Sulfated Fucans Research Articles

A draft genome of the brown alga, Cladosiphon okamuranus, S-strain: a platform for future studies of 'mozuku' biology.

The brown alga, Cladosiphon okamuranus (Okinawa mozuku), is economically one of the most important edible seaweeds, and is cultivated for market primarily in Okinawa, Japan. C. okamuranus constitutes a significant source of fucoidan, which has various physiological and biological activities. To facilitate studies of seaweed biology, we decoded the draft genome of C. okamuranus S-strain. The genome size of C. okamuranus was estimated as ∼140 Mbp, smaller than genomes of two other brown algae, Ectocarpus siliculosus and Saccharina japonica. Sequencing with ∼100× coverage yielded an assembly of 541 scaffolds with N50 = 416 kbp. Together with transcriptomic data, we estimated that the C. okamuranus genome contains 13,640 protein-coding genes, approximately 94% of which have been confirmed with corresponding mRNAs. Comparisons with the E. siliculosus genome identified a set of C. okamuranus genes that encode enzymes involved in biosynthetic pathways for sulfated fucans and alginate biosynthesis. In addition, we identified C. okamuranus genes for enzymes involved in phlorotannin biosynthesis. The present decoding of the Cladosiphon okamuranus genome provides a platform for future studies of mozuku biology.

Wenyingzhuangia fucanilytica sp. nov., a sulfated fucan utilizing bacterium isolated from shallow coastal seawater.

A Gram-staining-negative, rod-shaped, strictly aerobic, non-flagellated, non-gliding and yellow-pigmented bacterial strain, designated as strain CZ1127T, capable of utilizing sulfated fucan, was isolated from shallow coastal seawater of Jiaozhou Bay, Qingdao, PR China. Its taxonomic position was investigated by a polyphasic approach. CZ1127T grew at 15-37 °C (optimum, 25 °C), pH 5.5-9.0 (optimum, pH 6.5-7.5) and in the presence of 0-4 % (w/v) NaCl [optimum, 0-3 % (w/v)]. CZ1127T contained MK-6 as the sole menaquinone, and iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1ω7c/C16 : 1ω6c and iso-C15 : 0 3-OH as its major fatty acids. The DNA G+C content of strain CZ1127T was 32.2 mol%. Phylogenetic analysis based on the 16S rRNA gene indicated that strain CZ1127T clustered with members of the genus Wenyingzhuangia and was closely related to Wenyingzhuangia marina CGMCC 1.12162T (97.4 % 16S rRNA gene sequence similarity) and Wenyingzhuangia gracilariae KCTC 42246T (96.1 %). The DNA-DNA relatedness between strain CZ1127T and W. marina CGMCC 1.12162T was 41.9±1.5 %. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, CZ1127T represents a novel species of the genus Wenyingzhuangia, for which the name Wenyingzhuangia fucanilytica sp. nov. is proposed. The type strain is CZ1127T (=CCTCC AB 2015089T=KCTC 42864T).

Abstract B44: Sulfated fucan inhibits tumor interaction with endothelial cells and tumor growth: Possible contribution of an antivascular endothelial growth factor (VEGF) neutralizing activity

Abstract The primary interaction between tumor and endothelial cells is mediated by endothelial selectins and sialyl-Lewis carbohydrates found on cancer cell surface. Heparin, a sulfated polysaccharide, binds to P- and L-selectins, an effect that has been associated with attenuation of metastasis. However, due to its strong anticoagulant activity, its use in anti-tumor therapy is restricted. Sulfated fucans from sea urchins have simple, unique structures of linear chains of sulfated α-L-fucose in well-defined repetitive patterns. In this study, we tested five sulfated fucans from different species of sea urchins for their ability to interfere with the tumor-endothelium interaction and compared our findings to those obtained with heparin. Our results show that sulfated fucan from L. variegatus (FucSulf I), bearing an unique 2,4-disulfated fucose residue and low anticoagulant activity, inhibits the adhesion of tumor cells to endothelium, tumor transendothelial migration and endothelial cell tubulogenesis, both by inhibiting P-selectin mediated tumor cell adhesion, and VEGF-dependent signaling in endothelial cells. FucSulf I also inhibited proliferation of several types of tumor cell lines in vitro and tumor growth in vivo. Our results indicate this sulfated fucan is a potential antitumor agent and a possible therapeutic alternative to heparin. Citation Format: Viviane Mignone, Camila Castro Figueiredo, Aline Oliveira, Eliene Kozlowski, Lubor Borsig, Mauro Pavão, Paulo Mourão, Veronica Morandi. Sulfated fucan inhibits tumor interaction with endothelial cells and tumor growth: Possible contribution of an antivascular endothelial growth factor (VEGF) neutralizing activity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B44.

