Glycobiology vol. 13 no. 6 pp. 29R±40R, 2003 DOI: 10.1093/glycob/cwg058

REVIEW Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 Olivier Berteau2 and Barbara Mulloy1,3 egg jelly coat of sea urchins (Mulloy et al., 1994) and in the body wall of sea cucumber (Mourao~ and Bastos, 1987). 2Department of Chemistry, Swedish University of Agricultural Sciences, Arrheniusplan 8, P.O. Box 7015, SE-750 07 Uppsala, Sweden; and The fucans of brown algae, often called fucoidans, have 3National Institute for Biological Standards and Control, been known for some time to act as modulators of coagula- Blanche Lane, South Mimms, Potters Bar, Hertfordshire ENG 3QG, tion, as have other algal polysaccharides (Chargaff et al., United Kingdom 1936). Fucoidan preparations have been proposed as alter- Accepted on February 12, 2003 natives to the anticoagulant heparin, which is prepared from mammalian mucosa; being of vegetable origin they Sulfated fucans, frequently referred to simply as fucans, are less likely to contain infectious agents, such as viruses or constitute a class of polysaccharides first isolated in 1913. prions. Like heparin, it has been shown that fucoidans For many years fucans were regarded only as a potential affect many biological activities, such as inflammation, source of L-fucose, although their anticoagulant activity was cell proliferation and adhesion, viral infection, and fertiliza- known. Even as the potent effects of fucans on physiological tion (Boisson-Vidal et al., 1995). systems have become better characterized, structural studies However, relatively few studies have interpreted the bio- have lagged behind. Recently the search for new drugs has logical activity of fucoidans in terms of molecular structure. raised increased interest in sulfated fucans. In the past few Almost all biological studies use a commercially available, years, several structures of algal and invertebrate fucans have crude preparation of sulfated polysaccharides from Fucus been solved, and many aspects of their biological activity have vesiculosus rather than a purified fucoidan (Mulloy et al., been elucidated. From this work emerges a more interesting 1994). Recent insights into the structures of fucans from picture of this class of polysaccharides than was previously different plant and animal species may help explain their suspected. The availability of purified fucans and fucan frac- mode of activity, whether as research reagents or as poten- tions with simple, but varied structures, in conjunction with tial therapeutics. the development of new enzymatic tools, demonstrate that the The aim of this review is to give an up-to-date view of the biological properties of sulfated fucans are not only a simple physiological and structural properties of sulfated fucans function of their charge density but also are determined by from marine algae and invertebrates and to present a dis- detailed structural features. cussion of specific hydrolytic enzymes, which are expected to simplify structural and structure/function studies. Key words: fucan/fucoidan/fucoidin/sulfated fucan/ Algal fucoidans are present in several orders, mainly fucoidanase Fucales and Laminariales but also in Chordariales, Dictyo- tales, Dictyosiphonales, Ectocarpales, and Scytosiphonales (Table I). In fact they are widely present among all the Introduction brown algae (Phaeophyceae) so far investigated. On the other hand, fucoidans seem to be absent from green The first isolation of ``fucoidin'' from marine brown algae algae (Chlorophyceae), red algae (Rhodophyceae), and was reported 90 years ago (Killing, 1913). Thirty-five years golden algae (Xanthophyceae) and from freshwater algae later, evidence was published showing that fucans also and terrestrial plants. occur in marine invertebrates (Vasseur, 1948). These poly- The only other sources of sulfated fucan known to date saccharides, mainly constituted of sulfated L-fucose, are are marine invertebrates. The first report was made by easily extracted from the cell wall of brown algae (i.e., Vasseur (1948), who extracted a polysaccharide mainly con- Phaeophyceae) with hot water (Percival and Ross, 1950) stituted of sulfated methyl-pentose from eggs of sea urchin. or acid solution (Black, 1954) and can account for more Since then, sulfated fucans have been isolated from the egg than 40% of the dry weight of isolated cell walls (Kloareg, jelly coat of many species of sea urchin (Mulloy et al., 1994; 1984). In marine invertebrates, sulfated fucans occur in the Alves et al., 1997, 1998; Vilela-Silva et al., 1999, 2002) and from the body wall of another type of marine echinoderm, the sea cucumber Ludwigothurea grisea (Mourao~ and Bastos, 1To whom correspondence should be addressed; e-mail: 1987; Ribeiro et al., 1994). To date, naturally occurring [email protected] fucans without sulfate groups have never been reported.

Glycobiology vol. 13 no. 6 # Oxford University Press 2003; all rights reserved. 29R O. Berteau and B. Mulloy

Table I. Brown algae containing fucoidan

Species Order Reference

Cladosiphon okamuranus Chordariales Nagaoka et al., 1999 Chordaria flagelliformis, Ch. gracilis Chordariales Usov et al., 2001 Saundersella simplex Chordariales Usov et al., 2001 Desmarestia intermedia Desmarestiales Usov et al., 2001 Dictyosiphon foeniculaceus Dictyosiphonales Usov et al., 2001 Dictyota dichotoma Dictyotales Abdel-Fattah et al., 1978 Padina pavonica Dictyotales Mian and Percival, 1973 Spatoglossum schroederi Dictyotales Leite et al., 1998 Adenocystis utricularis Ectocarpales Ponce et al., 2003 Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 Pylayella littoralis Ectocarpales Usov et al., 2001 Ascophyllum nodosum Fucales Killing, 1913 Bifurcaria bifurcata Fucales Mian and Percival, 1973 Fucus.vesiculosus, F. spiralis, F. serratus, F. evanescens Fucales Killing, 1913; Black, 1954; Usov et al. 2001 Himanthalia lorea Fucales Mian and Percival, 1973 Hizikia fusiforme Fucales Dobashi et al., 1989 Pelvetia canaliculata, P. wrightii Fucales Anno, 1966; Mabeau et al., 1990 Sargassum stenophyllum, S. horneri, S. Kjellmanium, Fucales Yamamoto et al., 1984; Mabeau et al., 1990; S. muticum Duarte et al., 2001; Preeprame et al., 2001 Alaria fistulosa, A. marginata Laminariales Usov et al., 2001 Arthrothamnus bifidus Laminariales Usov et al., 2001 Chorda filum Laminariales Chizhov et al., 1999 Ecklonia kurome, E. cava Laminariales Nishino et al., 1991; Tanaka and Sorai, 1970 Eisenia bicyclis Laminariales Usui et al., 1980 Laminaria angustata, L. brasiliensis, L. cloustoni, L. digitata, Laminariales Killing, 1913; Black, 1954; de Reviers et al., 1983; L. japonica, L. religiosa, L. saccharina Maruyama et al., 1987; Rozkin et al., 1989; Kitamura et al., 1991; Pereira et al., 1999 Macrocystis integrifolia, M. pyrifera Laminariales Wort, 1954; Schweiger, 1962 Nereocystis luetkeana Laminariales Wort, 1954 Undaria pinnatifida Laminariales Mori et al., 1982 Petalonia fascia Scytosiphonales Usov et al., 2001 Scytosiphon lomentaria Scytosiphonales Usov et al., 2001

Nomenclature the generic term fucans, to designate all polysaccharides rich Killing baptized his polysaccharide fucoidin; 40 years later, in L-fucose, was commonly used (Percival and Ross, 1950). McNeely changed fucoidin to fucoidan, to conform with As separation and analytic techniques improved, different polysaccharide nomenclature (McNeely, 1959). The ques- types of sulfated polysaccharides were distinguished in tions of nomenclature and purity have always been linked fucan preparations. The first was ascophyllan or xylofuco- for algal fucoidans; early preparations contained large glycuronan, based on a backbone of uronic acid (mannuronic amounts of sugars other than fucose, such as galactose, acid) with fucose containing branches (3-O-D-xylosyl-L- mannose, xylose, or uronic acid, and sometimes even pro- fucose-4-sulfate) (Larsen et al., 1966; Kloareg et al., 1986). teins. Furthermore, their composition changed according to The other family isolated was sargassan or glycuronofuco- the algal species (Percival and Ross, 1950; Mian and glycan, based on linear chains of D-galactose with branches Percival, 1973), the extraction process (Mabeau et al., 1990), of L-fucose-3-sulfate or occasionally uronic acid (Percival, and the season of harvest and local climatic conditions 1968; Medcalf et al., 1978; Kloareg et al., 1986). Fucoidans, (Black, 1954; Von Holdt et al., 1955; Wort, 1955; Honya as originally defined, were identified as homofucans et al., 1999). Were these contaminants (Percival and Ross, (Kloareg et al., 1986). Many authors still use the outdated 1950; O'Neill, 1954; Bernardi and Springer, 1962) or part of term fucoidin. In some cases, authors create their own the polysaccharide (Schweiger, 1962; Anno et al., 1966)? nomenclature, such as fucansulfate (in one word) (Trento Some authors even considered suppressing the term of et al., 2001). The confusion is increased with some fucoidan (Larsen et al., 1966). To face these uncertainties, publications in which the term fucoidan is used to describe 30R Structure and biological properties of sulfated fucans

a complex polysaccharide containing only 20% to 60% A. Ascophyllum nodosum/Fucus vesiculosus/Fucus evanescens B. Ecklonia kurome L-fucose (Duarte et al., 2001). H H It seems wise and in agreement with IUPAC recommen- O O O dations to define sulfated fucan as a polysaccharide based H O CH3 H CH3 - OH mainly on sulfated L-fucose, with less than 10% other mono- H O3SO H HO H H -O SO saccharides. This term was applied to the sulfated fucans of H 3 H O H marine invertebrates (Ribeiro et al., 1994; Alves et al., 1998; O O H H O CH3 H Vilela-Silva et al., 1999), whereas the term fucoidan has been CH3 - OH H O3SO H H used for fucans extracted from algae. For clarity, and - H O3SO because the fucans extracted from those two sources differ, - OSO3 H this nomenclature will be adopted for this review. H

