Utilization of a particle gun DNA introduction system for the analysis of <Emphasis Type="Italic">cis</Emphasis>-regulatory elements controlling the spatial expression pattern of the arylsulfatase gene (<Emphasis Type="Italic">HpArs</Emphasis>) in sea urchin embryos

We applied a particle gun method to introduce DNA into fertilized sea urchin eggs for the analysis of cis-regulatory elements responsible for spatial gene expression during development. We introduced HpArs (sea urchin arylsulfatase gene) -GFP and HpArs-LacZfusion constructs into the fertilized eggs and obtained high expression levels of the fusion genes. Using this assay system, we demonstrated that a fragment of HpArs (-3,484 to +4,636) is sufficient for aboral ectoderm-specific expression, and that the region in the first intron from +406 to +1,993 contains the control elements responsible for the repression of the HpArs promoter activity in secondary mesenchyme cells.     

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.     

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.     

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.     

Ca-transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca²⁺ by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K⁺ leading to an enhancement of the Ca²⁺ transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca²⁺ at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K⁺ but not by Li⁺ and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K⁺. These data suggest that the regulation of the SERCA pump by K⁺ and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.     

A Unique 2-Sulfated ��-Galactan from the Egg Jelly of the Sea Urchin Glyptocidaris crenularis: CONFORMATION FLEXIBILITY VERSUS INDUCTION OF THE SPERM ACROSOME REACTION*

Sulfated polysaccharides from the egg jelly of sea urchins act as species-specific inducers of the sperm acrosome reaction, which is a rare molecular mechanism of carbohydrate-induced signal-transduction event in animal cells. The sea urchin polysaccharides differ in monosaccharide composition (l-fucose or l-galactose), glycosylation, and sulfation sites, but they are always in the α-anomeric configuration. Herein, structural analysis of the polysaccharide from the sea urchin Glyptocidaris crenularis surprisingly revealed a unique sulfated β-d-galactan composed by (3-β-d-Galp-2(OSO₃)-1→3-β-d-Galp-1)_n repeating units. Subsequently, we used the G. crenularis galactan to compare different 2-sulfated polysaccharides as inducers of the acrosome reaction using homologous and heterologous sperm. We also tested the effect of chemically over-sulfated galactans. Intriguingly, the anomeric configuration of the glycosidic linkage rather than the monosaccharide composition (galactose or fucose) is the preferential structural requirement for the effect of these polysaccharides on sea urchin fertilization. Nuclear magnetic resonance and molecular dynamics indicate that sulfated α-galactan or α-fucan have less dynamic structural behavior, exhibiting fewer conformational populations, with an almost exclusive conformational state with glycosidic dihedral angles Φ/Ψ = −102°/131°. The preponderant conformer observed in the sulfated α-galactan or α-fucan is not observed among populations in the β-form despite its more flexible structure in solution. Possibly, a proper spatial arrangement is required for interaction of the sea urchin-sulfated polysaccharides with the specific sperm receptor.The evolution of barriers to inter-specific hybridization is a crucial step in the fertilization of free-spawning marine invertebrates. In sea urchins the molecular recognition between sperm and egg ensures species recognition. The jelly coat surrounding sea urchin eggs is not a simple accessory structure; it is considerably complex on a molecular level and intimately involved in gamete recognition. It contains sulfated polysaccharides, sialoglycans, and peptides.Structural changes in the sulfated polysaccharide from the egg jelly of sea urchins modulate cell-cell recognition and species specificity leading to exocytosis of the acrosomal vesicle, the acrosome reaction. This is a crucial event for the recognition between male and female gametes, leading to the fertilization success, and is also what prevents intercrosses. The sulfated polysaccharide from the egg jelly recognizes its specific receptor present in the sperm. Apart from the sialoglycans that act in synergy with the sulfated polysaccharides, other components of the egg jelly do not possess acrosome reaction-inducing activity (1). The sulfated polysaccharide-mediated mechanism of sperm-egg recognition co-exists with that of bindin and its receptor in the egg (2–4).The sulfated polysaccharides from sea urchin show species-specific structures composed of repetitive units (mono-, tri-, and tetrasaccharides) that differ in the monosaccharide backbone (l-fucose or l-galactose), glycosidic linkage (3- or 4-linked), and sulfation (2- and/or 4-sulfation). However, they are always in the α-enantiomeric configuration (4, 5). Previous studies from our laboratory have demonstrated that sea urchin-sulfated polysaccharides induce the acrosome reaction in a species-specific way. In some cases the sperm from a certain species of sea urchin recognizes the sulfated polysaccharide containing a similar structure from a different species. For example, the egg jelly from Strongylocentrotus franciscanus contains a 2-sulfated, 3-linked α-fucan, but the sperm from this species recognizes a heterologous 2-sulfated, 3-linked α-galactan from Echinometra lucunter (6).We now extended our studies to the sulfated polysaccharides of the sea urchin Glyptocidaris crenularis (7). Surprisingly, we observed that this species contains a unique sulfated β-d-galactan composed of repetitive disaccharide units alternating 2-sulfated and non-sulfated 3-linked units. This polymer is markedly distinct from all other sea urchin-sulfated polysaccharides described so far that are composed of units on α-l-configuration. Furthermore, this sea urchin does not contain sialoglycans, which are commonly found in the echinoderm egg jelly.We used this new sulfated β-galactan to investigate the acrosome reaction in a further molecular detail using homologous and heterologous sperm. We tested three 2-sulfated polysaccharides that differ in their conformation (α or β) and monosaccharide composition (galactose or fucose) as inducers of the sperm acrosome reaction. We aimed to establish the structure versus biological activity of the echinoderm polysaccharides, including structural features at a conformational level.     

