Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide

Olivier Berteau2 and Barbara Mulloy1,3

2 Department of Chemistry, Swedish University of Agricultural Sciences, Arrheniusplan 8, P.O. Box 7015, SE-750 07 Uppsala, Sweden
3 National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire ENG 3QG, United Kingdom

accepted on February 12, 2003


    Abstract
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 Abstract
 Introduction
 Nomenclature
 Structural diversity
 Physiological properties of...
 Biological properties of fucans
 Enzymes active on sulfated...
 Conclusions
 References
 
Sulfated fucans, frequently referred to simply as fucans, constitute a class of polysaccharides first isolated in 1913. For many years fucans were regarded only as a potential source of L-fucose, although their anticoagulant activity was known. Even as the potent effects of fucans on physiological systems have become better characterized, structural studies have lagged behind. Recently the search for new drugs has raised increased interest in sulfated fucans. In the past few years, several structures of algal and invertebrate fucans have been solved, and many aspects of their biological activity have been elucidated. From this work emerges a more interesting picture of this class of polysaccharides than was previously suspected. The availability of purified fucans and fucan fractions with simple, but varied structures, in conjunction with the development of new enzymatic tools, demonstrate that the biological properties of sulfated fucans are not only a simple function of their charge density but also are determined by detailed structural features.

Key words: fucan / fucoidan / fucoidin / sulfated fucan / fucoidanase


    Introduction
 Top
 Abstract
 Introduction
 Nomenclature
 Structural diversity
 Physiological properties of...
 Biological properties of fucans
 Enzymes active on sulfated...
 Conclusions
 References
 
The first isolation of "fucoidin" from marine brown algae was reported 90 years ago (Killing, 1913Go). Thirty-five years later, evidence was published showing that fucans also occur in marine invertebrates (Vasseur, 1948Go). These polysaccharides, mainly constituted of sulfated L-fucose, are easily extracted from the cell wall of brown algae (i.e., Phaeophyceae) with hot water (Percival and Ross, 1950Go) or acid solution (Black, 1954Go) and can account for more than 40% of the dry weight of isolated cell walls (Kloareg, 1984Go). In marine invertebrates, sulfated fucans occur in the egg jelly coat of sea urchins (Mulloy et al., 1994Go) and in the body wall of sea cucumber (Mourão and Bastos, 1987Go). The fucans of brown algae, often called fucoidans, have been known for some time to act as modulators of coagulation, as have other algal polysaccharides (Chargaff et al., 1936Go). Fucoidan preparations have been proposed as alternatives to the anticoagulant heparin, which is prepared from mammalian mucosa; being of vegetable origin they are less likely to contain infectious agents, such as viruses or prions. Like heparin, it has been shown that fucoidans affect many biological activities, such as inflammation, cell proliferation and adhesion, viral infection, and fertilization (Boisson-Vidal et al., 1995Go).

However, relatively few studies have interpreted the biological activity of fucoidans in terms of molecular structure. Almost all biological studies use a commercially available, crude preparation of sulfated polysaccharides from Fucus vesiculosus rather than a purified fucoidan (Mulloy et al., 1994Go). Recent insights into the structures of fucans from different plant and animal species may help explain their mode of activity, whether as research reagents or as potential therapeutics.

The aim of this review is to give an up-to-date view of the physiological and structural properties of sulfated fucans from marine algae and invertebrates and to present a discussion of specific hydrolytic enzymes, which are expected to simplify structural and structure/function studies.

Algal fucoidans are present in several orders, mainly Fucales and Laminariales but also in Chordariales, Dictyotales, Dictyosiphonales, Ectocarpales, and Scytosiphonales (Table I). In fact they are widely present among all the brown algae (Phaeophyceae) so far investigated. On the other hand, fucoidans seem to be absent from green algae (Chlorophyceae), red algae (Rhodophyceae), and golden algae (Xanthophyceae) and from freshwater algae and terrestrial plants.


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Table I. Brown algae containing fucoidan

 
The only other sources of sulfated fucan known to date are marine invertebrates. The first report was made by Vasseur (1948)Go, who extracted a polysaccharide mainly constituted of sulfated methyl-pentose from eggs of sea urchin. Since then, sulfated fucans have been isolated from the egg jelly coat of many species of sea urchin (Mulloy et al., 1994Go; Alves et al., 1997Go, 1998Go; Vilela-Silva et al., 1999Go, 2002Go) and from the body wall of another type of marine echinoderm, the sea cucumber Ludwigothurea grisea (Mourão and Bastos, 1987Go; Ribeiro et al., 1994Go). To date, naturally occurring fucans without sulfate groups have never been reported.


    Nomenclature
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 Abstract
 Introduction
 Nomenclature
 Structural diversity
 Physiological properties of...
 Biological properties of fucans
 Enzymes active on sulfated...
 Conclusions
 References
 
Killing baptized his polysaccharide fucoidin; 40 years later, McNeely changed fucoidin to fucoidan, to conform with polysaccharide nomenclature (McNeely, 1959)Go. The questions of nomenclature and purity have always been linked for algal fucoidans; early preparations contained large amounts of sugars other than fucose, such as galactose, mannose, xylose, or uronic acid, and sometimes even proteins. Furthermore, their composition changed according to the algal species (Percival and Ross, 1950Go; Mian and Percival, 1973Go), the extraction process (Mabeau et al., 1990Go), and the season of harvest and local climatic conditions (Black, 1954Go; Von Holdt et al., 1955Go; Wort, 1955Go; Honya et al., 1999Go). Were these contaminants (Percival and Ross, 1950Go; O'Neill, 1954Go; Bernardi and Springer, 1962Go) or part of the polysaccharide (Schweiger, 1962Go; Anno et al., 1966)Go? Some authors even considered suppressing the term of fucoidan (Larsen et al., 1966)Go. To face these uncertainties, the generic term fucans, to designate all polysaccharides rich in L-fucose, was commonly used (Percival and Ross, 1950)Go.

As separation and analytic techniques improved, different types of sulfated polysaccharides were distinguished in fucan preparations. The first was ascophyllan or xylofucoglycuronan, based on a backbone of uronic acid (mannuronic acid) with fucose containing branches (3-O-D-xylosyl-L-fucose-4-sulfate) (Larsen et al., 1966Go; Kloareg et al., 1986Go). The other family isolated was sargassan or glycuronofucoglycan, based on linear chains of D-galactose with branches of L-fucose-3-sulfate or occasionally uronic acid (Percival, 1968Go; Medcalf et al., 1978Go; Kloareg et al., 1986Go). Fucoidans, as originally defined, were identified as homofucans (Kloareg et al., 1986Go). Many authors still use the outdated term fucoidin. In some cases, authors create their own nomenclature, such as fucansulfate (in one word) (Trento et al., 2001Go). The confusion is increased with some publications in which the term fucoidan is used to describe a complex polysaccharide containing only 20% to 60% L-fucose (Duarte et al., 2001Go).

It seems wise and in agreement with IUPAC recommendations to define sulfated fucan as a polysaccharide based mainly on sulfated L-fucose, with less than 10% other monosaccharides. This term was applied to the sulfated fucans of marine invertebrates (Ribeiro et al., 1994Go; Alves et al., 1998Go; Vilela-Silva et al., 1999Go), whereas the term fucoidan has been used for fucans extracted from algae. For clarity, and because the fucans extracted from those two sources differ, this nomenclature will be adopted for this review.


    Structural diversity
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 Abstract
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 Nomenclature
 Structural diversity
 Physiological properties of...
 Biological properties of fucans
 Enzymes active on sulfated...
 Conclusions
 References
 
Algal sulfated fucans: the common fucales F. vesiculosus and A. nodosum
Percival and Ross (1950)Go described fucoidan from the common brown algae F. vesiculosus as a polysaccharide based on L-fucose with mainly {alpha}(1->2) glycosidic bonds and sulfate groups at position 4. They also found branches of sulfated fucose every five units. This model, supported by several other studies of F. vesiculosus fucoidan (O'Neill, 1954Go; Côté, 1959Go), was the only structural model available for fucoidan for more than 40 years, even though fucoidan from various algae was commonly used in biological studies.

In 1993 Patankar and co-workers reinvestigated the structure of F. vesiculosus fucoidan (the only one commercially available) (Patankar et al., 1993)Go. Their model differs in the nature of the main glycosidic bond: {alpha}(1->3) instead of {alpha}(1->2). The position of the sulfate, in accord with the previous model, was found to be mainly in position 4.

