The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.     

Anticoagulant sulfated glycosaminoglycans in the tissues of the primitive chordate Styela plicata (Tunicata)

We performed a biochemical and histochemical study of sulfated glycosaminoglycans in the tissues of the ascidian Styela plicata. A highly sulfated dermatan sulfate and a heparin-like polymer, identified by incubation with specific lyases, occur at different concentrations in intestine, heart, pharynx, and cloak. Dermatan sulfate prevails in the pharynx, whereas the heparin-like polymer abounds in the intestine. Staining of tissues sections with the cationic dye 1,9-dimethylmethylene blue before and after incubation with specific lyases revealed that the dermatan sulfate occurs in the extracellular matrix, while the heparin-like polymer is located within cytoplasmic granules of cells in the lumen of intestine and pharynx. The dermatan sulfate has a similar disaccharide composition in all tissues studied, whereas the heparin-like polymer differs in sulfate content. A direct relationship between sulfate content of the heparin-like polymer and antithrombin activity was observed. Analysis of the repeating disaccharide units of the heparin-like polymer indicates the presence of relatively high amounts of the disulfated disaccharide namely DeltaUA-1-->4-GlcN(SO(4))-(6SO(4)), which may suggest the occurrence in ascidians of regulatory biosynthetic mechanisms different from those observed for heparin in mammals.     

Controlled sulfatation of natural anionic bacterial polysaccharides can yield agents with specific regenerating activity in vivo

The regenerating activities of chemically modified anionic bacterial polysaccharides by O-sulfonation were investigated using a in vivo model of rat injured muscle regeneration. Glucuronan (GA), a linear homopolysaccharide of -->4)-beta-D-GlcpA-(1--> residues partially acetylated at the C-3 and/or the C-2 position, and glucoglucuronan (GGA), a linear heteropolysaccharide of -->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1--> residues were sulfated. SO3-DMF sulfatation complex provided polysaccharides with different sulfur contents, however, a depolymerization occurred because we did not use large excess of pyridine to obtain pure modified polysaccharides. A regenerating activity on injured extensor digitorum longus (EDL) muscles on rats was obtained with these two sulfated anionic polymers. The position of sulfate groups on glucoglucuronan (primary or secondary alcohol) seems to have no influence on the biological activity by opposition to the degree of sulfatation both for the glucuronans and the glucoglucuronans. The yield of acetate groups in the glucuronan polymer modulated the specific activity.     

Sulfated fucans from the egg jellies of the closely related sea urchins Strongylocentrotus droebachiensis and Strongylocentrotus pallidus ensure species-specific fertilization.

Sulfated polysaccharides from egg jelly are the molecules responsible for inducing the sperm acrosome reaction in sea urchins. This is an obligatory event for sperm binding to, and fusion with, the egg. The sulfated polysaccharides from sea urchins have simple, well defined repeating structures, and each species represents a particular pattern of sulfate substitution. Here, we examined the egg jellies of the sea urchin sibling species Strongylocentrotus droebachiensis and Strongylocentrotus pallidus. Surprisingly, females of S. droebachiensis possess eggs containing one of two possible sulfated fucans, which differ in the extent of their 2-O-sulfation. Sulfated fucan I is mostly composed of a regular sequence of four residues ([4-alpha-l-Fucp-2(OSO3)-1-->4-alpha-l-Fucp-2(OSO3)-1-->4-alpha-l-Fucp-1-->4-alpha-l-Fucp-1]n), whereas sulfated fucan II is a homopolymer of 4-alpha-l-Fucp-2(OSO3)-1 units. Females of S. pallidus contain a single sulfated fucan with the following repeating structure: [3-alpha-l-Fucp-2(OSO3)-1-->3-alpha-l-Fucp-2(OSO3)-1-->3-alpha-l-Fucp-4(OSO3)-1-->3-alpha-l-Fucp-4(OSO3)-1]n. The egg jellies of these two species of sea urchins induce the acrosome reaction in homologous (but not heterologous) sperm. Therefore, the fine structure of the sulfated alpha-fucans from the egg jellies of S. pallidus and S. droebachiensis, which differ in their sulfation patterns and in the position of their glycosidic linkages, ensures species specificity of the sperm acrosome reaction and prevents interspecies crosses. In addition, our observations allow a clear appreciation of the common structural features among the sulfated polysaccharides from sea urchin egg jelly and help to identify structures that confer finer species specificity of recognition in the acrosome reaction.     

