NOVEL TETRASACCHARIDES ISOLATED FROM SQUID CARTILAGE CHONDROITIN SULFATE-E CONTAIN UNUSUAL SULFATED DISACCHARIDE UNITS GLCA(3-O-SULFATE)BETA-1-3GALNAC(6-O-SULFATE) OR GLCA(3-O-SULFATE)BETA-1-3GALNAC(4,6-O-DISULFATE)

We previously isolated novel tetrasaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K and dem onstrated that the disaccharide units containing 3-O-sulfated glucuron ic 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. (1986) J. Biol. Chem. 271, 26745-26754). The find ings indicated the necessity to re-evaluate the disaccharide compositi ons of chondroitin sulfate preparations purified from other biological sources and analyzed using the above enzyme, in this study, to evalua te squid cartilage chondroitin sulfate E a series of even-numbered oli gosaccharides were isolated after exhaustive digestion with sheep test icular hyaluronidase and subsequent fractionation by gel chromatograph y. The tetrasaccharide fraction was subfractionated by high performanc e liquid chromatography on an amine-bound silica column. Systematic st ructural analysis of five major fractions, h, l, m, n, and q, by fast atom bombardment mass spectrometry, enzymatic digestions in conjunctio n with capillary electrophoresis, and 500-MHz H-1 NMR spectroscopy rev ealed one disulfated, three trisulfated, and one diasulfated tetrasacc haride structure: fraction h, GlcA beta 1-3GalNAc(4S)beta 1-4GlcA beta 1-3GalNAc(4S); fraction l, GlcA(3S)beta 1-3GalNAc(6S)beta 1-4GlcA bet a 1-3GalNAc(4S) fraction m, GlcA(3S)beta 1-3GalNAc(4S)beta 1-4GlcA bet a 1-3GalNAc(4S); fraction n, GlcA beta 1-3GalNAc(4S.6S)beta 1-4GlcA be ta 1-3GaINAc(4S); and fraction q, GlcA(3S)beta 1-3GalNAc(4S,6S)beta 1- 4GlcA beta 1-3GalNAc(4S), where 3S, 4S, and 6S represent 3-O-, 4-O- an d 6-O-sulfate, respectively. The structures found in fractions h and m as well as the unsaturated counterpart of that found in fraction n ha ve beem 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), res pectively. These novel tetrasaccharide sequences are distinct from tho se found in other chondroitin sulfate isoforms and may play key roles in the biological functions and activities of chondroitin sulfate E no t only from squid cartilage but also from mammalian cells and tissues.