More recent studies have identified a different type of structure for fucoidan from the common Fucales Ascophyllum nodosum (Chevolot et al., 1999Go, 2001Go; Daniel et al., 1999Go, 2001Go). Several studies clearly show that this fucoidan possesses large proportions of both {alpha}(1->3) and {alpha}(1->4) glycosidic bonds (Daniel et al., 1999Go, 2001Go; Chevolot et al., 1999Go, 2001Go). A repeating structure of alternating {alpha}(1->3) and {alpha}(1->4) glycosidic bonds was determined for oligosaccharides (of about 8–14 monosaccharide units) prepared from A. nodosum fucoidan (Chevolot et al., 2001Go) (Figure 1A). As the characteristic nuclear magnetic resonance (NMR) spectrum of this repeating disaccharide is the major feature in the NMR spectra of whole fucoidan from both A. nodosum and F. vesiculosus (Pereira et al., 1999Go), it may be that the backbones of both fucoidans consist of this structure. Highly branched fractions may also be prepared from A. nodosum fucoidan (Marais and Joseleau, 2001Go), and minor features in the complex NMR spectra of algal fucoidans may well be due to these branches (Pereira et al., 1999Go). A. nodosum fucoidan has also been studied using specific enzymes, confirming the presence of high amounts of {alpha}(1->3) and {alpha}(1->4) glycosidic bonds (Daniel et al., 1999Go, 2001Go).



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Fig. 1. Common structures in fucoidans from brown algae. (A) The disaccharide repeating unit [4)-{alpha}-L-Fucp(2,3di-OSO3-)-(1->3)-{alpha}-L-Fucp(2OSO3-)-(1] of a fraction of A. nodosum fucoidan representing the most abundant structural feature of fucoidans from both A. nodosum and F. vesiculosus (Chevolot et al., 1999Go, 2001Go). The same structure has been identified in the fucoidan of F. evanescens (Bilan et al. 2002Go). (B) The 3-linked, preponderantly 4-sulfated fucoidan from E. kurome (Nishino and Nagumo, 1991Go). (C) The quasi-repeat unit identified in fucoidan from C. filum (Chizhov et al., 1999Go). Other substituents, such as O-acetyl, and branches are present in all these fucoidans and add considerably to their heterogeneity.

 
Algal sulfated fucans: other brown algae
The structure of fucoidan from another species, Ecklonia kurome, was published in 1991 (Nishino and Nagumo, 1991Go). NMR spectra of the polysaccharide were too complex to allow direct structure elucidation, probably due to structural heterogeneity. The average structure of this fucoidan is mainly 3-O-linked with sulfate groups at C-4, without excluding the presence of other sulfate groups or branches in position 2 (Figure 1B).

In 1999 the structures of fucoidans from three algae were published, Cladosiphon okamuranus (chordariales) (Nagaoka et al., 1999Go), Chorda filum (laminariales) (Chizhov et al., 1999Go), and Ascophyllum nodosum (fucales) (Chevolot et al., 1999Go; Daniel et al., 1999Go). Fucoidans from Cladosiphon okamuranus and Chorda filum present an average structure based on a backbone of L-fucose linked {alpha}(1->3) with most sulfation at position 4. Fucoidan from C. filum seems based on a regular hexasaccharide repeating unit, with some 2-O-sulfation (Figure 1C). Both fucoidans have some 2-O-acetylation, and the authors argue that O-acetylation is also present in other fucoidans, in particular those from F. vesiculosus, E. kurome, and L. brasiliensis (Chizhov et al., 1999Go). A fucoidan from L. brasiliensis has been studied by NMR spectroscopy and methylation analysis; its structure was not determined, but clear signs of a complex repeating unit were identified (Pereira et al., 1999Go). More recently, the structure of a high-molecular-weight fucoidan from Fucus evanescens, another fucales, was published and found to resemble A. nodosum fucoidan with, in addition, the presence of large amount of acetyl groups and maybe the presence of fucosyl branches (Bilan et al., 2002)Go.

Although there are clear links between algal species and fucoidan structure, there is insufficient evidence yet to establish any systematic correspondence between structure and algal order.

Sulfated fucans from marine invertebrates
It was known for many years that invertebrates possess sulfated fucans (Vasseur, 1948Go), but no structural study was performed on these compounds before 1987 (Mourão and Bastos, 1987Go). Two kinds of marine invertebrates have been investigated: one species of sea cucumber (Ludwigothurea grisea) (Ribeiro et al., 1994Go) and several species of sea urchins (Lytechinus variegatus, Arbacia lixula, Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, Strongylocentrotus pallidus, and Strongylocentrotus droebachiensis) (Mulloy et al., 1994Go; Alves et al., 1997Go, 1998Go; Vilela-Silva et al., 1999Go, 2002Go).

In contrast with the apparently complex algal fucoidans, they possess a clear regularity, allowing determination of their structures by high-field NMR (Mulloy et al., 1994Go). They are linear polysaccharides consisting of a regular repeating unit, either mono-, tri-, or tetrasaccharide (Figure 2), in which the glycosidic linkage is constant, and the repeating unit defined by a distinctive pattern of sulfate substitution. In general, each species has its own particular sulfated fucan. These egg-jelly polysaccharides are able to induce the acrosomal reaction (a change that sea urchin spermatozoa must undergo to fertilize the egg successfully) preferentially in sperm of the same species (Alves et al., 1997Go; Vilela-Silva et al., 2002Go). In this way, interspecific fertilization is avoided. Two species, S. purpuratus and S. droebachiensis, have been studied in which most individual females are able to produce only one of two different types of sulfated fucans (Alves et al., 1998Go; Vilela-Silva et al., 2002Go). However, 10% of the eggs of S. purpuratus contain both types of fucans. For S. droebachiensis the polymorphism seems to be correlated with the place of collection.



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Fig. 2. Repeating structures of sulfated fucans, and a sulfated L-galactan, from sea urchin egg jelly. In contrast with algal fucoidans, these polymers of L-fucose are homogeneous and unbranched and bear no substituents other than those shown. Sulfation pattern alone defines the repeat unit of these polysaccharides and in general each species has its own pattern, capable of interacting with spermatozoa preferentially from the same species. Some species (S. droebachiensis, S. purpuratus) may produce two distinct fucans. The two species A. lixula and S. droebachiensis share a fucan structure, but because these two species are well separated geographically there is little chance of cross-fertilization.

 
There are many species of sea urchin, and only a few egg-jelly sulfated fucans (and occasionally galactans) have been studied. It is also the case that sulfated fucan structure is not the only barrier to cross-fertilization. It is worth noting, however, that two species known to synthesize a sulfated fucan with the same structure (i.e. A. lixula and S. droebachiensis) are found in nonoverlapping geographical locations (from the tropical Atlantic Ocean and the Arctic Ocean, respectively). Cross-species induction of the acrosome reaction in the laboratory has shown that position of sulfation and linkage are both important for the interaction. It is interesting to note in this context that an L-galactan from the species Echinometra lucunter (Figure 2) may substitute for an L-fucan from S. franciscanus (Figure 2) of identical linkage and substitution pattern (Hirohashi et al., 2002Go).

Sperm membrane receptors for sea urchin egg-jelly fucans include REJ1 (receptor for egg jelly 1) (Vacquier and Moy, 1997Go) and maybe REJ3 (Mengerink et al., 2002Go). This interaction is unusual in that a pure carbohydrate, in the absence of any protein component, is capable of inducing signal transduction resulting in exocytosis (Vacquier and Moy, 1997Go). The high molecular weight of egg-jelly fucans is required for successful opening of both Ca2+ channels involved in the acrosome reaction, though partially hydrolysed fucan will bind to sperm receptors (Hirohashi and Vacquier, 2002Go).

Though we now have some data about the structure of these sulfated fucans, almost nothing is known about their conformations or how their regular spatial patterns of sulfate groups determine their biological properties (Gerbst et al., 2001Go, 2002).


    Physiological properties of sulfated fucans
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 Abstract
 Introduction
 Nomenclature
 Structural diversity
 Physiological properties of...
 Biological properties of fucans
 Enzymes active on sulfated...
 Conclusions
 References
 
Little is known about the role of most fucans in marine organisms, apart from the case of sea-urchin fertilization as described. The role of sulfated fucans in the body wall of sea cucumber is less well understood but may be in maintenance of the body wall's integrity (Mourão and Bastos, 1987Go; Ribeiro et al., 1994Go). For algae, some studies have shown a correlation between fucoidan content and the depth at which they grow—the closer algae are to the surface, the greater the fucoidan content (Black, 1954Go; Kloareg, 1981Go; Evans, 1989Go).

Furthermore, fucoidans appear to play a role in the algal cell wall organization (Kloareg and Quatrano, 1988Go; Bisgrove and Kropf, 2001Go) and could be involved in the cross-linkage of alginate and cellulose (Mabeau et al., 1990Go). Fucoidans may also be involved in the morphogenesis of algae embryos (Bisgrove and Kropf, 2001Go).


