An anticoagulant fucan sulfate with hexasaccharide repeating units from the sea cucumber Holothuria albiventer
Graphical abstract
Introduction
Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWHs) have been the cornerstones of antithrombotic treatment and prophylaxis for the last 70 years, which are the only type of sulfated polysaccharide currently used as anticoagulant drugs [1,2]. The commercial sources of heparins are mainly porcine intestinal mucosa and bovine lung, where the heparin content is low [3]. The possibility that concomitants such as prions and viruses may be carried by the biological products combined with the increasing demand for antithrombotic therapies indicates the necessity to research for alternative sources of anticoagulant agents [4].
Marine echinoderm and brown alga are abundant sources of anticoagulant polysaccharides, such as fucoidans and fucan sulfates [[4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]]. Brown algal polysaccharides, called fucoidans, usually are mixtures of several sulfated polymers with complex monosaccharide composition, thus they are structurally non-regular and heterogeneous [[4], [5], [6], [7], [8], [9], [10]], which hinders the clarification of their structure-activity relationship [10,11]. Distinguishing from fucoidans, the fucans from echinoderm such as sea cucumber and sea urchin, often known as fucan sulfates (FSs), are structurally more regular for they comprise only one kind of monosaccharide [[12], [13], [14], [15], [16], [17], [18]]. The chemical structures of these fucan sulfates were found to be species-specific [12,17]. Thus, each new sulfated polysaccharide purified from a certain sea cucumber or sea urchin would be a new compound with unique structures and, consequently, with potential novel biological activities.
Recently, when searching for new anticoagulant sulfated polysaccharides, we obtained two fucan sulfates from two species of sea cucumbers Holothuria edulis and Ludwigothurea grisea [16,17]. Both fucan sulfates have a unique structure composed of a central core of regular (1 → 2) and (1 → 3)-linked tetrasaccharide repeating units. Approximately 50% units of the FS from L. grisea (100% for H. edulis FS) contain side chains that are formed by nonsulfated fucose residues and linked to the O-4 positions of the central core. Anticoagulant activity assays indicated that the sea cucumber FSs can strongly inhibit human blood clotting through the intrinsic pathway of the coagulation cascade. Their distinctive structure with the tetrasaccharide repeating units contributes to the anticoagulant action [17].
Further exploration of sulfated polysaccharides from other sea cucumber species would provide a wider insight into the studies on their chemical structures and functional activities. In the present study, we discovered a new fucan sulfate from the sea cucumber Holothuria albiventer. The chemical structure was analyzed by chemical and instrumental methods such as Fourier transform infrared spectroscopy (FT−IR), high performance liquid chromatography (HPLC), monosaccharide composition analysis, and nuclear magnetic resonance (NMR) spectroscopies (1D 1H, 13C, 2D 1H/1H COSY, TOCSY, ROESY, 2D13C/1H HSQC and HMBC). Moreover, its effect on the clotting time of human plasma and intrinsic coagulation factor Xase complex were investigated. Our results may provide valuable information for understanding the structure-function relationships of the well-defined polysaccharides from invertebrate.
Section snippets
Purification and physicochemical characterization
The body wall of sea cucumber usually contains two types of sulfated polysaccharides, fucosylated chondroitin sulfate and fucan sulfate [[16], [17], [18], [19]]. The crude polysaccharides, with the yield of about 8.0% by dry weight, were extracted from the sea cucumber H. albiventer by the papain enzymolysis and alkaline hydrolysis [16]. The fucosylated chondroitin sulfate was partially removed by ethanol precipitation and KOAc salting out [19,20]. Then the crude fucan sulfate was further
Conclusions
In this study, we characterized the chemical structures of the fucan sulfate from the sea cucumber H. albiventer by chemical analysis, IR and 2D NMR spectroscopy techniques. Our data revealed that the sulfated polysaccharide is composed of regular α(1 → 3) linked hexasaccharide repeating units with a distinctive sulfation pattern. Anticoagulant assays indicated that the fucan sulfate possessed strong APTT and TT prolonging activities and intrinsic factor Xase inhibitory activity. The in vitro
Materials
Dried sea cucumber H. albiventer was purchased in seafood markets in Guangzhou city of Guangdong province (China). Amberlite FPA98Cl ion exchange resin was purchased from Rohm and Haas Company (USA). The monosaccharide standards including D-glucuronic acid (GlcA), D-glucose (Glc), D-galactose (Gal) and D-mannose (Man) were purchased from Alfa Aesar. L-rhamnose (Rha) was purchased from TCI. L-fucose (Fuc) and 1-phenyl-3-methyl-5-pyrazolone (PMP, 99%) were purchased from Sigma Chemical Co.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
This work was funded in part by the National Natural Science Foundation of China (81673330, 81703374, and 81773737), Yunnan Provincial Science and Technology Department in China (2016FA050 and 2014FB078), a grant from Youth Innovation Promotion Association (2017435), Kunming Institute of Botany (KIB2017011), Discovery, Evaluation and Transformation of Active Natural Compounds, Strategic Biological Resources Service Network Programme (ZSTH-020) of Chinese Academy of Sciences.
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