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 2.4.2.26, XT) initiates the biosynthesis of glycosaminoglycan lateral chai ns in proteoglycans by transfer of xylose from UDP-xylose to specific serin e residues of the core protein. In this study, we report the first isolatio n of the XT and present the first partial amino acid sequence of this enzym e. We purified XT 4,700-fold with 1% yield from serum-free JAR choriocarcin oma cell culture supernatant, The isolation procedure included a combinatio n of ammonium sulfate precipitation, heparin affinity chromatography, ion e xchange chromatography, and protamine affinity chromatography, Among other proteins an unknown protein was detected by matrix-assisted laser desorptio n ionization mass spectrometry-time of flight analysis in the purified samp le. The molecular mass of this protein was determined as 120 kDa by SDS-pol yacrylamide gel electrophoresis, The isolated protein was enzymatically cle aved by trypsin and endoproteinase Lys-C, Eleven peptide fragments were seq uenced by Edman degradation. Searches with the amino acid sequences in prot ein and EST data bases showed no homology to known sequences. XT was enrich ed by immunoaffinity chromatography with an immobilized antibody against a synthetic peptide deduced from the sequenced peptide fragments and was spec ifically eluted with the antigen. In addition, XT was purified alternativel y with an aprotinin affinity chromatography and was detected by Western blo t analysis in the enzyme-containing fraction.