Abstract
Fucosylated glycosaminoglycan (FG), a structurally complex glycosaminoglycan found up to now exclusively in sea cucumbers, has distinct anticoagulant properties, notably a strong inhibitory activity of intrinsic factor Xase complex (FXase). Knowledge of the FG structures could facilitate the development of a clinically effective intrinsic FXase inhibitor for anticoagulant drugs. Here, a new fucosylated glycosaminoglycan was obtained from the widely traded sea cucumber Bohadschia argus The precise structure was deduced as {→4)-[l-Fuc3S4S-α-(1→3)-]-d-GlcA-β-(1→3)-d-GalNAc4S6S-β-(1} through analysis of its chemical properties and homogeneous oligosaccharides purified from its β-eliminative depolymerized products. The B. argus FG with mostly 3,4-di-O-sulfated fucoses expands our knowledge on FG structural types. This β-elimination process, producing oligosaccharides with well-defined structures, is a powerful tool for analyzing the structure of complex FGs. Among these oligosaccharides, an octasaccharide displayed potent FXase inhibitory activity. Compared with oligosaccharides with various degrees of polymerization (3n and 3n - 1), our analyses reveal that the purified octasaccharide is the minimum structural unit responsible for the potent selective FXase inhibition, because the d-talitol in the nonsaccharide is unnecessary. The octasaccharide with 2,4-di-O-sulfated fucoses is more potent than that of one with 3,4-di-O-sulfated fucoses. Thus, sulfation patterns can play an important role in the inhibition of intrinsic factor Xase complex.
Highlights
Fucosylated glycosaminoglycan (FG), a structurally complex glycosaminoglycan found up to now exclusively in sea cucumbers, has distinct anticoagulant properties, notably a strong inhibitory activity of intrinsic factor Xase complex (FXase)
FG has been recognized to possess a backbone of chondroitin sulfate consisting of the disaccharide repeating units of glucuronic acid (GlcA) and GalNAc residues, and fucose (Fuc) branches linked to the O-3 of GlcA (1)
Compared with chemical structures of these oligosaccharides (Fig. 5), we found that at least three trisaccharide repeating units might be required both for the strong intrinsic anticoagulant activity and the potent selective inhibition of intrinsic factor Xase complex
Summary
Crude polysaccharides from B. argus were isolated by the methods of papain enzymolysis, alkaline hydrolysis, and ethanol precipitation (20). GalNAc and Fuc of the native FG were readily confirmed by protons with the distinctive values of 1.98 ppm (COCH3) and 1.28 ppm (CH3) in the 1H NMR spectrum, respectively (Fig. S3). Characteristic proton values of 1.23 and 1.93 ppm in the 1H NMR spectrum were assigned as CH3 of Fuc and COCH3 of GalNAc-ol (rA), respectively. Further analysis of the NMR spectra indicates that minor Fuc2S4S residues existing in these purified oligosaccharides are ␣-(133)–linked to GlcA To assess the anticoagulant activities, the native FG from B. argus, its depolymerized product dFG and compounds [1,2,3] were evaluated and compared with the nonasaccharide (R2) (14) and octasaccharide (R1) (16) from our previous reports. APTT-prolonging activities of homogeneous oligosaccharides 3, R1, and R2 were 47.43, 25.11, and 27.57 g/ml, respectively (Table 2) These results indicated potent intrinsic anticoagulant activities of these compounds.
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