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.     

Fucanomics and galactanomics: Current status in drug discovery,mechanisms of action and role of the well-defined structures

Background

With the recent advent of glycomics, many medically relevant glycans have been discovered. Sulfated fucans (SFs) and sulfated galactans (SGs) are one of these classes of glycans with increasing interest to both glycomics and medicine. Besides having very unique structures, some of these molecules exhibit a broad range of pharmacological actions. In certain cases, high levels of effectiveness may be reached when the proper structural requirements are found.

Scope of review

Here, we cover the fundamental biochemical mechanisms of some of these medicinal properties. We particularly focus on the beneficial activities of SFs and SGs in inflammation, hemostasis, vascular biology, and cancer.

Major conclusions

In these clinical systems, intermolecular complexes directly driven by electrostatic interactions of SFs and SGs with P- and L-selectins, chemokines, antithrombin, heparin cofactor II, thrombin, factor Xa, bFGF, and VEGF, overall govern the resultant therapeutic effects. In spite of that, the structural features of SFs and SGs have shown to be essential determinants for formation and stability of those molecular complexes, which consequently account to the differential levels of the biomedical responses.

General significance

Accurate structure–function relationships have mostly been achieved when SFs and SGs of well-defined structures are used for study. Therefore, these types of glycans have become of great usefulness to identify the chemical requirements needed to achieve satisfactory clinical responses.     

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.     

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.     

Solution NMR conformation of glycosaminoglycans

Nuclear magnetic resonance (NMR) spectroscopy has been giving a pivotal contribution to the progress of glycomics, mostly by elucidating the structural, dynamical, conformational and intermolecular binding aspects of carbohydrates. Particularly in the field of conformation, NOE resonances, scalar couplings, residual dipolar couplings, and chemical shift anisotropy offsets have been the principal NMR parameters utilized. Molecular dynamics calculations restrained by NMR-data input are usually employed in conjunction to generate glycosidic bond dihedral angles. Glycosaminoglycans (GAGs) are a special class of sulfated polysaccharides extensively studied worldwide. Besides regulating innumerous physiological processes, these glycans are also widely explored in the global market as either clinical or nutraceutical agents. The conformational aspects of GAGs are key regulators to the quality of interactions with the functional proteins involved in biological events. This report discusses the solution conformation of each GAG type analyzed by one or more of the above-mentioned methods.     

Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells.

The accumulation of an abnormal, protease-resistant form of the protein PrP (PrP-res) in hosts with scrapie and related transmissible spongiform encephalopathies appears to be important in disease pathogenesis. To gain insight into the mechanism of PrP-res accumulation and the in vivo antiscrapie activity of certain polyanions, we have studied effects of sulfated glycans on PrP metabolism in scrapie-infected neuroblastoma cells. Pentosan polysulfate, like the amyloid-binding dye Congo red, potently inhibited the accumulation of PrP-res in these cells without apparent effects on the metabolism of the normal isoform. The inhibition was due primarily to prevention of new PrP-res accumulation rather than destabilization of preexisting PrP-res. PrP-res accumulation remained depressed in the cultures after removal of the inhibitors. The activities of other sulfated glycans, nonsulfated polyanions, dextran, and DEAE-dextran were compared with those of pentosan polysulfate and Congo red. This comparison provided evidence that the density of sulfation and molecular size are factors influencing anti-PrP-res activity of sulfated glycans. The relative potencies of these compounds corresponded well with their previously determined antiscrapie activities in vivo, suggesting that the prophylactic effects of sulfated polyanions may be due to inhibition of PrP-res accumulation. Since PrP-res amyloid is known to contain sulfated glycosaminoglycans, we reason that these inhibitors may competitively block an interaction between PrP and endogenous glycosaminoglycans that is essential for its accumulation in a protease-resistant, potentially amyloidogenic state.     