H O O

C. Chorda filum H Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 Structural diversity CH3 1 H R O H Algal sulfated fucans: the common fucales F. vesiculosus R2O H and A. nodosum O H O H Percival and Ross (1950) described fucoidan from the com- CH3 3 H R O H mon brown algae F. vesiculosus as a polysaccharide based R2O H on L-fucose with mainly a(1 ! 2) glycosidic bonds and O H O H 1 - sulfate groups at position 4. They also found branches of CH3 R = SO3 or H or COCH3 2 1 sulfated fucose every five units. This model, supported by R O H R O H 2 - several other studies of F. vesiculosus fucoidan (O'Neill, H R = SO3 or H O H 1954; Cot^ e, 1959), was the only structural model available O H H R3 = for fucoidan for more than 40 years, even though fucoidan CH3 O 1 H H R O H CH from various algae was commonly used in biological studies. R2O 3 H 2 2 H R O H In 1993 Patankar and co-workers reinvestigated the struc- O H R O H O ture of F. vesiculosus fucoidan (the only one commercially CH3 OH H H R1O available) (Patankar et al., 1993). Their model differs in the 2 H nature of the main glycosidic bond: a(1 ! 3) instead of R O a(1 ! 2). The position of the sulfate, in accord with the H previous model, was found to be mainly in position 4. Fig. 1. Common structures in fucoidans from brown algae. (A) The More recent studies have identified a different type of disaccharide repeating unit [4)-a-L-Fucp(2,3di-OSO3 )-(1 ! 3)-a-L- structure for fucoidan from the common Fucales Ascophyl- Fucp(2OSO3 )-(1] of a fraction of A. nodosum fucoidan representing the lum nodosum (Chevolot et al., 1999, 2001; Daniel et al., most abundant structural feature of fucoidans from both A. nodosum and F. vesiculosus (Chevolot et al., 1999, 2001). The same structure has been 1999, 2001). Several studies clearly show that this fucoidan identified in the fucoidan of F. evanescens (Bilan et al. 2002). (B) The possesses large proportions of both a(1 ! 3) and a(1 ! 4) 3-linked, preponderantly 4-sulfated fucoidan from E. kurome (Nishino glycosidic bonds (Daniel et al., 1999, 2001; Chevolot et al., and Nagumo, 1991). (C) The quasi-repeat unit identified in fucoidan from 1999, 2001). A repeating structure of alternating a(1 ! 3) C. filum (Chizhov et al., 1999). Other substituents, such as O-acetyl, and branches are present in all these fucoidans and add considerably to their and a(1 ! 4) glycosidic bonds was determined for oligosac- heterogeneity. charides (of about 8±14 monosaccharide units) prepared from A. nodosum fucoidan (Chevolot et al., 2001) (Figure 1A). As the characteristic nuclear magnetic resonance (NMR) spectrum of this repeating disaccharide structural heterogeneity. The average structure of this is the major feature in the NMR spectra of whole fucoidan fucoidan is mainly 3-O-linked with sulfate groups at C-4, from both A. nodosum and F. vesiculosus (Pereira et al., without excluding the presence of other sulfate groups or 1999), it may be that the backbones of both fucoidans branches in position 2 (Figure 1B). consist of this structure. Highly branched fractions may In 1999 the structures of fucoidans from three algae also be prepared from A. nodosum fucoidan (Marais and were published, Cladosiphon okamuranus (chordariales) Joseleau, 2001), and minor features in the complex NMR (Nagaoka et al., 1999), Chorda filum (laminariales) (Chizhov spectra of algal fucoidans may well be due to these branches et al., 1999), and Ascophyllum nodosum (fucales) (Chevolot (Pereira et al., 1999). A. nodosum fucoidan has also been et al., 1999; Daniel et al., 1999). Fucoidans from studied using specific enzymes, confirming the presence of Cladosiphon okamuranus and Chorda filum present an average high amounts of a(1 ! 3) and a(1 ! 4) glycosidic bonds structure based on a backbone of L-fucose linked a(1 ! 3) (Daniel et al., 1999, 2001). with most sulfation at position 4. Fucoidan from C. filum seems based on a regular hexasaccharide repeating unit, Algal sulfated fucans: other brown algae with some 2-O-sulfation (Figure 1C). Both fucoidans The structure of fucoidan from another species, Ecklonia have some 2-O-acetylation, and the authors argue that O- kurome, was published in 1991 (Nishino and Nagumo, acetylation is also present in other fucoidans, in particular 1991). NMR spectra of the polysaccharide were too com- those from F. vesiculosus, E. kurome, and L. brasiliensis plex to allow direct structure elucidation, probably due to (Chizhov et al., 1999). A fucoidan from L. brasiliensis has 31R O. Berteau and B. Mulloy been studied by NMR spectroscopy and methylation ana- In contrast with the apparently complex algal fucoidans, lysis; its structure was not determined, but clear signs of a they possess a clear regularity, allowing determination of complex repeating unit were identified (Pereira et al., 1999). their structures by high-field NMR (Mulloy et al., 1994). More recently, the structure of a high-molecular-weight They are linear polysaccharides consisting of a regular fucoidan from Fucus evanescens, another fucales, was pub- repeating unit, either mono-, tri-, or tetrasaccharide lished and found to resemble A. nodosum fucoidan with, in (Figure 2), in which the glycosidic linkage is constant, and addition, the presence of large amount of acetyl groups and the repeating unit defined by a distinctive pattern of sulfate maybe the presence of fucosyl branches (Bilan et al., 2002). substitution. In general, each species has its own particular Although there are clear links between algal species and sulfated fucan. These egg-jelly polysaccharides are able to fucoidan structure, there is insufficient evidence yet to induce the acrosomal reaction (a change that sea urchin establish any systematic correspondence between structure spermatozoa must undergo to fertilize the egg successfully) and algal order. preferentially in sperm of the same species (Alves et al., 1997; Vilela-Silva et al., 2002). In this way, interspecific

Sulfated fucans from marine invertebrates fertilization is avoided. Two species, S. purpuratus and Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 It was known for many years that invertebrates possess S. droebachiensis, have been studied in which most indivi- sulfated fucans (Vasseur, 1948), but no structural study dual females are able to produce only one of two different was performed on these compounds before 1987 (Mourao~ types of sulfated fucans (Alves et al., 1998; Vilela-Silva et al., and Bastos, 1987). Two kinds of marine invertebrates 2002). However, 10% of the eggs of S. purpuratus contain have been investigated: one species of sea cucumber both types of fucans. For S. droebachiensis the polymorph- (Ludwigothurea grisea) (Ribeiro et al., 1994) and several ism seems to be correlated with the place of collection. species of sea urchins (Lytechinus variegatus, Arbacia There are many species of sea urchin, and only a few egg- lixula, Strongylocentrotus purpuratus, Strongylocentrotus jelly sulfated fucans (and occasionally galactans) have been franciscanus, Strongylocentrotus pallidus, and Strongylocen- studied. It is also the case that sulfated fucan structure is not trotus droebachiensis) (Mulloy et al., 1994; Alves et al., 1997, the only barrier to cross-fertilization. It is worth noting, 1998; Vilela-Silva et al., 1999, 2002). however, that two species known to synthesize a sulfated

Strongylocentrotus purpuratus Strongylocentrotus pallidus H H O H O II O CH I O 3 H H CH3 -O SO H HO H O 3 H HO H H H O -O SO CH3 3 O H H - H O R H O3SO H O H CH O 3 H HO CH H 3 - H H O3SO Strongylocentrotus - H HO H H O3SO droebachiensis I - O H H O R = OSO3 (80%) H O H CH3 O H or OH (20%) H - CH3 H O3SO H Arbacia lixula O HO - H O H O3SO H H CH -O SO 3 3 O H H H HO H O H H CH3 O H O - CH H O3SO H 3 OH H HO H HO H H O H O H CH 3 OH H Echinometra lucunter H - H O3SO H O H O H H CH3 OH H O O - Lytechinus variegatus H O3SO H H O H O CH2OH CH3 - H -O SO OH H HO H O3SO H HO 3 H H O H H O CH3 -O SO Strongylocentrotus franciscanus H 3 - H S. droebachiensis II O3SO H H O H O O H H H O CH O CH3 3 O H - H HO H CH3 HO H O3SO H -O SO 3 - H H O3SO H O H O H OH H CH3 - H O3SO H HO

