Structural heterogeneity among unique sulfated L-galactans from different species of ascidians (tunicates)

The sulfated polysaccharides that occur in the tunic of ascidians differ markedly in molecular weight and chemical composition. A high molecular weight fraction (F-1), which has a high galactose content and a strong negative optical rotation, is present in all species. Several structural differences were observed among the F-1 fractions obtained from three species of ascidians that were studied in detail. Large numbers of alpha-L-galactopyranose residues sulfated at position 3 and linked glycosidically through position 1----4 are present in F-1 from all three ascidians. However, alpha-L-galactopyranose units, 1----3-linked and partially sulfated at position 4, comprise about half of the sugar units in the central core of F-1 from Ascidian nigra. In addition, L-galactopyranose nonreducing end units occur in F-1 from Styela plicata and A. nigra, but comprise only a minor fraction of F-1 from Clavelina sp. The combination of these various component units gives a complex structure for F-1 from S. plicata and A. nigra, whereas F-1 from Clavelina sp. possesses a simpler structure. The structures of these ascidian glycans are unique among all previously described sulfated polysaccharides, since they are highly branched (except that from Clavelina sp), sulfated at position 3, and contain large amounts of L-galactose without its D-enantiomorph. These data show unusual examples of polyanionic glycans with structural function in animal tissues.     

Highly acidic glycans from sea cucumbers. Isolation and fractionation of fucose-rich sulfated polysaccharides from the body wall of Ludwigothurea grisea

The body wall of the sea cucumber contains high amounts of sulfated glycans, which differ in structure from glycosaminoglycans of animal tissues and also from the fucose-rich sulfated polysaccharides isolated from marine algae and from the jelly coat of sea urchin eggs. In Ludwigothurea grisea, glycans can be separated into three fractions which differ in molecular mass and chemical composition. The fraction containing a high-molecular-mass component has a high proportion of fucose and small amounts of amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction, which represents the major portion of the sea cucumber polysaccharides, contains besides fucose, approximately equimolar proportions of glucuronic acid and amino sugars, and has a sulfate content higher than that in the other two fractions. Both D and L-isomers of fucose are found in these polysaccharides, and the sulfate is linked to the O-3 position of the fucose residues. The attachment position of the sulfate groups to the glucuronic acid units and amino sugars is still undetermined. It is possible that these compounds are involved in maintaining the integrity of the sea cucumber's body wall, in analogy with the role of other macromolecules in the vertebrate connective tissue.     

Isolation, fractionation, and preliminary characterization of a novel class of sulfated glycans from the tunic of Styela plicata (Chordata Tunicata)

The sulfated glycans in the tunic of Styela plicata differ from the glycosaminoglycans of animal tissues and also from the sulfated polysaccharides isolated from marine algae. The ascidian glycans occur primarily as three fractions that differ markedly in molecular weight and chemical composition. The high molecular weight fraction encompasses a broad range of molecular weights but is chemically homogeneous and contains an unusual amount of galactose. The 20,000 molecular weight polysaccharide is rich in galactose and glucose while the 8,000 molecular weight fraction is rich in amino sugars and contains the neutral hexoses galactose, glucose, and mannose. All fractions contain large amounts of sulfate esters. The ascidians polysaccharides can be extracted from the tissue by proteolytic enzyme or by guanidine hydrochloride solutions. The high molecular weight fraction is preferentially extracted by papain while guanidine hydrochloride removes mainly the low molecular weight polysaccharides. We speculate that these sulfated glycans are essential for maintaining the structural integrity of the tunic, in analogy with the glycosaminoglycans of vertebrate connective tissues.     

Epimerization of D-glucose to L-galactose during the biosynthesis of a sulfated L-galactan in the ascidian tunic.