Effect of (1-->3)- and (1-->4)-linkages of fully sulfated polysaccharides on their anticoagulant activity

Chemically fully sulfated polysaccharides including xylan (-->4Xylbeta-(1-->4)Xylbeta1-->), amylose (-->4Glcalpha-(1-->4)Glcalpha1-->), cellulose (-->4Glcbeta-(1-->4)Glcbeta1-->), curdlan (-->3Glcbeta-(1-->3)Glcbeta1-->) and galactan (-->3Galbeta-(1-->3)Galbeta1-->), which have been isolated from Korean clam, were prepared, and their anticoagulant activity was investigated. The results strongly suggest that the activity might not be depending on anomeric configuration (alpha or beta) or monosaccharide species but on the glycosidic linkage, either (1-->3) or (1-->4). 1H NMR studies of these modified polysaccharides show that the neighboring sulfate groups at the C-2 and C-3 positions might have caused the conformational changes of each monosaccharide from 4C(1) to 1C(4). Furthermore, the effect of 6-sulfate residues on the anticoagulant activity was investigated using a specific desulfated reaction for the chemically fully sulfated polysaccharides. The 6-sulfate group is very important in determining anticoagulant activity of (1-->3)-linked polysaccharides, whereas the activity is not affected by presence or absence of the 6-sulfate group in (1-->4)-linked polysaccharides.     

Chemical synthesis of sulfated oligosaccharides with a beta-D-Gal-(1-->3)

The syntheses of two sulfated pentasaccharides: beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc-(1-->6)]-alpha-D-GalNAc-->OMe (1) and beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc6SO3Na-(1-->6)]-alpha-D-GalNAc-->OMe (2) by using Lewisx trisaccharides 12 and 16 as glycosyl donors are described. Sulfated oligosaccharides 1-2 and intermediate compounds are fully characterized by 2D 1H-1H DQF-COSY and 2D ROESY experiments.     

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.     

Sulfated modification of the polysaccharides obtained from defatted rice bran and their antitumor activities

Nine sulfated defatted rice bran polysaccharides (sRBPS), with various degrees of sulfation (DS) and carbohydrate content, were prepared by chlorosulfonic acid–pyridine (CSA–Pyr) method according to orthogonal test. Nine sulfated derivatives sRBPS were obtained and their antitumor activities were compared by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that when DS within the scope of 0.81–1.29, carbohydrate content in the range of 41.41–78.56%, sulfated derivatives exhibit relatively strong antitumor activity in vitro. The optimum modification conditions were reaction temperature of 70 °C, the ratio of chlorosulfonic acid to pyridine of 1:4 and the reaction time of 2 h.     

Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos

Six polysaccharides were extracted sequentially from the fresh sclerotium of Poria cocos cultivated in China using 0.9% NaCl (PCS1), hot water (PCS2), 0.5M NaOH (PCS3-I and PCS3-II), and 88% formic acid (PCS4-I and PCS4-II). Their chemical and physical characteristics were determined using infrared spectroscopy (IR), gas chromatography (GC), GC-MS methylation analysis, 13C NMR spectroscopy, elementary analysis (EA), protein analysis, size exclusion chromatography combined with laser light scattering (SEC-LLS), light scattering (LS), and viscometry. The results indicated that the polysaccharides PCS1, PCS2, and PCS3-I were heteropolysaccharides containing D-glucose, D-galactose, D-mannose, D-fucose, and D-xylose; the predominant monosaccharide was D-glucose except for PCS1 where it was D-galactose. PCS3-II, the main component of the sclerotium of P. cocos, was a linear (1-->3)-beta-D-glucan of high purity. PCS4-I consisted of (1-->3)-beta-D-glucan with some beta-(1-->6) linked branches. PCS4-II was mainly composed of (1-->3)-beta-D-glucan containing some glucose branches. The M(w) values of the six polysaccharides PCS1, PCS2, PCS3-I, PCS4-I in 0.2M NaCl aqueous solution, PCS3-II, and PCS4-II in dimethyl sulfoxide (Me(2)SO) were determined to be 11.6 x 10(4), 20.8 x 10(4), 17.1 x 10(4), 9.1 x 10(4), 12.3 x 10(4), and 21.1 x 10(4), respectively. The six polysaccharides in aqueous solution or Me(2)SO exist as flexible chains.     

Biochemical characterization of inner sugar chains of acrosome reaction-inducing substance in jelly coat of starfish eggs

