Development-dependent modification of the extracellular matrix by a sulphated glycoprotein in Volvox carteri

We report the chemical characterization of the highly sulphated glycoprotein SSG 185 from Volvox carteri. SSG 185 is a hydroxyproline-containing, extracellular glycoprotein. The sulphate residues are clustered within the parent saccharide structure of SSG 185, since on mercaptolysis all the sulphate residues are recovered in a small saccharide fragment containing mannose, arabinose and sulphate (in a molar ratio of 2). SSG 185 is a short-lived molecule, serving as a precursor for a high mol. wt. component of the extracellular matrix. Synthesis of SSG 185 is developmentally controlled. Different SSG 185 variants, with unknown modifications in the sulphated saccharide fragment, are synthesized at different developmental stages or under the influence of the sexual inducer. These modifications remain conserved in the aggregated state of SSG 185, indicating the development-dependent modification of the extracellular matrix.     

Pherophorins: a family of extracellular matrix glycoproteins fromVolvox structurally related to the sex-inducing pheromone

Pherophorins are extracellular matrix (ECM) glycoproteins from Volvox that share homology with the sex-inducing pheromone. A novel pherophorin (pherophorin III) was characterized both with respect to expression pattern and proteolytic processing in vivo. Furthermore, it was shown that the pherophorins represent a protein family of ECM glycoproteins exhibiting a modular composition: their N-terminally located domain is a homolog of a domain found in the ECM glycoprotein SSG 185. Together with SSG 185, pherophorin I is a main component of the cellular zone within the ECM. The Volvox genome contains a tandem arrangement of genes encoding pherophorin II-related polypeptides. Inhibition of proteolytic processing of pherophorin II and III in vivo appears to result in the suppression of sexual induction.Abbreviations ECM extracellular matrix - PCR polymerase chain reaction - RACE rapid amplification of cDNA ends We wish to thank Dr. R. Deutzmann (Universität Regensburg, Germany) for sequencing peptides. This work was supported by the Deutsche Forschungsgemeinschaft (SFB43).     

Further studies on the composition and structure of a fucoidan preparation from the brown alga Saccharina latissima

The polysaccharide composition of a fucoidan preparation isolated from the brown alga Saccharina latissima (formerly Laminaria saccharina) was reinvestigated. The preparation was fractionated by anion-exchange chromatography, and the fractions obtained were analyzed by chemical methods combined with NMR spectroscopy. Several 2D procedures, including HSQC, HMQC-TOCSY, and HMQC-NOESY, were used to obtain reliable structural information from the complex spectra, and the signal assignments were additionally confirmed by comparison with the literature spectra of the related polysaccharides and synthetic oligosaccharides. In accordance with the previous data, the main polysaccharide component was shown to be a fucan sulfate containing a backbone of 3-linked α-l-fucopyranose residues sulfated at C-4 and/or at C-2 and branched at C-2 by single sulfated α-l-fucopyranose residues. In addition, three other types of sulfated polysaccharide molecules were detected in the total fucoidan preparation: (i) a fucogalactan having a backbone of 6-linked β-d-galactopyranose residues branched mainly at C-4 and containing both terminal galactose and fucose residues; (ii) a fucoglucuronomannan having a backbone of alternating 4-linked β-d-glucopyranosyluronic acid and 2-linked α-d-mannopyranose residues with α-l-fucopyranose residues as single branches at C-3 of α-d-Manp; and (iii) a fucoglucuronan having a backbone of 3-linked β-d-glucopyranosyluronic acid residues with α-l-fucopyranose residues as single branches at C-4. Hence, even a single algal species may contain, at least in minor amounts, several sulfated polysaccharides differing in molecular structure. Partial resolution of these polysaccharides has been accomplished, but unambiguous evidence on their presence as separate entities was not obtained.     

