Sulfated fucans and galactans are strongly anionic polysaccharides found in marine organisms. Their structures vary among species, but their major features are conserved among phyla. Sulfated fucans are found in marine brown algae and echinoderms, whereas sulfated galactans occur in red and green algae, marine angiosperms, tunicates (ascidians), and sea urchins. Polysaccharides with 3-linked, beta-galactose units are highly conserved in some taxonomic groups of marine organisms and show a strong tendency toward 4-sulfation in algae and marine angiosperms, and 2-sulfation in invertebrates. Marine algae mainly express sulfated polysaccharides with complex, heterogeneous structures, whereas marine invertebrates synthesize sulfated fucans and sulfated galactans with regular repetitive structures. These polysaccharides are structural components of the extracellular matrix. Sulfated fucans and galactans are involved in sea urchin fertilization acting as species-specific inducers of the sperm acrosome reaction. Because of this function the structural evolution of sulfated fucans could be a component in the speciation process. The algal and invertebrate polysaccharides are also potent anticoagulant agents of mammalian blood and represent a potential source of compounds for antithrombotic therapies.
A polysaccharide isolated from the body wall of the sea cucumber Ludwigothurea grisea has a backbone like that of mammalian chondroitin sulfate: [4-beta-D-GlcA-1-->3-beta-D-GalNAc-1]n but substituted at the 3-position of the beta--glucuronic acid residues with sulfated alpha--fucopyranosyl branches (Vieira, R. P., Mulloy, B., and Mourão, P. A. S. (1991) J. Biol. Chem. 266, 13530-13536). Mild acid hydrolysis removes the sulfated alpha--fucose branches, and cleaved residues have been characterized by 1H NMR spectroscopy; the most abundant species is fucose 4-O-monosulfate, but 2,4- and 3, 4-di-O-sulfated residues are also present. Degradation of the remaining polysaccharide with chondroitin ABC lyase shows that the sulfated alpha-L-fucose residues released by mild acid hydrolysis are concentrated toward the non-reducing end of the polysaccharide chains; enzyme-resistant polysaccharide material includes the reducing terminal and carries acid-resistant -fucose substitution. The sulfated alpha-L-fucose branches confer anticoagulant activity on the polysaccharide. The specific activity of fucosylated chondroitin sulfate in the activated partial thromboplastin time assay is greater than that of a linear homopolymeric alpha-L-fucan with about the same level of sulfation; this activity is lost on defucosylation or desulfation but not on carboxyl-reduction of the polymer. Assays with purified reagents show that the fucosylated chondroitin sulfate can potentiate the thrombin inhibition activity of both antithrombin and heparin cofactor II.
Sulfated fucans are among the most widely studied of all the sulfated polysaccharides of non-mammalian origin that exhibit biological activities in mammalian systems. Examples of these polysaccharides extracted from echinoderms have simple structures, composed of oligosaccharide repeating units within which the residues differ by specific patterns of sulfation among different species. In contrast the algal fucans may have some regular repeating structure but are clearly more heterogeneous when compared with the echinoderm fucans. The structures of the sulfated fucans from brown algae also vary from species to species. We compared the anticoagulant activity of the regular and repetitive fucans from echinoderms with that of the more heterogeneous fucans from three species of brown algae. Our results indicate that different structural features determine not only the anticoagulant potency of the sulfated fucans but also the mechanism by which they exert this activity. Thus, the branched fucans from brown algae are direct inhibitors of thrombin, whereas the linear fucans from echinoderms require the presence of antithrombin or heparin cofactor II for inhibition of thrombin, as reported for mammalian glycosaminoglycans. The linear sulfated fucans from echinoderms have an anticoagulant action resembling that of mammalian dermatan sulfate and a modest action through antithrombin. A single difference of one sulfate ester per tetrasaccharide repeating unit modifies the anticoagulant activity of the polysaccharide markedly. Possibly the spatial arrangements of sulfate esters in the repeating tetrasaccharide unit of the echinoderm fucan mimics the site in dermatan sulfate with high affinity for heparin cofactor II.Since the description of high amounts of sulfated fucans in marine brown algae over 50 years ago (1), these polysaccharides have been widely tested for biological activities in different mammalian systems. Algal-sulfated fucans have anticoagulant activity as measured in several different assays (2-5) and have also venous antithrombotic activity (6). These polysaccharides are inhibitors of native (7) and recombinant (8) human immunodeficiency virus reverse transcriptase activity in vitro.
Thromboembolic diseases are increasing worldwide and always require anticoagulant therapy. We still need safer and more secure antithrombotic drugs than those presently available. Sulfated polysaccharides from marine organisms may constitute a new source for the development of such drugs. Investigation of these compounds usually attempts to reproduce the therapeutic effects of heparin. However, we may need to follow different routes, focusing particularly in the following aspects: (1) defining precisely the specific structures required for interaction of these sulfated polysaccharides with proteins of the coagulation system; (2) looking for alternative mechanisms of action, distinct from those of heparin; (3) identifying side effects (mostly pro-coagulant action and hypotension rather than bleeding) and preparing derivatives that retain the desired antithrombotic action but are devoid of side effects; (4) considering that sulfated polysaccharides with low anticoagulant action on in vitro assays may display potent effects on animal models of experimental thrombosis; and finally (5) investigating the antithrombotic effect of these sulfated polysaccharides after oral administration or preparing derivatives that may achieve this effect. If these aspects are successfully addressed, sulfated polysaccharides from marine organisms may conquer the frontier of antithrombotic therapy and open new avenues for treatment or prevention of thromboembolic diseases.
