Novel Tetrasaccharides Isolated from Squid Cartilage Chondroitin Sulfate E Contain Unusual Sulfated Disaccharide Units GlcA(3-O-sulfate)β1–3GalNAc(6-O-sulfate) or GlcA(3-O-sulfate)β1–3GalNAc(4,6-O-disulfate)

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 1 H NMR spectroscopy revealed one disulfated, three trisulfated, and one tetrasulfated tetrasaccharide structure: fraction h, GlcA␤1-3GalNAc(4S)␤1-4GlcA␤1-3GalNAc(4S); fraction l, GlcA-(3S)␤1-3GalNAc(6S)␤1-4GlcA␤1-3GalNAc(4S); fraction m, GlcA(3S)␤1-3GalNAc(4S)␤1-4GlcA␤1-3GalNAc(4S); fraction n, GlcA␤1-3GalNAc(4S,6S)␤1-4GlcA␤1-3Gal-NAc(4S); and fraction q, GlcA(3S)␤1-3GalNAc(4S,6S)␤1-4GlcA␤1-3GalNAc(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.

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

“…First, the presence of a 3-O sulfated glucuronic acid is rare, only observed in specific contexts within certain organisms (13). In addition, a tetrasulfated disaccharide repeat unit has not been isolated to date from animal tissues.…”

“…The delay for evolution of long range couplings was set with a J lr of 8 Hz. Samples for 13 C-NMR analysis were dissolved in D 2 O (99.9%) at 40 mg/mL and analysis was performed at 303K with a 400 MHz Bruker spectrometer equipped with a multinuclear probe.…”

Involvement of Human Natural Killer-1 (HNK-1) Sulfotransferase in the Biosynthesis of the GlcUA(3-O-sulfate)-Gal-Gal-Xyl Tetrasaccharide Found in α-Thrombomodulin from Human Urine

“…The sulfate group(s) on GalNAc residue(s) appears to have an inhibitory effect on the sulfotransferase activity. However, disaccharide structures composed of a 3-O-sulfated GlcUA and sulfated GalNAc residue, GlcUA(3-O-sulfate)-GalNAc(6-O-sulfate) and GlcUA(3-O-sulfate)-GalNAc (4,6-O-disulfate), have been detected in CS preparations from squid cartilage (38). In addition, GlcUA(3-O-sulfate)-GalNAc(4-O-sulfate) has been demonstrated in CS from king crab cartilage (37).…”

Structural Requirements of Heparan Sulfate for the Binding to the Tumor-derived Adhesion Factor/Angiomodulin That Induces Cord-like Structures to ECV-304 Human Carcinoma Cells

“…CS-E is expressed in a cell-type-and tissue-type-specific manner and in a developmentally regulated fashion (reviewed in Ref. 28). It remains to be determined whether CS-E is a physiological ligand of AGM and is involved in the expression of biological activities of AGM in vivo.…”