H

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. 32R Structure and biological properties of sulfated fucans fucan with the same structure (i.e. A. lixula and S. droeba- ``specific'' ligand for L- and P-selectins and macrophage chiensis) are found in nonoverlapping geographical loca- scavenger receptors. Some studies have been published, tions (from the tropical Atlantic Ocean and the Arctic however, on partly characterized fucoidan fractions, and Ocean, respectively). Cross-species induction of the acro- most of these concern the effects of fucoidan on blood some reaction in the laboratory has shown that position of coagulation. sulfation and linkage are both important for the interaction. It is interesting to note in this context that an L-galactan Anticoagulant and antithrombotic activity from the species Echinometra lucunter (Figure 2) may substi- F. vesiculosus fucoidan has a specific anticoagulant activity tute for an L-fucan from S. franciscanus (Figure 2) of iden- by the activated partial thromboplastin time assay of 9±13 tical linkage and substitution pattern (Hirohashi et al., 2002). U/mg, as compared with 167 U/mg for heparin (Nishino Sperm membrane receptors for sea urchin egg-jelly fucans et al., 1994), or 16 U/mg, as compared with 193 U/mg for include REJ1 (receptor for egg jelly 1) (Vacquier and Moy, heparin (Mourao~ and Pereira, 1999). A fucoidan from 1997) and maybe REJ3 (Mengerink et al., 2002). This inter- Laminaria brasiliensis has higher specific activity (30 U/mg), action is unusual in that a pure carbohydrate, in the absence though its sulfate content is lower (Mourao~ and Pereira, Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 of any protein component, is capable of inducing signal 1999). The high potency of heparin depends on a specific transduction resulting in exocytosis (Vacquier and Moy, pentasaccharide sequence with high affinity for the 1997). The high molecular weight of egg-jelly fucans is serine protease inhibitor antithrombin, a sequence that is 2‡ required for successful opening of both Ca channels obviously absent from fucoidan. However, purified fucoidan involved in the acrosome reaction, though partially hydro- fractions (from Ecklonia kurome [Nishino et al., 1999], lysed fucan will bind to sperm receptors (Hirohashi and A. nodosum [Millet et al., 1999], and Pelvetia caniculata Vacquier, 2002). [Colliec et al., 1994]) have activity mediated both by anti- Though we now have some data about the structure of thrombin and by another plasma serine protease inhibitor these sulfated fucans, almost nothing is known about (serpin), heparin cofactor II (HCII) (Colwell et al., 1999). their conformations or how their regular spatial patterns Heparin interacts with HCII by means of its regular repeating of sulfate groups determine their biological properties unit (Figure 3A), not the antithrombin-binding sequence. (Gerbst et al., 2001, 2002). These serpins act against several of the coagulation system

