Sulfated Galactans Research Articles

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.

Enzyme-Assisted Preparation of Furcellaran-Like κ-/β-Carrageenan

Carrageenans are sulfated galactans that are widely used in industrial applications for their thickening and gelling properties, which vary according to the amount and distribution of ester sulfate groups along the galactan backbone. To determine and direct the sulfation of κ-carrageenan moieties, we purified an endo-κ-carrageenan sulfatase (Q15XH1 accession in UniprotKB) from Pseudoalteromonas atlantica T6c extracts. Based on sequence analyses and exploration of the genomic environment of Q15XH1, we discovered and characterized a second endo-κ-carrageenan sulfatase (Q15XG7 accession in UniprotKB). Both enzymes convert κ-carrageenan into a hybrid, furcellaran-like κ-/β-carrageenan. We compared the protein sequences of these two new κ-carrageenan sulfatases and that of a previously reported ι-carrageenan sulfatase with other predicted sulfatases in the P. atlantica genome, revealing the existence of additional new carrageenan sulfatases.

Extraction process optimization of sulfated galactan-rich fractions from Hypnea musciformis in order to obtain antioxidant, anticoagulant, or immunomodulatory polysaccharides

The seaweed Hypnea musciformis synthesizes sulfated galactans and these galactans have been reported to have antioxidant, anticoagulant, and immunostimulatory properties. However, efficient extraction of these polysaccharides depends on the particular extraction method used, some of these polysaccharides are not extracted. Previously, we obtained galactans with different properties by using a simple extraction method. Here, we performed slight modifications to the extraction method, which led to variation in the type of polysaccharides obtained and their biological activity. We obtained four sulfated galactan-rich fractions named as FT4v, FT6v, FT8v, and FT10v. The chemical composition data showed that the fractions had high sugar and sulfate contents. Fraction FT4v exhibited the highest sulfate/sugar ratio (0.47), while FT6v (0.37), FT8v (0.39), and FT10v (0.39) presented lower sulfate/sugar ratios. Fractions FT4v and FT6v showed the highest antioxidant activity. Fraction FT4v also exhibited the greatest anticoagulant potential; however, other fractions presented marked immunomodulatory action. FT10v stimulated the highest levels of NO production whereas FT6v stimulated the highest expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in RAW cells. Fraction FT10v exhibited the greatest cytotoxic action of the obtained fractions against HeLa and 786 cell lines. In vitro tests showed that the sulfated galactan-rich fractions isolated from H. musciformis with low sulfate/sugar ratios, i.e., the fractions containing fewer sulfated polysaccharides (FT6v, FT8v, and FT10v), showed marked immunostimulatory action while the fraction with the high sulfate/sugar ratio (FT4v) exhibited improved anticoagulant activity. Therefore, our results suggest that different extraction conditions promote the acquisition of sulfated galactan-rich fractions from H. musciformis with different biological activities.

Sulfated galactans from Gracilaria fisheri bind to shrimp haemocyte membrane proteins and stimulate the expression of immune genes.

Previous studies demonstrated that sulfated galactans (SG) from Gracilaria fisheri (G. fisheri) exhibit immunostimulant activity in shrimp. The present study was conducted to test the hypothesis that SG stimulates signaling molecules of the immune response of shrimp by binding to receptors on the host cell membrane. Accordingly, we evaluated the ability of SG to bind to shrimp haemocytes and showed that SG bound to the shrimp haemocyte membrane (SHM), potentially to specific receptors. Furthermore, this binding was associated with an activation of immune response genes of shrimp. Data from confocal laser scanning micrographs revealed that FITC-labeled SG bound to haemocytes. Far western blot analysis demonstrated that SHM peptides, with molecular sizes of 13, 14, 15, 17, and 25 kDa, were associated with SG. Peptide sequence analysis of the isolated bands using LC-MS/MS and NCBI blast search revealed the identity of the 13, 14, and 17 kDa peptides as lipopolysaccharide and β-1,3-glucan binding protein (LGBP). SG induced the expression of immune related genes and downstream signaling mediators of LGBP including IMD, IKKs, NF-κB, antimicrobial peptides (crustin and PEN-4), the antiviral immunity (dicer), and proPO system (proPO-I and proPO-II). A LGBP neutralizing assay with anti-LGBP antibody indicated a decrease in SG-induced expression of LGBP downstream signaling mediators and the immune related genes. In conclusion, this study demonstrated that the SG-stimulated immune activity in haemocytes is mediated, in part, through the LGBP, and IMD-NF-κB pathway.

