Structural features of sulfated glycans from the tunic of Styela plicata (Chordata-Tunicata). A unique occurrence of L-galactose in sulfated polysaccharides

The sulfated polysaccharides in the tunic of Styela plicata occur as three fractions that differ markedly in molecular mass and chemical composition. The high-molecular-mass fraction has a high galactose content and a strong negative optical rotation while the low-molecular-mass fractions have a higher proportion of amino sugars and glucose. The galactose occurs in these polysaccharides entirely in the L-enantiomeric form. Although L-galactose is a constituent of several polysaccharides, this is the first report of sulfated polysaccharides that contain high amounts of L-galactose, and that lack the D enantiomorph of this sugar. Furthermore, the structure of the high-molecular-mass fraction, which is composed mainly of a core of alpha-L-galactopyranose residues, sulfated at position 3, linked glycosidically though position 1----4, and with non-sulfated L-galactopyranose non-reducing end-units, is unique among other previously described sulfated glycans. These data are of considerable interest as they show an unusual example of possible variants of polyanionic glycans with structure function in living tissues.     

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.     

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.     

Observations on the mechanism of the reversible epimerization of GDP-D-mannose to GDP-L-galactose by an enzyme from Chlorella pyrenoidosa.

An enzyme fraction from the green alga Chlorella pyrenoidosa that catalyzes the reversible epimerization of guanosine 5'-diphosphate D-mannose to guanosine 5'-diphosphate L-galactose brings about the incorporation of tritium from tritium-labeled water into the hexosyl moieties of those sugar nucleotides. The hexoses were degraded by periodate oxidation whereby the tritium was found to be equally distributed between carbon atoms 3 and 5. That observation was taken to imply that the epimerizations proceed via ene-diol intermediates.     

Expression of two different sulfated fucans by females of Lytechinus variegatus may regulate the seasonal variation in the fertilization of the sea urchin

The egg jellies of sea urchins contain sulfated polysaccharides with unusual structures, composed of linear chains of l-fucose or l-galactose with well-defined repetitive units. The specific pattern of sulfation and the position of the glycosidic bond vary among sulfated polysaccharides from different species. These polysaccharides show species specificity in inducing the acrosome reaction, which is a critical event for fertilization. Females of the sea urchin Lytechinus variegatus spawn eggs containing a sulfated fucan with the repetitive sequence [3-alpha-L-Fucp-2(OSO(3))-1 --> 3-alpha-L-Fucp-4(OSO(3))-1 --> 3-alpha-L-Fucp-2,4(OSO(3))-1 --> 3-alpha-L-Fucp-2(OSO(3))-1](n). We now observe that, close to winter, a period of decreased fertility for the sea urchin, the females synthesize a distinct sulfated fucan with a simple structure, composed of 4-sulfated, 3-linked alpha-fucose residues. This sulfated fucan is inactive when tested in vitro for the acrosome reaction using homologous sperm. The amount of egg jellies spawned by females (and their constituent sulfated polysaccharides) varied greatly throughout the year. Apparently, there is a correlation between the temperature of the sea water and the expression of the 4-sulfated, 3-linked sulfated fucan. Overall, we described the occurrence of two isotypes of sulfated fucan in the egg jelly of the sea urchin L. variegatus, which differ in their biological activity and may be involved in the periodicity of the reproductive cycle of the invertebrate.     

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.     

