In vitro assays of orphan glycosyltransferases and their application to identify Notch xylosyltransferases

Maya K Sethi; Falk F R Buettner; Angel Ashikov; Hans Bakker

doi:10.1007/978-1-62703-465-4_23

In vitro assays of orphan glycosyltransferases and their application to identify Notch xylosyltransferases

Methods Mol Biol. 2013:1022:307-20. doi: 10.1007/978-1-62703-465-4_23.

Authors

Maya K Sethi¹, Falk F R Buettner, Angel Ashikov, Hans Bakker

Affiliation

¹ Department of Cellular Chemistry, Hannover Medical School, Hannover, Germany.

PMID: 23765671
DOI: 10.1007/978-1-62703-465-4_23

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cloning, Molecular / methods
Enzyme Assays / methods*
Glycosyltransferases / genetics
Glycosyltransferases / isolation & purification
Glycosyltransferases / metabolism*
Humans
Hydrolysis
Pentosyltransferases / genetics
Pentosyltransferases / isolation & purification
Pentosyltransferases / metabolism*
Polysaccharides / chemistry
Polysaccharides / metabolism
Protein Structure, Tertiary
Receptors, Notch / chemistry
Receptors, Notch / genetics
Receptors, Notch / metabolism*
Substrate Specificity
UDP Xylose-Protein Xylosyltransferase

Substances

Polysaccharides
Receptors, Notch
Glycosyltransferases
Pentosyltransferases