Physiological properties of sulfated fucans A. Heparin Little is known about the role of most fucans in marine - CH2OSO3 organisms, apart from the case of sea-urchin fertilization as B. Fucoidan fraction from A. nodosum described. The role of sulfated fucans in the body wall of sea O H H H H cucumber is less well understood but may be in maintenance H H O of the body wall's integrity (Mourao~ and Bastos, 1987; O OH O O O H H CH3 Ribeiro et al., 1994). For algae, some studies have shown COOH - - H O3SO H a correlation between fucoidan content and the depth at OH H H NH2SO3 HO which they growÐthe closer algae are to the surface, the H H O H H OSO - O greater the fucoidan content (Black, 1954; Kloareg, 1981; 3 H Evans, 1989). CH3 - Furthermore, fucoidans appear to play a role in the algal H O3SO H cell wall organization (Kloareg and Quatrano, 1988; OSO - H Bisgrove and Kropf, 2001) and could be involved in the 3 cross-linkage of alginate and cellulose (Mabeau et al., 1990). Fucoidans may also be involved in the morphogen- C. Galactan from B. occidentalis esis of algae embryos (Bisgrove and Kropf, 2001). CH2OH O O CH OH 1 2 3 - R3O 2 R , R , R = H or SO3 H O O H 1 - Biological properties of fucans H H H R as SO3 > 66% 2 H OR H 3 H 2 - Fucoidans have a wide spectrum of activity in biological H OR R as SO3 > 33% systems. Besides their well-attested anticoagulant and H OR1 antithrombotic activity, they act on the inflammation and immune systems, have antiproliferative and antiadhesive Fig. 3. Disaccharide structures associated with anticoagulant polysaccharides. (A) The main repeating unit [4)-a-L- effect on cells, protect cells from viral infection, and can IdopA2OSO3 (1 ! 3)-a-D-GlcpNSO3±6OSO3 (1 ! ] found in the widely interfere with mechanisms involved in fertilization. All of used anticoagulant polysaccharide heparin. This structure does not account these properties have been reviewed by Boisson-Vidal et al. for the main part of heparin's anticoagulant activity, as a potentiator of (1995). Most studies have used a commercial preparation of antithrombin, but has activity by other routes such as activation of heparin cofactor II. (B) The repeating unit [4)-a-L-Fucp(2,3di-OSO3 )-(1 ! 3)- F. vesiculosus fucoidan, which has been shown to contain a-L-Fucp(2OSO3 )-(1 ! ] from A. nodosum (also F. vesiculosus and heteropolysaccharides of various kinds besides those F. evanescens) (Chevolot et al., 1999, 2001). (C) The repeat unit based consisting predominantly of sulfate and fucose (Nishino on the backbone [4)-a-D-Galp-(1 ! 3)-b-D-Galp-(1 ! ] of a galactan with et al., 1994). This mixture has been used, for example, as a high anticoagulant activity isolated from the red alga B. occidentalis. 33R O. Berteau and B. Mulloy proteases, including thrombin, Factor Xa, and Factor IXa structures are involved. Other linear polysaccharides, such (Mauray et al., 1998). All of these factors may be involved as heparin, share this property, but the fucose branches of in the ability of fucoidan to prevent venous thrombosis native selectin ligands bring to mind the branched struc- (Millet et al., 1999). The release of tissue factor pathway tures in fucoidans. inhibitor from endothelium, which is stimulated by fucoi- The interaction between fucoidan and selectins has phy- dan more potently than by heparin, may also have an siological consequences that could be therapeutically useful; antithrombotic effect (Giraux et al., 1998a). for example, perfusion with fucoidan can reduce neutrophil Anticoagulant and antithrombotic activities of fucoidan infiltration and myocardial injury after ischemia/reperfu- fractions (A. nodosum) increase with increasing molecular sion (Omata et al., 1997). Also, leukocyte accumulation weight and sulfate content. However, fractions in which the occurs in the same circumstances (Ritter et al., 1998). native pattern of sulfation was intact were more potent than Injection of fucoidan into sensitized mice before hapten fractions of equivalent molecular weight and overall degree challenge can reduce contact hypersensitivity reaction of sulfation in which this pattern had been disrupted by (Nasu et al., 1997). Recruitment of leukocytes into cereb- partial desulfation (Boisson-Vidal et al., 2000). Fucoidans rospinal fluid in a meningitis model is reduced by fucoidan Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 may also promote fibrinolysis by potentiating plasminogen (Granert et al., 1999), as is IL-1 production in a similar activators (Nishino et al., 2000). The predominant pattern model (Ostergaard et al., 2000). Neutrophil (Shimaoka of sulfation in A. nodosum fucoidan is the trisulfated dis- et al., 1996) and eosinophil (Teixera and Hellewell, 1997) accharide motif (Figure 3B), similar to that found in migration to sites of inflammation are also inhibited by heparin, which also has a trisulfated disaccharide repeat fucoidan. All of these studies seem to indicate the potential unit (Figure 3A). A heavily sulfated substituted disacchar- of fucoidan as an anti-inflammatory agent. However, ide is also the repeat unit of a highly anticoagulant galactan Verdrengh and co-workers (2000) have observed that, isolated from red algae (Farias et al., 2000) (Figure 3C). although fucoidan treatment led to less severe symptoms Correlation of the sulfation patterns of less highly sul- in the early stages of S. aureus±triggered arthritis in mice, fated fucans and galactans with their anticoagulant activ- delayed recruitment of phagocytes decreased clearance of ities (Mourao~ and Pereira, 1999; Pereira et al., 2002) has bacteria. used invertebrate fucans as shown in Figure 2. The Two recent studies use fucoidan in studies of selectins structural homogeneity of these compounds allows clear in the release of haematopoietic blood cells from bone conclusions to be drawn. For example, a 3-linked, regularly marrow. Both fucoidan (Sweeney et al., 2000) and a pure, 2-O-sulfated galactan has anticoagulant activity lacking linear sulfated fucan (Frenette and Weiss, 2000) have in the corresponding 3-linked, 2-O-sulfated fucan or a been shown to stimulate mobilization of stem/progenitor 4-linked, regularly 3-O-sulfated galactan. This study estab- cells in vivo; they are also effective in selectin-deficient lished definitively that regular, linear sulfated fucans mice and therefore must be capable of acting in a selectin- express anticoagulant activity, which is not simply a func- independent fashion. This selectin-independent mechanism tion of charge density but depends critically on the exact may well be related to the release of stromal-derived structure of the polysaccharide. factor 1, a potent chemoattractant, from bone marrow into the circulation on administration of fucoidan (Sweeney Platelet activation et al., 2002). Fucoidan has also been used to probe the involvement of Fucoidan fractions of varying molecular weight and degree L- and P-selectin in thrombus formation in vivo (Thorlacius of sulfation all induced platelet aggregation in vitro (Durig et al., 2000). Although both fucoidan and an anti-P-selectin et al., 1997). In this case low sulfate fractions were most antibody abolished P-selectin function, only fucoidan was potent, showing that anticoagulation and platelet activation able to prevent thrombus formation, presumably through differ in their structural dependence. On the other hand, a its anticoagulant activity. study in baboons found fucoidan to be a potent inhibitor of platelet aggregation in vivo (Alwayn et al., 2000). Macrophage scavenger receptors Selectins Macrophage scavenger receptors are a group of proteins Selectins are a group of lectins found on the surface of that, as the name suggests, are involved in the uptake by leukocytes (L-selectins) platelets (P-selectins), and endothe- macrophages of items for disposal, such as oxidised lipo- lial cells (P- and E-selectins). They interact with oligosac- proteins, damaged cells, or invading microorganisms. charides clustered on cell surfaces (Lasky, 1995) during the Fucoidan is a ligand for at least some of these receptors, margination and rolling of leukocytes prior to firm adhe- triggering a protein kinase±dependent signaling pathway sion, extravasation, and migration to a site of infection. (Hsu et al., 1998). Binding of neutrophil-derived azurocidin Fucoidan can act as a ligand for either L- or P-selectins, (also known as heparin-binding protein) to monocytes is both of which interact