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Metastasis is responsible for the majority of cancer-associated deaths, though only a very small number of tumor cells are able to efficiently complete all the steps of that process. Tumor cell survival in the bloodstream is one of the limiting aspects of the metastatic cascade. The formation of tumor cell-platelet complexes that promote tumor cell survival is facilitated by the binding of P-selectin on activated platelets to sialyl Lewis-containing oligosaccharides on the surface of tumor cells. Inhibition of this interaction has been shown to attenuate metastasis. Heparin is a potent selectin inhibitor and is capable to block platelet-tumor cell complex formation, thereby attenuating metastasis. Similarly, other sulfated polysaccharides isolated from marine invertebrates attenuate metastasis by a P-selectin-mediated mechanism. In this work, we investigated the selectin-dependent antimetastatic activity of sea urchin sulfated polysaccharides with slight structural differences: a sulfated fucan from Strongylocentrotus franciscanus; a sulfated fucan from Strongylocentrotus droebachiensis; and a sulfated galactan from Echinometra lucunter. The results demonstrate that these fucans and the galactan have different antiselectin activities despite being very similar molecules. Therefore, they may be interesting tools for studies on the structure-function relationship or even for future treatments.

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Here we have performed an nuclear magnetic resonance-based study on the ring and chain conformations as well as dynamics of oligosaccharides generated by acid hydrolysis on two structurally related glycans, a 3-linked 2-sulfated alpha-L-galactan and a 3-linked 2-sulfated alpha-L-fucan. Results derived from scalar couplings have confirmed the 1C4 chair configuration to both alpha-L-fucose and alpha-L-galactose, and a similar solution 3D structure for the oligosaccharide chains of both sulfated glycans as seen on the basis of NOE patterns. Measurements of spin-relaxation rates have suggested, however, a slight difference dynamical property to these glycans. The fucose-based oligosaccharides showed an enhanced dynamical property if compared to the galactose-based oligosaccharides of same anomericity, sugar configuration, glycosidic bond and sulfation type. This distinction solely on the dynamical aspect has been driven therefore by the different sugar composition of the two studied sulfated glycans.

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This work found the occurrence of a distinct sialic acid-rich polysaccharide in the sperm surface of the sea urchin Lytechinus variegatus, which differed significantly from a similar molecule found in the egg jelly. The sperm polysaccharide extracted by protease digestion was purified using anion exchange chromatography and characterized using agarose gel electrophoresis, gas chromatography/mass spectrometry and NMR spectroscopy. This polysaccharide was highly sulfated and composed almost exclusively of N-acetylneuraminic acid. In contrast, the sialic acid-rich polysaccharide from the egg jelly was composed of N-glycolylneuraminic acid and contains several other hexoses in its structure. This new molecule could help to characterize in further detail the mechanism of fertilization in the sea urchin model system. Sulfated polysaccharides from the jelly coat of sea urchins showed species-specificity in inducing the sperm acrosome reaction, providing an example of a signal transduction event regulated by the sulfated polysaccharide. The new sialic acid-rich polysaccharide found in the sperm head could represent a new molecule involved in the biology of the sea urchin fertilization.

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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 alpha-anomeric configuration. Herein, structural analysis of the polysaccharide from the sea urchin Glyptocidaris crenularis surprisingly revealed a unique sulfated beta-d-galactan composed by (3-beta-d-Galp-2(OSO(3))-1-->3-beta-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 alpha-galactan or alpha-fucan have less dynamic structural behavior, exhibiting fewer conformational populations, with an almost exclusive conformational state with glycosidic dihedral angles Phi/Psi = -102 degrees /131 degrees . The preponderant conformer observed in the sulfated alpha-galactan or alpha-fucan is not observed among populations in the beta-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.

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The eggs of sea urchins are covered by a jelly coat, which contains high concentrations of sulfated polysaccharides. These carbohydrates show species-specificity in inducing the sperm acrosome reaction. Several studies about the egg jelly of sea urchins have been published, but there is no information about the composition of the seminal fluid of these echinoderms. Here we report for the first time the occurrence of complex sulfated polysaccharides in the seminal fluid of the sea urchin Lytechinus variegatus. These polysaccharides occur as three fractions that differ mostly in their carbohydrate/protein ratios. The native molecular masses of the polymers are very high (> or = 200 kDa) but, after digestion with papain the size decreases to approximately 8 kDa. All fractions have a similar carbohydrate composition, containing mostly galactose, glucosamine and mannose. The heterogeneous sulfated polysaccharides differ from vertebrate glycosaminoglycans and also from all previously described polysaccharides from invertebrates. The physiological role of the sulfated carbohydrates from seminal fluid is not yet determined. However, by analogy with the effects proposed for some glycoproteins found in vertebrate seminal fluid, it may be possible that the sulfated polysaccharides from invertebrate are also involved in fertilization process.

