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

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.