with sulfated oligosaccharides. Most inhibited in the presence of fucoidan, which also abrogates likely, fucoidan is acting like heparin or heparan sulfate the enhancing effect of azurocidin on lipopolysaccharide- (HS), presenting a spatial pattern of sulfated saccharide induced tumour necrosis factor-a (TNF-a) production. structures that imitates the clustering of sulfated, sialylated, This is taken as evidence that the azurocidin receptor on and fucosylated oligosaccharides on the cell surface. monocytes may be related to the scavenger receptors Because purified fucoidans are not usually used in studies (Heinzelmann et al., 1998). However, the same study also of this type, however, it is difficult to define which fucan observed that fucoidan can itself stimulate TNF-a release 34R Structure and biological properties of sulfated fucans from monocytes. It is important not to be overly simplistic Like many other sulfated polysaccharides, fucoidan can in approach when dealing with a polyvalent reagent such as inhibit virus infection of cells. This has recently been demon- fucoidan. strated for Herpes simplex, cytomegalovirus, and human immunodeficiency virus (Hoshino et al., 1998) as well as Cell growth, migration, and adhesion bovine viral diarrhea virus (Iqbal et al., 2000), probably by Like heparin, fucoidan has antiproliferative effects on vas- competing with cell surface HS for binding to the virus. cular smooth muscle cells (SMCs). A fucoidan fraction from A. nodosum was more active than heparin (Logeart et al., 1997); fucoidans were internalized by cells and per- Enzymes active on sulfated fucans haps transported to the nucleus. Patel and co-workers (2002) were able to distinguish between the modes of activ- Although almost nothing is known about the biosynthesis ity of fucoidan and heparin; fucoidan was active even for of algal fucoidans and invertebrate sulfated fucans, heparin-resistant SMCs. In this study, crude commercial enzymes capable of degrading such polysaccharides have fucoidan was more active than the purified material, indi- been isolated from several marine species. Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 cating that some highly active fraction was discarded in Specific enzymatic methods can be used to provide the purification, another indication that specific structures tailored oligosaccharides for biological studies, as well as within these complex mixtures can be linked to particular simplified samples from which it is possible to deduce the biological effects. Fucoidan can also modulate proliferation structure of the original fucoidan. Currently employed of fibroblasts, and here again it has been shown that anti- methods of chemical modifications prior to analysis (hydro- proliferative and anticoagulant fucoidan structures are lysis, desulfation, deacetylation) often require strong basic different (Haroun-Bouhedja et al., 2000). The situation for or acidic condition at high temperature, which can modify endothelial cells is complex and depends on the agent used the polysaccharide. For example, acetyl groups have been to stimulate proliferation; heparin and fucoidan affect found in almost all the algal fucoidans studied in recent growth and migration differently (Giraux et al., 1998b). years (C. filum, C. okamuranus, F. evanescens) and may be present in all the algal fucoidans but removed during the Fertilization preparation process (Chizov et al., 1999). At least two kinds of glycosidase act on fucans, ``fucan In view of the importance of egg-jelly fucans for sea urchin sulfate hydrolase'' or fucoidanase EC 3.2.1.44, and a-L- fertilization (see Sulfated fucans from marine invertebrates), fucosidase EC 3.2.1.51 (EC 3.2.1.63, EC 3.2.1.111, or EC it is interesting to note that mammalian fertilization also 3.2.1.127 depending on specificity for the glycosidic link- involves oligosaccharides in the zona pellucida surrounding age). If the activity of a-L-fucosidases is easily described as the egg and that these are fucosylated (Johnston et al., 1998) the release of L-fucose from the nonreducing end of a poly- and sulfated (Moreno et al., 2001). Fucoidan, in common saccharide, it is less easy to define fucoidanase activity. with other sulfated glycans, is a powerful inhibitor of sperm Fucoidanase can produce the cleavage of glycosidic binding to oviductal monolayers (Talevi and Gualtieri, bonds in the core of the polysaccharide, leading to rapid 2001). reduction of the molecular weight (i.e., endo-fucoidanase), or on the edge of the polysaccharide, releasing some oligo- Parasites and viruses saccharides with a slow decrease of the molecular weight Sulfated polysaccharides are antimalarial in vitro, inhibiting (i.e., exo-fucoidanase) (Tanaka and Sorai, 1970; Furukawa the invasion of free Plasmodium falciparum parasites into et al., 1992a). Fucoidanase has been reported mostly in erythrocytes (Clark et al., 1997). A thrombospondin-related marine mollusks and marine bacteria (Table II) but is also adhesion protein is implicated in host cell invasion; this present in other marine invertebrates (Burtseva et al., 2000). binds to fucoidan and heparin and probably to cell surface One of the difficulties in purification of fucoidanase is the HS (McCormick et al., 1999). Fucoidan and low-molecular- absence of simple tests to identify and quantify this kind of weight fucoidan, but not desulfated fucoidan, inhibit enzyme. Methods proposed have included reduction of Plasmodium berghei development in Hep G2 cells and spor- viscosity (Kitamura et al., 1992; Furukawa et al., 1992a), ozoite invasion of Chinese hamster ovary cells (Ying et al., increase in reducing sugar (Yaphe and Morgan, 1959; 1997). Hepatocytes bear a particularly highly sulfated HS Thanassi and Nakada, 1967), precipitation with albumin that is thought to be instrumental in the clearance by the (Morinaga et al., 1981; Descamps et al., 1998), or size liver of circulating sporozoites. However, sulfated polysac- exclusion chromatography (Daniel et al., 1999). However, charides can enhance adhesion of infected erythrocytes to fucoidans of various origins have been used that may cells bearing CD36 (McCormick et al., 2000). Chondroitin present different structures varying in their sensitivity to sulfate A has been identified as a cell-surface receptor for hydrolysis (Table II). P. falciparum-infected erythrocytes (Rogerson et al., 1995), Yaphe and Morgan (1959) first reported the ability of two with which neither fucoidan nor other highly sulfated poly- marine bacteria, Pseudomonas atlantica and Pseudomonas saccharides can compete. carrageenova, to produce reducing sugars (i.e., hydrolysis) Another widespread parasite, Toxoplasma gondii, is also from fucoidan. Later, several species of abalone (Haliotis affected by fucoidans. In this case low concentrations sp.) were found able to degrade fucoidan, producing fuco- can enhance infection of fibroblasts in culture, though oligosaccharides (Thanassi and Nakada, 1967). higher concentrations are inhibitory (Ortega-Barria and More recently, two species of Pectinidae have been Boothroyd, 1999). investigated, Patinopecten yessoensis and Pecten maximus 35R O. Berteau and B. Mulloy

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

Generation of

Fuco- L-fucose Sulfated Reduction Species oligosaccharidea fucose of viscosity Origin of fucoidan substrate Reference

Bacteria Pseudomonas atlantica ‡ Fucus vesiculosus Yaphe and Morgan, 1959 Pseudomonas carrageenova ‡ Fucus vesiculosus Yaphe and Morgan, 1959 Vibrio sp. ‡ Kjellmaniella crassifolia Furukawa et al., 1992 Pseudoalteromonas citrea ‡ ND Laminaria cichoriodes Bakunina et al., 2002 Molluscs Haliotis rufescens ‡ ‡ ND ‡ Fucus gardneri Thanassi and Nakada, 1967 Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 Haliotis corrugata ‡ ‡ ND ‡ Fucus gardneri Thanassi and Nakada, 1967 Haliotis gigantea ‡ ‡ Ecklonia cava Tanaka and Sorai, 1970 Patinopecten yessoensis ‡ ND ‡ Nemacystus decipens Kitamura et al., 1992 Pecten maximus ‡ ‡ ND ND Ascophyllum nodosum Daniel et al., 1999

ND: Not determined; ‡: release of L-fucose or of sulfated L-fucose; : their absence. Burtseva and co-workers (2000) have also shown that crude protein extract from many species of marine invertebrates (echinoderms, arthropods, and molluscs) are able to hydrolyze various fucoidans. aFuco-oligosaccharide means the isolation or the evidence of oligosaccharide by liquid chromatography.

