MS analysis of chondroitin polymerization: Effects of Mn 2+ ions on the stability of UDP-sugars and chondroitin synthesis

• Published in: Analytical biochemistry Vol. 365; no. 1; pp. 62 - 73
• Main Authors: Sugiura, Nobuo, Shimokata, Satoshi, Watanabe, Hideto, Kimata, Koji
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 2007
• Subjects: Chondroitin; Glycosaminoglycan; MALDI–TOF MS; Manganese ion; Polymerase; UDP-GlcA; Polymerase; MALDI–TOF MS; UDP-GlcA; Glycosaminoglycan; Chondroitin; Manganese ion
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2007.02.023

Chondroitin polymerase from Escherichia coli strain K4 (K4CP) synthesizes chondroitin (CH) polysaccharides by the alternate addition of N-acetyl- d-galactosamine (GalNAc) and d-glucuronic acid (GlcA) to acceptor CH oligosaccharides in the presence of Mn 2+ ions. In this study, we applied matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI–TOF MS) for the further characterization of the products synthesized by K4CP from CH hexasaccharide as an initial acceptor and UDP-GalNAc and UDP-GlcA as donors. The analysis identified individual CH chains of various lengths and enabled the calculation of their average molecular weights. The ion peaks of the CH chains synthesized in the short-time reactions demonstrated not only the alternate addition of GlcA and GalNAc but also the more frequent transfer of GlcA and GalNAc, consistent with our previous kinetic data. In contrast, the MS spectra of the chains synthesized in the long-time reaction showed that CH chains containing GalNAc at the nonreducing ends were more abundant than those containing GlcA. We found that this inconsistency was due to the preferential decomposition of UDP-GlcA by Mn 2+ ions. We defined the optimal conditions to yield further elongation of the CH chains that have nearly equal numbers of GlcA and GalNAc residues at the nonreducing ends.