Marine Non-Glycosaminoglycan Sulfated Glycans as Potential Pharmaceuticals.

Sulfated fucans (SFs) and sulfated galactans (SGs) are currently the marine non-glycosaminoglycan (GAG) sulfated glycans most studied in glycomics. These compounds exhibit therapeutic effects in several pathophysiological systems such as blood coagulation, thrombosis, neovascularization, cancer, inflammation, and microbial infections. As analogs of the largely employed GAGs and due to some limitations of the GAG-based therapies, SFs and SGs comprise new carbohydrate-based therapeutics available for clinical studies. Here, the principal structural features and the major mechanisms of action of the SFs and SGs in the above-mentioned pathophysiological systems are presented. Discussion is also given on the current challenges and the future perspectives in drug development of these marine glycans.

Anticoagulant and antithrombotic activities of modified xylofucan sulfate from the brown alga Punctaria plantaginea

Selectively and totally sulfated (1→3)-linked linear homofucans bearing ∼20 monosaccharide residues on average have been prepared from the branched xylofucan sulfate isolated from the brown alga Punctaria plantaginea. Anticoagulant and antithrombotic properties of the parent biopolymer and its derivatives were assessed in vitro. Highly sulfated linear fucan derivatives were shown to inhibit clot formation in APTT assay and ristocetin induced platelets aggregation, while the partially sulfated analogs were inactive. In the experiments with purified proteins, fucan derivatives with degree of sulfation of ∼2.0 were found to enhance thrombin and factor Xa inhibition by antithrombin III. The effect of sulfated fucans on thrombin inhibition, which was similar to those of heparinoid Clexane® (enoxaparin) and of a fucoidan from the brown alga Saccharina latissima studied previously, can be explained by the multicenter interaction and formation of a ternary complex thrombin–antithrombin III–polysaccharide. The possibility of such complexation was confirmed by computer docking study.

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.

Monoclonal antibodies directed to fucoidan preparations from brown algae.

Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance.

Sulfated glycans in sea urchin fertilization.

Fertilization is a controlled cell-cell interaction event that ultimately leads to the union of the gametes involved in reproduction. Fertilization is characterized by three major steps: (i) sperm binding to the extracellular matrix that coats the egg, inducing thereby the acrosome reaction; (ii) penetration of the acrosome-reacted sperm through the egg coat until its contact with the egg plasma membrane; and (iii) adhesion and fusion of the cell membranes of both gametes and the interchange of genetic materials. The acrosome reaction in the first step is important because it ensures that fertilization occurs only between gametes of homologous species. This specificity is primarily driven by the structure of egg jelly coat glycans recognized by a lectin-like binding protein (receptor) in the sperm membrane. Sea urchin fertilization is the best model utilizedfor understanding carbohydrate-mediated acrosome reactions. This report aims at describing the biochemical basis of regulatory mechanisms exerted by sea urchin sulfated fucans and galactans of well-defined chemical structures on the egg-sperm recognition process during fertilization of this invertebrate. Flagellasialin, a sulfated polysialic acid-containing glycoprotein found in sea urchin sperm flagella, is another sulfated glycan example also involved in fertilization of the echinoderm.

Anti-HSV1 activity of brown algal polysaccharides and possible relevance to the treatment of Alzheimer's disease

Herpes simplex virus type 1 (HSV1) induces the formation of the characteristic abnormal molecules of Alzheimer's disease (AD) brains, beta-amyloid, and abnormally phosphorylated, AD-like tau (P-tau). Formation of these molecules is inhibited by treatment with the antiviral agent acyclovir (ACV), which prevents viral DNA replication. A totally different mechanism of antiviral action against herpes simplex viruses is shown by sulfated fucans. The antiviral activity of sulfated fucans from five brown algae (Scytothamnus australis, Marginariella boryana, Papenfussiella lutea, Splachnidium rugosum and Undaria pinnatifida) was investigated in relation to the HSV1-induced formation of beta-amyloid, and AD-like tau. Antiviral activity was also related to specific structural features of these polysaccharides.Four sulfated fucan extracts each prevented the accumulation of HSV1-induced beta-amyloid and AD-like tau in HSV1-infected Vero cells. The structures of these extracts had some similarities but also key differences, indicating that a number of structural features can cause antiviral activity.The most active sulfated fucan combined with acyclovir was particularly effective, so may be particularly suitable for further experimental testing in order to develop treatment protocols for AD patients, with the aim of slowing or stopping disease progression.