Ca2+-transport in sea urchin unfertilized eggs: regulation by endogenous sulfated polysaccharides and K+

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca(2+) by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K(+) leading to an enhancement of the Ca(2+) transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca(2+) at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K(+) but not by Li(+) and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K(+). These data suggest that the regulation of the SERCA pump by K(+) and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.     

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.     

Structure, biology, evolution, and medical importance of sulfated fucans and galactans

Sulfated fucans and galactans are strongly anionic polysaccharides found in marine organisms. Their structures vary among species, but their major features are conserved among phyla. Sulfated fucans are found in marine brown algae and echinoderms, whereas sulfated galactans occur in red and green algae, marine angiosperms, tunicates (ascidians), and sea urchins. Polysaccharides with 3-linked, beta-galactose units are highly conserved in some taxonomic groups of marine organisms and show a strong tendency toward 4-sulfation in algae and marine angiosperms, and 2-sulfation in invertebrates. Marine algae mainly express sulfated polysaccharides with complex, heterogeneous structures, whereas marine invertebrates synthesize sulfated fucans and sulfated galactans with regular repetitive structures. These polysaccharides are structural components of the extracellular matrix. Sulfated fucans and galactans are involved in sea urchin fertilization acting as species-specific inducers of the sperm acrosome reaction. Because of this function the structural evolution of sulfated fucans could be a component in the speciation process. The algal and invertebrate polysaccharides are also potent anticoagulant agents of mammalian blood and represent a potential source of compounds for antithrombotic therapies.     

Sox regulates transcription of the sea urchin arylsulfatase gene

A 50 bp region from -194 bp to -144 bp of the arylsulfatase gene (HpArs) of the sea urchin, Hemicentrotus pulcherrimus, is related to the temporally regulated expression of this gene. This region contains a Sox (Sry-related HMG box)-binding site, and the introduction of sequence mutations to this site significantly reduced the activity of the HpArs promoter, even in the presence of the C15 enhancer, which consists of HpOtx and CAAT motifs. A protein that binds to the Sox-binding site in the 50 bp region of the HpArs gene was detected in nuclear extracts of mesenchyme blastulae and a protein synthesized in vitro using SoxB1 cDNA of another sea urchin, Strongylocentrotus purpuratus, also bound to this Sox site. These results suggest that HpSox, which is maternally expressed and remains abundant by the pluteus stage, is clearly implicated in regulation of the HpArs gene. The presence of a negatively acting cis element in this 50 bp region has also been detected.     

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.     