Inhibition of thrombin by sulfated polysaccharides isolated from green algae

Eight different sulfated polysaccharides were isolated from Chlorophyta. All exhibited thrombin inhibition through a heparin cofactor II (HCII)-dependent pathway, and their effects on the inhibition of thrombin were more potent than those of heparin or dermatan sulfate. In particular, remarkably potent thrombin inhibition was found for the sulfated polysaccharides isolated from the Codiales. In the presence of these sulfated polysaccharides, both the recombinant HCII (rHCII) variants Lys(173)-->Leu and Arg(189)-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHCII. This result indicates that the binding site of HCII for each of these eight sulfated polysaccharides is different from the heparin- or dermatan sulfate-binding site. All the sulfated polysaccharides but RS-2 significantly stimulated the inhibition of thrombin by an N-terminal deletion mutant of HCII (rHCII-Delta74). Furthermore, hirudin(54-65) decreased only 2-5-fold the rate of thrombin inhibition by HCII stimulated by the sulfated polysaccharides, while HD22, a single-stranded DNA aptamer that binds exosite II of thrombin, produced an approximately 10-fold reduction in this rate. These results suggest that, unlike heparin and dermatan sulfate, the sulfated polysaccharides isolated from Chlorophyta activate HCII primarily by an allosteric mechanism different from displacement and template mechanisms.     

Sulfated polysaccharides from marine sponges: conspicuous distribution among different cell types and involvement on formation of in vitro cell aggregates

Marine sponges (Porifera) display an ancestral type of cell-cell adhesion, based on carbohydrate-carbohydrate interaction. The aim of the present work was to investigate further details of this adhesion by using, as a model, the in vitro aggregation of dissociated sponge cells. Our results showed the participation of sulfated polysaccharides in this cell-cell interaction, as based on the following observations: (1) a variety of sponge cells contained similar sulfated polysaccharides as surface-associated molecules and as intracellular inclusions; (2) ³⁵S-sulfate metabolic labeling of dissociated sponge cells revealed that the majority (two thirds) of the total sulfated polysaccharide occurred as a cell-surface-associated molecule; (3) the aggregation process of dissociated sponge cells demanded the active de novo synthesis of sulfated polysaccharides, which ceased as cell aggregation reached a plateau; (4) the typical well-organized aggregates of sponge cells, known as primmorphs, contained three cell types showing sulfated polysaccharides on their cell surface; (5) collagen fibrils were also produced by the primmorphs in order to fill the extracellular spaces of their inner portion and the external layer surrounding their entire surface. Our data have thus clarified the relevance of sulfated polysaccharides in this system of in vitro sponge cell aggregation. The molecular basis of this system has practical relevance, since the culture of sponge cells is necessary for the production of molecules with biotechnological applications.     

Evaluating the possible anticoagulant and antioxidant effects of sulfated polysaccharides from the tropical green alga Caulerpa cupressoides var. flabellata

Seaweeds are a source of several biopolymers widely used in cosmetics, food, and pharmaceuticals. Among them are sulfated polysaccharides, which have several biological/pharmacological activities, such as antioxidant and anticoagulant activities. In the present study, four sulfated polysaccharides, denominated CCB-F0.3, CCB-F0.5, CCB-F1.0, and CCB-F2.0, were obtained from the chlorophyte Caulerpa cupressoides var. flabellata through proteolytic digestion, followed by acetone fractionation and molecular sieving in Sephadex G-100. Chemical analyses showed that CCB-F0.5 had the highest sulfate/sugar ratio (0.73), whereas CCB-F1.0 exhibited the lowest ratio (0.23). Polysaccharides from C. cupressoides displayed a heterogeneous constitution of monosaccharides, with galactose as the main sugar unit (except for CCB-F2.0). The presence of sulfated polysaccharides was confirmed by electrophoretic and infrared analyses. Sulfated polysaccharides showed no activity in superoxide and hydroxyl radical scavenging; however, they did demonstrate total antioxidant capacity and ferrous chelating activity. Caulerpa polysaccharides also exhibited anticoagulant activity in the intrinsic (activated partial thromboplastin time (aPTT) test) and extrinsic pathway (prothrombin time (PT) test). In the aPTT test, all polysaccharides displayed considerable dose-dependent activity. A significant result was the aPTT activity of the polysaccharides CCB-F0.3 and CCB-F0.5, which was similar to that of Clexane?, a commercial low molecular weight heparin. In addition, CCB-F0.3 and CCB-F0.5 showed PT activity. Sulfated polysaccharides from C. cupressoides are therefore promising antioxidant agents in preventing the formation of reactive oxygen species and for their possible use in anticoagulant therapy.     