Impact of sulfation pattern on the conformation and dynamics of sulfated fucan oligosaccharides as revealed by NMR and MD.

Sulfated fucans from sea urchin egg jelly express well-defined chemical structures that vary with species. This species specificity regulates the sperm acrosome reaction, a critical step to assure intra-specific fertilization. In addition, these polysaccharides are involved in other biological activities such as anticoagulation. Although sulfation patterns are relevant to the levels of response in both activities, conformation and dynamics of these glycans are also contributing factors. However, data about these features of sulfated fucans are very rare. To address this, we have employed nuclear magnetic resonance experiments combined with molecular dynamics on structurally defined oligosaccharides derived from two sulfated fucans. The results have indicated that the oligosaccharides are flexible in solution. Ring conformation of their composing units displays just the (1)C4 chair configuration. In a particular octasaccharide, composed of two tetrasaccharide sequences, inter-residual hydrogen bonds play a role to decrease dynamics in these repeating units. Conversely, the linking disaccharide [-3)-α-L-Fucp-2(OSO3(-))-(1-3)-α-L-Fucp-4(OCO3(-))-(1-] located right between the two tetrasaccharide units has amplified motions suggested to be promoted by electrostatic repulsion of sulfates on opposite sides of the central glycosidic bond. This conjunction of information about conformation and dynamics of sulfated fucan oligosaccharides provides new insights to explain how these glycans behave free in solution and influenced by sulfation patterns. It may also serve for future studies concerning structure-function relationship of sulfated fucans, especially those involving sea urchin fertilization and anticoagulation.

Sea, carbohydrates and clotting: a triad on the road of drug discovery.

Cardiovascular diseases (CDs) are the principal cause of death in the world. Anticoagulation is the commonest therapeutic strategy for treatments of CDs in clinical settings. Although possessed of numerous downsides, heparin is the main clinical anticoagulant/antithrombotic agent used so far. Novel sulfated polysaccharides like the marine dermatan sulfate, sulfated fucans and galactans are also able to block clot and thrombus formation. These relatively new marine glycans call special attention mostly due to their unique structures and distinct mechanisms of action. This structural uniqueness is seen by the peculiar aspect of these polysaccharides being made of clear and regular sulfation patterns. The structures have been reported only in polysaccharides from marine invertebrates like sea urchins and cucumbers. This report intends to prove the promising combination of the triad sea-carbohydrates-clotting in drug discovery of the cardiovascular field.

Molecular characterization and immunomodulatory activity of sulfated fucans from Agarum cribrosum

The sulfated-fucans, known as fucoidans, were isolated from Agarum cribrosum and fractionated using ion-exchange chromatography to determine their molecular characteristics and in vitro immunomodulatory activity. The crude and fractionated fucoidans (F1 and F2) consisted mostly of carbohydrates (52.4–56.0%), sulfates (12.7–23.0%) and uronic acid (14.1–21.8%), with a small amount of proteins (3.9–9.3%), and included various levels of fucose (44.0–46.7%), mannose (18.9–26.8%), galactose (16.8–33.0%), xylose (10.7–17.0%) and glucose (3.5–9.5%). The crude and fractionated fucans contained one or two subfractions with average molecular weights (Mw) ranging from 110.1×103 to 2420×103g/mol. The fractionated fucoidan, especially the F1 fraction, strongly stimulated murine macrophages (Raw 264.7 cells), producing a considerable amount of nitric oxide (NO) and inducing expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10) transcripts by activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) pathways. The maximally immunoenhancing F1 fraction was mainly composed of (1→3)-linked fucose, (1→2)-linked mannose and (1→4)-linked glucuronic acid with sulfates at C-2 or both the C-2 and C-4 positions in (1→2,3)- and (1→2,3,4)-linked fucose residues.