The sulfated polysaccharides occurring in the tunic of ascidians are unique among known sulfated polysaccharides in that their major constituent sugar is galactose, which occurs exclusively in the L-enantiomeric form. In vitro incorporation experiments using tunic slices incubated with 14C-labeled sugars revealed that cells from this tissue epimerize D-isomers of hexose into L-galactose during the biosynthesis of their constituent polysaccharides. Compared with other hexoses, the precursor D-[14C]glucose has the highest rate of incorporation and produces the highest proportion of L-galactose units. This metabolic pathway is distinct from the epimerization of D-mannose to L-galactose through its guanosine 5'-diphosphate nucleotide, described previously in an alga and in a snail. Therefore, the epimerization of D-glucose to L-galactose in the ascidian tunic occurs through a novel metabolic route, which involves inversion of the configuration of carbon atoms 2, 3, and 5 of the hexosyl moieties.     

Occurrence of a unique fucose-branched chondroitin sulfate in the body wall of a sea cucumber

The sulfated polysaccharides in the body wall of the sea cucumber occur as three fractions that differ markedly in molecular mass and chemical composition. The fraction containing a high molecular mass component has a high proportion of fucose and small amounts of galactose and amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction (F-2), which represents the major portion of the sea cucumber-sulfated polysaccharides, contains approximately equimolar quantities of glucuronic acid, N-acetyl galactosamine, and fucose, and has a sulfate content higher than that in the other two fractions. The structure of fraction F-2 was examined in detail. This polysaccharide has an unusual structure composed of a chondroitin sulfate-like core, containing side chain disaccharide units of sulfated fucopyranosyl linked to approximately half of the glucuronic acid moieties through the O-3 position of the acid. These unusual fucose branches obstruct the access of chondroitinases to the chondroitin sulfate core of F-2. However, after partial acid hydrolysis, which removes the sulfated fucose residues from the polymer, fraction F-2 is degraded by chondroitinases into 6-sulfated and nonsulfated disaccharides.     

Study of antioxidant activities of sulfated polysaccharides from Laminaria japonica

The composition, molecular weight and in vitro antioxidant activity of various sulfated polysaccharides obtained by anion exchange chromatography, acid hydrolysis and radical process degradation of the crude sulfated polysaccharide extracted from Laminaria japonica were compared. The low sulfated F-A2, with a peak-molecular weight (Mp) of 5–15 kDa, 14.5% sulfated ester and 21.8% glucuronic acid, exhibited a very strong antioxidant activity on superoxide and hydroxyl radicals, with activity even higher than that of large molecular weight fractions F-A and F-B. However, highly sulfated fractions with a peak-molecular weight below 15 kDa had much lower antioxidant activities than other fractions. These results indicated that the sulfate group of the low molecular weight fractions represents a physical block for the reaction with oxygen radicals. The chemical properties and antioxidant activities of sulfated polysaccharide fractions obtained by radical process degradation of crude sulfated polysaccharide were quite different from those obtained by acid hydrolysates. By radical process degradation, the high molecular weight was decreased to give LM2 (Mp 8 kDa) and LM1 (Mp 1.5 kDa), with a yield of 40% and 15%, respectively. LM2 was enriched with fucose and sulfated ester, while containing low amounts of glucuronic acid. The antioxidant activity showed that LM2 was unable to scavenge either superoxide or hydroxyl radical, which suggested that radical process degradation targeted mainly ascopyllan-like species rich in glucuronic acid, while the fraction rich in sulfated l-fucose remained unchanged. However, LM1 with Mp 1.5 kDa still retained apparent scavenging ability for superoxide radical, although it contained no glucuronic acid and certain amounts of galactose and mannose as main neutral sugars. These result suggest that the antioxidant activity of sulfated polysaccharides is apparently related not only to molecular weight and sulfated ester content, as previously determined, but also to glucuronic acid and fucose content.     