Ultrastructure of acidic polysaccharides from the cell walls of brown algae

We have studied the ultrastructure of acidic polysaccharides from the cell walls of brown algae using a variety of electron microscopy techniques. Polysaccharides from Padina gymnospora present self assembled structures, forming trabecular patterns. Purified fractions constituted by alginic acid and sulfated fucan also form well-organized ultrastructures, but the pattern of organization varies depending on the polysaccharide species. Alginic acid presents sponge-like structures. Sulfated fucan exhibits particles with polygonal forms with a polycrystalline structure. These particles are in fact constituted by sulfated fucan molecules since they are recognized by a lectin specific for alpha-l-fucosyl residues. X-ray microanalysis reveal that S is a constituent element, as expected for sulfated groups. Finally, an exhaustive purified sulfated fucan shows the same ultrastructure formed by polygonal forms. Furthermore, elemental analyses of acidic polysaccharides indicate that they retain Zn, when algae were collected from a contaminated area. This observation is supported by direct quantification of heavy metal in the biomass and also in the solubilized polysaccharides compared with the algae from a non-contaminated site. We conclude that these molecules have specific ultrastructure and elemental composition; and act as metal binder for the nucleation and precipitation of heavy metals when the algae are exposed to a metal contaminated environment.     

Effect of Sulfated Modification on the Molecular Characteristics and Biological Activities of Polysaccharides from Hypsizigus marmoreus

The effect of sulfated modification on polysaccharides from Hypsizigus marmoreus was examined by determining their molecular structures and bioactivities. The sulfation, which was implemented by using an orthogonal array design, produced polysaccharides with varying degrees of substitution (DS) ranging from 0.11 to 1.06. The sulfated polysaccharides exhibited a lower average molecular weight (M _w) and considerably higher radius of gyration (R _g) than those of native polysaccharide, suggesting that the conformation of the sulfated polysaccharides had been changed towards a more extended type. The inhibitory activity toward cancer cell growth was enhanced by treating with the sulfated polysaccharides by up to 34%, as compared to the native polysaccharide. In addition, treating with the sulfated polysaccharides increased the nitric oxide (NO) and cytokine (IL-1β and TNF-α) release to levels comparable to those detected in the positive control, lipopolysaccharide (LPS), suggesting that the sulfated polysaccharides might have strong immunomodulatory activity.     

Isolation of Polysaccharides Sulfated during Early Embryogenesis in Fucus

Beginning 10 hours after fertilization, zygotes of Fucus distichus L. Powell incorporate (35)S into polysaccharides as a sulfate ester of fucose. These sulfated polysaccharides are sequestered in only the rhizoid cell of the two-celled embryo and can serve as a marker of cellular differentiation. Zygotes were pulsed at different times after fertilization with Na(2) (35)SO(4) to identify and isolate the fucans localized within the region of cytoplasm destined to become the rhizoid cell. Low molecular weight pools of (35)S were saturated within 60 minutes, with the greatest incorporation into ethanol-soluble and insoluble fractions occurring with 0.1 mm Na(2)SO(4) in the artificial sea water medium. At the time of rhizoid formation, four fucose-containing polysaccharide fractions incorporated (35)S. When each fraction was subjected to diethylaminoethyl chromatography, two components were eluted with KCl that contained over 84% of the fucose and 93% of the (35)S of the particular fraction. Highvoltage paper electrophoresis of each fraction also resulted in the separation of these two major components. Both components from each of the four fractions behaved identically when separated by diethylaminoethyl chromatography and paper electrophoresis. By comparing the incorporation of (35)S into the polysaccharide fractions at 4 and 16 hours after fertilization, the fucan-sulfate components that are localized in the cytoplasm at the time of rhizoid formation were isolated. Although sulfated polysaccharides in brown algae are reported to be very heterogeneous in terms of their sugar composition and complexes with other heteropolymers, we propose that there are two major components that are sulfated during early embryogenesis in Fucus. The location of these two sulfated polysaccharides in different chemical fractions may reflect their subcellular localization (e.g., cytoplasmic vesicles or cell walls), or their association with other heteropolymers.     