Fucoidans as a platform for new anticoagulant drugs discovery

AbstractAnionic fucose-containing polysaccharides (fucoidans of brown seaweeds, sulfated fucans and fucosylated chondroitin sulfates of invertebrates) are attracting a rapidly growing research interest due to different types of their biological activity discovered in recent years. In particular, algal fucoidans are characterized by large structural variations depending on the species used for their isolation and by the lack of structural regularity due to random distribution of both carbohydrate and non-carbohydrate substituents along the polymer chains. These features make it difficult to find distinct correlations between structural elements and biological properties of polysaccharides. Nevertheless, there is expectation that systematic structural and biochemical studies of fucoidans will form a basis for the development of new drugs. Herewith we summarize our recent results on the influence of fucoidan structure on blood coagulation.

Applications of mass spectrometry to structural analysis of marine oligosaccharides.

Marine oligosaccharides have attracted increasing attention recently in developing potential drugs and biomaterials for their particular physical and chemical properties. However, the composition and sequence analysis of marine oligosaccharides are very challenging for their structural complexity and heterogeneity. Mass spectrometry (MS) has become an important technique for carbohydrate analysis by providing more detailed structural information, including molecular mass, sugar constituent, sequence, inter-residue linkage position and substitution pattern. This paper provides an overview of the structural analysis based on MS approaches in marine oligosaccharides, which are derived from some biologically important marine polysaccharides, including agaran, carrageenan, alginate, sulfated fucan, chitosan, glycosaminoglycan (GAG) and GAG-like polysaccharides. Applications of electrospray ionization mass spectrometry (ESI-MS) are mainly presented and the general applications of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) are also outlined. Some technical challenges in the structural analysis of marine oligosaccharides by MS have also been pointed out.

Chemical and enzymatic fractionation of cell walls from Fucales: insights into the structure of the extracellular matrix of brown algae.

Brown algae are photosynthetic multicellular marine organisms evolutionarily distant from land plants, with a distinctive cell wall. They feature carbohydrates shared with plants (cellulose), animals (fucose-containing sulfated polysaccharides, FCSPs) or bacteria (alginates). How these components are organized into a three-dimensional extracellular matrix (ECM) still remains unclear. Recent molecular analysis of the corresponding biosynthetic routes points toward a complex evolutionary history that shaped the ECM structure in brown algae. Exhaustive sequential extractions and composition analyses of cell wall material from various brown algae of the order Fucales were performed. Dedicated enzymatic degradations were used to release and identify cell wall partners. This approach was complemented by systematic chromatographic analysis to study polymer interlinks further. An additional structural assessment of the sulfated fucan extracted from Himanthalia elongata was made. The data indicate that FCSPs are tightly associated with proteins and cellulose within the walls. Alginates are associated with most phenolic compounds. The sulfated fucans from H. elongata were shown to have a regular α-(1→3) backbone structure, while an alternating α-(1→3), (1→4) structure has been described in some brown algae from the order Fucales. The data provide a global snapshot of the cell wall architecture in brown algae, and contribute to the understanding of the structure-function relationships of the main cell wall components. Enzymatic cross-linking of alginates by phenols may regulate the strengthening of the wall, and sulfated polysaccharides may play a key role in the adaptation to osmotic stress. The emergence and evolution of ECM components is further discussed in relation to the evolution of multicellularity in brown algae.

Anticoagulant motifs of marine sulfated glycans.

Sulfated polysaccharides, like the glycosaminoglycan (GAG) heparin, are known to exhibit anticoagulant properties when certain structural features are present. The structural requirement for this action is well-established for heparin, in which a pentasaccharide motif plays a key role for keeping the high-affinity interaction to antithrombin. Over the last years of this glycomic era, several novel anticoagulant sulfated glycans have been described. Those from marine sources have been awakening special attention mainly because of their impressive anticoagulant effects together with structural uniqueness. The commonest of these glycans are the sulfated fucans (SFs), the sulfated galactans (SGs), and the marine invertebrate GAGs like the fucosylated chondroitin sulfate and ascidian dermatan sulfate. Since these marine sulfated glycans do not bear within their polymeric chains the specific pentasaccharide motif of heparin, other structural features must be necessary to trigger the anticoagulant effect. The objective of this report is to present the anticoagulant motifs of the marine SFs, SGs and GAGs.