Inhibition of PrPSc formation by synthetic O-sulfated glycopyranosides and their polymers

Sulfated glycosaminoglycans (GAGs) and sulfated glycans inhibit formation of the abnormal isoform of prion protein (PrPSc) in prion-infected cells and prolong the incubation time of scrapie-infected animals. Sulfation of GAGs is not tightly regulated and possible sites of sulfation are randomly modified, which complicates elucidation of the fundamental structures of GAGs that mediate the inhibition of PrPSc formation. To address the structure-activity relationship of GAGs in the inhibition of PrPSc formation, we screened the ability of various regioselectively O-sulfated glycopyranosides to inhibit PrPSc formation in prion-infected cells. Among the glycopyranosides and their polymers examined, monomeric 4-sulfo-N-acetyl-glucosamine (4SGN), and two glycopolymers, poly-4SGN and poly-6-sulfo-N-acetyl-glucosamine (poly-6SGN), inhibited PrPSc formation with 50% effective doses below 20 microg/ml, and their inhibitory effect became more evident with consecutive treatments. Structural comparisons suggested that a combination of an N-acetyl group at C-2 and an O-sulfate group at either O-4 or O-6 on glucopyranoside might be involved in the inhibition of PrPSc formation. Furthermore, polymeric but not monomeric 6SGN inhibited PrPSc formation, suggesting the importance of a polyvalent configuration in its effect. These results indicate that the synthetic sulfated glycosides are useful not only for the analysis of structure-activity relationship of GAGs but also for the development of therapeutics for prion diseases.     

Prospects for the use of sulfated polysaccharides from brown seaweeds as vaccine adjuvants

The data from the Russian and foreign literature on the effects of brown seaweed-derived sulfated polysaccharides (fucoidans) used as potential vaccine adjuvants to enhance the anti-infection and anti-tumor immune response are discussed in the present review. Due to their low toxicity, high biocompatibility, safety, and good tolerability by macroorganisms, as well as their mechanisms of immunomodulatory activity, fucoidans can be considered as promising adjuvants to administer in the composition of prophylactic and therapeutic vaccines. Fucoidans are agonists to receptors of innate immunity and are potent inducers of the cellular and humoral immune response, which is an important factor to be taken into account in the development of vaccines against various pathogens, including viruses, as well as anti-tumor vaccines. The results of numerous studies in which sulfated polysaccharides were tested as components of experimental vaccines, as presented in this review, show that these substances can be used as safe and effective adjuvants.     

A dot blot assay for sulfated glycosaminoglycans

A dot blot assay for detection of low amounts of heparin and sulfated glycosaminoglycans (GAGs) is described. The detection range is between 25 ng/ml and 1000 ng/ml of heparin. The assay is based on the interference of sulfated GAGs with the binding of a synthetic ligand (described in this paper) to defined receptors like collagen type V and histones. Ligand binding to type V collagen was suppressed specifically by heparin, but not by other sulfated GAGs like heparin sulfate and chondroitin sulfate. Ligand binding to histones was suppressed most strongly by heparin, but also by chondroitin sulfate. Hyaluronic acid did not interfere.     

Interaction of 70-kDa heat shock protein with glycosaminoglycans and acidic glycopolymers

Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments.     

Review: An overview about the structure–function relationship of marine sulfated homopolysaccharides with regular chemical structures

Efforts in both structural and biological studies of sulfated polysaccharides from marine organisms have increased significantly over the last 10 years. Marine invertebrates have been demonstrated to be a source of glycans with particularly well‐defined chemical structures, although ordered structural patterns can also be found occasionally in algal sources such as red seaweeds. Clear and regular structural features are essential for a good understanding of the biological activities of these marine homopolysaccharides of which sulfated fucans and sulfated galactans are the most studied. Herein, the main structural features (sugar type, sulfation and glycosylation sites, and orientational binding preferences) of both sulfated fucans and galactans are individually reviewed with regard to their specific contributions to two frequently described biological functions: the acrosome reaction (a physiological event of sea‐urchin fertilization), and the anticoagulant and antithrombotic activities (an alternative and highly desirable pharmacological application). © 2009 Wiley Periodicals, Inc. Biopolymers 91: 601–609, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Sulfated glycosaminoglycans in two hematophagous arthropod vectors of Chagas disease, Triatoma brasiliensis and Rhodnius prolixus (Hemiptera: Reduviidae)

