Comparative characterisation of recombinant invertebrate and vertebrate peptide O-Xylosyltransferases.
Chondroitin and heparan sulphates have key functions in animal development and their synthesis is initiated by the action of UDP-alpha-D-xylose:proteoglycan core protein beta-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.
Brunner
Andrea
A
Department für Chemie der, Universität für Bodenkultur, Muthgasse 18, A-1190, Wien, Austria.
Kolarich
Daniel
D
Voglmeir
Josef
J
Paschinger
Katharina
K
Wilson
Iain B H
IB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D053667:Syndecans / Q000737:chemistry / Q000378:metabolism; D000097608:UDP Xylose-Protein Xylosyltransferase
D018076:DNA, Complementary; D019812:Heparan Sulfate Proteoglycans; D010430:Pentosyltransferases; D010446:Peptide Fragments; D011994:Recombinant Proteins; D053667:Syndecans; D000097608:UDP Xylose-Protein Xylosyltransferase; C071927:perlecan