Proteins and Carbohydrates from Red Seaweeds: Evidence for Beneficial Effects on Gut Function and Microbiota.

Based on their composition, marine algae, and namely red seaweeds, are good potential functional foods. Intestinal mucosal barrier function refers to the capacity of the intestine to provide adequate containment of luminal microorganisms and molecules. Here, we will first outline the component of seaweeds and will summarize the effects of these on the regulation of mucosal barrier function. Special attention will be paid to unique components of red seaweeds: proteins and derived peptides (e.g., phycobiliproteins, glycoproteins that contain “cellulose binding domains”, phycolectins and the related mycosporine-like amino acids) together with polysaccharides (e.g., floridean starch and sulfated galactans, such as carrageenans, agarans and “dl-hybrid”) and minerals. These compounds have been shown to exert prebiotic effects, to regulate intestinal epithelial cell, macrophage and lymphocyte proliferation and differentiation and to modulate the immune response. Molecular mechanisms of action of peptides and polysaccharides are starting to be elucidated, and evidence indicating the involvement of epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), Toll-like receptors (TLR) and signal transduction pathways mediated by protein kinase B (PKB or AKT), nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) will also be summarized. The need for further research is clear, but in vivo experiments point to an overall antiinflammatory effect of these algae, indicating that they can reinforce membrane barrier function.

Carrageenans from nuclear phases of subantartic Mazzaella laminarioides (Gigartinales, Rhodophyta) and graft copolymerization of alkali-modified carrageenan with acrylamide

Aqueous extraction of cystocarpic, tetrasporic, and vegetative individuals of Mazzaella laminariodes collected in the Magellan Ecoregion, Southern Chile, afforded sulfated galactans. Analysis by Fourier transform infrared spectroscopy (FT-IR) and by 1H and 13C NMR spectroscopy indicated that purified extracts from cystocarpic (CP) and vegetative individuals (VP) were mixtures of κ/ι-type carrageenans and their precursors μ- and ν-carrageenans, whereas the aqueous extract from tetrasporic individuals was a λ-type carrageenan. Alkaline treatment of CP and VP polysaccharides produced modified polysaccharides with lower content of sulfate groups and with a concomitant increase of 3,6-anhydrogalactose content; analysis by 2D 1H and 13C NMR indicated the presence of hybrid κ-ι-carrageenans. Copolymerization of alkali-treated CP carrageenan with acrylamide was achieved by using ceric ammonium nitrate as the initiator; studies by NMR spectroscopy indicated grafting of polyacrylamide at position C-6 of β-galactopyranosyl residues. Reaction of modified CP with acrylamide assisted by microwave irradiation in the presence of ammonium persulfate afforded a copolymer grafted at position C-2 of β-galactopyranosyl residues.

Sulfated Galactan from Palisada flagellifera Inhibits Toxic Effects of Lachesis muta Snake Venom.