    Biological properties of fucans
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Fucoidans have a wide spectrum of activity in biological systems. Besides their well-attested anticoagulant and antithrombotic activity, they act on the inflammation and immune systems, have antiproliferative and antiadhesive effect on cells, protect cells from viral infection, and can interfere with mechanisms involved in fertilization. All of these properties have been reviewed by Boisson-Vidal et al. (1995)Go. Most studies have used a commercial preparation of F. vesiculosus fucoidan, which has been shown to contain heteropolysaccharides of various kinds besides those consisting predominantly of sulfate and fucose (Nishino et al., 1994Go). This mixture has been used, for example, as a "specific" ligand for L- and P-selectins and macrophage scavenger receptors. Some studies have been published, however, on partly characterized fucoidan fractions, and most of these concern the effects of fucoidan on blood coagulation.

Anticoagulant and antithrombotic activity
F. vesiculosus fucoidan has a specific anticoagulant activity by the activated partial thromboplastin time assay of 9–13 U/mg, as compared with 167 U/mg for heparin (Nishino et al., 1994Go), or 16 U/mg, as compared with 193 U/mg for heparin (Mourão and Pereira, 1999Go). A fucoidan from Laminaria brasiliensis has higher specific activity (30 U/mg), though its sulfate content is lower (Mourão and Pereira, 1999Go). The high potency of heparin depends on a specific pentasaccharide sequence with high affinity for the serine protease inhibitor antithrombin, a sequence that is obviously absent from fucoidan. However, purified fucoidan fractions (from Ecklonia kurome [Nishino et al., 1999Go], A. nodosum [Millet et al., 1999Go], and Pelvetia caniculata [Colliec et al., 1994Go]) have activity mediated both by antithrombin and by another plasma serine protease inhibitor (serpin), heparin cofactor II (HCII) (Colwell et al., 1999Go). Heparin interacts with HCII by means of its regular repeating unit (Figure 3A), not the antithrombin-binding sequence. These serpins act against several of the coagulation system proteases, including thrombin, Factor Xa, and Factor IXa (Mauray et al., 1998Go). All of these factors may be involved in the ability of fucoidan to prevent venous thrombosis (Millet et al., 1999Go). The release of tissue factor pathway inhibitor from endothelium, which is stimulated by fucoidan more potently than by heparin, may also have an antithrombotic effect (Giraux et al., 1998aGo).



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Fig. 3. Disaccharide structures associated with anticoagulant polysaccharides. (A) The main repeating unit [4)-{alpha}-L-IdopA2OSO3-(1->3)-{alpha}-D-GlcpNSO3–6OSO3-(1->] found in the widely used anticoagulant polysaccharide heparin. This structure does not account for the main part of heparin's anticoagulant activity, as a potentiator of antithrombin, but has activity by other routes such as activation of heparin cofactor II. (B) The repeating unit [4)-{alpha}-L-Fucp(2,3di-OSO3-)-(1->3)- {alpha}-L-Fucp(2OSO3-)-(1->] from A. nodosum (also F. vesiculosus and F. evanescens) (Chevolot et al., 1999Go, 2001Go). (C) The repeat unit based on the backbone [4)-{alpha}-D-Galp-(1->3)-ß-D-Galp-(1->] of a galactan with high anticoagulant activity isolated from the red alga B. occidentalis.

 
Anticoagulant and antithrombotic activities of fucoidan fractions (A. nodosum) increase with increasing molecular weight and sulfate content. However, fractions in which the native pattern of sulfation was intact were more potent than fractions of equivalent molecular weight and overall degree of sulfation in which this pattern had been disrupted by partial desulfation (Boisson-Vidal et al., 2000Go). Fucoidans may also promote fibrinolysis by potentiating plasminogen activators (Nishino et al., 2000Go). The predominant pattern of sulfation in A. nodosum fucoidan is the trisulfated disaccharide motif (Figure 3B), similar to that found in heparin, which also has a trisulfated disaccharide repeat unit (Figure 3A). A heavily sulfated substituted disaccharide is also the repeat unit of a highly anticoagulant galactan isolated from red algae (Farias et al., 2000Go) (Figure 3C).

Correlation of the sulfation patterns of less highly sulfated fucans and galactans with their anticoagulant activities (Mourão and Pereira, 1999Go; Pereira et al., 2002Go) has used invertebrate fucans as shown in Figure 2. The structural homogeneity of these compounds allows clear conclusions to be drawn. For example, a 3-linked, regularly 2-O-sulfated galactan has anticoagulant activity lacking in the corresponding 3-linked, 2-O-sulfated fucan or a 4-linked, regularly 3-O-sulfated galactan. This study established definitively that regular, linear sulfated fucans express anticoagulant activity, which is not simply a function of charge density but depends critically on the exact structure of the polysaccharide.

Platelet activation
Fucoidan fractions of varying molecular weight and degree of sulfation all induced platelet aggregation in vitro (Durig et al., 1997Go). In this case low sulfate fractions were most potent, showing that anticoagulation and platelet activation differ in their structural dependence. On the other hand, a study in baboons found fucoidan to be a potent inhibitor of platelet aggregation in vivo (Alwayn et al., 2000Go).

Selectins
Selectins are a group of lectins found on the surface of leukocytes (L-selectins) platelets (P-selectins), and endothelial cells (P- and E-selectins). They interact with oligosaccharides clustered on cell surfaces (Lasky, 1995Go) during the margination and rolling of leukocytes prior to firm adhesion, extravasation, and migration to a site of infection. Fucoidan can act as a ligand for either L- or P-selectins, both of which interact with sulfated oligosaccharides. Most likely, fucoidan is acting like heparin or heparan sulfate (HS), presenting a spatial pattern of sulfated saccharide structures that imitates the clustering of sulfated, sialylated, and fucosylated oligosaccharides on the cell surface. Because purified fucoidans are not usually used in studies of this type, however, it is difficult to define which fucan structures are involved. Other linear polysaccharides, such as heparin, share this property, but the fucose branches of native selectin ligands bring to mind the branched structures in fucoidans.

The interaction between fucoidan and selectins has physiological consequences that could be therapeutically useful; for example, perfusion with fucoidan can reduce neutrophil infiltration and myocardial injury after ischemia/reperfusion (Omata et al., 1997Go). Also, leukocyte accumulation occurs in the same circumstances (Ritter et al., 1998Go). Injection of fucoidan into sensitized mice before hapten challenge can reduce contact hypersensitivity reaction (Nasu et al., 1997Go). Recruitment of leukocytes into cerebrospinal fluid in a meningitis model is reduced by fucoidan (Granert et al., 1999Go), as is IL-1 production in a similar model (Ostergaard et al., 2000Go). Neutrophil (Shimaoka et al., 1996Go) and eosinophil (Teixera and Hellewell, 1997Go) migration to sites of inflammation are also inhibited by fucoidan. All of these studies seem to indicate the potential of fucoidan as an anti-inflammatory agent. However, Verdrengh and co-workers (2000)Go have observed that, although fucoidan treatment led to less severe symptoms in the early stages of S. aureus–triggered arthritis in mice, delayed recruitment of phagocytes decreased clearance of bacteria.

Two recent studies use fucoidan in studies of selectins in the release of haematopoietic blood cells from bone marrow. Both fucoidan (Sweeney et al., 2000Go) and a pure, linear sulfated fucan (Frenette and Weiss, 2000Go) have been shown to stimulate mobilization of stem/progenitor cells in vivo; they are also effective in selectin-deficient mice and therefore must be capable of acting in a selectin-independent fashion. This selectin-independent mechanism may well be related to the release of stromal-derived factor 1, a potent chemoattractant, from bone marrow into the circulation on administration of fucoidan (Sweeney et al., 2002Go).

Fucoidan has also been used to probe the involvement of L- and P-selectin in thrombus formation in vivo (Thorlacius et al., 2000Go). Although both fucoidan and an anti-P-selectin antibody abolished P-selectin function, only fucoidan was able to prevent thrombus formation, presumably through its anticoagulant activity.

Macrophage scavenger receptors
Macrophage scavenger receptors are a group of proteins that, as the name suggests, are involved in the uptake by macrophages of items for disposal, such as oxidised lipoproteins, damaged cells, or invading microorganisms. Fucoidan is a ligand for at least some of these receptors, triggering a protein kinase–dependent signaling pathway (Hsu et al., 1998Go). Binding of neutrophil-derived azurocidin (also known as heparin-binding protein) to monocytes is inhibited in the presence of fucoidan, which also abrogates the enhancing effect of azurocidin on lipopolysaccharide-induced tumour necrosis factor-{alpha} (TNF-{alpha}) production. This is taken as evidence that the azurocidin receptor on monocytes may be related to the scavenger receptors (Heinzelmann et al., 1998Go). However, the same study also observed that fucoidan can itself stimulate TNF-{alpha} release from monocytes. It is important not to be overly simplistic in approach when dealing with a polyvalent reagent such as fucoidan.