(Kitamura et al., 1992; Daniel et al., 1999); both molluscs F. Vesiculosus fucoidan was purified from the fungus are able to hydrolyze fucoidan efficiently to produce Fusarium oxysporum (Yamamoto et al., 1986). This enzyme oligosaccharides. is able to release only a few units of L-fucose from fucoidan, To date only two fucoidanases, with apparently no com- as might be expected because of branches and the presence mon structural features, have been purified to homogeneity of sulfate groups (more than one per fucose unit). More (Furukawa et al., 1992a; Kitamura et al., 1992). Nothing is recently an a-L-fucosidase was purified from the common known about the mechanism of fucoidanases, and it is scallop P. maximus. Surprisingly, this glycosidase is able difficult to compare literature results because degradation to release fucose from A. nodosum fucoidan but failed to studies have been conducted using fucoidans of different hydrolyze F. vesiculosus (Berteau et al., 2002). The differ- origin (Table II). The first report of such an enzyme in ence of activity toward both fucoidans must arise because of structural studies used a fucoidanase from P. maximus structural differences, although no clear specificity was (Daniel et al., 1999). The protein extract obtained from demonstrated using simple oligosaccharides. this scallop is active not only on A. nodosum fucoidan but The structural basis for biological and physiological also, and in a more efficient way, on the linear sulfated properties of sulfated fucans depends crucially on the pre- fucans from L. variegatus and L. grisea (Berteau unpub- sence of sulfate groups. Both their density and their specific lished data); thus, it could be more accurate to call these positions influence biological properties, as discussed. To enzymes sulfated fucan hydrolase. date the only way to assess the importance of position of Bacterial fucoidanases have attracted more recent study sulfate groups is to try to compare related fucans. Sulfatases (Morinaga et al., 1981; Bakunina et al., 2000, 2002), and at (EC 3.1.6) are enzymes able to remove sulfate groups and least one strain of bacteria has been patented for its ability could therefore be an invaluable tool for such studies. Only to produce fucoidan oligosaccharides without purification minor reports have appeared in the literature concerning of the fucoidanase (Descamps et al., 1998). sulfatases able to hydrolyze fucoidan (Lloyd and Lloyd, Comparing the properties and structures of sulfated 1963; Furukawa and Fujikawa, 1984; Furukawa et al., fucans from invertebrates and algae, it seems that L-fucose 1992b). Nothing is known about their mechanism and branches might play a key role in biological properties. The properties, particularly whether they can act alone or need use of a-L-fucosidase to remove these branches could help glycosidases to achieve significant desulfation of fucan. assess this hypothesis. To date three a-L-fucosidases have Only one study has shown a sulfatase able to act specifically been reported able to release fucose from fucoidan. The first on some sulfate groups of fucoidan (Daniel et al., 2001). a-L-fucosidase described was purified from abalone and This enzyme partially purified from the scallop P. maximus seemed able to hydrolyze Ecklonia cava fucoidan comple- is able to release sulfate groups present at position 2 of tely to L-fucose and sulfated L-fucose (Tanaka and Sorai, monosulfated L-fucose, or of mono- and disulfated compo- 1970). However, it is doubtful that this kind of glycosidase nents of the disaccharide from A. nodosum fucoidan. In alone can fully hydrolyze such a complex polysaccharide. conjunction with the a-L-fucosidase purified from the The second a-L-fucosidase reported able to hydrolyze same mollusc it seems possible to increase the degradation

36R Structure and biological properties of sulfated fucans of fucans (Berteau et al., 2002). These enzymes are therefore Anno, K., Terahata, H., Hayashi, Y., and Seno, N. (1966) Isolation and unique tools to produce selectively modified fucans, which purification of fucoidin from brown seaweed Pelvetia wrightii. Agr. could allow direct determination of the involvement of Biol. Chem., 30, 495±499. Bakunina,I.Y.,Shevchenko,L.S.,Nedashkovskaya,O.I.,Shevchenko,N.M., branches and sulfate groups in their biological activity. Alekseeva, S.A., Mikhailov, V.V., and Zvyagintseva, T.N. (2000) Screening of marine bacteria for fucoidanases. Microbiology (Moscow), 69, 303±308. Conclusions Bakunina, I.Y., Nedashkovskaia, O.I., Alekseeva, S.A., Ivanova, E.P., Romanenko, L.A., Gorshkova, N.M., Isakov, V.V., Zviagintseva, T.N., Recent structural studies present fucans as a more orderly and Mikhailov, V.V. (2002) Degradation of fucoidan by the marine family of polysaccharides than was previously thought. The protebacterium Pseudoalteromonas citrea. Microbiology, 71, 41±47. fucoidans of brown algae are complex and heterogeneous, Bernardi, G. and Springer, G.F. (1962) Properties of highly purified fucan. J. Biol. Chem., 237, 75±80. but recent structural analyses have revealed the presence of Berteau O., McCort I., Goasdoue N., Tissot B., and Daniel R. (2002) ordered repeat units for fucoidans from several species. In Characterization of a new alpha-L-fucosidase isolated from the marine contrast, sulfated fucans from marine invertebrates have mollusk Pecten maximus that catalyzes the hydrolysis of alpha-L-fucose simple, regular structures; in the case of sea urchin egg- from algal fucoidan (Ascophyllum nodosum). Glycobiology, 12, 273±282. Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 jelly fucans, the regular pattern of sulfate substitution 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 plays a part in ensuring species specificity of fertilization. brown seaweed Fucus evanescens C.Ag. Carbohydr. Res., 337, 719±730. Sulfated fucans and fucoidans have many potent biologi- Bisgrove, S.R. and Kropf, D.L. (2001) Cell wall deposition during cal activities due to their ability to imitate patterns of sulfate morphogenesis in fucoid algae. Planta, 212, 648±658. substitution on glycosaminoglycans and other sulfated glu- Black, W.A.P. (1954) The seasonal variation in the combined L-fucose cans. Because most studies of biological activity are carried content of the common British laminariaceae and fucaceae. J. Sci. out using a relatively crude fucoidan preparation, it is not Food Agr., 5, 445±448. Boisson-Vidal, C., Haroun, F., Ellouali, M., Blondin, C., Fischer, A.M., easy at present to determine the relationships between activ- De Agostini, A., and Jozefonvicz, J. (1995) Biological activities of ity and structure. However, it has become clear that at least polysaccharide from marine algae. Drugs Fut., 20, 1237±1249. some of these various activities are not merely a function of Boisson-Vidal, C., Chaubet, F., Chevolot, L., Sinquin, C., Theveniaux, J., high charge density but have distinct structural specificities. Millet, J., Sternberg, C., Mulloy, B., and Fischer, A.M. (2000) The availability of fucans with well-defined structures and Relationship between antithrombotic activities of fucans and their of enzymes able to modify these polysaccharides will bring structure. Drug Devel. Res., 51, 216±224. Burtseva, Y.V., Kusaikin, M.I., Sova, V.V., Shevchenko, N.M., clarity to analysis of their biological properties and allow Skobun, A.S., and Zvyagintseva, T.N. (2000) Distribution of fucoidan the design of new experiments. Future conformational stu- hydrolases and some glycosidases among marine invertebrates. Russ. J. dies of well-defined fucan structures should lead to better Mar. Biol., 26, 453±456. understanding of the biological properties of fucoidans. Chargaff, E., Bancroft, F.W., and Brown, M.S. (1936) Studies on the The complexity and heterogeneity of fucoidans, and their chemistry of blood coagulation II. On the inhibition of blood clotting by substances of high molecular weight. J. Biol. Chem., 115, 155±156. wide range of activities in mammalian systems, are not Chevolot, L., Foucault, A., Kervarec, N., Sinquin, C., Fisher, A.M., and attractive within current paradigms of drug development. Boisson-Vidal, C. (1999) Further data on the structure of brown The combination of structural elucidation and assignment seaweed fucans: relationships with anticoagulant activity. Carbohyd. of biological activity to specific structural features can only Res., 319, 154±165. improve the potential of sulfated fucans and fucoidans both Chevolot, L., Mulloy, B., Ratiskol, J., Foucault, A., and Colliec-Jouault, S. as therapeutic agents and as research reagents. (2001) A disaccharide repeat unit is the major structure in fucoidans from two species of brown algae. Carbohyd. Res., 330, 529±535. 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. Abbreviations (1999) A study of fucoidan from the brown seaweed Chorda filum. Carbohyd. Res., 320, 108±119. HS, heparan sulfate; NMR, nuclear magnetic resonance; Clark, D.L., Su, S.D., and Davidson, E.A. (1997) Saccharide anions as SMC, smooth muscular cell; TNF-a, tumor necrosis inhibitors of the malaria parasite. Glycoconj. J., 14, 473±479. factor-a. 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. References Colwell, N.S., Grupe, M.J., and Tollefsen, D.M. (1999) Amino acid residues of heparin cofactor II required for stimulation of thrombin Abdel-Fattah, A.F., Hussein, M.M.D., and Fouad, S.T. (1978) Carbohy- inhibition by sulphated polyanions. Biochim. Biophys. Acta, 1431, drates of the brown seaweed Dictyota dichotoma. Phytochemistry, 17, 148±156. 741±743. Cot^ e, R.H. (1959) Disaccharides from fucoidin. J. Chem. Soc., 2248±2254. Alves, A.P., Mulloy, B., Diniz, J.A., and Mourao, P.A.S. (1997) Sulfated Daniel, R., Berteau, O., Jozefonvicz, J., and Goasdoue, N. (1999) polysaccharides from the egg jelly layer are species-specific inducers Degradation of algal (Ascophyllum nodosum) fucoidan by an enzymatic of acrosomal reaction in sperms of sea urchins. J. Biol. Chem., 272, activity contained in digestive glands of the marine mollusc Pecten 6965±6971. maximus. Carbohyd. Res., 322, 291±297. Alves, A.P., Mulloy, B., Moy, G.W., Vacquier, V.D., and Mourao, P.A.S. Daniel, R., Berteau, O., Chevolot, L., Varenne, A., Gareil, P., and (1998) Females of the sea urchin Strongylocentrotus purpuratus differ Goasdoue, N. (2001) Regioselective desulfation of sulfated in the structure of their egg jelly sulphated fucans. Glycobiology, 8, L-fucopyranoside by a new sulfoesterase from the marine mollusk 939±946. Pecten maximus: application to the structural study of algal fucoidan Alwayn, I.P., Appel, J.Z., Goepfert, C., Buhler, L., Cooper, D.K., and (Ascophyllum nodosum). Eur. J. Biochem., 268, 5617±5628. Robson, S.C. (2000) Inhibition of platelet aggregation in baboons: de Reviers, B., Mabeau, S., and Kloareg, B. (1983) Essai d'interpretation therapeutic implications for xenotransplantation. Xenotransplantation, de la structure des fucoidanes en liaison avec leur localisation dans la 7, 247±257. paroi des pheophycees. Crit. Rev. Biochem., 4, 55±62. 37R O. Berteau and B. Mulloy