Chemical characteristic of an anticoagulant-active sulfated polysaccharide from Enteromorpha clathrata

A sulfated polysaccharide FEP from marine green alga Enteromorpha clathrata was extracted with hot water and further purified by ion-exchange and size-exclusion chromatography. Results of chemical and spectroscopic analyses showed that FEP was a high arabinose-containing sulfated polysaccharide with sulfate ester of 31.0%, and its average molecular weight was about 511kDa. The backbone of FEP was mainly composed of (1→4)-linked β-l-arabinopyranose residues with partially sulfate groups at the C-3 position. In vitro anticoagulant assay indicated that FEP effectively prolonged the activated partial thromboplastin time and thrombin time. The investigation demonstrated that FEP was a novel sulfated polysaccharide with different chemical characteristics from other sulfated polysaccharides from marine algae, and could be a potential source of anticoagulant.     

Chemical characteristic and anticoagulant activity of the sulfated polysaccharide isolated from Monostroma latissimum (Chlorophyta)

A polysaccharide was isolated from marine green algae Monostroma latissimum, and its chemical characteristic and anticoagulant activity were investigated. The results demonstrated that the polysaccharide was high rhamnose-containing sulfated polysaccharide, and was mainly composed of 1,2-linked l-rhamnose residues with sulfate groups substituted at positions C-3 and/or C-4. The sulfated polysaccharide exhibited high anticoagulant activities by assays of the activated partial thromboplastin time (APTT) and thrombin time (TT). The anticoagulant property of the sulfated polysaccharide was mainly attributed to powerful potentiation thrombin by heparin cofactor II.     

A preponderantly 4-sulfated, 3-linked galactan from the green alga Codium isthmocladum

The green algae of the genus Codium have recently been demonstrated to be an important source of sulfated galactans from the marine environment. Here, a sulfated galactan was isolated from the species Codium isthmocladum and its structure was studied by a combination of chemical analyses and NMR spectroscopy. Two fractions (SG 1, approximately 14 kDa, and SG 2, approximately 20 kDa) were derived from this highly polydisperse and heterogeneous polysaccharide. Both exhibited similar structures in (1)H 1D NMR spectra. The structural features of SG 2 and its desulfated derivative were analyzed by COSY, TOCSY, DEPT-HSQC, HSQC, and HMBC. This sulfated galactan is composed preponderantly of 4-sulfated, 3-linked beta-D-galactopyranosyl units. In minor amounts, it is sulfated and glycosylated at C-6. Pyruvate groups are also found, forming five-membered cyclic ketals as 3,4-O-(1'carboxy)-ethylidene-beta-D-galactose residues. A comparison of sulfated galactans from different marine taxonomic groups revealed similar backbones of 3-beta-D-Galp-1.     

Isolation and characterization of an antitumor active agar-type polysaccharide of Gracilaria dominguensis

Cold water extraction of the red alga Gracilaria dominguensis, followed by cetyltrimethylammonium bromide fractionation, gave a highly sulfated, agar-type polysaccharide which inhibited the transplantation of Ehrlich ascites carcinoma in mice. The structure of the polysaccharide has been investigated by methylation analysis, and 1H- and 13C-n.m.r. spectroscopy, and was shown to be mainly composed of alternating (1----3)-linked beta-D-galactopyranosyl 6-sulfate and (1----4)-linked 3,6-anhydro-alpha-L-galactopyranosyl residues.     

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.     

A phosphodiester bridge between two arabinose residues as a structural element of an extracellular glycoprotein of Volvox carteri

The sulphated glycoprotein SSG 185 is the monomeric precursor of a highly aggregated structural element in the extracellular matrix of the multicellular green alga Volvox carteri. A phosphodiester of arabinose was isolated from a saccharide fragment of SSG 185. The structure of this phosphodiester was investigated by methylation analysis, 13C-NMR, photometric methods and enzymatic assays and identified as D-Araiota-5-phospho-5-D-Araiota. The function of this phosphodiester bridge as a crosslink of different carbohydrate chains in SSG 185 is discussed.     