Cell growth, migration, and adhesion
Like heparin, fucoidan has antiproliferative effects on vascular smooth muscle cells (SMCs). A fucoidan fraction from A. nodosum was more active than heparin (Logeart et al., 1997Go); fucoidans were internalized by cells and perhaps transported to the nucleus. Patel and co-workers (2002)Go were able to distinguish between the modes of activity of fucoidan and heparin; fucoidan was active even for heparin-resistant SMCs. In this study, crude commercial fucoidan was more active than the purified material, indicating that some highly active fraction was discarded in the purification, another indication that specific structures within these complex mixtures can be linked to particular biological effects. Fucoidan can also modulate proliferation of fibroblasts, and here again it has been shown that antiproliferative and anticoagulant fucoidan structures are different (Haroun-Bouhedja et al., 2000Go). The situation for endothelial cells is complex and depends on the agent used to stimulate proliferation; heparin and fucoidan affect growth and migration differently (Giraux et al., 1998b)Go.

Fertilization
In view of the importance of egg-jelly fucans for sea urchin fertilization (see Sulfated fucans from marine invertebrates), it is interesting to note that mammalian fertilization also involves oligosaccharides in the zona pellucida surrounding the egg and that these are fucosylated (Johnston et al., 1998Go) and sulfated (Moreno et al., 2001Go). Fucoidan, in common with other sulfated glycans, is a powerful inhibitor of sperm binding to oviductal monolayers (Talevi and Gualtieri, 2001Go).

Parasites and viruses
Sulfated polysaccharides are antimalarial in vitro, inhibiting the invasion of free Plasmodium falciparum parasites into erythrocytes (Clark et al., 1997Go). A thrombospondin-related adhesion protein is implicated in host cell invasion; this binds to fucoidan and heparin and probably to cell surface HS (McCormick et al., 1999Go). Fucoidan and low-molecular-weight fucoidan, but not desulfated fucoidan, inhibit Plasmodium berghei development in Hep G2 cells and sporozoite invasion of Chinese hamster ovary cells (Ying et al., 1997Go). Hepatocytes bear a particularly highly sulfated HS that is thought to be instrumental in the clearance by the liver of circulating sporozoites. However, sulfated polysaccharides can enhance adhesion of infected erythrocytes to cells bearing CD36 (McCormick et al., 2000Go). Chondroitin sulfate A has been identified as a cell-surface receptor for P. falciparum-infected erythrocytes (Rogerson et al., 1995Go), with which neither fucoidan nor other highly sulfated polysaccharides can compete.

Another widespread parasite, Toxoplasma gondii, is also affected by fucoidans. In this case low concentrations can enhance infection of fibroblasts in culture, though higher concentrations are inhibitory (Ortega-Barria and Boothroyd, 1999Go).

Like many other sulfated polysaccharides, fucoidan can inhibit virus infection of cells. This has recently been demonstrated for Herpes simplex, cytomegalovirus, and human immunodeficiency virus (Hoshino et al., 1998Go) as well as bovine viral diarrhea virus (Iqbal et al., 2000Go), probably by competing with cell surface HS for binding to the virus.


    Enzymes active on sulfated fucans
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 Abstract
 Introduction
 Nomenclature
 Structural diversity
 Physiological properties of...
 Biological properties of fucans
 Enzymes active on sulfated...
 Conclusions
 References
 
Although almost nothing is known about the biosynthesis of algal fucoidans and invertebrate sulfated fucans, enzymes capable of degrading such polysaccharides have been isolated from several marine species.

Specific enzymatic methods can be used to provide tailored oligosaccharides for biological studies, as well as simplified samples from which it is possible to deduce the structure of the original fucoidan. Currently employed methods of chemical modifications prior to analysis (hydrolysis, desulfation, deacetylation) often require strong basic or acidic condition at high temperature, which can modify the polysaccharide. For example, acetyl groups have been found in almost all the algal fucoidans studied in recent years (C. filum, C. okamuranus, F. evanescens) and may be present in all the algal fucoidans but removed during the preparation process (Chizov et al., 1999Go).

At least two kinds of glycosidase act on fucans, "fucan sulfate hydrolase" or fucoidanase EC 3.2.1.44, and {alpha}-L-fucosidase EC 3.2.1.51 (EC 3.2.1.63, EC 3.2.1.111, or EC 3.2.1.127 depending on specificity for the glycosidic linkage). If the activity of {alpha}-L-fucosidases is easily described as the release of L-fucose from the nonreducing end of a polysaccharide, it is less easy to define fucoidanase activity.

Fucoidanase can produce the cleavage of glycosidic bonds in the core of the polysaccharide, leading to rapid reduction of the molecular weight (i.e., endo-fucoidanase), or on the edge of the polysaccharide, releasing some oligosaccharides with a slow decrease of the molecular weight (i.e., exo-fucoidanase) (Tanaka and Sorai, 1970Go; Furukawa et al., 1992aGo). Fucoidanase has been reported mostly in marine mollusks and marine bacteria (Table II) but is also present in other marine invertebrates (Burtseva et al., 2000Go).


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Table II. Properties of fucoidanase purified or partially purified from various species

 
One of the difficulties in purification of fucoidanase is the absence of simple tests to identify and quantify this kind of enzyme. Methods proposed have included reduction of viscosity (Kitamura et al., 1992Go; Furukawa et al., 1992aGo), increase in reducing sugar (Yaphe and Morgan, 1959Go; Thanassi and Nakada, 1967Go), precipitation with albumin (Morinaga et al., 1981Go; Descamps et al., 1998Go), or size exclusion chromatography (Daniel et al., 1999Go). However, fucoidans of various origins have been used that may present different structures varying in their sensitivity to hydrolysis (Table II).

Yaphe and Morgan (1959)Go first reported the ability of two marine bacteria, Pseudomonas atlantica and Pseudomonas carrageenova, to produce reducing sugars (i.e., hydrolysis) from fucoidan. Later, several species of abalone (Haliotis sp.) were found able to degrade fucoidan, producing fuco-oligosaccharides (Thanassi and Nakada, 1967Go).

More recently, two species of Pectinidae have been investigated, Patinopecten yessoensis and Pecten maximus (Kitamura et al., 1992Go; Daniel et al., 1999Go); both molluscs are able to hydrolyze fucoidan efficiently to produce oligosaccharides.

To date only two fucoidanases, with apparently no common structural features, have been purified to homogeneity (Furukawa et al., 1992aGo; Kitamura et al., 1992Go). Nothing is known about the mechanism of fucoidanases, and it is difficult to compare literature results because degradation studies have been conducted using fucoidans of different origin (Table II). The first report of such an enzyme in structural studies used a fucoidanase from P. maximus (Daniel et al., 1999Go). The protein extract obtained from this scallop is active not only on A. nodosum fucoidan but also, and in a more efficient way, on the linear sulfated fucans from L. variegatus and L. grisea (Berteau unpublished data); thus, it could be more accurate to call these enzymes sulfated fucan hydrolase.

Bacterial fucoidanases have attracted more recent study (Morinaga et al., 1981Go; Bakunina et al., 2000Go, 2002Go), and at least one strain of bacteria has been patented for its ability to produce fucoidan oligosaccharides without purification of the fucoidanase (Descamps et al., 1998Go).

Comparing the properties and structures of sulfated fucans from invertebrates and algae, it seems that L-fucose branches might play a key role in biological properties. The use of {alpha}-L-fucosidase to remove these branches could help assess this hypothesis. To date three {alpha}-L-fucosidases have been reported able to release fucose from fucoidan. The first {alpha}-L-fucosidase described was purified from abalone and seemed able to hydrolyze Ecklonia cava fucoidan completely to L-fucose and sulfated L-fucose (Tanaka and Sorai, 1970Go). However, it is doubtful that this kind of glycosidase alone can fully hydrolyze such a complex polysaccharide. The second {alpha}-L-fucosidase reported able to hydrolyze F. Vesiculosus fucoidan was purified from the fungus Fusarium oxysporum (Yamamoto et al., 1986Go). This enzyme is able to release only a few units of L-fucose from fucoidan, as might be expected because of branches and the presence of sulfate groups (more than one per fucose unit). More recently an {alpha}-L-fucosidase was purified from the common scallop P. maximus. Surprisingly, this glycosidase is able to release fucose from A. nodosum fucoidan but failed to hydrolyze F. vesiculosus (Berteau et al., 2002Go). The difference of activity toward both fucoidans must arise because of structural differences, although no clear specificity was demonstrated using simple oligosaccharides.