L-六碳糖形成机制的研究进展

糖及其衍生物在许多初级或次级代谢过程中发挥着重要作用。糖结构与功能的多样性和糖在疾病诊断与治疗中的重要性推动了糖生物学的快速发展。D型糖,尤其是D-六碳糖在糖中占据着主导地位,L-六碳糖也是许多重要糖蛋白复合物、多糖及抗生素的组成成分。了解L-六碳糖的形成机制有助于理性改造糖的结构并开发其应用价值。L-六碳糖通常由3,5位差向异构酶或5位差向异构酶催化D-六碳糖的C5位异构化形成,这种转变赋予了糖在构型上的多样性,并在许多天然产物中起决定生物活性的作用。对3,5位差向异构酶和5位差向异构酶的功能及晶体结构的研究揭示了L-六碳糖的形成机制。本文综述了L-六碳糖形成过程中不同类型的3,5-位差向异构酶和5-位差向异构酶的催化机制,揭示L-六碳糖在生理和医药领域的重要意义。     

Rising from the sea: correlations between sulfated polysaccharides and salinity in plants

High salinity soils inhibit crop production worldwide and represent a serious agricultural problem. To meet our ever-increasing demand for food, it is essential to understand and engineer salt-resistant crops. In this study, we evaluated the occurrence and function of sulfated polysaccharides in plants. Although ubiquitously present in marine algae, the presence of sulfated polysaccharides among the species tested was restricted to halophytes, suggesting a possible correlation with salt stress or resistance. To test this hypothesis, sulfated polysaccharides from plants artificially and naturally exposed to different salinities were analyzed. Our results revealed that the sulfated polysaccharide concentration, as well as the degree to which these compounds were sulfated in halophytic species, were positively correlated with salinity. We found that sulfated polysaccharides produced by Ruppia maritima Loisel disappeared when the plant was cultivated in the absence of salt. However, subjecting the glycophyte Oryza sativa Linnaeus to salt stress did not induce the biosynthesis of sulfated polysaccharides but increased the concentration of the carboxylated polysaccharides; this finding suggests that negatively charged cell wall polysaccharides might play a role in coping with salt stress. These data suggest that the presence of sulfated polysaccharides in plants is an adaptation to high salt environments, which may have been conserved during plant evolution from marine green algae. Our results address a practical biological concept; additionally, we suggest future strategies that may be beneficial when engineering salt-resistant crops.     

A 2-sulfated, 3-linked alpha-L-galactan is an anticoagulant polysaccharide

Marine alga is an abundant source of sulfated polysaccharides with potent anticoagulant activity. However, several attempts to identify the specific structural features in these compounds, which confer the biological activity, failed due to their complex, heterogeneous structure. We isolated and characterized several sulfated alpha-L-galactans and sulfated alpha-L-fucans from marine invertebrates. In contrast to the algal fucans and galactans, these invertebrate polysaccharides have a simple structure, composed of well-defined units of oligosaccharides. We employed two of these compounds to elucidate their structure-anticoagulant action relationship. Our results indicate that a 2-sulfated, 3-linked alpha-L-galactan, but not an alpha-L-fucan, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaces thrombin. Thus, the anticoagulant activity of sulfated galactan and sulfated fucan is not merely a consequence of their charge density. The interaction of these polysaccharides with coagulation cofactors and their target proteases are specific. Identification of specific structural requirements in sulfated galactans and sulfated fucans necessary for interaction with coagulation cofactors is an essential step for a more rational approach to develop new anticoagulant and antithrombotic drugs.     

