One- and Two-Dimensional 1H-NMR Characterization of Two Series of Sulfated Disaccharides Prepared from Chondroitin Sulfate and Heparan Sulfate/Heparin by Bacterial Eliminase Digestion1

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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.

Section: Fig 4 Negative Ion Mode Fab Mass Spectra Of Underivatized supporting

confidence: 81%

Section: -Mhzmentioning

confidence: 99%

Section: Fig 4 Negative Ion Mode Fab Mass Spectra Of Underivatized mentioning

confidence: 99%

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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)

Kinoshita

Yamada

Haslam

et al. 1997

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“…Sep-Pak ® Plus Accell TM QMA anion-exchange cartridges were from Waters Corp. (Milford, MA). Unsaturated disaccharides derived from heparin and HS were prepared as described previously (26). The following 2AB-derivatives of the authentic unsaturated tetra-and hexasaccharides of the GAG-protein linkage region were prepared from various CS preparations as previously described (27)…”

Section: Methodsmentioning

confidence: 99%

Determination of the Glycosaminoglycan-Protein Linkage Region Oligosaccharide Structures of Proteoglycans from Drosophila melanogaster and Caenorhabditis elegans

Yamada

Okada

Ueno

et al. 2002

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Caenorhabditis elegans and Drosophila melanogaster are relevant models for studying the roles of glycosaminoglycans (GAG) during the development of multicellular organisms. The genome projects of these organisms have revealed the existence of multiple genes related to GAG-synthesizing enzymes. Although the putative genes encoding the enzymes that synthesize the GAG-protein linkage region have also been identified, there is no direct evidence that the GAG chains bind covalently to core proteins. This study aimed to clarify whether GAG chains in these organisms are linked to core proteins through the conventional linkage region tetrasaccharide sequence found in vertebrates and whether modifications by phosphorylation and sulfation reported for vertebrates are present also in invertebrates. The linkage region oligosaccharides were isolated from C. elegans chondroitin in addition to D. melanogaster heparan and chondroitin sulfate after digestion with the respective bacterial eliminases and were then derivatized with a fluorophore 2-aminobenzamide. Their structures were characterized by gel filtration and anion-exchange high performance liquid chromatography in conjunction with enzymatic digestion and matrix-assisted laser desorption ionization time-of-flight spectrometry, which demonstrated a uniform linkage tetrasaccharide structure of -GlcUA-Gal-Gal-Xyl-or -GlcUA-Gal-Gal-Xyl(2-O-phosphate)-for C. elegans chondroitin and D. melanogaster CS, respectively. In contrast, the unmodified and phosphorylated counterparts were demonstrated in heparan sulfate of adult flies at a molar ratio of 73:27, and in that of the immortalized D. melanogaster S2 cell line at a molar ratio of 7:93, which suggests that the linkage region in the fruit fly first becomes phosphorylated uniformly on the Xyl residue and then dephosphorylated. It has been established here that GAG chains in both C. elegans and D. melanogaster are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide sequence, suggesting that indispensable functions of the linkage region in the GAG synthesis have been well conserved during evolution. Mutations affecting the genes encoding putative proteins related to GAG biosynthetic enzymes have also been described for these organisms. Mutations in the tout velu (ttv) gene of D. melanogaster cause defects in Hedgehog movement in mosaic wing discs (1, 6). The ttv gene is a putative ortholog of vertebrate EXT1, which encodes a heparan polymerase and is associated with the hereditary multiple exostoses syndrome in humans (7). The pipe gene, which affects dorsal-ventral patterning of D. melanogaster development, encodes a homolog of vertebrate HS 2-O-sulfotransferase (2, 8). The sugarless and sulfateless genes, both of which affect the fibroblast growth factor signaling during the D. melanogaster development, en-

“…2-Aminobenzamide (2AB) was purchased from Nacalai Tesque (Kyoto, Japan). Unsaturated disaccharides derived from Hep and HS were prepared as described previously (26). The following 2AB-derivatives of the authentic unsaturated tetrasaccharides of the GAG-protein linkage region were prepared as described previously (24) from various CS preparations:…”

mentioning

confidence: 99%

Structural Characterization of Heparan Sulfate and Chondroitin Sulfate of Syndecan-1 Purified from Normal Murine Mammary Gland Epithelial Cells

Ueno¹,

Yamada²,

Zako³

et al. 2001

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Syndecan-1, present on the surfaces of normal murine mammary gland epithelial cells, is a transmembrane hybrid proteoglycan, which bears glycosaminoglycan (GAG) side chains of heparan sulfate (HS) and chondroitin sulfate (CS). Purified syndecan-1 ectodomains were analyzed for disaccharide composition and the GAG-protein linkage region after digestion with bacterial lyases. The HS chains contained predominantly a nonsulfated unit with smaller proportions of two monosulfated, two disulfated, and a trisulfated unit, whereas CS chains were demonstrated for the first time to bear GlcUA-GalNAc(4-O-sulfate) as a major component as well as GlcUA-GalNAc, GlcUA-GalNAc(6-O-sulfate), and an E disaccharide unit GlcUA-GalNAc(4,6-O-disulfate) as minor yet appreciable components. Two kinds of linkage region tetrasaccharides, GlcUA-Gal-Gal-Xyl and GlcUA-Gal-Gal-Xyl(2-O-phosphate), were found for the HS chains in a molar ratio of 55:45. In marked contrast, an additional sulfated tetrasaccharide, GlcUA-Gal(4-Osulfate)-Gal-Xyl, was demonstrated only for the CS chains, and the unmodified phosphorylated and sulfated components were present at a molar ratio of 55:26: 19. The present study thus provided conclusive evidence for the hypothesis that 4-O-sulfation of Gal is peculiar to CS chains in contrast to the phosphorylation of Xyl, which is common to both HS and CS chains. These modifications may be required for biosynthetic maturation of the linkage region tetrasaccharide sequence, which is a prerequisite for creating the repeating disaccharide region of GAG chains and/or biosynthetic selective chain assembly of CS and HS chains.