Effect of Enzyme Preparation from the Marine Mollusk Littorina kurila on Fucoidan from the Brown Alga Fucus distichus

A fucoidanase preparation from the marine mollusk Littorina kurila cleaved some glycosidic bonds in fucoidan from the brown alga Fucus distichus, but neither fucose nor lower oligosaccharides were produced. The main product isolated from the incubation mixture was a polysaccharide built up of disaccharide repeating units -->3)-alpha-L-Fucp-(2,4-di-SO3(-))-(1-->4)-alpha-L-Fucp-(2SO3(-))-(1-->, the structure coinciding with the idealized formula proposed for the initial substance. A polymer fraction with the same carbohydrate chain but sulfated only at positions 2 and nonstoichiometrically acetylated at positions 3 and 4 of fucose residues was isolated as a minor component. It is suggested that the native polysaccharide should contain small amounts of non-sulfated and non-acetylated fucose residues, and only their glycosidic bonds are cleaved by the enzyme. The enzymatic hydrolysis showed that irregular regions of the native polysaccharide containing acetylated and partially sulfated repeating units were assembled in blocks.     

STRUCTURE AND DISTRIBUTION OF GLUCOMANNAN AND SULFATED GLUCAN IN THE CELL WALLS OF THE RED ALGA KAPPAPHYCUS ALVAREZII (GIGARTINALES, RHODOPHYTA)

Two new polysaccharides were isolated from the cell walls of the carrageenan producing red seaweed Kappaphycus alvarezii (Doty) Doty. They were characterized by chemical analyses, enzymatic degradations, and nuclear magnetic resonance spectroscopy. One was a 4.0 M NaOH soluble β-(1,4)- d -glucomannan that mostly precipitated upon neutralization and dialysis. It was composed of about 82 residues, and 70% of its glucose and mannose were released by a commercial cellulase enzyme complex. The disaccharide β- d -Man (1→4) d -Glc was recovered from the hydrolysate during the first hours of degradation and confirmed the chemical structure of the polysaccharide. The other polysaccharide was extracted with 1.5 M NaOH and was identified as a sulfated glucan of degree of polymerization of about 180 1,4-linked β-glucose containing 10% 1,3-linkages. The sulfate was located on C-6 of 64% of the 4-linked glucose residues. A third alkali-soluble polysaccharide rich in galactose was also detected. The distribution of the glucomannan and galactose containing polysaccharides was inversely related to the algal cell size. Potential functions of these alkali-soluble polymers are discussed in the context of cell wall polysaccharide assembly.     

Effect of fully sulfated glycosaminoglycans on pulmonary artery smooth muscle cell proliferation.

Fully sulfated heparin and other glycosaminoglycans, namely heparan, chondroitin, and dermatan sulfates, and hyaluronan have been prepared by using sulfur trioxide under mild chemical conditions. All these derivatives were assayed for antiproliferative activity on cultured bovine pulmonary artery smooth muscle cells (BPASMCs). No appreciable difference was found between heparin and fully sulfated heparin. Chondroitin and dermatan sulfates actually stimulated BPASMCs growth but full sulfonation made them strongly antiproliferative. Native hyaluronan was not antiproliferative but became strongly so after sulfonation. Neither acharan sulfate nor N-sulfoacharan sulfate had any antiproliferative activity. This suggests that O-sulfonation of the polysaccharide is critical for antiproliferative activity, whereas N-sulfonation of glucosamine residues is not.     

The structure of the glycerophosphate-containing O-specific polysaccharide from Escherichia coli 0130

A phosphorylated O-specific polysaccharide was obtained by mild acidic degradation of the lipopolysaccharide from the intestinal bacterium Escherichia coli 0130 and characterized by the methods of chemical analysis, including dephosphorylation, and 1H and 13C NMR spectroscopy. The polysaccharide was shown to be composed of branched tetrasaccharide repeating units containing two N-acetyl-D-galactosamine residues, D-galactose, D-glucose, and glycerophosphate residues (one of each). The polysaccharide has the following structure, which is unique among the known bacterial polysaccharides.     

The molecular characterization of the polysaccharide gum from Laguncularia racemosa

A polysaccharide isolated from the exudate of Laguncularia racemosa, (Combreta-ceae) has been investigated using Smith-degradation, methylation analysis, hydrolysis, and ¹³C-NMR spectroscopy. The backbone of the structure is constituted of uronic acids, galactose and rhamnose. A complex pentasaccharide, constituted of these sugars, was isolated from the original gum and degradation products. This oligosaccharide is, probably, the main structural feature of the investigated polysaccharide. On the other hand, according to chemical and spectral evidence rhamnose is present, predominantly as internal residues. Arabinosyl (pyranosyl and furanosyl) residues and some galactosyl, glucuronic acid and 4-0-methyl--D-glucuronic acid residues are located in branches.     

