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Comparative characterisation of recombinant invertebrate and vertebrate peptide O-Xylosyltransferases
- Source :
- Glycoconjugate Journal. 23:543-554
- Publication Year :
- 2006
- Publisher :
- Springer Science and Business Media LLC, 2006.
-
Abstract
- 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.
- Subjects :
- Spectrometry, Mass, Electrospray Ionization
DNA, Complementary
Syndecans
Xylosyltransferase
Molecular Sequence Data
Peptide
Perlecan
In Vitro Techniques
Biochemistry
Pichia
Substrate Specificity
Syndecan 1
Pichia pastoris
Species Specificity
Animals
Humans
Amino Acid Sequence
Pentosyltransferases
Caenorhabditis elegans
Molecular Biology
Chromatography, High Pressure Liquid
chemistry.chemical_classification
Binding Sites
Base Sequence
biology
Cell Biology
biology.organism_classification
Peptide Fragments
Recombinant Proteins
carbohydrates (lipids)
Caenorhabditis
Drosophila melanogaster
chemistry
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
biology.protein
Heparan Sulfate Proteoglycans
Subjects
Details
- ISSN :
- 15734986 and 02820080
- Volume :
- 23
- Database :
- OpenAIRE
- Journal :
- Glycoconjugate Journal
- Accession number :
- edsair.doi.dedup.....831ab6eb4f1f980eb8089f12f29e3c2e