First isolation of human UDP-D-xylose: proteoglycan core protein beta-D-xylosyltransferase secreted from cultured JAR choriocarcinoma cells.
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.
Kuhn
J
J
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstrasse 11, 32545 Bad Oeynhausen, Germany.
Götting
C
C
Schnölzer
M
M
Kempf
T
T
Brinkmann
T
T
Kleesiek
K
K
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002822:Choriocarcinoma; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000737:chemistry; D006801:Humans; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011479:Protamines / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured; D000097608:UDP Xylose-Protein Xylosyltransferase
D000906:Antibodies; D006493:Heparin; D010430:Pentosyltransferases; D011479:Protamines; D000097608:UDP Xylose-Protein Xylosyltransferase