Abstract
Human UDP-d-xylose:proteoglycan core protein beta-d-xylosyltransferase (EC, XT) initiates the biosynthesis of glycosaminoglycan lateral chains in proteoglycans by transfer of xylose from UDP-xylose to specific serine residues of the core protein. In this study, we report the first isolation of the XT and present the first partial amino acid sequence of this enzyme. We purified XT 4,700-fold with 1% yield from serum-free JAR choriocarcinoma cell culture supernatant. The isolation procedure included a combination of ammonium sulfate precipitation, heparin affinity chromatography, ion exchange chromatography, and protamine affinity chromatography. Among other proteins an unknown protein was detected by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis in the purified sample. The molecular mass of this protein was determined as 120 kDa by SDS-polyacrylamide gel electrophoresis. The isolated protein was enzymatically cleaved by trypsin and endoproteinase Lys-C. Eleven peptide fragments were sequenced by Edman degradation. Searches with the amino acid sequences in protein and EST data bases showed no homology to known sequences. XT was enriched by immunoaffinity chromatography with an immobilized antibody against a synthetic peptide deduced from the sequenced peptide fragments and was specifically eluted with the antigen. In addition, XT was purified alternatively with an aprotinin affinity chromatography and was detected by Western blot analysis in the enzyme-containing fraction.
Highlights
Transfer nonspecific antibody-binding sites were blocked with 2% bovine serum albumin in 0.1 M Tris-HCl, pH 7.2, for 1 h at room temperature
Step 2: Heparin Affinity Chromatography on POROS 20 HE—4 ml of XT-enriched solution from step 1 was loaded onto the POROS 20 HE column
About 50% of the XT activity of an applied sample was bound (Fig. 3, panel A) when a partially purified XT sample obtained by heparin affinity chromatography was loaded onto the column. 58% of the adsorbed XT activity was eluted with 150 mM NaCl, and the rest was eluted with 12 mM HCl
Summary
Dried UltraDOMA-PF medium was obtained from BioWhittaker (Vervier, Belgium) and aqua ad injecta from Braun (Melsungen, Germany). Serological pipettes, and sterile tubes were purchased from Becton Dickinson (Heidelberg, Germany). Ultrafiltration cells, YM1 membranes, and polyvinylidene difluoride membranes (Immobilon P) were purchased from Millipore (Eschborn, Germany). The MALDI mass spectrometer Reflex II was from Bruker Daltonik GmbH (Bremen, Germany) and protein sequencer Procise 494 cLC was purchased from PE Biosystems (Framingham, MA). The synthetic peptide CSRQKELLKRKLEQQEK and the rabbit antiserum were purchased from BioScience (Gottingen, Germany). Peroxidase-conjugated affinipure F(abЈ) fragment goat anti-rabbit IgG (HϩL) was purchased from Dianova (Hamburg, Germany). N-Glycosidase F was obtained from Roche Molecular Biochemicals (Mannheim, Germany). All other chemicals were of analytical grade and obtained from Merck (Darmstadt, Germany)
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