In Brazil, snakebites are a public health problem and accidents caused by Lachesis muta have the highest mortality index. Envenomation by L. muta is characterized by systemic (hypotension, bleeding and renal failure) and local effects (necrosis, pain and edema). The treatment to reverse the evolution of all the toxic effects is performed by injection of antivenom. However, such therapy does not effectively neutralize tissue damage or any other local effect, since in most cases victims delay seeking appropriate medical care. In this way, alternative therapies are in demand, and molecules from natural sources have been exhaustively tested. In this paper, we analyzed the inhibitory effect of a sulfated galactan obtained from the red seaweed Palisada flagellifera against some toxic activities of L. muta venom. Incubation of sulfated galactan with venom resulted in inhibition of hemolysis, coagulation, proteolysis, edema and hemorrhage. Neutralization of hemorrhage was also observed when the galactan was administered after or before the venom injection; thus mimicking a real in vivo situation. Moreover, the galactan blocked the edema caused by a phospholipase A2 isolated from the same venom. Therefore, the galactan from P. flagellifera may represent a promising tool to treat envenomation by L. muta as a coadjuvant for the conventional antivenom.

Polysaccharides of algae 67. Carrageenan from Pacific red alga Turnerella mertensiana (Gigartinales, Rhodophyta)

A sulfated galactan composed of nearly equimolar amounts of d-galactose, 3,6-anhydro-d-galactose, and sulfate was isolated from the red alga Turnerella mertensiana collected in the Sea of Japan. The structures of native polysaccharide and its alkaline modification products were studied by NMR spectroscopy. The polysaccharide molecules were shown to contain a linear carbohydrate chain consisting of alternating 3-linked β-d-galactopyranose 4-sulfate and 4-linked 3,6-anhydro-α-d-galactopyranose residues (known as к-carrageenan), which is typical of carrageenans, but the regularity of polymer structure is masked by the presence of some 3,6-anhydro-α-d-galactose 2-sulfate (ι-carrageenan units) and α-D-galactose 6-sulfate (µ-carrageenan units) instead of 3,6-anhydro-α-d-galactose. Upon addition of potassium chloride (up to 4%) to a solution of the native polysaccharide, about half of the substance transforms into gel. The gel-forming fraction is к-ι-µ-hybrid carrageenan with the ∼65 : 15 : 20 ratio of к-, ι-, and µ-units. The non-gelling fraction contains the к-, ι-, and µ-units at the ratio of ∼46 : 12 : 42. The gel-forming carrageenan product free of µ-units can be otained in ∼30% yield (based on the dry biomass) by alkaline treatment of the alga prior to extraction of the polysaccharide.

Treatment with sulphated galactan inhibits macrophage chemotaxis and reduces intraplaque macrophage content in atherosclerotic mice

Experimental data from animal models and clinical studies support connections between the haemostasis and inflammation in atherogenesis. These interfaces among inflammation and thrombogenesis have been suggested as targets for pharmacological intervention to reduce disease progression. We hypothesize that the recently discovered antithrombotic drug Sulphated Galactan (SG) (isolated from the red marine alga Acanthophora muscoides) might reduce atherosclerotic plaque vulnerability and inflammatory gene expression in 10-week aged apolipoprotein E deficient (ApoE−/−) mice under high-cholesterol diet for additional 11weeks. Then, the underlying cellular mechanisms were investigated in vitro. SG (10mg/kg) or Vehicle was subcutaneously injected from week 6 until week 11 of the diet. Treatment with SG reduced intraplaque macrophage and Tissue Factor (TF) content as compared to Vehicle-treated animals. Intraplaque TF co-localized and positively correlated with macrophage rich-areas. No changes on atherosclerotic plaque size, and other intraplaque features of vulnerability (such as lipid, neutrophil, MMP-9 and collagen contents) were observed. Moreover, mRNA expression of MMPs, chemokines and genetic markers of Th1/2/reg/17 lymphocyte polarization within mouse aortic arches and spleens was not affected by SG treatment. In vitro, treatment with SG dose-dependently reduced macrophage chemotaxis without affecting TF production. Overall, the chronic SG treatment was well tolerated. In conclusion, our results indicate that SG treatment reduced intraplaque macrophage content (by impacting on cell recruitment) and, concomitantly, intraplaque TF content of potential macrophage origin in atherosclerotic mice.

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.

Structural Characterization of a Hybrid Carrageenan-Like Sulfated Galactan from a Marine Red Alga Furcellaria lumbricalis.