Structures of sulfated oligosaccharides in human trachea mucin glycoproteins

The structures of high molecular weight sulfated oligosaccharide chains in mucins purified from the sputum of a patient with cystic fibrosis and blood group H determinant were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by ion exchange chromatography on DEAE-Agarose and a fraction containing multisulfated chains was further purified by lectin affinity chromatography to completely remove small amounts of sialylated chains. A major sulfated oligosaccharide fraction containing chains with an average of 160 to 200 sugar residues was isolated by gel filtration on BioGel P-10 columns and individual subfractions were characterized by methylation analysis, periodate oxidation and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GldNAc in a ratio of 1:2:2.1 and only one galactosaminitol residue for every 160-to 200 sugar residues. The average molecular weight of oligosaccharide chains in these fractions was between 27,000 and 40,000 daltons. Structural analysis showed that these high molecular weight chains contained varying amounts of the repeating unit shown in the following oligosaccharide. Only one in about every 10 repeating units contained sulfate esters.Several shorter chains which contain 2 to 3 sulfate esters were also isolated from this multisulfated oligosaccharide fraction. The structures proposed for these oligosaccharides indicate that they are lower molecular weight chains with the same general structure as those found in the high molecular weight sulfated oligosaccharides. Taken collectively, the results of these studies show that a major sulfated oligosaccharide fraction in resporatory mucin purified from the mucus of patients with cystic fibrosis contains high molecular weight branched chains that consist of a repeating oligosaccharide sequence with sulfate linked to the 6 positions of galactose and possibly GlcNAc residues in the side chains.     

Anticoagulant motifs of marine sulfated glycans

Sulfated polysaccharides, like the glycosaminoglycan (GAG) heparin, are known to exhibit anticoagulant properties when certain structural features are present. The structural requirement for this action is well-established for heparin, in which a pentasaccharide motif plays a key role for keeping the high-affinity interaction to antithrombin. Over the last years of this glycomic era, several novel anticoagulant sulfated glycans have been described. Those from marine sources have been awakening special attention mainly because of their impressive anticoagulant effects together with structural uniqueness. The commonest of these glycans are the sulfated fucans (SFs), the sulfated galactans (SGs), and the marine invertebrate GAGs like the fucosylated chondroitin sulfate and ascidian dermatan sulfate. Since these marine sulfated glycans do not bear within their polymeric chains the specific pentasaccharide motif of heparin, other structural features must be necessary to trigger the anticoagulant effect. The objective of this report is to present the anticoagulant motifs of the marine SFs, SGs and GAGs.     

Chemical characters and antioxidative properties of sulfated polysaccharides from Laminaria japonica

A low molecular weight sulfated polysaccharide (L-A) was prepared bymild acid hydrolysis of crude fucoidan (F-A) from Laminaria japonica. In comparison with F-A, L-A had a lower proportion of fucose residues,but a similar proportion of sulfate. The galactose content of both fractionswas relatively high, especially for L-A (57%). The antioxidant propertiesof the two polysaccharides were studied using two low-density lipoproteinoxidation systems. L-A had a stronger effect against low-density lipoproteinoxidation in both systems. F-A inhibited the AAPH-induced low-densitylipoprotein oxidation, but had little effect on the Cu⁺⁺-inducedsystem due to its large molecular mass. The active sulfated fraction L-A,containing galactose, mannose and fucose (about 9: 2: 2) is reported herefor the first time.     

STRUCTURE OF EXTRACELLULAR POLYSACCHARIDES PRODUCED BY A SOIL CRYPTOMONAS SP. (CRYPTOPHYCEAE)1

The Hindak strain of a Cryptomonas species (Cryptophyceae) produces extracellular polysaccharides. Because there is no information on the structure of these compounds in the Cryptophyceae we conducted structural studies. Gas–liquid chromatographic analyses showed that the polysaccharide is composed of fucose, rhamnose, xylose, mannose, glucose, galactose, galacturonic acid, glucuronic acid, and traces of 3-O-methyl galactose. The polysaccharide was separated into two subtractions by ion-exchange chromatography. Fraction A consisted mainly of 1,3-linked galactose units and 1,4-linked galacturonic acid. Unlike fraction B, fraction A did not have xylose, 3-O-methyl galactose, or glucuronic acid. Also, its degree of branching was low compared to that of fraction B. Only traces of sulfate were present infraction A, but fraction B was 10–15% sulfated. Protein was approximately 1% in both fractions. These polysaccharides appear to be a novel type of polymer in algae.     