Functions of Chondroitin Sulfate and Heparan Sulfate in the Developing Brain

Chondroitin sulfate and heparan sulfate proteoglycans are major components of the cell surface and extracellular matrix in the brain. Both chondroitin sulfate and heparan sulfate are unbranched highly sulfated polysaccharides composed of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine, and glucuronic acid and N-acetylglucosamine, respectively. During their biosynthesis in the Golgi apparatus, these glycosaminoglycans are highly modified by sulfation and C5 epimerization of glucuronic acid, leading to diverse heterogeneity in structure. Their structures are strictly regulated in a cell type-specific manner during development partly by the expression control of various glycosaminoglycan-modifying enzymes. It has been considered that specific combinations of glycosaminoglycan-modifying enzymes generate specific functional microdomains in the glycosaminoglycan chains, which bind selectively with various growth factors, morphogens, axon guidance molecules and extracellular matrix proteins. Recent studies have begun to reveal that the molecular interactions mediated by such glycosaminoglycan microdomains play critical roles in the various signaling pathways essential for the development of the brain.     

Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchyme cell activity in the sea urchin embryo

The relationship between ³⁵SO₄ incorporation into acid mucopolysaccharides and the appearance and activity of the primary mesenchyme cells has been studied in the sea urchin, Lytechinus pictus. The ratio of the uptake of ³⁵SO₄ to its incorporation into cetylpyridinium chloride precipitable material varies over a wide range during early development, with the smallest ratio, therefore the greatest sulfation activity, being found at the early mesenchyme blastula stage. The types of mucopolysaccharides produced have not been identified, but are heterogeneous. At the mesenchyme blastula stage nearly 90% of the polysaccharides produced become sulfated. When embryos develop in sulfate-free sea water to the mesenchyme blastula stage there is a 70% decrease in the incorporation of ³H-acetate into polysaccharides and a 13-fold decrease in the ratio of sulfated to nonsulfated polysaccharides produced. Embryos raised in sulfate-free sea water develop normally to the mesenchyme blastula stage at which time there is an accumulation in the blastocoel of primary mesenchyme cells that do not migrate. The surface of the primary mesenchyme cells of sulfate-deficient embryos has a smooth appearance in the scanning electron microscope, while the surface of these cells in control embryos is rough, possibly reflecting the presence of an extracellular coat. It is suggested that there is a correlation between sulfated polysaccharide synthesis, cell surface morphology and cell movement.     

Biosynthesis of heparin. O-sulfation of D-glucuronic acid units

Incubation of a microsomal fraction from murine mastocytoma, with UDP-[1-3H]GlcA, UDP-GlcNAc, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), yielded labeled, N-sulfated polysaccharides, in which most of the incorporated O-sulfate groups were located at C2 of L-iduronic acid and at C6 of D-glucosamine units. Analysis by anion-exchange high pressure liquid chromatography of disaccharides, generated by deaminative cleavage of these polysaccharides, revealed that, in addition, an appreciable portion of the -GlcNSO3-HexA-GlcNSO3- sequences in the intact polymers contained O-sulfated (at C2 or C3) D-glucuronic acid units. Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE-cellulose, showed that glucuronosyl 2/3-O-sulfate accounted for approximately 12% of the total incorporated O-sulfate groups. With [35S]PAPS (at a low total PAPS concentration) as an alternative source of label, the sulfated glucuronic acid residues were again detectable, albeit in much smaller amounts (1.8% of the total O-sulfate groups). Incorporation of label from UDP-[5-3H]GlcA was retained by the O-sulfated glucuronic acid units, thus demonstrating that these components had in fact been formed by sulfation of glucuronic acid residues and not by "back epimerization" of sulfated iduronic acid units. Structural analysis of polysaccharide intermediates at various stages of biosynthetic polymer modification, separated by ion-exchange chromatography, showed O-sulfation of glucuronic and iduronic acid units to appear simultaneously and before the 6-O-sulfation of glucosamine residues.