Descamps, V., Klarszinsky, O., Barbeyron, T., Cloarec, B., Fritig, B., Hoshino, T., Hayashi, T., Hayashi, K., Hamada, J., Lee, J.B., and Jouber, J.M., Plesse, B., and Yvin, J.C. (1998) Fuco-oligosaccharides, Sankawa, U. (1998) An antivirally active sulfated polysaccharide from enzyme pour leur preparation a partir de fucanes, bacterie productrice de Sargassum horneri (TURNER) C. AGARDH. Biol. Pharm. Bull., 21, l'enzyme et applications des fuco-oligosaccharides a la protection des 730±734. plantes. Brevet, FR 2 783 523. Hsu H.Y., Hajjar D.P., Khan K.M., and Falcone D.J. (1998) Ligand Dobashi K., Nishino T., Fujihara M., and Nagumo, T. (1989) Isolation binding to macrophage scavenger receptor-A induces urokinase-type and preliminary characterization of fucose-containing sulfated poly- plasminogen activator expression by a protein kinase-dependent saccharides with blood-anticoagulant activity from the brown seaweed signaling pathway. J. Biol. Chem., 273, 1240±1246. Hizikia fusiforme. Carbohydr. Res., 194, 315±320. Iqbal, M., Flick-Smith, H., and McCauley, J.W. (2000) Interactions of Duarte, M.E.R., Cardoso, M.A., Noseda, M.D., and Cerezo, A.S. (2001) bovine viral diarrhoea virus glycoprotein E(rns) with cell surface Structural studies on fucoidans from the brown seaweed Sargassum glycosaminoglycans. J. Gen. Virol., 81, 451±459. stenophyllum. Carbohyd. Res., 333, 281±293. Johnston, D.S., Wright, W.W., Shaper, J.H., Hokke, C.H., Van den Durig, J., Bruhn, T., Zurborn, K.H., Gutensohn, K., Bruhn, H.D., and Eijnden, D.H., and Joziasse, D.H. (1998) Murine sperm-zona binding, Beress, L. (1997) Anticoagulant fucoidan fractions from Fucus a fucosyl residue is required for a high affinity sperm-binding ligand. A vesiculosus induce platelet activation in vitro. Thromb. Res., 85, 479±491. second site on sperm binds a nonfucosylated, beta-galactosyl-capped Evans, L.V. (1989) Mucilaginous substances from macroalgae: an oligosaccharide. J. Biol. Chem., 273, 1888±1895. overview. Symp. Soc. Exp. Biol., 43, 455±461. Killing, H. (1913) Zur biochemie der Meersalgen. Z. Physiol. Chem., 83, Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 Farias, W.R.L., Valente, A.P., Pereira, M.S., and Mourao, P.A.S. (2000) 171±197. Structure and anticoagulant activity of sulfated galactansÐIsolation Kitamura, K., Matsuo, M., and Yasui, T. (1992) Enzymic degradation of of a unique sulfated galactan from the red algae Botryocladia fucoidan by fucoidanase from the hepatopancreas of Patinopecten occidentalis and comparison of its anticoagulant action with that of yessoensis. Biosci. Biotech. Biochem., 56, 490±494. sulfated galactans from invertebrates. J. Biol. Chem., 275, 29299±29307. Kloareg, B. (1981) Structure et role^ ecophysiologique des parois des algues Frenette, P.S. and Weiss, L. (2000) Sulfated glycans induce rapid littorales: contribution a la resistance aux variations de salinite. hematopoietic progenitor cell mobilization: evidence for selectin- Physiol. Veg., 17, 731±747. dependent and independent mechanisms. Blood, 96, 2460±2468. Kloareg, B. (1984) Isolation and analysis of cell walls of the brown marine Furukawa, S. and Fujikawa, T. (1984) Growth and fucoidan sulfatase algae Pelvetia canaliculata and Ascophyllum nodosum. Physiol. Veg., production in fucoidan-utilizing bacteria from sea sand. Nippon 22, 47±56. Nogeik. Kaishi, 11, 1123±1126. Kloareg, B. and Quatrano, R.S. (1988) Structure of the cell walls of Furukawa, S.I., Fujikawa, T., Koga, D., and Ide, A. (1992a) Purification marine algae and ecophysiological functions of the matrix poly- and some properties of exo-type fucoidanases from Vibrio sp. N-5. saccharides. Oceanogr. Mar. Biol. Annu. Rev., 26, 259±315. Biosci. Biotech. Biochem., 56, 1829±1834. Kloareg, B., Demarty, M., and Mabeau, S. (1986) Polyanionic character- Furukawa, S.I., Fujikawa, T., Koga, D., and Ide, A. (1992b) Production istic of purified sulphated homofucans from brown algae. Int. J. Biol. of fucoidan-degrading enzymes, fucoidanase, and fucoidan sulfatase Macromol., 8, 380±386. by Vibrio sp. N-5. Nippon Suisan Gakk., 58, 1499±1503. Larsen, B., Haug, A., and Painter, T.J. (1966) Sulphated polysaccharides Gerbst, A.G., Ustuzhanina, N.E., Grachev, A.A., Khatuntseva, E.A., in brown algaeÐI. Isolation and preliminary characterisation of three Tsvetkov, D.E., Whitfield, D.M., Berces, A., and Nifantiev, N.E. sulphated polysaccharides from Ascophyllum nodosum (L.) Le JOL. (2001) Synthesis, NMR, and conformational studies of fucoidan Acta Chem. Scand., 20, 219±230. fragments 3. J. Carbohyd. Chem., 20, 821±831. Lasky, L.A. (1995) Selectin-carbohydrate interactions and the initiation of Gerbst, A.G., Ustuzhanina, N.E., Grachev, A.A., Zlotina, N.S., the inflammatory response. Annu. Rev. Biochem., 64, 113±139. Khatuntseva, E.A., Tsvetkov, D.E., Shashkov, A.S., Usov, A.I., and Leite,E.L.,Medeiros,M.G.L.,Rocha,H.A.O.,Farias,G.G.M.,daSilva,L.F., Nifantiev, N.E. (2002) Synthesis, NMR, and conformational studies of Chavante, S.F., de Abreu, L.D., Dietrich, C.P., and Nader, H.B. (1998) fucoidan fragments 4. J. Carbohyd. Chem., 2(1), 313±324. Structure and pharmacological activities ofa sulfated xylofucoglucuronan Giraux, J.L., Matou, S., Bros, A., Tapon-Bretaudiere, J., Letourneur, D., from the alga Spatoglossum schroederi. Plant Sci., 132, 215±228. and Fischer, A.M. (1998a) Modulation of human endothelial cell Lloyd, P.F. and Lloyd, K.O. (1963) Sulphatases and sulphated proliferation and migration by fucoidan and heparin. Eur. J. Cell. polysaccharide in the viscera of marine mollusc. Nature, 199, 287. Biol., 77, 352±359. Logeart, D., Prigent-Richard, S., Boisson-Vidal, C., Chaubet, F., Durand, Giraux, J.L., Tapon-Bretaudiere, J., Matou, S., and Fischer, A.M. (1998b) P., Jozefonvicz, J., and Letourneur, D. (1997) Fucans, sulfated Fucoidan, as heparin, induces tissue factor pathway inhibitor polysaccharides extracted from brown seaweeds, inhibit vascular release from cultured human endothelial cells. Thromb. Haemost., 80, smooth muscle cell proliferation. II. Degradation and molecular 692±695. weight effect. Eur. J. Cell. Biol., 74, 385±390. Granert, C., Raud, J., Waage, A., and Lindquist, L. (1999) Effects of Mabeau, S., Kloareg, B., and Joseleau, J.P. (1990) Fractionation and polysaccharide fucoidin on cerebrospinal fluid interleukin-1 and tumor analysis of fucans from brown algae. Phytochemistry, 29, 2441±2445. necrosis factor alpha in Pneumococcal meningitis in the rabbit. Infect. Marais, M.-F. and Joseleau, J.-P. (2001) A fucoidan fraction from Immun., 67, 2071±2074. Ascophyllum nodosum. Carbohydr. Res., 336, 155±159. Haroun-Bouhedja, F., Ellouali, M., Sinquin, C., and Boisson-Vidal, C. Maruyama, H., Nakajima, J., and Yamamoto, I. (1987) A study on the (2000) Relationship between sulfate groups and biological activities of anticoagulant and fibrinolytic activities of a crude fucoidan from the fucans. Thromb. Res., 100, 453±459. edible brown seaweed Laminaria religiosa, with special reference to its Heinzelmann, M., Polk, H.C.J., and Miller, F.N. (1998) Modulation of inhibitory effect on the growth of sarcoma-180 ascites cells subcuta- lipopolysaccharide-induced monocyte activation by heparin-binding neously implanted into mice. Kitasato Arch. Exp. Med., 60, 105±121. protein and fucoidan. Infect. Immun., 66, 5842±5847. Mauray, S., de Raucourt, E., Talbot, J.C., Dachary-Prigent, J., Hirohashi, N. and Vacquier, V. D. (2002) High molecular mass egg fucose Jozefowicz, M., and Fischer, A.M. (1998) Mechanism of factor IXa sulfate polymer is required for opening both Ca2‡ channels involved in inhibition by antithrombin in the presence of unfractionated and low triggering the sea urchin sperm acrosome reaction. J. Biol. Chem., 277, molecular weight heparins and fucoidan. Biochim. Biophys. Acta, 1387, 1182±1189. 184±194. Hirohashi, N., Vilela-Silva, A.E.S., Mourao,~ P.A.S., and Vacquier, V.D. McCormick, C.J., Tuckwell, D.S., Crisanti, A., Humphries, M.J., and (2002) Structural requirements for species-specific induction of the Hollingdale, M.R. (1999) Identification of heparin as a ligand for sperm acrosome reaction by sea urchin egg sulfated fucan. Biochem. the A-domain of Plasmodium falciparum thrombospondin-related Biophys. Res. Commun., 298, 403±407. adhesion protein. Mol. Biochem. Parasitol., 100, 111±124. Honya, M., Mori, M., Anzai, M., Araki, Y., and Nisizawa, K. (1999) McCormick, C.J., Newbold, C.I., and Berendt, A.R. (2000) Sulfated Monthly changes in the content of fucans, their constituent sugars glycoconjugates enhance CD36-dependent adhesion of Plasmodium and sulphate in cultured Laminaria japonica. Hydrobiologia, 398/399, falciparum-infected erythrocytes to human microvascular endothelial 411±416. cells. Blood, 96, 327±333.

