Zi Li

Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan

Significance Selective inhibition of the intrinsic coagulation pathway is a promising strategy for developing safer anticoagulants without serious bleeding consequences. We prepared and identified a series of oligosaccharides as inhibitors of the intrinsic tenase, which is the final and rate-limiting enzyme complex in the intrinsic coagulation pathway and is an attractive but less explored target for anticoagulants due to the lack of a pure selective inhibitor. Analysis of these purified oligosaccharides reveals the precise structure of fucosylated glycosaminoglycan. Among these oligosaccharides, nonasaccharide is the minimum fragment that retains potent anticoagulant activity by selective inhibition of the intrinsic tenase while avoiding adverse effects and, thus, it may pave the way for the development of better treatments for thromboembolic diseases. Selective inhibition of the intrinsic coagulation pathway is a promising strategy for developing safer anticoagulants that do not cause serious bleeding. Intrinsic tenase, the final and rate-limiting enzyme complex in the intrinsic coagulation pathway, is an attractive but less explored target for anticoagulants due to the lack of a pure selective inhibitor. Fucosylated glycosaminoglycan (FG), which has a distinct but complicated and ill-defined structure, is a potent natural anticoagulant with nonselective and adverse activities. Herein we present a range of oligosaccharides prepared via the deacetylation–deaminative cleavage of FG. Analysis of these purified oligosaccharides reveals the precise structure of FG. Among these fragments, nonasaccharide is the minimum fragment that retains the potent selective inhibition of the intrinsic tenase while avoiding the adverse effects of native FG. In vivo, the nonasaccharide shows 97% inhibition of venous thrombus at a dose of 10 mg/kg in rats and has no obvious bleeding risk. This nonasaccharide may therefore serve as a novel promising anticoagulant.

Anti-HIV-1 activity and structure-activity-relationship study of a fucosylated glycosaminoglycan from an echinoderm by targeting the conserved CD4 induced epitope

BACKGROUND Fucosylated glycosaminoglycan (FG) is a novel glycosaminoglycan with a chondroitin sulfate-like backbone and fucose sulfate branches. The aim of this study is to investigate the mechanism and structure-activity relationships (SAR) of FG for combating HIV-1 infection. METHODS Anti-HIV activities of FGs were assessed by a cytopathic effect assay and an HIV-1 p24 detection assay. The biomolecule interactions were explored via biolayer interferometry technology. The SAR was established by comparing its anti-HIV-1 activities, conserved CD4 induced (CD4i) epitope-dependent interactions and anticoagulant activities. RESULTS FG efficiently and selectively inhibited the X4- and R5X4-tropic HIV-1 infections in C8166 cells with little cytotoxicity against C8166 cells and PBMCs. Our data indicated that FG bound to gp120 with nanomolar affinity and may interact with CD4i of gp120. Additionally, the CD4i binding affinity of FG was higher than that of dextran sulfate. SAR studies suggested that the unique sulfated fucose branches account for the anti-HIV-1 activity. The molecular size and present carboxyl groups of FG may also play important roles in various activities. Notably, several FG derivatives showed higher anti-HIV-1 activities and much lower anticoagulant activities than those of heparin. CONCLUSIONS FG exhibits strong activity against X4- and R5X4-tropic HIV-1 infections. The mechanism may be related to targeting CD4i of gp120, which results in inhibition of HIV-1 entry. The carboxyl group substituted derivatives of FG (8.5-12.8kDa), might display high anti-HIV-1 activity and low anticoagulant activity. GENERAL SIGNIFICANCE Our data supports further the investigation of FG derivatives as novel HIV-1 entry inhibitors targeting CD4i.

Preparation and characterization of O-acylated fucosylated chondroitin sulfate from sea cucumber.

Fucosylated chondroitin sulfate (FuCS), a kind of complex glycosaminoglycan from sea cucumber, has potent anticoagulant activity. In order to understand the relationship between structures and activity, the depolymerized FuCS (dFuCS) was chosen to prepare its derivates by selective substitution at OH groups. Its O-acylation was carried out in a homogeneous way using carboxylic acid anhydrides. The structures of O-acylated derivatives were characterized by NMR. The results indicated that the 4-O-sulfated fucose residues may be easier to be acylated than the other ones in the sulfated fucose branches. But the O-acylation was always accompanied by the β-elimination, and the degree of elimination was higher as that of acylation was higher. The results of clotting assay indicated that the effect of partial O-acylation of the dFuCS on their anticoagulant potency was not significant and the O-acylation of 2-OH groups of 4-O-sulfated fucose units did not affect the anticoagulant activity.