We have characterized the fine structure of sulfated polysaccharides from the egg jelly layer of three species of sea urchins and tested the ability of these purified polysaccharides to induce the acrosome reaction in spermatozoa. The sea urchin Echinometra lucunter contains a homopolymer of 2-sulfated, 3-linked ␣-L-galactan. The species Arbacia lixula and Lytechinus variegatus contain linear sulfated ␣-L-fucans with regular tetrasaccharide repeating units. Each of these sulfated polysaccharides induces the acrosome reaction in conspecific but not in heterospecific spermatozoa. These results demonstrate that species specificity of fertilization in sea urchins depends in part on the fine structure of egg jelly sulfated polysaccharide.Successful fertilization by free-spawning organisms such as sea urchins can occur only if a series of constraints are overcome before the sperm ever makes contact with the egg (1). First, males and females must synchronize the time of release of their gametes (2). Once spawned, the sperm must find and interact with an egg of the correct species. A further event necessary for successful fertilization is induction of the acrosome reaction in the sperm (3, 4), which involves fusion of the acrosomal vesicle membrane with the plasma membrane. This results in exocytosis of the vesicle contents, which include proteases and bindin. Concomitantly, actin in the subacrosomal region of the sperm polymerizes and causes extension of the tip of the sperm. As a consequence of these two events, bindin is localized to the outside of the tip of the process where it can then interact with an egg protein (3, 4).The acrosome reaction is induced when the sperms contact the egg jelly layer. The sea urchin egg is surrounded by a transparent gelatinous layer composed mainly of sulfated fucan, sialoprotein, and other glycoproteins or peptides (5). Previous attempts to identify the acrosome reaction inducer in sea urchin egg jelly have suggested that all the activity resides in the sulfated fucan (6, 7). In addition, these authors suggested that the jelly coat preparations of some species of sea urchins are totally species-specific and induce the acrosome reaction only in homologous sperm. On the basis of these observations, it was suggested that the specificity of induction of the acrosome reaction might reside in structural differences in the carbohydrate linkages and/or location and degree of sulfation of the polysaccharide (6, 7). Recent studies suggest that a glycoprotein or peptide, tightly associated with the sulfated fucan, was also involved in acrosome reaction induction (8 -11).In this study, we isolated, purified, and characterized the fine structure of the sulfated polysaccharides from the egg jelly coat of three species of sea urchins. These compounds have simple, well-defined repeating structures that from each species present a particular pattern of sulfate substitution. Purified polysaccharide from the egg jelly induces the acrosome reaction in sperm from the same species of sea urchin and not f...
We attempted to identify the specific structural features in sulfated galactans and sulfated fucans that confer anticoagulant activity. For this study we employed a variety of invertebrate polysaccharides with simple structures composed of well-defined units of oligosaccharides. Our results indicate that a 2-O-sulfated, 3-linked alpha-L-galactan, but not a alpha-L-fucan with a similar molecular size, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaced thrombin. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked alpha-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units. The presence of 2-O-sulfated fucose residues always had a deleterious effect on anticoagulant activity. Overall, our results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.
Sulfated alpha-L-fucans from brown algae (also known as fucoidan) have complex and heterogeneous structures but recent studies revealed the occurrence of ordered repeat units in the sulfated fucans from several species. Even in these cases, the presence of highly branched portions and the complex distributions of sulfate and acetyl groups highlight the heterogeneity of algal fucans. Another source of sulfated alpha-L-fucans (and their parental compounds sulfated alpha-L-galactans and fucosylated chondroitin sulfate) is marine invertebrates. The invertebrate polysaccharides have simple, ordered structures, which differ in the specific patterns of sulfation and/or position of the glycosidic linkages within their repeating units. The algal and invertebrate sulfated fucans have potent anticoagulant activity, mediated by antithrombin and/or heparin cofactor II. As most of the studies were carried out with algal fucans it was not easy to trace a structure versus activity relationship. This aspect was clarified as studies were extended to invertebrate polysaccharides. These definitively established that regular, linear sulfated alpha-L-fucans and sulfated alpha-L-galactans express anticoagulant activity, which is not simply a function of charge density, but depends critically on the pattern of sulfation and monosaccharide composition. Sulfated alpha-L-fucans and fucosylated chondroitin sulfate also express antithrombotic activity when tested on in vivo models of venous and arterial thrombosis in experimental animals. These polysaccharides constitute potential therapeutic compounds as alternative to heparin and may help to design structure-based drugs with specific activity on each type of thrombosis episode and few side effects. They can also serve as research reagents to investigate and distinguish among a variety of interrelated events, such as coagulation, bleeding, thrombosis and platelet aggregation.
We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of ϳ15 to ϳ45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.
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