The structural basis for biological and physiological properties of sulfated fucans depends crucially on the presence of sulfate groups. Both their density and their specific positions influence biological properties, as discussed. To date the only way to assess the importance of position of sulfate groups is to try to compare related fucans. Sulfatases (EC 3.1.6) are enzymes able to remove sulfate groups and could therefore be an invaluable tool for such studies. Only minor reports have appeared in the literature concerning sulfatases able to hydrolyze fucoidan (Lloyd and Lloyd, 1963Go; Furukawa and Fujikawa, 1984Go; Furukawa et al., 1992bGo). Nothing is known about their mechanism and properties, particularly whether they can act alone or need glycosidases to achieve significant desulfation of fucan. Only one study has shown a sulfatase able to act specifically on some sulfate groups of fucoidan (Daniel et al., 2001Go). This enzyme partially purified from the scallop P. maximus is able to release sulfate groups present at position 2 of monosulfated L-fucose, or of mono- and disulfated components of the disaccharide from A. nodosum fucoidan. In conjunction with the {alpha}-L-fucosidase purified from the same mollusc it seems possible to increase the degradation of fucans (Berteau et al., 2002Go). These enzymes are therefore unique tools to produce selectively modified fucans, which could allow direct determination of the involvement of branches and sulfate groups in their biological activity.


    Conclusions
 Top
 Abstract
 Introduction
 Nomenclature
 Structural diversity
 Physiological properties of...
 Biological properties of fucans
 Enzymes active on sulfated...
 Conclusions
 References
 
Recent structural studies present fucans as a more orderly family of polysaccharides than was previously thought. The fucoidans of brown algae are complex and heterogeneous, but recent structural analyses have revealed the presence of ordered repeat units for fucoidans from several species. In contrast, sulfated fucans from marine invertebrates have simple, regular structures; in the case of sea urchin egg-jelly fucans, the regular pattern of sulfate substitution plays a part in ensuring species specificity of fertilization.

Sulfated fucans and fucoidans have many potent biological activities due to their ability to imitate patterns of sulfate substitution on glycosaminoglycans and other sulfated glucans. Because most studies of biological activity are carried out using a relatively crude fucoidan preparation, it is not easy at present to determine the relationships between activity and structure. However, it has become clear that at least some of these various activities are not merely a function of high charge density but have distinct structural specificities.

The availability of fucans with well-defined structures and of enzymes able to modify these polysaccharides will bring clarity to analysis of their biological properties and allow the design of new experiments. Future conformational studies of well-defined fucan structures should lead to better understanding of the biological properties of fucoidans.

The complexity and heterogeneity of fucoidans, and their wide range of activities in mammalian systems, are not attractive within current paradigms of drug development. The combination of structural elucidation and assignment of biological activity to specific structural features can only improve the potential of sulfated fucans and fucoidans both as therapeutic agents and as research reagents.

Special acknowledgment: The authors would like to acknowledge the contribution of Dr. R. Daniel to this review.

1 To whom correspondence should be addressed; e-mail: bmulloy{at}nibsc.ac.uk Back


    Abbreviations
 
HS, heparan sulfate; NMR, nuclear magnetic resonance; SMC, smooth muscular cell; TNF-{alpha}, tumor necrosis factor-{alpha}


    References
 Top
 Abstract
 Introduction
 Nomenclature
 Structural diversity
 Physiological properties of...
 Biological properties of fucans
 Enzymes active on sulfated...
 Conclusions
 References
 
Abdel-Fattah, A.F., Hussein, M.M.D., and Fouad, S.T. (1978) Carbohydrates of the brown seaweed Dictyota dichotoma. Phytochemistry, 17, 741–743.[CrossRef][ISI]

Alves, A.P., Mulloy, B., Diniz, J.A., and Mourao, P.A.S. (1997) Sulfated polysaccharides from the egg jelly layer are species-specific inducers of acrosomal reaction in sperms of sea urchins. J. Biol. Chem., 272, 6965–6971.[Abstract/Free Full Text]

Alves, A.P., Mulloy, B., Moy, G.W., Vacquier, V.D., and Mourao, P.A.S. (1998) Females of the sea urchin Strongylocentrotus purpuratus differ in the structure of their egg jelly sulphated fucans. Glycobiology, 8, 939–946.[Abstract/Free Full Text]

Alwayn, I.P., Appel, J.Z., Goepfert, C., Buhler, L., Cooper, D.K., and Robson, S.C. (2000) Inhibition of platelet aggregation in baboons: therapeutic implications for xenotransplantation. Xenotransplantation, 7, 247–257.[CrossRef][ISI][Medline]

Anno, K., Terahata, H., Hayashi, Y., and Seno, N. (1966) Isolation and purification of fucoidin from brown seaweed Pelvetia wrightii. Agr. Biol. Chem., 30, 495–499.[ISI]

Bakunina, I.Y., Shevchenko, L.S., Nedashkovskaya, O.I., Shevchenko, N.M., Alekseeva, S.A., Mikhailov, V.V., and Zvyagintseva, T.N. (2000) Screening of marine bacteria for fucoidanases. Microbiology (Moscow), 69, 303–308.[ISI]

Bakunina, I.Y., Nedashkovskaia, O.I., Alekseeva, S.A., Ivanova, E.P., Romanenko, L.A., Gorshkova, N.M., Isakov, V.V., Zviagintseva, T.N., and Mikhailov, V.V. (2002) Degradation of fucoidan by the marine protebacterium Pseudoalteromonas citrea. Microbiology, 71, 41–47.[CrossRef][ISI]

Bernardi, G. and Springer, G.F. (1962) Properties of highly purified fucan. J. Biol. Chem., 237, 75–80.[Free Full Text]

Berteau O., McCort I., Goasdoue N., Tissot B., and Daniel R. (2002) Characterization of a new alpha-L-fucosidase isolated from the marine mollusk Pecten maximus that catalyzes the hydrolysis of alpha-L-fucose from algal fucoidan (Ascophyllum nodosum). Glycobiology, 12, 273–282.[Abstract/Free Full Text]

Bilan, M.I., Grachev, A.A., Ustuzhanina, N.E., Shashkov, A.S., Nifantiev, N.E., and Usov, A.I. (2002) Structure of a fucoidan from the brown seaweed Fucus evanescens C.Ag. Carbohydr. Res., 337, 719–730.[CrossRef][ISI][Medline]

Bisgrove, S.R. and Kropf, D.L. (2001) Cell wall deposition during morphogenesis in fucoid algae. Planta, 212, 648–658.[CrossRef][ISI][Medline]

Black, W.A.P. (1954) The seasonal variation in the combined L-fucose content of the common British laminariaceae and fucaceae. J. Sci. Food Agr., 5, 445–448.[ISI]

Boisson-Vidal, C., Haroun, F., Ellouali, M., Blondin, C., Fischer, A.M., De Agostini, A., and Jozefonvicz, J. (1995) Biological activities of polysaccharide from marine algae. Drugs Fut., 20, 1237–1249.

Boisson-Vidal, C., Chaubet, F., Chevolot, L., Sinquin, C., Theveniaux, J., Millet, J., Sternberg, C., Mulloy, B., and Fischer, A.M. (2000) Relationship between antithrombotic activities of fucans and their structure. Drug Devel. Res., 51, 216–224.[CrossRef][ISI]

Burtseva, Y.V., Kusaikin, M.I., Sova, V.V., Shevchenko, N.M., Skobun, A.S., and Zvyagintseva, T.N. (2000) Distribution of fucoidan hydrolases and some glycosidases among marine invertebrates. Russ. J. Mar. Biol., 26, 453–456.[CrossRef]

Chargaff, E., Bancroft, F.W., and Brown, M.S. (1936) Studies on the chemistry of blood coagulation II. On the inhibition of blood clotting by substances of high molecular weight. J. Biol. Chem., 115, 155–156.

Chevolot, L., Foucault, A., Kervarec, N., Sinquin, C., Fisher, A.M., and Boisson-Vidal, C. (1999) Further data on the structure of brown seaweed fucans: relationships with anticoagulant activity. Carbohyd. Res., 319, 154–165.[CrossRef][ISI][Medline]

Chevolot, L., Mulloy, B., Ratiskol, J., Foucault, A., and Colliec-Jouault, S. (2001) A disaccharide repeat unit is the major structure in fucoidans from two species of brown algae. Carbohyd. Res., 330, 529–535.[CrossRef][ISI][Medline]

Chizhov, A.O., Dell, A., Morris, H.R., Haslam, S.M., McDowell, R.A., Shashkov, A.S., Nifant'ev, N.E., Khatuntseva, E.A., and Usov, A.I. (1999) A study of fucoidan from the brown seaweed Chorda filum. Carbohyd. Res., 320, 108–119.[CrossRef][ISI][Medline]

Clark, D.L., Su, S.D., and Davidson, E.A. (1997) Saccharide anions as inhibitors of the malaria parasite. Glycoconj. J., 14, 473–479.[CrossRef][ISI][Medline]

Colliec, S., Boisson-Vidal, C., and Jozefonvicz, J. (1994) A low molecular weight fucoidan fraction from the brown seaweed Pelvetia caniculata. Phytochemistry, 35, 697–700.[CrossRef][ISI]

Colwell, N.S., Grupe, M.J., and Tollefsen, D.M. (1999) Amino acid residues of heparin cofactor II required for stimulation of thrombin inhibition by sulphated polyanions. Biochim. Biophys. Acta, 1431, 148–156.[ISI][Medline]

Côté, R.H. (1959) Disaccharides from fucoidin. J. Chem. Soc., 2248–2254.

