Comparative characterisation of recombinant invertebrate and vertebrate peptide O-Xylosyltransferases |
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Authors: | Andrea Brunner Daniel Kolarich Josef Voglmeir Katharina Paschinger Iain B H Wilson |
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Institution: | 1. Department für Chemie der, Universit?t für Bodenkultur, Muthgasse 18, A-1190, Wien, Austria
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Abstract: | Chondroitin and heparan sulphates have key functions in animal development and their synthesis is initiated by the action
of UDP-α-D-xylose:proteoglycan core protein β-D-xylosyltransferase (EC 2.4.2.26). cDNAs encoding this enzyme have been previously
cloned from mammalian species; this in turn facilitated identification of corresponding Caenorhabditis
elegans (sqv-6) and Drosophila
melanogaster (oxt) genes. In the present study, we report the expression in Pichia pastoris and subsequent assay using either MALDI-TOF MS or RP-HPLC of recombinant forms of the Caenorhabditis xylosyltransferase SQV-6 and the human xylosyltransferase I, in addition to extending our previous studies on the xylosyltransferase
from Drosophila. The enzyme activities were tested with a number of peptide substrates based on portions of the human bikunin, human perlecan
and Drosophila syndecan core peptides. Whereas a variant of the latter, containing two Ser-Gly motifs was only modified on one of these
motifs, the perlecan peptide with three Ser-Gly motifs could be multiply modified in vitro. Using this substrate, we could
for the first time follow, by mass spectrometry, the xylosylation of a peptide with multiple xylosyltransferase acceptor motifs. |
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Keywords: | Xylosyltransferase Glycosaminoglycan |
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