Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues.

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.

The structure of a unique fucose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the 0-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position 0-3 of the @-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-su~fo-@-D-g~ucuronosy~ residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-a-L-fucopyranosyl units glycosidically linked through position 1 + 2 to 4-O-sulfo-c~-~-fucopyranose. Finally, the presence of unusual 416-disulfated disaccharide units, together with the common 6-sulfated and nonsulfated units, was detected in the chondroitin sulfate core of this polysaccharide.
During the last few years we have searched for sulfated polysaccharides in different invertebrate connective tissues. The main purpose of such studies is to compare these polysaccharides with the well known glycosaminoglycans that occur in vertebrate tissues and to relate their structure with physicochemical and biological properties. In previous studies, * This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq), Fundaciio Banco do Brad (FBB), Fundaciio de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Financiadora de Estudos e Projetos (FI-NEP). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence and reprint requests should be addressed.
In the body wall of a sea cucumber, we found that the main fraction of sulfated polysaccharide has a chondroitin sulfatelike structure, containing unexpectedly large numbers of a-Lfucopyranose branches linked to position 3 of the p-D-glucuronic acid residues (7). Some other chondroitin sulfates containing branches have been described. Thus, a chondroitin sulfate containing small amounts of glucose branches substituted at carbon 6 of the hexosamine moieties was previously found in squid cartilage (8). In addition, a polysaccharide consisting of a desulfated chondroitin backbone, to which pfructofuranose is linked at carbon 3 of the D-glucuronic acid, was isolated from the capsular polysaccharide of a uropathogenic Escherichia coli (9). The squid glycosaminoglycan can be degraded by prolonged incubation with chondroitinase ABC (8). In contrast, the a-L-fucopyranose and p-fructofuranose branches obstruct the access of the chondroitinases to the chondroitin core. However, after partial acid hydrolysis, which removes these branches, the polymers obtained are degraded by chondroitinases (7,9).
In the present work, we report new data on the structure of the fucose-branched chondroitin sulfate from the sea cucumber body wall. Methylation and chondroitinase degradation of the native and chemically modified polysaccharide, together with immunoreaction with a monoclonal antibody, suggest that besides the fucose branches, this chondroitin contains sulfate esters a t position 3 of the p-D-glucuronic acid. In analogy with the fucose branched units, the 3-0-sulfo-p-Dglucuronosyl residues are resistant to chondroitinase degradation.  (Table V) suggests that the fucopyranosyl side chains are glycosidically linked through position 1 + 2. Regarding the site of sulfation, the methylation (Table 111) and the 'H NMR (Table V and Fig. 4) studies suggest high heterogeneity. After partial acid hydrolysis (lower portion of the figure), which removes the sulfated fucose residues from the polysaccharide at the positions labeled I , the polymer is susceptible to chondroitinase AC at the positions labeled 2, forming 4/6-disulfated, 6-sulfated, and nonsulfated disac- cucumber. This polysaccharide is totally resistant to chondroitinase degradation. However, after partial hydrolysis with acid (I, Fig. 7), which removes the sulfated fucose residues (7), the polymer is partially degraded by chondroitinase (2, Fig. 7) into 4/6-disulfated, 6-sulfated, and nonsulfated disaccharides ( Fig. 3 and Table 111). The desulfated polysaccharide is also partially degraded by chondroitinase, whereas after combined desulfation and defucosylation, the polymer is almost totally degraded by the enzyme (Fig. 2). Overall, these results suggest that fucose branches and sulfate esters obstruct the access of chondroitinase to the chondroitin core of the sea cucumber polysaccharide.
Comparison of the methylated derivatives obtained from native, desulfated, and defucosylated carboxyl-reduced chondroitin from sea cucumber suggests that the fucose branches and sulfate esters are linked to the 0-3 position of the p-Dglucuronic acid units from the chondroitin core (Table 111). The presence of 3-O-sulfo-~-D-glucuronosyl residues is also confirmed by immunoreaction of the native polymer with monoclonal antibody anti-Leu-7 (Fig. 6).
Although different types of sulfate esters had been described in glycosaminoglycans, this is the first report of a GalNAc bearing a sulfate ester at position 6 and at positions 4 and 6, respectively, of the hexosamine moiety. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. glucuronic acid residue sulfated a t position 0-3. Another rare feature of the sea cucumber chondroitin sulfate is the presence of 4/6-disulfated galactosamine units.
Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, the 'H NMR studies ( Fig. 4 and Table V) suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-c~-~-fucopyranosyl units glycosidically linked through position 1 + 2 to 4-O-sulfo-a-~-fucopyranose (Table I11 and Fig. 4).
The biological relevance of this unusual polysaccharide is still unclear. Possibly, the presence of fucose branches and sulfate esters linked to the 0-3 position of the P-D-glUCUrOniC acid, which make this glycosaminoglycan resistant to degradation by hyaluronidase and chondroitinase, serves to prevent digestion of the sea cucumber body wall by microorganisms present in the marine environment.

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Desulfatioai Desulfation of the polyeamharide wae performad as described previously (7 Uethylation studies, Previously we reported that fucose branchea in the chondroitin sulfate from sea cucumber obstruct the access Of chondroitinase AC or ABC to the chondroitin sulfate C O~, and that after defucosylation the polymer is degraded by these enzymes to form saturated and unsaturated disaccharides (7). However, the experiments of Pig. 2 and Table I 1     The fucose residues show considerable heterogeneity in the native polysaccharide (Fig. 4 A ) . Five different fucose H -1 Signals and Seven slightly different H-5/H-6 pairs are distinguishable in the COSY spectrum and perhaps to the presence of some monosaccharide fucose SideChaine in the (data nor shown). This heterogeneity may be due to Variations in sulfation, "dtiVe polymer.