Daniel, R., Berteau, O., Jozefonvicz, J., and Goasdoue, N. (1999) Degradation of algal (Ascophyllum nodosum) fucoidan by an enzymatic activity contained in digestive glands of the marine mollusc Pecten maximus. Carbohyd. Res., 322, 291–297.[CrossRef][ISI]

Daniel, R., Berteau, O., Chevolot, L., Varenne, A., Gareil, P., and Goasdoue, N. (2001) Regioselective desulfation of sulfated L-fucopyranoside by a new sulfoesterase from the marine mollusk Pecten maximus: application to the structural study of algal fucoidan (Ascophyllum nodosum). Eur. J. Biochem., 268, 5617–5628.[Abstract/Free Full Text]

de Reviers, B., Mabeau, S., and Kloareg, B. (1983) Essai d'interprétation de la structure des fucoidanes en liaison avec leur localisation dans la paroi des phéophycées. Crit. Rev. Biochem., 4, 55–62.

Descamps, V., Klarszinsky, O., Barbeyron, T., Cloarec, B., Fritig, B., Jouber, J.M., Plesse, B., and Yvin, J.C. (1998) Fuco-oligosaccharides, enzyme pour leur préparation à partir de fucanes, bacterie productrice de l'enzyme et applications des fuco-oligosaccharides à la protection des plantes. Brevet, FR 2 783 523.

Dobashi K., Nishino T., Fujihara M., and Nagumo, T. (1989) Isolation and preliminary characterization of fucose-containing sulfated polysaccharides with blood-anticoagulant activity from the brown seaweed Hizikia fusiforme. Carbohydr. Res., 194, 315–320.[CrossRef][ISI][Medline]

Duarte, M.E.R., Cardoso, M.A., Noseda, M.D., and Cerezo, A.S. (2001) Structural studies on fucoidans from the brown seaweed Sargassum stenophyllum. Carbohyd. Res., 333, 281–293.[CrossRef][ISI][Medline]

Durig, J., Bruhn, T., Zurborn, K.H., Gutensohn, K., Bruhn, H.D., and Beress, L. (1997) Anticoagulant fucoidan fractions from Fucus vesiculosus induce platelet activation in vitro. Thromb. Res., 85, 479–491.[CrossRef][ISI][Medline]

Evans, L.V. (1989) Mucilaginous substances from macroalgae: an overview. Symp. Soc. Exp. Biol., 43, 455–461.[Medline]

Farias, W.R.L., Valente, A.P., Pereira, M.S., and Mourao, P.A.S. (2000) Structure and anticoagulant activity of sulfated galactans—Isolation of a unique sulfated galactan from the red algae Botryocladia occidentalis and comparison of its anticoagulant action with that of sulfated galactans from invertebrates. J. Biol. Chem., 275, 29299–29307.[Abstract/Free Full Text]

Frenette, P.S. and Weiss, L. (2000) Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms. Blood, 96, 2460–2468.[Abstract/Free Full Text]

Furukawa, S. and Fujikawa, T. (1984) Growth and fucoidan sulfatase production in fucoidan-utilizing bacteria from sea sand. Nippon Nogeik. Kaishi, 11, 1123–1126.

Furukawa, S.I., Fujikawa, T., Koga, D., and Ide, A. (1992a) Purification and some properties of exo-type fucoidanases from Vibrio sp. N-5. Biosci. Biotech. Biochem., 56, 1829–1834.[ISI]

Furukawa, S.I., Fujikawa, T., Koga, D., and Ide, A. (1992b) Production of fucoidan-degrading enzymes, fucoidanase, and fucoidan sulfatase by Vibrio sp. N-5. Nippon Suisan Gakk., 58, 1499–1503.[ISI]

Gerbst, A.G., Ustuzhanina, N.E., Grachev, A.A., Khatuntseva, E.A., Tsvetkov, D.E., Whitfield, D.M., Berces, A., and Nifantiev, N.E. (2001) Synthesis, NMR, and conformational studies of fucoidan fragments 3. J. Carbohyd. Chem., 20, 821–831.[CrossRef][ISI]

Gerbst, A.G., Ustuzhanina, N.E., Grachev, A.A., Zlotina, N.S., Khatuntseva, E.A., Tsvetkov, D.E., Shashkov, A.S., Usov, A.I., and Nifantiev, N.E. (2002) Synthesis, NMR, and conformational studies of fucoidan fragments 4. J. Carbohyd. Chem., 2(1), 313–324.

Giraux, J.L., Matou, S., Bros, A., Tapon-Bretaudiere, J., Letourneur, D., and Fischer, A.M. (1998a) Modulation of human endothelial cell proliferation and migration by fucoidan and heparin. Eur. J. Cell. Biol., 77, 352–359.[ISI][Medline]

Giraux, J.L., Tapon-Bretaudiere, J., Matou, S., and Fischer, A.M. (1998b) Fucoidan, as heparin, induces tissue factor pathway inhibitor release from cultured human endothelial cells. Thromb. Haemost., 80, 692–695.[ISI][Medline]

Granert, C., Raud, J., Waage, A., and Lindquist, L. (1999) Effects of polysaccharide fucoidin on cerebrospinal fluid interleukin-1 and tumor necrosis factor alpha in Pneumococcal meningitis in the rabbit. Infect. Immun., 67, 2071–2074.[Abstract/Free Full Text]

Haroun-Bouhedja, F., Ellouali, M., Sinquin, C., and Boisson-Vidal, C. (2000) Relationship between sulfate groups and biological activities of fucans. Thromb. Res., 100, 453–459.[CrossRef][ISI][Medline]

Heinzelmann, M., Polk, H.C.J., and Miller, F.N. (1998) Modulation of lipopolysaccharide-induced monocyte activation by heparin-binding protein and fucoidan. Infect. Immun., 66, 5842–5847.[Abstract/Free Full Text]

Hirohashi, N. and Vacquier, V. D. (2002) High molecular mass egg fucose sulfate polymer is required for opening both Ca2+ channels involved in triggering the sea urchin sperm acrosome reaction. J. Biol. Chem., 277, 1182–1189.[Abstract/Free Full Text]

Hirohashi, N., Vilela-Silva, A.E.S., Mourão, P.A.S., and Vacquier, V.D. (2002) Structural requirements for species-specific induction of the sperm acrosome reaction by sea urchin egg sulfated fucan. Biochem. Biophys. Res. Commun., 298, 403–407.[CrossRef][ISI][Medline]

Honya, M., Mori, M., Anzai, M., Araki, Y., and Nisizawa, K. (1999) Monthly changes in the content of fucans, their constituent sugars and sulphate in cultured Laminaria japonica. Hydrobiologia, 398/399, 411–416.[CrossRef]

Hoshino, T., Hayashi, T., Hayashi, K., Hamada, J., Lee, J.B., and Sankawa, U. (1998) An antivirally active sulfated polysaccharide from Sargassum horneri (TURNER) C. AGARDH. Biol. Pharm. Bull., 21, 730–734.[ISI][Medline]

Hsu H.Y., Hajjar D.P., Khan K.M., and Falcone D.J. (1998) Ligand binding to macrophage scavenger receptor-A induces urokinase-type plasminogen activator expression by a protein kinase-dependent signaling pathway. J. Biol. Chem., 273, 1240–1246.[Abstract/Free Full Text]

Iqbal, M., Flick-Smith, H., and McCauley, J.W. (2000) Interactions of bovine viral diarrhoea virus glycoprotein E(rns) with cell surface glycosaminoglycans. J. Gen. Virol., 81, 451–459.[Abstract/Free Full Text]

Johnston, D.S., Wright, W.W., Shaper, J.H., Hokke, C.H., Van den Eijnden, D.H., and Joziasse, D.H. (1998) Murine sperm-zona binding, a fucosyl residue is required for a high affinity sperm-binding ligand. A second site on sperm binds a nonfucosylated, beta-galactosyl-capped oligosaccharide. J. Biol. Chem., 273, 1888–1895.[Abstract/Free Full Text]

Killing, H. (1913) Zur biochemie der Meersalgen. Z. Physiol. Chem., 83, 171–197.