Structural Analysis and Anticoagulant Activities of the Novel Sulfated Fucan Possessing a Regular Well-Defined Repeating Unit from Sea Cucumber

Sulfated fucans, the complex polysaccharides, exhibit various biological activities. Herein, we purified two fucans from the sea cucumbers Holothuria edulis and Ludwigothurea grisea. Their structures were verified by means of HPGPC, FT-IR, GC–MS and NMR. As a result, a novel structural motif for this type of polymers is reported. The fucans have a unique structure composed of a central core of regular (1→2) and (1→3)-linked tetrasaccharide repeating units. Approximately 50% of the units from L. grisea (100% for H. edulis fucan) contain sides of oligosaccharides formed by nonsulfated fucose units linked to the O-4 position of the central core. Anticoagulant activity assays indicate that the sea cucumber fucans strongly inhibit human blood clotting through the intrinsic pathways of the coagulation cascade. Moreover, the mechanism of anticoagulant action of the fucans is selective inhibition of thrombin activity by heparin cofactor II. The distinctive tetrasaccharide repeating units contribute to the anticoagulant action. Additionally, unlike the fucans from marine alga, although the sea cucumber fucans have great molecular weights and affluent sulfates, they do not induce platelet aggregation. Overall, our results may be helpful in understanding the structure-function relationships of the well-defined polysaccharides from invertebrate as new types of safer anticoagulants.

Structural Elucidation and Biological Activity of a Highly Regular Fucosylated Glycosaminoglycan from the Edible Sea Cucumber Stichopus herrmanni

Edible sea cucumbers are widely used as a health food and medicine. A fucosylated glycosaminoglycan (FG) was purified from the high-value sea cucumber Stichopus herrmanni. Its physicochemical properties and structure were analyzed and characterized by chemical and instrumental methods. Chemical analysis indicated that this FG with a molecular weight of ∼64 kDa is composed of N-acetyl-d-galactosamine, d-glucuronic acid (GlcA), and l-fucose. Structural analysis clarified that the FG contains the chondroitin sulfate E-like backbone, with mostly 2,4-di-O-sulfated (85%) and some 3,4-di-O-sulfated (10%) and 4-O-sulfated (5%) fucose side chains that link to the C3 position of GlcA. This FG is structurally highly regular and homogeneous, differing from the FGs of other sea cucumbers, for its sulfation patterns are simpler. Biological activity assays indicated that it is a strong anticoagulant, inhibiting thrombin and intrinsic factor Xase. Our results expand the knowledge on structural types of FG and illustrate its biological activity as a functional food material.

Structural analysis and biological activity of a highly regular glycosaminoglycan from Achatina fulica

Edible snails have been widely used as a health food and medicine in many countries. A unique glycosaminoglycan (AF-GAG) was purified from Achatina fulica. Its structure was analyzed and characterized by chemical and instrumental methods, such as Fourier transform infrared spectroscopy, analysis of monosaccharide composition, and 1D/2D nuclear magnetic resonance spectroscopy. Chemical composition analysis indicated that AF-GAG is composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) and its average molecular weight is 118kDa. Structural analysis clarified that the uronic acid unit in glycosaminoglycan (GAG) is the fully epimerized and the sequence of AF-GAG is →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. Although its structure with a uniform repeating disaccharide is similar to those of heparin and heparan sulfate, this GAG is structurally highly regular and homogeneous. Anticoagulant activity assays indicated that AF-GAG exhibits no anticoagulant activities, but considering its structural characteristic, other bioactivities such as heparanase inhibition may be worthy of further study.

Precise structures of fucosylated glycosaminoglycan and its oligosaccharides as novel intrinsic factor Xase inhibitors

Selective inhibition of the endogenous coagulation pathway is a promising strategy for developing new anticoagulants. Fucosylated glycosaminoglycan (FG), a structurally complex glycosaminoglycan, has distinct anticoagulant properties, especially the strong intrinsic factor Xase inhibitory activity that is recognized as a new target with potential physiological and therapeutic applications. Detailed knowledge of FG structures is necessary for developing a clinically effective intrinsic FXase inhibitor. However, challenges remain to elucidate FG structures as a basis for pharmaceutical development. Herein, using the highly selective β-elimination method, oligosaccharides with regular structures were prepared from the depolymerization products. Analysis of oligosaccharides further confirmed the precise structural sequence of the FG. Furthermore, biological activity assay suggested that these pure homogeneous oligosaccharides, particularly an octasaccharide, exhibit strong inhibition of the intrinsic coagulation pathway by inhibiting human intrinsic factor Xase. Our finding is significant for discovery of a new class of anticoagulant agents as intrinsic factor Xase inhibitors.