negative regulatory role in the Notch signaling pathway, and reduced activity of XXYLT1 would lead to enhanced Notch signaling [18]. XXYLT1 participated in the biosynthesis of Glc-O-type sugar chains and encodes xylosyltransferase activity [19]. After XXYLT1 activities, it transfers the second xylose to O-glucosylated EGF repeats of Notch, and modulated Notch activation [16].…”