Kitamura, K., Matsuo, M., and Yasui, T. (1992) Enzymic degradation of fucoidan by fucoidanase from the hepatopancreas of Patinopecten yessoensis. Biosci. Biotech. Biochem., 56, 490–494.[ISI]

Kloareg, B. (1981) Structure et rôle écophysiologique des parois des algues littorales: contribution à la résistance aux variations de salinité. Physiol. Vég., 17, 731–747.[ISI]

Kloareg, B. (1984) Isolation and analysis of cell walls of the brown marine algae Pelvetia canaliculata and Ascophyllum nodosum. Physiol. Vég., 22, 47–56.[ISI]

Kloareg, B. and Quatrano, R.S. (1988) Structure of the cell walls of marine algae and ecophysiological functions of the matrix polysaccharides. Oceanogr. Mar. Biol. Annu. Rev., 26, 259–315.

Kloareg, B., Demarty, M., and Mabeau, S. (1986) Polyanionic characteristic of purified sulphated homofucans from brown algae. Int. J. Biol. Macromol., 8, 380–386.[CrossRef][ISI]

Larsen, B., Haug, A., and Painter, T.J. (1966) Sulphated polysaccharides in brown algae—I. Isolation and preliminary characterisation of three sulphated polysaccharides from Ascophyllum nodosum (L.) Le JOL. Acta Chem. Scand., 20, 219–230.[ISI]

Lasky, L.A. (1995) Selectin-carbohydrate interactions and the initiation of the inflammatory response. Annu. Rev. Biochem., 64, 113–139.[CrossRef][ISI][Medline]

Leite, E.L., Medeiros, M.G.L., Rocha, H.A.O., Farias, G.G.M., da Silva, L.F., Chavante, S.F., de Abreu, L.D., Dietrich, C.P., and Nader, H.B. (1998) Structure and pharmacological activities of a sulfated xylofucoglucuronan from the alga Spatoglossum schroederi. Plant Sci., 132, 215–228.[CrossRef][ISI]

Lloyd, P.F. and Lloyd, K.O. (1963) Sulphatases and sulphated polysaccharide in the viscera of marine mollusc. Nature, 199, 287.

Logeart, D., Prigent-Richard, S., Boisson-Vidal, C., Chaubet, F., Durand, P., Jozefonvicz, J., and Letourneur, D. (1997) Fucans, sulfated polysaccharides extracted from brown seaweeds, inhibit vascular smooth muscle cell proliferation. II. Degradation and molecular weight effect. Eur. J. Cell. Biol., 74, 385–390.[ISI][Medline]

Mabeau, S., Kloareg, B., and Joseleau, J.P. (1990) Fractionation and analysis of fucans from brown algae. Phytochemistry, 29, 2441–2445.[CrossRef][ISI]

Marais, M.-F. and Joseleau, J.-P. (2001) A fucoidan fraction from Ascophyllum nodosum. Carbohydr. Res., 336, 155–159.[CrossRef][ISI][Medline]

Maruyama, H., Nakajima, J., and Yamamoto, I. (1987) A study on the anticoagulant and fibrinolytic activities of a crude fucoidan from the edible brown seaweed Laminaria religiosa, with special reference to its inhibitory effect on the growth of sarcoma-180 ascites cells subcutaneously implanted into mice. Kitasato Arch. Exp. Med., 60, 105–121.[Medline]

Mauray, S., de Raucourt, E., Talbot, J.C., Dachary-Prigent, J., Jozefowicz, M., and Fischer, A.M. (1998) Mechanism of factor IXa inhibition by antithrombin in the presence of unfractionated and low molecular weight heparins and fucoidan. Biochim. Biophys. Acta, 1387, 184–194.[ISI][Medline]

McCormick, C.J., Tuckwell, D.S., Crisanti, A., Humphries, M.J., and Hollingdale, M.R. (1999) Identification of heparin as a ligand for the A-domain of Plasmodium falciparum thrombospondin-related adhesion protein. Mol. Biochem. Parasitol., 100, 111–124.[CrossRef][ISI][Medline]

McCormick, C.J., Newbold, C.I., and Berendt, A.R. (2000) Sulfated glycoconjugates enhance CD36-dependent adhesion of Plasmodium falciparum-infected erythrocytes to human microvascular endothelial cells. Blood, 96, 327–333.[Abstract/Free Full Text]

McNeely, W.H. (1959) Fucoidan. In Whistler, R.L. (ed), Industrial gums. Academic Press, New York, pp. 117–125.

Medcalf, D.G., Schneider, T.L., and Barnett, R.W. (1978) Structural features of a novel glucuronogalactofucan from Ascophyllum nodosum. Carbohyd. Res., 66, 167–171.[CrossRef][ISI]

Mengerink, K.J., Moy, G.W., and Vacquier, V.D. (2002) suREJ3, a polycystin-1 protein, is cleaved at the GPS domain and localizes to the acrosomal region of sea urchin sperm. J. Biol. Chem., 277, 943–948.[Abstract/Free Full Text]

Mian, A.J. and Percival, E. (1973) Carbohydrates of the brown seaweeds Himanthalia lorea, Bifurcaria bifurcata, and Padina pavonia. Carbohyd. Res., 26, 133–146.[CrossRef][ISI]

Millet, J., Jouault, S.C., Mauray, S., Theveniaux, J., Sternberg, C., Boisson, V.C., and Fischer, A.M. (1999) Antithrombotic and anticoagulant activities of a low molecular weight fucoidan by the subcutaneous route. Thromb. Haemost., 81, 391–395.[ISI][Medline]

Moreno, R., Orihuela, P., and Barros, C. (2001) Differential effects of polysulphates between mouse and hamster during in vitro fertilization. Andrologia, 33, 19–25.[CrossRef][ISI][Medline]

Mori, H., Kamei, H., Nishide, E., and Nisizawa, K. (1982) Sugar constituents of some sulfated polysaccharides from the sporophylls of wakame (Undaria pinnatifida) and their biological activities. In Marine algae in pharmaceutical science. Walter de Gruyter, Berlin and New York, pp. 109–121.

Morinaga, T., Araki, T., Ito, M., and Kitamikado, M. (1981) A search for fucoidan-degrading bacteria in coastal sea environments of Japan. Bull. Japan. Soc. Sci. Fish., 47, 621–625.[ISI]

Mourão, P.A.S. and Bastos, I.G. (1987) Highly acidic glycans from sea cucumbers. Eur. J. Biochem., 166, 639–645.[Abstract]

Mourão, P.A.S. and Pereira, M.S. (1999) Searching for alternatives to heparin: sulfated fucans from marine invertebrates. Trends Cardiovasc. Med., 9, 225–232.[CrossRef][Medline]

Mulloy, B., Ribeiro, A.C., Alves, A.P., Vieira, R.P., and Mourao, P.A.S. (1994) Sulfated fucans from echinoderms have a regular tetrasaccharide repeating unit defined by specific patterns of sulfation at the O-2 and O-4 positions. J. Biol. Chem., 269, 22113–22123.[Abstract/Free Full Text]

Nagaoka, M., Shibata, H., Kimura-Takagi, I., Hashimoto, S., Kimura, K., Makino, T., Aiyama, R., Ueyama, S., and Yokokura, T. (1999) Structural study of fucoidan from Cladosiphon okamuranus Tokida. Glycoconj. J., 16, 19–26.[CrossRef][ISI][Medline]

Nasu, T., Fukuda, Y., Nagahira, K., Kawashima, H., Noguchi, C., and Nakanishi, T. (1997) Fucoidin, a potent inhibitor of L-selectin function, reduces contact hypersensitivity reaction in mice. Immunol. Lett., 59, 47–51.[CrossRef][ISI][Medline]

Nishino, T. and Nagumo, T. (1991) Structural characterization of a new anticoagulant fucan sulfate from the brown seaweed Ecklonia kurome. Carbohyd. Res., 211, 77–90.[CrossRef][ISI][Medline]

Nishino, T., Nishioka, C., Ura, H., and Nagumo, T. (1994) Isolation and partial characterization of a novel amino sugar-containing fucan sulfate from commercial Fucus vesiculosus fucoidan. Carbohydr. Res., 255, 213–224.[CrossRef][ISI][Medline]

Nishino, T., Fukuda, A., Nagumo, T., Fujihara, M., and Kaji, E. (1999) Inhibition of the generation of thrombin and factor Xa by a fucoidan from the brown seaweed Ecklonia kurome. Thromb. Res., 96, 37–49.[CrossRef][ISI][Medline]

Nishino, T., Yamauchi, T., Horie, M., Nagumo, T., and Suzuki, H. (2000) Effects of a fucoidan on the activation of plasminogen by u-PA and t-PA. Thromb. Res., 99, 623–634.[CrossRef][ISI][Medline]

Omata, M., Matsui, N., Inomata, N., and Ohno, T. (1997) Protective effects of polysaccharide fucoidin on myocardial ischemia-reperfusion injury in rats. J. Cardiovasc. Pharmacol., 30, 717–724.[CrossRef][ISI][Medline]

O'Neill, A.N. (1954) Degradative studies on fucoidin. J. Am. Chem. Soc., 76, 5074–5076.[ISI]