The characterization of sulfated glycosaminoglycans (GAGs) in hematophagous arthropod vectors in general has been limited, with the exception of the studies in the triatomine Rhodnius prolixus. Heparan sulfate (HS) and chondroitin sulfate (CS) were previously identified and structurally characterized in extracts of whole bodies of fourth instar larvae of R. prolixus. Recently, we showed the expression of these two sulfated GAGs in specific body tissues of adult males and females and in embryos of R. prolixus. In the present work, we identified and compared the sulfated GAG composition in specific tissues of adult insects and in embryos of another triatomine species, Triatoma brasiliensis. Sulfated GAGs were isolated from the fat body, intestinal tract, and the reproductive tracts of adult insects and from embryos. Only HS and CS were found in the tissues analyzed. The present results extend the initial observations on the sulfated GAG composition in R. prolixus by showing that these molecules are widely distributed among internal organs of triatomines. These observations may be useful for future investigations aiming to evaluate the possible implication of these compounds in physiological events that take place in a specific organ(s) in these insects.     

Protective effect of fucoidans from tropical seaweeds against oxidative stress in HepG2 cells

Fucoidans are sulfated polysaccharides with proven pharmacological effects localized in the cell wall of marine brown algae. The majority of studies have been performed with temperate brown algal species, but in recent years, the evaluation of species from tropical areas has been growing. The aim of this study was to determine the protective effect of fucoidans extracted from the tropical brown seaweeds Dictyota ciliolata, Padina sanctae-crucis, and Sargassum fluitans, against oxidative stress (OS). The D. ciliolata fucoidan (FDc) exhibited the highest reactive oxygen species (ROS) scavenging activity (26%), followed by P. sanctae-crucis fucoidan (FPs) (22%) and S. fluitans fucoidan (FSf) (14%). No cytotoxic effect was detected for any of the extracted fucoidans at a concentration of 2 mg mL^?1. Not only did the fucoidans tested show protective effect against OS by reducing ROS generation, but they also increased the glutathione (GSH) level and restored catalase (CAT) activity. Fucoidans obtained from tropical seaweeds could be used as a potential natural ingredient for functional foods.     

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer''s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.     

Lysosomal storage of sulfated glycosaminoglycans in renal interstitial cells of rats treated with tilorone

Summary This investigation provides histochemical evidence for lysosomal storage of sulfated glycosaminoglycans (GAGs) in the interstitial cells of the renal cortex and in macrophage-like cells of the renal medullary zones of rats chronically treated with the drug tilorone. This compound is known to interfere with lysosomal degradation of sulfated GAGs; therefore cells that develop GAG-storage can be assumed to be involved in the turnover of GAGs. In view of this consideration, the most remarkable and still unexplained finding was that the intrinsic interstitial cells in the papilla, which is known to be particularly rich in sulfated GAGs, did not show the cytological symptoms of lysosomal GAG-storage. The present findings may stimulate further studies focused on the cellular sites of turnover of the sulfated GAGs present in the renal medullary interstitium.     

Transversely isotropic distribution of sulfated glycosaminoglycans in human medial collateral ligament: A quantitative analysis

Decorin and its associated glycosaminoglycan (GAG) side chain, dermatan sulfate (DS), play diverse roles in soft tissue formation and potentially aid in the mechanical integrity of the tissue. Deeper understanding of the distribution and orientation of the GAGs on a microscopic level may help elucidate the structure/function relationship of these important molecules. The hypothesis of the present study was that sulfated GAGs are aligned with transversely isotropic material symmetry in human medial collateral ligament (MCL) with the collagen acting as the axis of symmetry. To test the hypothesis, sulfated GAGs were visualized using transmission electron microscopy (TEM). Three orthogonal anatomical planes were examined to evaluate GAG distributions against symmetry criteria. GAG populations were differentiated using targeted enzyme digestion. Results suggest that sulfated GAGs including DS, chondroitin sulfates A and C, as well as other sub-populations assume transversely isotropic distributions in human MCL. Sulfated GAGs in the plane normal to the collagen axis were found to be isotropic with no preferred orientation. GAGs in the two planes along the collagen axis did not statistically differ and exhibited apparent bimodal distributions, favoring orthogonal distributions with over half at other angles with respect to collagen. A previously developed model, GAGSim3D, was used to interpret potential TEM artifacts. The data collected herein provide refined inputs to micro-scale models of the structure/function relationship of sulfated GAGs in soft tissues.     