Acetylated methylmannose polysaccharide of Streptomyces.

A polysaccharide composed of 3-O-methyl-D-mannose and D-mannose in a molar ratio of approximately 10:1 and containing 3 to 4 esterified acetyl residues has been isolated from Streptomyces griseus. This acetylated methylmannose polysaccharide (AMMP) is similar to the methylmannose polysaccharide (MMP) of Mycobacterium smegmatis (Gray, G. R., and Ballou, C. E. (1971) J. Biol. Chem. 246, 6835-6842) in its size and composition, the absence of acidic or basic groups, and the lack of a reducing end. It is different, however, in its content of esterified acetyl residues, and it is slightly different in its structure and in its gel filtration properties. The structure of AMMP has been established by proton magnetic resonance spectroscopy, and by combinations of methylation analysis and Smith degradation utilizing non-radioactively labeled polysaccharide and [3H]methyl-labeled polysaccharide obtained from cells grown in the presence of L-[methyl-3H]methionine. It is concluded that AMMP is a linear, nonreducing, neutral polysaccharide composed of a terminal D-mannose residue linked alpha(1 leads to 4) to a chain of 10 consecutive alpha(1 leads to 4)-linked 3-O-methyl-D-mannose residues. The reducing terminal 3-O-methyl-D-mannose residue exists, at least in part, as its alpha-methyl glycoside. The positions of attachment of the ester residues have not been established.     

Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent?

The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked beta-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked alpha-fucose and one or two nonsulfated, 4-linked beta-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.     

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.     

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.     

PRESENCE OF A SULFATED POLYSACCHARIDE IN THE EXTRACELLULAR MATRIX OF PLATYDORINA CAUDATA (VOLVOCALES,CHLOROPHYTA)1

Evidence for the presence of a sulfated polysaccharide component within the extracellular matrix of Platydorina caudata Kofoid is presented. In situ staining with alcian blue and toluidine blue O indicates accumulation of a sulfated polysaccharide in the matrix. The entire matrix was readily solubilized by a hot aqueous extraction and a sulfated proteoglycan complex was isolated. Thin-layer chromatography of hydrolysates and infrared analysis and chemical desulfation of the intact molecule indicate that the polysaccharide component is principally an arabinogalactan with ester-linked sulfate groups. Protease treatment of the extract revealed two distinct bands separable on cellulose acetate electrophoresis. The slower moving component was a sulfated glycoprotein while the faster moving component was a sulfated mucopolysaccharide essentially free of protein. This is the first report of specific chemical analyses and electrophoretic separation of a sulfated polysaccharide within the matrix of a member of the Volvocales. The cytochemistry and electrophoretic patterns of the P. caudata preparation are compared with the same type of extract made from Chlamydomonas reinhardtii Dang. The possible evolutionary significance of the electrophoretic patterns is presented.     

Self-assembly and cross-linking of Volvox extracellular matrix glycoproteins are specifically inhibited by Ellman's reagent.

A major impediment to the biochemical characterization of extracellular matrices from algae (as well as higher plants) is the extensive covalent cross-linking that exists in the matrix, rendering most components insoluble and resistant to conventional extraction procedures. In the multicellular green alga Volvox, biogenesis of the extracellular matrix (ECM) is initiated immediately after the process of embryonic inversion. At this stage of development, the sulfhydryl reagent 5, 5'-dithio-bis(2-nitrobenzoic acid), known as Ellman's reagent, interferes in a highly specific manner with ECM biogenesis. Treated post-inversion embryos are no longer able to assemble an intact ECM and consequently dissociate into a suspension of single cells. Dissociated cells remain viable and continue to secrete ECM proteins into the growth medium, as documented by the identification of several members of the pherophorin family. Cross-linked ECM polymers such as sulfated surface glycoprotein 185 remain in a soluble state. Thus, treatment with Ellman's reagent opens a simple approach for the isolation and characterization of otherwise inaccessible monomeric precursors.