Carrageenans are sulfated galactan isolated from marine red algae with different disaccharide forms. There are also some hybrid carrageenan-like oligomers, which are reported to possess a number of bioactivities. Here, we describe a method to study the structural characterization of a carrageenan-like sulfated galactan FB1 extracted from the red seaweed Furcellaria lumbricalis. We show the process of the general analysis of FB1, including the molecular weight, sulfate content, total sugar content, protein content, and 3,6-anhydrogalactose (3,6-AnG) content analyses. The fine structure identification methods, including desulfation and methylation, nuclear magnetic resonance (NMR), and electrospray ionization collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS), are also described in detail.

Hydrogen peroxide released from Pyropia yezoensis induced by oligo-porphyrans: Mechanisms and effect

Porphyran, a sulfated galactan, is the main cellular polysaccharide in the marine red alga Pyropia yezoensis. In this study, oligo-porphyrans, obtained by acid hydrolysis of porphyran, were investigated for their H2O2-inducing abilities in the defense responses of P. yezoensis. Oligo-porphyrans with average molecular weights (MWs) lower than 1.43 kDa had H2O2-inducing abilities. In contrast, oligo-porphyrans with average MWs of 6.12 kDa triggered no response. The active oligo-porphyrans were fractioned by anion-exchange chromatography. We found that two distinct mechanisms might be involved in the oligo-porphyran-induced H2O2 release in P. yezoensis. Mixtures of mono-sulfated oligogalactans with degrees of polymerization (DPs) ranging from 1 to 3 might induce the response through the oxidation of cellular oligosaccharides, which enable P. yezoensis to resist rotting caused by dense incubation. Mixtures of oligoporphyrans, consisting of 4 similar to 7 monosaccharide residues and 2 similar to 3 sulfate groups, might induce the generation of H2O2 by activation of NADPH oxidase, leading to an oxidative burst in P. yezoensis. The elicitor activity of oligo-porphyrans thus depends on their molecular size.

Chemical structure and anticoagulant activity of highly pyruvylated sulfated galactans from tropical green seaweeds of the order Bryopsidales.

Sulfated and pyruvylated galactans were isolated from three tropical species of the Bryopsidales, Penicillus capitatus, Udotea flabellum, and Halimeda opuntia. They represent the only important sulfated polysaccharides present in the cell walls of these highly calcified seaweeds of the suborder Halimedineae. Their structural features were studied by chemical analyses and NMR spectroscopy. Their backbone comprises 3-, 6-, and 3,6-linkages, constituted by major amounts of 3-linked 4,6-O-(1'-carboxy)ethylidene-d-galactopyranose units in part sulfated on C-2. Sulfation on C-2 was not found in galactans from other seaweeds of this order. In addition, a complex sulfation pattern, comprising also 4-, 6-, and 4,6-disulfated galactose units was found. A fraction from P. capitatus, F1, showed a moderate anticoagulant activity, evaluated by general coagulation tests and also kinetics of fibrin formation was assayed. Besides, preliminary results suggest that one of the possible mechanisms involved is direct thrombin inhibition.

Bioactive polysaccharides from marine algae

Abstract Laminaran, fucoidan and sulfated galactan were extracted from the seaweeds Desmarestia distans , Lessonia vadosa , and Gigartina skottsbergii , respectively. Modified polysaccharides were prepared by desulfation, depolymerization, and sulfation. Antioxidant, anticoagulant and immunostimulating activities of native and modified polysaccharides were assayed in vitro . The oversulfated derivatives showed high antioxidant capacity towards oxygen radical assay; however, no direct relation between sulfate content and antioxidant capacity was found. Oversulfated polysaccharides presented higher antioxidant capacity towards hydroxyl radicals than the native polysaccharide. Regarding the ABTS •+ radical cation assay moderate inhibition values (35.1–3.4%) were observed. The anticoagulant activity of native and modified polysaccharides was measured using the activated partial thromboplastin time assay; the native sulfated galactan from G. skottsbergii presented the highest value, close to that shown by heparin at similar concentrations. The immunostimulating activity of polysaccharides was measured through their effects on bone marrow-derived mice dendritic cell maturation. The native sulfated galactan from G. skottsbergii presented good dose-dependent activity inducing increased levels of MHC class II in dendritic cells.