Monthly fluctuations in the content and monosaccharide composition of fucoidan from Undaria pinnatifida sporophylls from northern Patagonia

The invasive brown seaweed Undaria pinnatifida was first recorded in 1992 in Golfo Nuevo, northern Patagonia, Argentina (hereafter Undaria). Like other brown seaweeds, Undaria synthesizes fucoidans, a unique class of sulfated polysaccharides, which display an array of biological activities and have important commercial value. In this work we have measured the content, sulfate, and monosaccharide composition of fucoidans in Undaria sporophylls from Golfo Nuevo. These results were analyzed in relation to harvest month and development stage of algal thalli. Samples were collected between November 2015 and March 2016 and classified according to morphological traits into previously defined development stages. Acid extraction (0.01 M HCl, pH 2) was carried out at room temperature and at 70 °C. Average fucoidan content was 18.1% dry wt. and slightly increased with month progression and in senescent individuals. Average sulfate content in fucoidan extracts was 20.3% dry wt. decreasing with month and development. Predominant sugars were fucose and galactose averaging a total of 91 mol% of neutral sugars. This study confirms that sporophyll fucoidans from Patagonian Undaria are sulfated galactofucans. Fucoidan content significantly increases from 14.5 to 19.2% dry wt. with sporophyte development. Slight increments with month progression are not statistically significant. Molar proportion of neutral sugars is constant between maturity stages and varies slightly with month progression. Sulfate content of fucoidan decreased significantly with development and month progression. Pooled with previous reports about abundance and seasonality of Undaria in Patagonia, our results suggest that sporophylls could be harvested for fucoidan production at least during 5 months between November and March.

     

Identification and tissue-specific distribution of sulfated glycosaminoglycans in the blood-sucking bug Rhodnius prolixus (Linnaeus)

We have previously characterized heparan sulfate (HS) as the major ovarian sulfated glycosaminoglycan (GAG) in females of Rhodnius prolixus, while chondroitin sulfate (CS) was the minor component. Using histochemical procedures we found that GAGs were concentrated in the ovarian tissue but not found inside the oocytes. Here, we extend our initial observations of GAG expression in R. prolixus by characterizing these molecules in other organs: the fat body, intestinal tract, and the reproductive tracts. Only HS and CS were found in the three organs analyzed, however CS was the major GAG species in these tissues. We also determined the compartmental distribution of GAGs in these organs by histochemical analysis using 1,9-dimethylmethylene blue, and evaluated the specific distribution of CS within both male and female reproductive tracts by immunohistochemistry using an anti-CS antibody. We also determined the GAG composition in eggs at days 0 and 6 of embryonic development. Only HS and CS were found in eggs at day 6, while no sulfated GAGs were detected at day 0. Our results demonstrate that HS and CS are the only sulfated GAG species expressed in the fat body and in the intestinal and reproductive tracts of Rhodnius male and female adults. Both sulfated GAGs were also identified in Rhodnius embryos. Altogether, these results show no qualitative differences in the sulfated GAG composition regarding tissue-specific or development-specific distribution.     

Sulfated glycans and elevated temperature stimulate PrP(Sc)-dependent cell-free formation of protease-resistant prion protein

A conformational conversion of the normal, protease- sensitive prion protein (PrP-sen or PrP(C)) to a protease-resistant form (PrP-res or PrP(Sc)) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP-res deposits in vivo, suggesting that they may facilitate PrP-res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP-res accumulation and serve as prophylactic anti-TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP-res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell-free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP-res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP-sen that may potentiate its PrP-res-induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.