38R Structure and biological properties of sulfated fucans

McNeely, W.H. (1959) Fucoidan. In Whistler, R.L. (ed), Industrial gums. Patankar, M.S., Oehninger, S., Barnett, T., Williams, R.L., and Clark, G.F. Academic Press, New York, pp. 117±125. (1993) A revised structure for fucoidan may explain some of its Medcalf, D.G., Schneider, T.L., and Barnett, R.W. (1978) Structural biological activities. J. Biol. Chem., 268, 21770±21776. features of a novel glucuronogalactofucan from Ascophyllum nodosum. Patel, M.K., Mulloy, B., Gallagher, K.L., O'Brien, L., and Hughes, A.D. Carbohyd. Res., 66, 167±171. (2002) The antimitogenic action of the sulphated polysaccharide Mengerink, K.J., Moy, G.W., and Vacquier, V.D. (2002) suREJ3, a fucoidan differs from heparin in human vascular smooth muscle cells. polycystin-1 protein, is cleaved at the GPS domain and localizes Thromb. Haemost., 87, 149±154. to the acrosomal region of sea urchin sperm. J. Biol. Chem., 277, Percival, E. (1968) Glucuronoxylofucan, a cell-wall component of 943±948. Ascophyllum nodosum. Carbohyd. Res., 7, 272±283. Mian, A.J. and Percival, E. (1973) Carbohydrates of the brown seaweeds Percival, E.G.V. and Ross, A.G. (1950) The isolation and purification of Himanthalia lorea, Bifurcaria bifurcata, and Padina pavonia. Carbohyd. fucoidin from brown seaweeds. J. Chem. Soc., 717±720. Res., 26, 133±146. Pereira, M.S., Mulloy, B., and Mourao, P.A.S. (1999) Structure and Millet, J., Jouault, S.C., Mauray, S., Theveniaux, J., Sternberg, C., anticoagulant activity of sulfated fucans. J. Biol. Chem., 274, Boisson, V.C., and Fischer, A.M. (1999) Antithrombotic and anti- 7656±7667. coagulant activities of a low molecular weight fucoidan by the Pereira, M.S., Melo, F.R., and Mourao,~ P.A.S. (2002) Is there a subcutaneous route. Thromb. Haemost., 81, 391±395. correlation between structure and anticoagulant action of sulfated Moreno, R., Orihuela, P., and Barros, C. (2001) Differential effects of galactans and sulfated fucans? Glycobiology, 12, 573±580. Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 polysulphates between mouse and hamster during in vitro fertilization. Ponce, N.M.A., Pujol, C.A., Damonte, E.B., Flores, M.L., and Andrologia, 33, 19±25. Stortz, C.A. (2003) Fucoidans from the brown seaweed Adenocystis Mori, H., Kamei, H., Nishide, E., and Nisizawa, K. (1982) Sugar utricularis: extraction methods, antiviral activity and structural studies. constituents of some sulfated polysaccharides from the sporophylls of Carbohydr. Res., 338, 153±165. wakame (Undaria pinnatifida) and their biological activities. In Marine Preeprame, S., Hayashi, K., Lee, J.B., Sankawa, U., and Hayashi, T. algae in pharmaceutical science. Walter de Gruyter, Berlin and (2001) A novel antivirally active fucan sulfate derived from an New York, pp. 109±121. edible brown alga, Sargassum horneri. Chem. Pharm. Bull., 49, Morinaga, T., Araki, T., Ito, M., and Kitamikado, M. (1981) A search for 484±485. fucoidan-degrading bacteria in coastal sea environments of Japan. Ribeiro, A.C., Vieira, R.P., Mourao, P.A.S., and Mulloy, B. (1994) A Bull. Japan. Soc. Sci. Fish., 47, 621±625. sulfated a-L-fucan from sea cucumber. Carbohyd. Res., 255, 225±240. Mourao,~ P.A.S. and Bastos, I.G. (1987) Highly acidic glycans from sea Ritter, L.S., Copeland, J.G., and McDonagh, P.F. (1998) Fucoidin cucumbers. Eur. J. Biochem., 166, 639±645. reduces coronary microvascular leukocyte accumulation early in Mourao,~ P.A.S. and Pereira, M.S. (1999) Searching for alternatives to reperfusion. Ann. Thorac. Surg., 66, 2063±2072. heparin: sulfated fucans from marine invertebrates. Trends Cardiovasc. Rogerson, S.J., Chaiyaroj, S.C., Ng, K., Reeder, J.C., and Brown, G.V. Med., 9, 225±232. (1995) Chondroitin sulfate A is a cell surface receptor for Plasmodium Mulloy, B., Ribeiro, A.C., Alves, A.P., Vieira, R.P., and Mourao, P.A.S. falciparum-infected erythrocytes. J. Exp. Med., 182, 15±20. (1994) Sulfated fucans from echinoderms have a regular tetrasacchar- Rozkin, M.Y., Levina, M.N., Kameneva, N.S., Usov, A.I., and ide repeating unit defined by specific patterns of sulfation at the O-2 Yefimov, V.S. (1989) Investigation of the mechanism of the anti- and O-4 positions. J. Biol. Chem., 269, 22113±22123. coagulant action of fucoidans. Farmakol. i toksik., 3, 48±51. Nagaoka, M., Shibata, H., Kimura-Takagi, I., Hashimoto, S., Kimura, K., Schweiger, R.G. (1962) Methanolysis of fucoidan. I. Preparation of Makino, T., Aiyama, R., Ueyama, S., and Yokokura, T. (1999) methyl a-L-fucoside and L-fucose. J. Org. Chem., 27, 4267±4272. Structural study of fucoidan from Cladosiphon okamuranus Tokida. Shimaoka, M., Ikeda, M., Iida, T., Taenaka, N., Yoshiya, I., and Glycoconj. J., 16, 19±26. Honda, T. (1996) Fucoidin, a potent inhibitor of leukocyte rolling, Nasu, T., Fukuda, Y., Nagahira, K., Kawashima, H., Noguchi, C., and prevents neutrophil influx into phorbol-ester-induced inflammatory Nakanishi, T. (1997) Fucoidin, a potent inhibitor of L-selectin sites in rabbit lungs. Am. J. Respir. Crit. Care. Med., 153, 307±311. function, reduces contact hypersensitivity reaction in mice. Immunol. Sweeney, E.A., Priestley, G.V., Nakamoto, B., Collins, R.G., Lett., 59, 47±51. Beaudet, A.L., and Papayannopoulou, T. (2000) Mobilization of Nishino, T. and Nagumo, T. (1991) Structural characterization of a new stem/progenitor cells by sulfated polysaccharides does not require anticoagulant fucan sulfate from the brown seaweed Ecklonia kurome. selectin presence. Proc. Natl Acad. Sci. USA, 97, 6544±6549. Carbohyd. Res., 211, 77±90. Sweeney, E.A., Lortat-Jacob, H., Priestley, G.V., Nakamoto, B., and Nishino, T., Nishioka, C., Ura, H., and Nagumo, T. (1994) Isolation and Papayannopoulou, T. (2002) Sulfated polysaccharides increase plasma partial characterization of a novel amino sugar-containing fucan levels of SDF-1 in monkeys and mice: involvement in mobilization of sulfate from commercial Fucus vesiculosus fucoidan. Carbohydr. Res., stem/progenitor cells. Blood, 99, 44±51. 255, 213±224. Talevi, R. and Gualtieri, R. (2001) Sulfated glycoconjugates are powerful Nishino, T., Fukuda, A., Nagumo, T., Fujihara, M., and Kaji, E. (1999) modulators of bovine sperm adhesion and release from the oviductal Inhibition of the generation of thrombin and factor Xa by a fucoidan epithelium in vitro. Biol. Reprod., 64, 491±498. from the brown seaweed Ecklonia kurome. Thromb. Res., 96, 37±49. Tanaka, K. and Sorai, S. (1970) Hydrolysis of fucoidan by abalone liver Nishino, T., Yamauchi, T., Horie, M., Nagumo, T., and Suzuki, H. (2000) a-L-fucosidase. FEBS Lett., 9, 45±48. Effects of a fucoidan on the activation of plasminogen by u-PA and Teixeira, M.M. and Hellewell, P. (1997) The effect of the selectin binding t-PA. Thromb. Res., 99, 623±634. polysaccharide fucoidin on eosinophil recruitment in vivo. Brit. J. Omata, M., Matsui, N., Inomata, N., and Ohno, T. (1997) Protective Pharmacol., 120, 1059±1066. effects of polysaccharide fucoidin on myocardial ischemia-reperfusion Thanassi, N.M. and Nakada, H. (1967) Enzymic degradation of fucoidan injury in rats. J. Cardiovasc. Pharmacol., 30, 717±724. by enzymes from the hepatopancreas of abalone, Haliotus species. O'Neill, A.N. (1954) Degradative studies on fucoidin. J. Am. Chem. Soc., Arch. Biochem. Biophys., 118, 172±177. 76, 5074±5076. Thorlacius, H., Vollmar, B., Seyfert, U.T., Vestweber, D., and Ortega-Barria, E. and Boothroyd, J.C. (1999) A Toxoplasma lectin-like Menger, M.D. (2000) The polysaccharide fucoidan inhibits micro- activity specific for sulfated polysaccharides is involved in host cell vascular thrombus formation independently from P- and L-selectin infection. J. Biol. Chem., 274, 1267±1276. function in vivo. Eur. J. Clin. Invest., 30, 804±810. Ostergaard, C., Yieng-Kow, R.V., Benfield, T., Frimodt-Moller, N., Trento, F., Cattaneo, F., Pescador, R., Porta, R., and Ferro, L. (2001) Espersen, F., and Lundgren, J.D. (2000) Inhibition of leukocyte entry Antithrombin activity of an algal polysaccharide. Thromb. Res., 102, into the brain by the selectin blocker fucoidin decreases interleukin-1 457±465. (IL-1) levels but increases IL-8 levels in cerebrospinal fluid during Usov, A.I., Smirnova, G.P., and Klochkova, N.G. (2001) Algae experimental Pneumococcal meningitis in rabbits. Infect. Immun., 68, polysaccharides. 55. Polysaccharide composition of some brown 3153±3157. Kamchatka algae. Bioorg. Khim., 27, 444±448.

39R O. Berteau and B. Mulloy

Usui, T., Asari, K., and Mizuno, T. (1980) Isolation of highly purified Von Holdt, M.M., Ligthelm, S.P., and Nunn, J.R. (1955) South African ``fucoidan'' from Eisenia bicyclis and its anticoagulant and antitumor seaweeds: seasonal variations in the chemical composition of some activities. Agric. Biol. Chem., 44, 1965±1966. phaeophyceae. J. Sci. Food Agr., 6, 193±197. Vacquier, V.D. and Moy, G.W. (1997) The fucose sulfate polymer of egg Wort, D.J. (1955) The seasonal variation in chemical composition of jelly binds to sperm REJ and is the inducer of the sea urchin sperm Macrocystis integrifolia and Neroecystis luetkeana in British Colombia acrosome reaction. Dev. Biol., 192, 125±135. coastal waters. Can. J. Bot., 33, 323±340. Vasseur, E. (1948) Chemical studies on the jelly coat of the sea-urchin egg. Yamamoto, K., Tsuji, Y., Kumagai, H., and Tochikura, T. (1986) Acta Chem. Scand., 2, 900±913. Induction and purification of a-L-fucosidase from Fusarium Verdrengh, M., Erlandsson-Harris, H., and Tarkowski, A. (2000) Role of oxysporum. Agric. Biol. Chem., 50, 1689±1695. selectins in experimental Staphylococcus aureus-induced arthritis. Yaphe, W. and Morgan, K. (1959) Hydrolysis of fucoidin by Pseudomonas Eur. J. Immunol., 30, 1606±1613. atlantica and Pseudomonas carrageenovora. Nature, 463, 761±762. Vilela-Silva, A.C., Alves, A.P., and Valente, A.P. (1999) Structure of the Ying, P., Shakibaei, M., Patankar, M.S., Clavijo, P., Beavis, R.C., sulfated a-L-fucan from the egg jelly coat of the sea urchin Clark, G.F., and Frevert, U. (1997) The malaria circumsporozoite pro- Strongylocentrotus franciscanus: patterns of preferential 2-O- and 4- tein: interaction of the conserved regions I and II-plus with heparin- O-sulfation determine sperm cell recognition. Glycobiology, 9, 927±933. like oligosaccharides in heparan sulfate. Exp. Parasitol., 85, 168±182. 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 Downloaded from https://academic.oup.com/glycob/article/13/6/29R/572950 by guest on 02 October 2021 closely related sea urchins Strongylocentrotus droebachiensis and Special acknowledgment: The authors would like to Strongylocentrotus pallidus ensure species-specific fertilization. J. Biol. acknowledge the contribution of Dr. R. Daniel to this Chem., 277, 379±387. review.

40R