A wide diversity of sulfated polysaccharides are synthesized by different species of marine sponges

Sulfated polysaccharides were extracted from four species of marine sponges by exhaustive papain digestion. These compounds were purified by anion-exchange and gel-filtration chromatography. Analysis of the purified polysaccharides revealed a species-specific variation in their chemical composition and also in their molecular masses. In the species Aplysina fulva we found a sulfated glucan with a glycogen-like structure. The other three species contained sulfated polysaccharides with variable proportions of galactose, fucose, arabinose and hexuronic acid and also with different degrees of sulfation. Although the complex nature of these polysaccharides did not allow complete structure determination, we detected the occurrence of 4-sulfated residues of fucose and arabinose in the species Dysidea fragilis. The biological role of these sulfated polysaccharides requires further investigation. They may be involved in the species-specific aggregation of sponge cells and/or in the structural integrity of sponge, resembling the proteoglycans of mammalian connective tissues.     

In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera

Water-soluble sulfated polysaccharides extracted from Enteromorpha prolifera and fractionated using ion-exchange chromatography (crude, F₁, F₂ and F₃ fractions) were investigated to determine their in vitro and in vivo immunomodulatory activities. The sulfated polysaccharides, especially the F₁ and F₂ fractions, stimulated a macrophage cell line, Raw 264.7, inducing considerable nitric oxide (NO) and various cytokine production via up-regulated mRNA expression. The in vivo experiment results show that the sulfated polysaccharides (the crude and F₂ fractions) significantly increased Con A-induced splenocyte proliferation, revealing their potential comitogenic activity. In addition, IFN-γ and IL-2 secretions were considerably increased by the F₂ fraction without altering the release of IL-4 and IL-5. This implies that the F₂ fraction can activate T cells by up-regulating Th-1 response and that Th-1 cells might be the main target cells of the F₂ fraction. These in vitro and in vivo results suggest that the sulfated polysaccharides are strong immunostimulators.     

Structural characterization of sulfated arabinans extracted from Cladophora glomerata Kützing and their macrophage activation

Water-soluble sulfated heteropolysaccharides were extracted from Cladophora glomerata Kützing and fractionated by ion-exchange chromatography, which yielded two subfractions, F₁ and F₂. The crude and fractionated polysaccharides (F₁ and F₂) mostly consisted of carbohydrates (62.8–74.5%) with various amounts of proteins (9.00–17.3%) and sulfates (16.5–23.5%), including different levels of arabinose (41.7–54.4%), galactose (13.5–39.0%), glucose (0.80–10.6%), xylose (6.84–13.4%), and rhamnose (0.20–2.83%). Based on the size exclusion chromatography (SEC) profiles, the crude and fractions mainly contained one peak with shoulders having molecular weight (M_w) ranges of 358–1,501 × 10³. The F₁ fraction stimulated RAW264.7 cells to produce considerable amounts of nitric oxide and cytokines compared to the crude and F₂ fraction. The backbone of the most potent immunostimulating fraction (F₁) was α-(1→4)-L-arabinopyranoside with galactose and xylose residues as branches at O-2 position, and sulfates mainly at O-2 position as well.     