The inception of the acrosome reaction (AR) in the starfish Asterias amurensis is perceived to be strongly associated with sulfated polysaccharide chains derived from an extremely large proteoglycan-like molecule called AR-inducing substance (ARIS), in which one of the sugar fragments, named fragment 1 (Fr. 1), was composed of the repeating units of [-->4]-beta-D-Xylp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Fucp-4 (SO3-)-(1-->3)- alpha-L-Fucp-4(SO3-)-(1-->4)-alpha-L-Fucp-(1-->)n. In the current study, this sugar chain is inferred to link to the peptide part by O-glycosidic linkage through a sugar chain with different structural features from Fr. 1. This inner sugar portion of ARIS was isolated as Fr. 2 from the sonicated products of pronase digest of ARIS. Fr. 2, which retains AR-inducing activity to an admirable extent and has an apparent molecular size of 400 kDa, is composed of Gal, Xyl, Fuc, GalNAc, and GlcNAc in a molar ratio of 5:1:5:4:2 with O-sulfate substitutions at Gal-4, Gal-2, Gal-2,3 and Gal-2,4 (disulfated), Fuc-4, and GlcNAc-6. The study of Fr. 2 revealed that the major portion of the inner sugar chain of ARIS is composed of the heptasaccharide units of -->3)-Galp-(1-->3)-Fucp-(1-->3)-Galp-(1-->4)-GalNAcp-(1-->4)-GlcNAcp-6(SO3-)-(1-->6)-Galp-4(SO3-)-(1-->4)-GalNAcp-(1-->. This new structure of inner sugar chains of ARIS is elucidated by using electrospray ionization MS along with tandem mass analysis, sugar composition analysis, and methylation analysis of the sugar fragments obtained by acid-catalyzed resin-based partial hydrolysis of Fr. 2. Furthermore, this study corroborates that the sulfate groups are solely liable to the anionic character of ARIS, which ought to be present in the sugar chains of ARIS for its biological activity.     

The structure of the core region of the lipopolysaccharide from Klebsiella pneumoniae O3. 3-deoxy-alpha-D-manno-octulosonic acid (alpha-Kdo) residue in the outer part of the core, a common structural element of Klebsiella pneumoniae O1, O2, O3, O4, O5, O8, and O12 lipopolysaccharides.

The structure of lipid A-core region of the lipopolysaccharide (LPS) from Klebsiella pneumoniae serotype O3 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of the LPS: [carbohydrate structure see text] where P is H or alpha-Hep; J is H or beta-GalA; R is H or P (in the deacylated oligosaccharides).Screening of the LPS from K. pneumoniae O1, O2, O4, O5, O8, and O12 using deamination showed that they also contain alpha-Hep-(1-->4)-alpha-Kdo-(2-->6)-GlcN and alpha-Kdo-(2-->6)-GlcN fragments.     

Glycosaminoglycans from fish swim bladder: isolation,structural characterization and bioactive potential

The swim bladder of fish is an internal gas-filled organ that allows fish to control their buoyancy and swimming depth. Fish maws (the dried swim bladders of fish) have been used over many centuries as traditional medicines, tonics and a luxurious gourmet food in China and Southeast Asia. Little is known about the structural information of polysaccharides comprising this important functional material of fish tissue. In the present study, the total glycosaminoglycan (GAG) from fish maw was characterized. Two GAGs were identified, chondroitin sulfate (CS, having a molecular weight of 18–40 kDa) and heparan sulfate (HS), corresponding to 95% and 5% of the total GAG, respectively. Chondroitinase digestion showed that the major CS GAG was composed of ΔUA-1 → 3-GalNAc4S (59.7%), ΔUA-1 → 3-GalNAc4,6S (36.5%), ΔUA-1 → 3-GalNAc6S (2.2%) and ΔUA-1 → 3-GalNAc (1.6%) disaccharide units. ¹H–NMR analysis and degradation with specific chondroitinases, both CS-type A/C and CS-type B were present in a ratio of 1.4:1. Analysis using surface plasmon resonance showed that fibroblast growth factor (FGF)-2 bound to the CS fraction (K_D = 136 nM). These results suggest that this CS may be involved in FGF-signal pathway, mediating tissue repair, regeneration and wound healing. The CS, as the major GAG in fish maw, may have potential pharmacological activity in accelerating wound healing.     

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.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions.     

Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity

A linear sulfated fucan with a regular repeating sequence of [3)-alpha-L-Fucp-(2SO4)-(1-->3)-alpha-L-Fucp-(4SO4)-(1-->3)-alpha-L-Fucp-(2,4SO4)-(1-->3)-alpha-L-Fucp-(2SO4)-(1-->]n is an anticoagulant polysaccharide mainly due to thrombin inhibition mediated by heparin cofactor II. No specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid. Surprisingly, the linear sulfated fucan was cleaved by mild acid hydrolysis on an ordered sequence. Initially a 2-sulfate ester of the first fucose unit is selectively removed. Thereafter the glycosidic linkage between the nonsulfated fucose residue and the subsequent 4-sulfated residue is preferentially cleaved by acid hydrolysis, forming oligosaccharides with well-defined size. The low-molecular-weight derivatives obtained from the sulfated fucan were employed to determine the requirement for interaction of this polysaccharide with heparin cofactor II and to achieve complete thrombin inhibition. The linear sulfated fucan requires significantly longer chains than mammalian glycosaminoglycans to achieve anticoagulant activity. A slight decrease in the molecular size of the sulfated fucan dramatically reduces its effect on thrombin inactivation mediated by heparin cofactor II. Sulfated fucan with approximately 45 tetrasaccharide repeating units binds to heparin cofactor II but is unable to link efficiently the plasma inhibitor and thrombin. This last effect requires chains with approximately 100 or more tetrasaccharide repeating units. We speculate that the template mechanism may predominate over the allosteric effect in the case of the linear sulfated fucan inactivation of thrombin in the presence of heparin cofactor II.     

