Abstract
We previously isolated novel tetrasaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K and demonstrated that the disaccharide units containing 3-O-sulfated glucuronic acid were decomposed by chondroitinase ABC digestion (Sugahara, K., Tanaka, Y., Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M., Morris, H. R., and Dell, A. (1996) J. Biol. Chem. 271, 26745-26754). The findings indicated the necessity to re-evaluate the disaccharide compositions of chondroitin sulfate preparations purified from other biological sources and analyzed using the above enzyme. In this study, to evaluate squid cartilage chondroitin sulfate E a series of even-numbered oligosaccharides were isolated after exhaustive digestion with sheep testicular hyaluronidase and subsequent fractionation by gel chromatography. The tetrasaccharide fraction was subfractionated by high performance liquid chromatography on an amine-bound silica column. Systematic structural analysis of five major fractions, h, l, m, n, and q, by fast atom bombardment mass spectrometry, enzymatic digestions in conjunction with capillary electrophoresis, and 500-MHz 1H NMR spectroscopy revealed one disulfated, three trisulfated, and one tetrasulfated tetrasaccharide structure: fraction h, GlcAbeta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S); fraction l, GlcA(3S)beta1-3GalNAc(6S)beta1-4GlcAbeta1-3GalNAc(4S); fraction m, GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S); fraction n, GlcAbeta1-3GalNAc(4S,6S)beta1-4GlcAbeta1-3GalNAc(4S); and fraction q, GlcA(3S)beta1-3GalNAc(4S,6S)beta1-4GlcAbeta1-3GalNAc(4S), where 3S, 4S, and 6S represent 3-O-, 4-O- and 6-O-sulfate, respectively. The structures found in fractions h and m as well as the unsaturated counterpart of that found in fraction n have been reported, whereas those in fractions l and q are novel in that they contained unusual disulfated and trisulfated disaccharide units where GlcA(3S) is directly linked to GalNAc(6S) and GalNAc(4S,6S), respectively. These novel tetrasaccharide sequences are distinct from those found in other chondroitin sulfate isoforms and may play key roles in the biological functions and activities of chondroitin sulfate E not only from squid cartilage but also from mammalian cells and tissues.
Highlights
The previous study on the GlcA(3S)-containing oligosaccharides isolated from king crab cartilage Chondroitin sulfate (CS)-K indicated that the disaccharide units containing GlcA(3S) were decomposed by the action of chondroitinase ABC [44] and that re-evaluation of disaccharide compositions was required for CS preparations that were purified from other biological sources and analyzed using the above enzyme
Differential Susceptibility of Various CS Isoforms to Chondroitinase ABC—Various individual CS isoforms were digested with chondroitinase ABC, and the reactions were monitored by absorbance at 232 nm caused by ⌬HexA produced by the eliminase action of the enzyme
The digestion degree was calculated based on the comparison of the amounts of the resultant unsaturated oligosaccharides estimated by the absorption at 232 nm and the uronic acid values determined for each parent CS isoform by the carbazole reaction
Summary
Materials—CS isoform preparations (super special grade), including squid cartilage CS-E, whale cartilage CS-A, shark cartilage CS-C, and shark cartilage CS-D, were purchased from Seikagaku Corp., Tokyo, Japan. The digestion rate was calculated based on the amount of the resultant unsaturated oligosaccharides relative to the uronic acid value determined for each CS isoform by the carbazole reaction [49] (Fig. 1). Preparation of Oligosaccharide Fractions—A commercial squid cartilage CS-E (100 mg) was digested with 10 mg (approximately 15,000 National formulary units) of sheep testicular hyaluronidase in a total volume of 2.0 ml of 50 mM sodium phosphate buffer, pH 6.0, containing 150 mM NaCl (1 National formulary unit corresponds to the amount of the enzyme which hydrolyzes 74 g of hyaluronate/min) [51, 52]. For derivatization of oligosaccharide fractions, samples (1 nmol each) were first digested with chondroitinase AC-II as described above and concentrated to dryness in a vacuum concentrator. Tetrasaccharides for NMR analysis were repeatedly exchanged in 2H2O with intermediate lyophilization
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