NMR structural biology of sulfated glycans

Sulfated fucans, sulfated galactans, and glycosaminoglycans are extensively studied worldwide in terms of both structure and biomedical functions. Liquid-state nuclear magnetic resonance (NMR) spectroscopy is the most employed analytical technique in structural analysis of these sulfated glycans. This is due to the fact that NMR-based analyses enable a series of achievements such as (i) accurate structure characterization/determination; (ii) measurements of parameters regarding molecular motion (dynamics); (iii) assessment of the 3D structures (usually assisted by computational techniques of Molecular Modeling and/or Molecular Dynamics) of the composing monosaccharides (ring conformers) and the overall conformational states of the glycan chains either free in solution or bound to proteins; and (iv) analysis of the resultant intermolecular complexes with functional proteins through either the protein or the carbohydrate perspective. In this review, after a general introduction about the principal NMR parameters utilized for achieving this set of structural information, discussion is given on NMR-based studies of some representative sulfated fucans, sulfated galactans, and glycosaminoglycans. Due to the growing number of studies concerning both structure and function of sulfated glycans and the widely use of NMR spectroscopy in such studies, a review paper discussing (i) the most experiments employed for analysis, (ii) procedures used in data interpretation, and (iii) the general aspects of the sulfated glycans, is timely in the literature.     

Occurrence of sulfated galactans in marine angiosperms: evolutionary implications

We report for the first time that marine angiosperms (seagrasses) possess sulfated polysaccharides, which are absent in terrestrial and freshwater plants. The structure of the sulfated polysaccharide from the seagrass Ruppia maritima was determined. It is a sulfated D-galactan composed of the following regular tetrasaccharide repeating unit: [3-beta-D-Gal-2(OSO3)-1-->4-alpha-D-Gal-1-->4-alpha-D-Gal-1-->3-beta-D-Gal-4(OSO3)-1-->]. Sulfated galactans have been described previously in red algae and in marine invertebrates (ascidians and sea urchins). The sulfated galactan from the marine angiosperm has an intermediate structure when compared with the polysaccharides from these two other groups of organisms. Like marine invertebrate galactan, it expresses a regular repeating unit with a homogenous sulfation pattern. However, seagrass galactan contains the D-enantiomer of galactose instead of the L-isomer found in marine invertebrates. Like red algae, the marine angiosperm polysaccharide contains both alpha and beta units of D-galactose; however, these units are not distributed in an alternating order, as in algal galactan. Sulfated galactan is localized in the plant cell walls, mostly in rhizomes and roots, indicative of a relationship with the absorption of nutrients and of a possible structural function. The occurrence of sulfated galactans in marine organisms may be the result of physiological adaptations, which are not correlated with phylogenetic proximity. We suggest that convergent adaptation, due to environment pressure, may explain the occurrence of sulfated galactans in many marine organisms.     

Isolation and characterization of a highly sulfated heparan sulfate from ascidian test cells.

Several sulfated polysaccharides have been isolated from the test cells of the ascidian Styela plicata. The preponderant polysaccharide is a highly sulfated heparan sulfate with the following disaccharide composition: (1) UA(2SO4)-1-->4 GlcN(SO4)(6SO4), 53%; (2) UA(2SO4)-1-->4-GlcN(SO4), 22%; (3) UA-1-->4-GlcNAc(6SO4), 14% and (4) UA-1-->4-GlcN(SO4), 11%. Two others unidentified sulfated polysaccharides and a glycogen polymer are also present in the ascidian eggs. Histochemistry with the cationic dye 1,9-dimethyl-methylene blue and biochemical analysis of the 35S-sulfate incorporation into the eggs reveal that the sulfated glycans are present exclusively in the test cells. Possibly these sulfated polysaccharides are involved in important functions of these cells, such as to confer an external and hydrophilic layer which protect the eggs and the larvae of ascidians.     

Dual and antagonic therapeutic effects of sulfated glycans

Sulfated glycans currently explored in medicine like glycosaminoglycans (GAGs) or those of potential medical application like algal sulfated galactans (SGs) and fucoidans exhibit significant effects in numerous pathophysiological systems. According to the structure of these sulfated glycans, sample concentration and the method utilized in the approach opposite effects can be achieved. The effects aimed at down-regulating the events usually dominate. These effects are expected in most clinical endeavors. However, the effects capable of accelerating the events can be also beneficial in certain circumstances. Besides discoursing about the paradoxical effects of sulfated glycans in coagulation/thrombosis, angiogenesis, inflammation and microbial infections; this report aims primarily at highlighting the possible contribution of the neglected activities of some well-known sulfated glycans in up-regulating the events of these pathophysiological systems. The representative sulfated glycans taken here are the mammalian-derived GAGs, the unique holothurian GAG, the red algal SGs and the brown algal fucoidans. The current discussion is highly relevant in light of the future strategies for developing novel sulfated glycan-based therapies.     