A mucous secretion in the malpighian tubes of a neotropical bumblebee,Bombus atratus Franklin

Summary A filamentous secretion composed of carboxylated and sulfated acid glycosaminoglycans (AGAG), neutral polysaccharides, and protein(s) appears in the lumen of the Malpighian tubes of the fully grown larvae of the bumblebee,Bombus atratus Franklin. A well-ordered macromolecular array was demonstrated specially for the carboxylated AGAG components of this secretion, based on their linear dichroism and birefringence properties. It is suggested that the carboxylated AGAG macromolecules can acquire a helical conformation when present at the lumen of the organ. The mucous secretion elaborated by the Malpighian tubes ofB. atratus is excreted from the larvae in the form of condensed filaments. Its function remains unclear. Globules with concentric lamination containing protein and neutral polysaccharides were seen detaching from the apical border of the epithelial cells of the Malpighian tubes of the fully grown larvae. It could not be established whether they contribute for the elaboration of the filamentous secretion. Morphologically similar globules have been assumed in some other insect groups to originate from cytolysomes. Urate crystals surrounded by a halo of calcium granules were also found in the lumen of the Malpighian tubes ofB. atratus.     

Early events in sea urchin metamorphosis,description and analysis

The larval epithelium of the sea urchin, Lytechinus pictus, consists of squamous cells and bands of columnar epithelial cells bearing cilia. During metamorphosis this tissue undergoes a series of rapid, complex changes. Through the scanning and transmission electron microscope, we describe and analyse these changes. The changes can be divided into three steps. (1) The larval arms bend away from the left side of the larva, exposing the urchin rudiment. Cells which are identical to smooth muscle cells are in a position to bring about this bending. (2) The squamous epithelial cells assume a cuboidal shape. This change in shape results in the collapse of the larval epithelium onto the presumptive aboral surface. These cells possess a subapical band of microfilaments. The cellular shape change but not the bending of the arms is reversibly inhibited by Cytochalasin B. These observations suggest a mechanism for this change. (3) The former lining of the vestibule of the urchin rudiment comes to lie over the collapsed larval tissue and forms the adult epithelium. At this point, after only one hour, the larva has assumed the external shape of an adult sea urchin.     

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.      

In vitro anti-influenza virus activities of sulfated polysaccharide fractions from <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis>

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.     

A new extracellular matrix protein of the sea urchin embryo with properties of a substrate adhesion molecule

Summary A new embryonic extracellular matrix protein has been purified from eggs of the sea urchin Paracentrotus lividus. The molecule is a 210 kD dimer consisting of two 105 kD subunits that are held together by S-S bridges. In the unfertilized egg, the protein is found within granules uniformly distributed throughout the cytoplasm. After the egg is fertilized, the antigen is polarized to the apical surface of ectodermal and endodermal cells during all of the developmental stages examined, until the pluteus larva is formed. The protein promotes the adhesion of blastula cells to the substrate and is antigenically distinct from echinonectin, a well characterized substrate adhesion molecule. This report adds a new candidate to the list of known extracellular matrix molecules for the regulation of differentiation and morphogenesis in the sea urchin embryo. Offprint requests to: V. Matranga     

Upstream element of the sea urchin arylsulfatase gene serves as an insulator.

Insulator DNAs functionally isolate neighboring genes by blocking interactions between distal cis-regulatory elements and promoters. Here we report that a DNA fragment located in the upstream region of sea urchin, H. pulcherrimus, arylsulfatase (HpArs) gene blocks the interaction of the Ars enhancer when positioned between the enhancer and the target promoter, in an orientation dependent manner. The Ars insulator works only 3' to 5' direction and has no significant stimulatory or inhibitory effects on its own promoter. In transgenic Drosophila, the Ars insulator blocks the interaction between even-skipped stripe enhancer and its target promoter. The insulation mechanism operates also unidirectionally in Drosophila. We also show that the efficiency of transformation of HeLa cells is enhanced when the integrated gene is flanked by the Ars insulator, suggesting the sea urchin insulator overcomes the position-dependent transgene expression in mammalian cells. These results demonstrate that the mechanism of action of the insulator has been conserved throughout evolution.     

Antiviral Activities of Marine Pseudomonas Polysaccharides and Their Oversulfated Derivatives

A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide. Among the antiviral activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration is 1.4 μg/ml). Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration is 11.0 μg/ml; for another, 2.9 μg/ml). Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1. No cytotoxicity of these products was noted against host cells at the 50% cytotoxic concentration of 100 μg/ml, except that naturally occurring sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8–21 μg/ml. Received May 1, 1998; accepted July 24, 1998.     

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.