Water soluble sulfated-fucans with immune-enhancing properties from Ecklonia cava

Water-soluble sulfated fucans isolated from Ecklonia cava were fractionated using an anion-exchange chromatography to investigate their molecular characteristics and immunomodulating activities. The crude fucoidan extract and purified fractions (EF1, EF2, and EF3) consisted mostly of different ratios of neutral sugars, proteins, sulfates, uronic acids, and their monosaccharide compositions were also significantly different. The backbone of the most immunoenhancing fraction, EF2, was mainly linked by (1→3)-linked fucopyranosyl and (1→4)-linked mannopyranosyl residues with sulfates at C-4 of fucopyranosyl units. The molecular weights of the crude fucoidan extract and purified fractions ranged from 8.3×103 to 442.6×103g/mol. The crude extract, EF1 and EF2 stimulated RAW264.7 cells to produce considerable amounts of nitric oxide and cytokines. The treatment of cells with the sulfated fucans induced the degradation of Iκ-B and the phosphorylation of MAPK in RAW264.7 cells, implying that they might stimulate RAW264.7 cells through the activation of NF-κB and MAPK pathways.

Specific sulfation and glycosylation-a structural combination for the anticoagulation of marine carbohydrates.

Based on considered achievements of the last 25 years, specific combinations of sulfation patterns and glycosylation types have been proved to be key structural players for the anticoagulant activity of certain marine glycans. These conclusions were obtained from comparative and systematic analyses on the structure-anticoagulation relationships of chemically well-defined sulfated polysaccharides of marine invertebrates and red algae. These sulfated polysaccharides are known as sulfated fucans (SFs), sulfated galactans (SGs) and glycosaminoglycans (GAGs). The structural combinations necessary for the anticoagulant activities are the 2-sulfation in α-L-SGs, the 2,4-di-sulfation in α-L-fucopyranosyl units found as composing units of certain sea-urchin and sea-cucumber linear SFs, or as branching units of the fucosylated chondroitin sulfate, a unique GAG from sea-cucumbers. Another unique GAG type from marine organisms is the dermatan sulfate isolated from ascidians. The high levels of 4-sulfation at the galactosamine units combined with certain levels of 2-sulfation at the iduronic acid units is the anticoagulant structural requirements of these GAGs. When the backbones of red algal SGs are homogeneous, the anticoagulation is proportionally dependent of their sulfation content. Finally, 4-sulfation was observed to be the structural motif required to enhance the inhibition of thrombin via heparin cofactor-II by invertebrate SFs.

Marine medicinal glycomics

Glycomics is an international initiative aimed to understand the structure and function of the glycans from a given type of cell, tissue, organism, kingdom or even environment, as found under certain conditions. Glycomics is one of the latest areas of intense biological research. Glycans of marine sources are unique in terms of structure and function. They differ considerably from those of terrestrial origin. This review discusses the most known marine glycans of potential therapeutic properties. They are chitin, chitosan, and sulfated polysaccharides named glycosaminoglycans, sulfated fucans, and sulfated galactans. Their medical actions are very broad. When certain structural requirements are found, these glycans can exhibit beneficial effects in inflammation, coagulation, thrombosis, cancer growth/metastasis, and vascular biology. Both structure and therapeutic mechanisms of action of these marine glycans are discussed here in straight context with the current glycomic age through a project suggestively named marine medicinal glycomics.

How to analyze the anticoagulant and antithrombotic mechanisms of action in fucanome and galactanome?

Through the perspective of the current glycomics age, fucanomics and galactanomics denote the international projects concerned with the studies of the biomedically active marine sulfated fucose- or galactose-composed polysaccharides, named sulfated fucans (SFs), and sulfated galactans(SGs), respectively. SFs and SGs are isolated from algae or marine invertebrates. The range of therapeutic actions of SFs and SGs is impressively broad. When certain structural requirements are found, some SFs and SGs may exhibit beneficial properties in inflammation, nociception, hemostasis (coagulation and thrombosis), vascular biology (angiogenesis), oncology, oxidative-stress, and virus infections. Although many biomedical applications for SFs and SGs have been pointed out over the past two decades, only inflammation, hemostasis, cancer, and vascular biology have their mechanisms of action satisfactorily elucidated. In addition, advanced structure-function relationships have been achieved only for the anticoagulant and antithrombotic activities, in which glycans of well-defined structures have been assayed. Because of this, the activities of SFs and SGs in stopping the clot and thrombus formation represent the closest therapeutic areas of having these glycans truly explored for drug development. Here, through an analytical viewpoint, we present the common methods and protocols employed to achieve such advanced structure-function relationships of SFs and SGs in anticoagulation and antithrombosis.