In vitro antioxidant activities of sulfated polysaccharide fractions extracted from Corallina officinalis

Sulfated polysaccharides (F1, F2) from seaweed Corallina officinalis were isolated through anion-exchange column chromatography. Their chemical characteristics were determined by GC, HPLC, FT-IR and UV spectra. F1 and F2 contained only two monosaccharides, namely galactose and xylose. The antioxidant activities of F1, F2 and the de-sulfated polysaccharides (DF-1, DF-2) in vitro were investigated, including hydroxyl radicals scavenging effect, superoxide radical scavenging capacity, DPPH radical activity and reducing power. As expected, antioxidant assay showed that the two sulfated polysaccharide fractions (F1, F2) possessed considerable antioxidant properties and had more excellent abilities than de-sulfated polysaccharides (DF-1, DF-2).     

The structure of sulfated polysaccharides ensures a carbohydrate-based mechanism for species recognition during sea urchin fertilization

The evolution of barriers to inter-specific hybridization is a crucial step in the fertilization of free spawning marine invertebrates. In sea urchins, molecular recognition between sperm and egg ensures species recognition. Here we review the sulfated polysaccharide-based mechanism of sperm-egg recognition in this model organism. The jelly surrounding sea urchin eggs is not a simple accessory structure; it is molecularly complex and intimately involved in gamete recognition. It contains sulfated polysaccharides, sialoglycans and peptides. The sulfated polysaccharides have unique structures, composed of repetitive units of alpha-L-fucose or alpha-L-galactose, which differ among species in the sulfation pattern and/or the position of the glycosidic linkage. The egg jelly sulfated polysaccharides show species-specificity in inducing the sperm acrosome reaction, which is regulated by the structure of the saccharide chain and its sulfation pattern. Other components of the egg jelly do not possess acrosome reaction inducing activity, but sialoglycans act in synergy with the sulfated polysaccharide, potentiating its activity. The system we describe establishes a new view of cell-cell interaction in the sea urchin model system. Here, structural changes in egg jelly polysaccharides modulate cell-cell recognition and species-specificity leading to exocytosis of the acrosome. Therefore, sulfated polysaccharides, in addition to their known functions as growth factors, coagulation factors and selectin binding partners, also function in fertilization. The differentiation of these molecules may play a role in sea urchin speciation.     

Sulfated O-linked glycans of the vitelline coat as ligands in gamete interaction in the ascidian, Halocynthia roretzi

In the ascidian Halocynthia roretzi, sperm-egg binding is probably mediated through the interaction between alpha-L-fucosidase present on the sperm surface and anionic saccharide chains of the egg vitelline coat. To characterize biologically active glycans, total glycans were chemically released from the glycopeptide fraction of the vitelline coat. The fraction of uncharged glycans and two fractions of negatively charged glycans were separated by diethylaminoethyl-anion exchange chromatography. In a competitive inhibition assay of fertilization, both anionic fractions showed inhibitory activity, with more anionic glycans being most potent, while uncharged glycans were biologically inactive. Chemical desulfation combined with a competitive inhibition assay of fertilization and ion analysis determined that sulfate groups were responsible for anionic character and crucial for biological activity. Monosaccharide analysis of anionic fractions showed a high content of N-acetylgalactosamine, galactose, xylose and the presence of arabinose, mannose, N-acetylglucosamine, glucose and rhamnose. Glycans were O-linked and galactose and xylose residues were detected at reducing termini. Linkage analysis suggested that 1,4-linked xylose, 1,3-linked galactose and N-acetylgalactosamine residues, substituted to different degrees by sulfate groups on the C-3 and C-4 carbons, respectively, constituted the core structures of anionic glycans.