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.
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.
Gotoh
Masanori
M
Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Open Space Laboratory C-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Sato
Takashi
T
Akashima
Tomohiro
T
Iwasaki
Hiroko
H
Kameyama
Akihiko
A
Mochizuki
Hideo
H
Yada
Toshikazu
T
Inaba
Niro
N
Zhang
Yan
Y
Kikuchi
Norihiro
N
Kwon
Yeon-Dae
YD
Togayachi
Akira
A
Kudo
Takashi
T
Nishihara
Shoko
S
Watanabe
Hideto
H
Kimata
Koji
K
Narimatsu
Hisashi
H
D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D002807:Chondroitin / Q000096:biosynthesis*; D002851:Chromatography, High Pressure Liquid; D020723:Glucuronic Acid / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D014018:Tissue Distribution
D011993:Recombinant Fusion Proteins; D020723:Glucuronic Acid; D002807:Chondroitin; D017350:N-Acetylgalactosaminyltransferases; D000116:Acetylgalactosamine