Embryos of the sea urchin Strongylocentrotus purpuratus synthesize a dermatan sulfate enriched in 4-O- and 6-O-disulfated galactosamine units.

Unfertilized eggs of the sea urchin Strongylocentrotus purpuratus are surrounded by a gelatinous layer rich in sulfated fucan. Shortly after fertilization this polysaccharide disappears, but 24 h later the embryos synthesize high amounts of dermatan sulfate concomitantly with the mesenchyme blastula-early gastrula stage when the larval gut is forming. This glycosaminoglycan has the same backbone structure [4-alpha-L-IdoA-1-->3-beta-D-GalNAc-1](n) as the mammalian counterpart but possesses a different sulfation pattern. It has a high content of 4-O- and 6-O-disulfated galactosamine units. In addition, chains of this dermatan sulfate are considerable longer than those of vertebrate tissues. Adult sea urchin tissues contain high concentrations of sulfated polysaccharides, but dermatan sulfate is restricted to the adult body wall where it accounts for approximately 20% of the total sulfated polysaccharides. In addition, sulfation at the 4-O-position decreases markedly in the dermatan sulfate from adult sea urchin when compared with the glycan from larvae. Overall, these results demonstrate the occurrence of dermatan sulfates with unique sulfation patterns in this marine invertebrate. The physiological implication of these oversulfated dermatan sulfates is unclear. One hypothesis is that interactions between components of the extracellular matrix in marine invertebrates occur at higher salt concentrations than in vertebrates and therefore require glycosaminoglycans with increased charge density.     

Enzymatic synthesis of chondroitin 4-sulfate with well-defined structure

Synthesis of chondroitin sulfate (ChS) with well-defined structure was achieved for the first time by hyaluronidase-catalyzed polymerization. N-Acetylchondrosine (GlcAbeta(1-->3)GalNAc) oxazoline derivatives sulfated at C4 (1a), C6 (1b), and both C4 and C6 (1c) in the GalNAc unit were synthesized as transition state analogue substrate monomers for hyaluronidase (HAase) catalysis. Compound 1a was effectively polymerized by the enzyme, giving rise to synthetic ChS sulfated perfectly at the C4 position in all N-acetylgalactosamine units (Ch4S, 2a) in good yields. Molecular weights (Mn) of 2a ranged from 4000 to 18,400, which were controlled by varying reaction conditions. Compounds 1b and 1c were not catalyzed by the enzyme, affording the corresponding disaccharides through the oxazoline ring-opening without formation of polysaccharides.     

Generation of anti-factor Xa active, 3-O-sulfated glucosamine-rich sequences by controlled desulfation of oversulfated heparins.

In the framework of a project aimed at generating heparin-like sulfation patterns and biological activities in biotechnological glycosaminoglycans, different approaches have been considered for simulating the alpha(1-->4)-linked 2-O-sulfated L-iduronic acid (IdoA2SO(3))-->N,6-O-sulfated D-glucosamine (GlcNSO(3)6SO(3)) disaccharide sequences prevalent in mammalian heparins. Since the direct approach of sulfating totally O-desulfated heparins, taken as model compounds for C-5-epimerized sulfaminoheparosan (N-deacetylated, N-sulfated Escherichia coli K5 polysaccharide), preferentially afforded heparins O-sulfated at C-3 instead than at C-2 of the iduronate residues, leading to products with low anticoagulant activities, the problem of re-generating a substantial proportion of the original IdoA2SO(3) residues was circumvented by performing controlled solvolytic desulfation (with 9:1 v/v DMSO-MeOH) of extensively sulfated heparins. The order of desulfation of major residues of heparin GlcN and IdoA and of the minor one D-glucuronic acid was: GlcNSO(3)>GlcN6SO(3)>IdoA3SO(3) congruent with GlcA2SO(3) congruent with GlcN3SO(3)>IdoA2SO(3) congruent with GlcA3SO(3). Starting from a 'supersulfated' low-molecular weight heparin, we obtained products with up to 40% of iduronate residues O-sulfated exclusively at C-2 and up to 40% of their glucosamine residues O-sulfated at both C-6 and C-3. Upon re-N-sulfation, these products displayed an in vitro antithrombotic activity (expressed as anti-factor Xa units) comparable with those of current low-molecular weight heparins.     

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.