Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants
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Escherichia coli strain K4 expresses a chondroitin (CH)-polymerizing enzyme (K4CP) that contains two glycosyltransferase active domains. K4CP alternately transfers glucuronic acid (GlcA) and N-acetyl-galactosamine (GalNAc) residues using UDP-GlcA and UDP-GalNAc donors to the nonreducing end of a CH chain acceptor. Here we generated two K4CP point mutants substituted at the UDP-sugar binding motif (DXD) in the glycosyltransferase active domains, which showed either glycosyltransferase activity of the intact domain and retained comparable activity after immobilization onto agarose beads. The mutant enzyme-immobilized beads exhibited an addition of GlcA or GalNAc to GalNAc or GlcA residue at the nonreducing end of CH oligosaccharides and sequentially elongated pyridylamine-conjugated CH (PA-CH) chain by the alternate use. The sequential elongation up to 16-mer was successfully achieved as assessed by fluorescent detection on a gel filtration chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI potential lift tandem TOF mass spectrometry (MALDI-LIFT-TOF/TOF MS/MS) analyses in the negative reflection mode. This method provides exactly defined CH oligosaccharide derivatives, which are useful for studies on glycosaminoglycan functions.
KeywordsChondroitin Stepwise glycosylation MALDI-TOF MS MS/MS fragmentation Pyridylamination
CH polymerase from Escherichia coli strain K4
- MALDI-TOF MS
matrix-assisted laser desorption/ionization and time-of-flight mass spectrometry
- MALDI-LIFT-TOF/TOF MS/MS
matrix-assisted laser desorption/ionization and potential lift tandem time-of-flight mass spectrometry
We are grateful to Hiroshi Maeda and Yuniko Shibata, Seikagaku Corporation for kindly providing desulfated CH. We thank Minoru Fukayama, Aichi Medical University and Takashi Nirasawa and Nobuyuki Shimura, Bruker Daltonics for assistance in MALDI-TOF MS and MALDI-LIFT-TOF/TOF MS/MS analyses. This work was supported by a special research fund from Seikagaku Corporation by the New Energy and Industrial Technology Development Organization (NEDO) and by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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