Ortega-Barria, E. and Boothroyd, J.C. (1999) A Toxoplasma lectin-like activity specific for sulfated polysaccharides is involved in host cell infection. J. Biol. Chem., 274, 1267–1276.[Abstract/Free Full Text]

Ostergaard, C., Yieng-Kow, R.V., Benfield, T., Frimodt-Moller, N., Espersen, F., and Lundgren, J.D. (2000) Inhibition of leukocyte entry into the brain by the selectin blocker fucoidin decreases interleukin-1 (IL-1) levels but increases IL-8 levels in cerebrospinal fluid during experimental Pneumococcal meningitis in rabbits. Infect. Immun., 68, 3153–3157.[Abstract/Free Full Text]

Patankar, M.S., Oehninger, S., Barnett, T., Williams, R.L., and Clark, G.F. (1993) A revised structure for fucoidan may explain some of its biological activities. J. Biol. Chem., 268, 21770–21776.[Abstract/Free Full Text]

Patel, M.K., Mulloy, B., Gallagher, K.L., O'Brien, L., and Hughes, A.D. (2002) The antimitogenic action of the sulphated polysaccharide fucoidan differs from heparin in human vascular smooth muscle cells. Thromb. Haemost., 87, 149–154.[ISI][Medline]

Percival, E. (1968) Glucuronoxylofucan, a cell-wall component of Ascophyllum nodosum. Carbohyd. Res., 7, 272–283.[CrossRef][ISI]

Percival, E.G.V. and Ross, A.G. (1950) The isolation and purification of fucoidin from brown seaweeds. J. Chem. Soc., 717–720.

Pereira, M.S., Mulloy, B., and Mourao, P.A.S. (1999) Structure and anticoagulant activity of sulfated fucans. J. Biol. Chem., 274, 7656–7667.[Abstract/Free Full Text]

Pereira, M.S., Melo, F.R., and Mourão, P.A.S. (2002) Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans? Glycobiology, 12, 573–580.[Abstract/Free Full Text]

Ponce, N.M.A., Pujol, C.A., Damonte, E.B., Flores, M.L., and Stortz, C.A. (2003) Fucoidans from the brown seaweed Adenocystis utricularis: extraction methods, antiviral activity and structural studies. Carbohydr. Res., 338, 153–165.[CrossRef][ISI][Medline]

Preeprame, S., Hayashi, K., Lee, J.B., Sankawa, U., and Hayashi, T. (2001) A novel antivirally active fucan sulfate derived from an edible brown alga, Sargassum horneri. Chem. Pharm. Bull., 49, 484–485.[CrossRef][ISI][Medline]

Ribeiro, A.C., Vieira, R.P., Mourao, P.A.S., and Mulloy, B. (1994) A sulfated {alpha}-L-fucan from sea cucumber. Carbohyd. Res., 255, 225–240.[CrossRef][ISI][Medline]

Ritter, L.S., Copeland, J.G., and McDonagh, P.F. (1998) Fucoidin reduces coronary microvascular leukocyte accumulation early in reperfusion. Ann. Thorac. Surg., 66, 2063–2072.[Abstract/Free Full Text]

Rogerson, S.J., Chaiyaroj, S.C., Ng, K., Reeder, J.C., and Brown, G.V. (1995) Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes. J. Exp. Med., 182, 15–20.[Abstract]

Rozkin, M.Y., Levina, M.N., Kameneva, N.S., Usov, A.I., and Yefimov, V.S. (1989) Investigation of the mechanism of the anticoagulant action of fucoidans. Farmakol. i toksik., 3, 48–51.

Schweiger, R.G. (1962) Methanolysis of fucoidan. I. Preparation of methyl {alpha}-L-fucoside and L-fucose. J. Org. Chem., 27, 4267–4272.[ISI]

Shimaoka, M., Ikeda, M., Iida, T., Taenaka, N., Yoshiya, I., and Honda, T. (1996) Fucoidin, a potent inhibitor of leukocyte rolling, prevents neutrophil influx into phorbol-ester-induced inflammatory sites in rabbit lungs. Am. J. Respir. Crit. Care. Med., 153, 307–311.[Abstract]

Sweeney, E.A., Priestley, G.V., Nakamoto, B., Collins, R.G., Beaudet, A.L., and Papayannopoulou, T. (2000) Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence. Proc. Natl Acad. Sci. USA, 97, 6544–6549.[Abstract/Free Full Text]

Sweeney, E.A., Lortat-Jacob, H., Priestley, G.V., Nakamoto, B., and Papayannopoulou, T. (2002) Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells. Blood, 99, 44–51.[Abstract/Free Full Text]

Talevi, R. and Gualtieri, R. (2001) Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro. Biol. Reprod., 64, 491–498.[Abstract/Free Full Text]

Tanaka, K. and Sorai, S. (1970) Hydrolysis of fucoidan by abalone liver {alpha}-L-fucosidase. FEBS Lett., 9, 45–48.[CrossRef][ISI][Medline]

Teixeira, M.M. and Hellewell, P. (1997) The effect of the selectin binding polysaccharide fucoidin on eosinophil recruitment in vivo. Brit. J. Pharmacol., 120, 1059–1066.[Abstract]

Thanassi, N.M. and Nakada, H. (1967) Enzymic degradation of fucoidan by enzymes from the hepatopancreas of abalone, Haliotus species. Arch. Biochem. Biophys., 118, 172–177.[ISI]

Thorlacius, H., Vollmar, B., Seyfert, U.T., Vestweber, D., and Menger, M.D. (2000) The polysaccharide fucoidan inhibits microvascular thrombus formation independently from P- and L-selectin function in vivo. Eur. J. Clin. Invest., 30, 804–810.[CrossRef][ISI][Medline]

Trento, F., Cattaneo, F., Pescador, R., Porta, R., and Ferro, L. (2001) Antithrombin activity of an algal polysaccharide. Thromb. Res., 102, 457–465.[CrossRef][ISI][Medline]

Usov, A.I., Smirnova, G.P., and Klochkova, N.G. (2001) Algae polysaccharides. 55. Polysaccharide composition of some brown Kamchatka algae. Bioorg. Khim., 27, 444–448.[Medline]

Usui, T., Asari, K., and Mizuno, T. (1980) Isolation of highly purified "fucoidan" from Eisenia bicyclis and its anticoagulant and antitumor activities. Agric. Biol. Chem., 44, 1965–1966.[ISI]

Vacquier, V.D. and Moy, G.W. (1997) The fucose sulfate polymer of egg jelly binds to sperm REJ and is the inducer of the sea urchin sperm acrosome reaction. Dev. Biol., 192, 125–135.[CrossRef][ISI][Medline]

Vasseur, E. (1948) Chemical studies on the jelly coat of the sea-urchin egg. Acta Chem. Scand., 2, 900–913.[ISI]

Verdrengh, M., Erlandsson-Harris, H., and Tarkowski, A. (2000) Role of selectins in experimental Staphylococcus aureus-induced arthritis. Eur. J. Immunol., 30, 1606–1613.[CrossRef][ISI][Medline]

Vilela-Silva, A.C., Alves, A.P., and Valente, A.P. (1999) Structure of the sulfated {alpha}-L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus: patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition. Glycobiology, 9, 927–933.[Abstract/Free Full Text]

Vilela-Silva, A.C., Castro, M.O., Valente, A.P., Biermann, C.H., and Mourao, P.A.S. (2002) Sulfated fucans from the egg jellies of the closely related sea urchins Strongylocentrotus droebachiensis and Strongylocentrotus pallidus ensure species-specific fertilization. J. Biol. Chem., 277, 379–387.[Abstract/Free Full Text]

Von Holdt, M.M., Ligthelm, S.P., and Nunn, J.R. (1955) South African seaweeds: seasonal variations in the chemical composition of some phaeophyceae. J. Sci. Food Agr., 6, 193–197.

Wort, D.J. (1955) The seasonal variation in chemical composition of Macrocystis integrifolia and Neroecystis luetkeana in British Colombia coastal waters. Can. J. Bot., 33, 323–340.

Yamamoto, K., Tsuji, Y., Kumagai, H., and Tochikura, T. (1986) Induction and purification of {alpha}-L-fucosidase from Fusarium oxysporum. Agric. Biol. Chem., 50, 1689–1695.[ISI]

Yaphe, W. and Morgan, K. (1959) Hydrolysis of fucoidin by Pseudomonas atlantica and Pseudomonas carrageenovora. Nature, 463, 761–762.

Ying, P., Shakibaei, M., Patankar, M.S., Clavijo, P., Beavis, R.C., Clark, G.F., and Frevert, U. (1997) The malaria circumsporozoite protein: interaction of the conserved regions I and II-plus with heparin-like oligosaccharides in heparan sulfate. Exp. Parasitol., 85, 168–182.[CrossRef][ISI][Medline]