Enzymatic Synthesis of Chondroitin with a Novel Chondroitin Sulfate N-Acetylgalactosaminyltransferase That Transfers N-Acetylgalactosamine to Glucuronic Acid in Initiation and Elongation of Chondroitin Sulfate Synthesis
| Autor: | Norihiro Kikuchi, Hisashi Narimatsu, Akira Togayachi, Yan Zhang, Shoko Nishihara, Niro Inaba, Yeon-Dae Kwon, Takashi Kudo, Hideo Mochizuki, Takashi Sato, Toshikazu Yada, Masanori Gotoh, Koji Kimata, Tomohiro Akashima, Akihiko Kameyama, Hideto Watanabe, Hiroko Iwasaki |
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| Rok vydání: | 2002 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Acetylgalactosamine Recombinant Fusion Proteins Molecular Sequence Data Biochemistry N-Acetylgalactosamine chemistry.chemical_compound Glucuronic Acid Glycosyltransferase Humans Tetrasaccharide Chondroitin Tissue Distribution Amino Acid Sequence Chondroitin sulfate Molecular Biology Chromatography High Pressure Liquid chemistry.chemical_classification biology Chemistry Glycosyltransferase Gene Cell Biology Glucuronic acid Molecular biology Enzyme biology.protein N-Acetylgalactosaminyltransferases Sequence Alignment |
| Zdroj: | Journal of Biological Chemistry. 277:38189-38196 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m203619200 |
| Popis: | We found a novel glycosyltransferase gene having a hypothetical beta 1,4-galactosyltransferase motif (GenBank accession number ) by a BLAST search and cloned its full-length open reading frame using the 5'-rapid amplification of cDNA ends method. The truncated form was expressed in insect cells as a soluble enzyme. It transferred N-acetylgalactosamine, not galactose, to para-nitrophenyl-beta-glucuronic acid. The N-acetylgalactosamine-glucuronic acid linkage has been identified only in chondroitin sulfate; therefore, we examined its chondroitin elongation and initiation activities. N-Acetylgalactosaminyltransferase activity was observed toward chondroitin poly- and oligosaccharides, chondroitin sulfate oligosaccharides, and linkage tetrasaccharide (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), and the chondroitin polysaccharide and linkage tetrasaccharide were better acceptor substrates than the others. Northern blot analysis and quantitative real-time PCR analysis revealed that its 4-kb transcripts were highly expressed in thyroid and placenta, although they were ubiquitously expressed in various tissues and cells. These results suggest that this enzyme has N-acetylgalactosaminyltransferase activity in both the elongation and initiation of chondroitin sulfate synthesis. Furthermore, we performed enzymatic synthesis of chondroitin pentasaccharide in vitro. In one tube reaction with four enzymes, beta 1,4-galactosyltransferase-VII, beta 1,3-galactosyltransferase-VI, glucuronyltransferase-I, and this enzyme, and a synthetic xylose-peptide acceptor, the structure GalNAc-GlcA-Gal-Gal-Xyl-peptide was constructed. This is the first report of a chondroitin pentasaccharide constructed with recombinant glycosyltransferases in vitro. |
| Databáze: | OpenAIRE |
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