The antitumor activity of a red alga polysaccharide complexes carrying 5-fluorouracil

Porphyran is a sulfated galactan isolated from red algae Porphyra haitanensis, and have been reported to have many kinds of biological activities such as antitumor activity. In order to provide a water-soluble macromolecule prodrug of 5-Fu showing slow release of 5-Fu and reducing side-effect, we carried out fixation of 5-Fu to porphyran at 6-position. In this study, the antitumor and immunomodulation activities of low MW porphyran carrying 5-Fu on transplanted S180 tumor mice were studied. Weight of immune organ, proliferation ratio of lymphocyte concentration of TNF-α and NO from the transplanted S180 tumor mice were also determined. Results indicated that the conjugate could enhance antitumor activity of 5-Fu and improve immunocompetence damaged by 5-Fu.

Molecular weight between entanglements for κ- and ι-carrageenans in an ionic liquid

The molecular weight between entanglements (Me) for κ- and ι-carrageenans, sulfated galactans, was examined in concentrated solutions using an ionic liquid 1-butyl-3-methylimidazolium acetate as a solvent. The dynamic viscoelasticity data for the solutions measured at different temperatures were overlapped according to the time–temperature superposition principle, and the obtained master curves exhibited the flow and rubbery plateau zones, being typical of concentrated polymer solutions having entanglement coupling. The values of Me for κ- and ι-carrageenans in the solutions were determined from the plateau moduli. Then the values of Me in the molten state (Me,melt) estimated as a material constant to be 6.6×103 and 7.2×103, respectively. The close values of Me,melt for κ- and ι-carrageenans indicate that 4-sulfate group of ι-carrageenan are not so influential for the entanglement network. Compared with agarose, a non-sulfate galactan, carrageenans have larger values of average spacing between entanglements.

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.

Red seaweed derived polysaccharides, a novel marine resource for bio-ethanol production

Seaweed biomass could be a renewable feed stock source for bio-ethanol production. Kappaphycus alvarezii is a promising alternative feedstock for the production of renewable energy sources because of its high content of red seaweed derived polysaccharides (RSDP) and its abundance nationwide. Based on the success of the production of bio-ethanol from seaweed cellulose biomass, the purpose of this study was to verify the feasibility of converting RSDP (mainly composed of sulfated galactan) to fermentable reducing sugar for bio-ethanol production. In this study, RSDP, pretreated with methanolysis to remove sulfate radicals, had a better desulfuration effect than the method of organic solvolytic desulfation. The results of single factor and orthogonal experiments were conducted, and the optimal parameters for the methanolysis of RSDP were: 0.5% of substrate concentration, 1.0M HCl-methanol, temperature of 80 °C, and 20 h. The desulfuration rate and the total-sugars yield reached 93% and 91%, respectively. Using Infrared Spectra, it was proven that the sulfate radical content was reduced sharply and that the 3, 6-anhydro bond was broken. Above all, saccharification liquids of desulfurated RSDP inoculated with Saccharomyces cerevisiae L4 for ethanol fermentation could produce 0.27 g ethanol/g RSDP. The results could bring bright prospects of bio-ethanol conversion from red seaweed biomass.

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.

Sulfated galactans isolated from the red seaweed Gracilaria fisheri target the envelope proteins of white spot syndrome virus and protect against viral infection in shrimp haemocytes

The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from Penaeus monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability and viral load were determined. Haemocytes treated with WSSV-SG pre-mix showed decreased CPE, viral load and cell mortality from the viral infection. Solid-phase virus-binding assays revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26 and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from Gracilaria fisheri elicits its anti-WSSV activity by binding to viral proteins that are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for viral binding.