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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.

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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.

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Spawning marine invertebrates are excellent models for studying fertilization and reproductive isolating mechanisms. To identify variation in the major steps in sea urchin gamete recognition, we studied sperm activation in three closely related sympatric Strongylocentrotus species. Sperm undergo acrosomal exocytosis upon contact with sulfated polysaccharides in the egg-jelly coat. This acrosome reaction exposes the protein bindin and is therefore a precondition for sperm binding to the egg. We found that sulfated carbohydrates from egg jelly induce the acrosome reaction species specifically in S. droebachiensis and S. pallidus. There appear to be no other significant barriers to interspecific fertilization between these two species. Other species pairs in the same genus acrosome react nonspecifically to egg jelly but exhibit species-specific sperm binding. We thus show that different cell-cell communication systems mediate mate recognition among very closely related species. By comparing sperm reactions to egg-jelly compounds from different species and genera, we identify the major structural feature of the polysaccharides required for the specific recognition by sperm: the position of the glycosidic bond of the sulfated alpha-L-fucans. We present here one of the few examples of highly specific pure-carbohydrate signal transduction. In this system, a structural change in a polysaccharide has far-reaching ecological and evolutionary consequences.

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Brain-specific chondroitin sulfate (CS) proteoglycan (PG) DSD-1-PG/6B4-PG/phosphacan isolated from neonatal mouse brains exhibits neurite outgrowth-promoting activity toward embryonic rat and mouse hippocampal neurons in vitro through the so-called DSD-1 epitope embedded in its glycosaminoglycan side chains. Oversulfated CS variants, CS-D from shark cartilage and CS-E from squid cartilage, also possess similar activities. We have proposed that the neuritogenic property of the DSD-1 epitope may be attributable to a distinct CS structure characterized by the disulfated D disaccharide unit [GlcUA(2S)-GalNAc(6S)]. In this study, we assessed neuritogenic potencies of various oversulfated dermatan sulfate (DS) preparations purified from hagfish notochord, the bodies of two kinds of ascidians and embryonic sea urchin, which are characterized by the predominant disulfated disaccharide units of [IdoUA-GalNAc(4S,6S)] (68%), [IdoUA(2S)-GalNAc(4S)] (66%) plus [IdoUA(2S)-GalNAc(6S)] (5%), [IdoUA(2S)-GalNAc (6S)] (>90%), and [IdoUA-GalNAc(4S,6S)] (74%), respectively. They exerted marked neurite outgrowth-promoting activities, resulting in distinct morphological features depending on the individual structural features. Such activities were not observed for a less sulfated DS preparation derived from porcine skin, which has a monosulfated disaccharide unit [IdoUA-Gal-NAc(4S)] as a predominant unit. The neurite outgrowth-promoting activities of these oversulfated DS preparations and DSD-1-PG were eliminated by the specific enzymatic cleavage of GalNAc-IdoUA linkages characteristic of DS using chondroitinase B. In addition, chemical analysis of the glycosaminoglycan side chains of DSD-1-PG revealed the DS-type structures. These observations suggest potential novel neurobiological functions of oversulfated DS structures and may reflect the physiological neuritogenesis during brain development by mammalian oversulfated DS structures exemplified by the DSD-1 epitope.

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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.

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The sulfated fucan (SF) of egg jelly induces the acrosome reaction (AR) of sea urchin sperm. Strongylocentrotus franciscanus (Sf) SF is sulfated only at the 2-position. Strongylocentrotus purpuratus (Sp) has two SF isotypes, each one being female specific. One is rich in sulfate at both the 2- and 4-positionS (SF-1), and the other is rich in sulfate at the 4-position, but not the 2-position (SF-2). Sf SF is poor at inducing the AR of Sp sperm, presumably due to lack of 4-sulfation. Sp SF-1 is better at inducing the AR of Sf sperm than Sp SF-2, hypothetically due to increased 2-sulfation. Chemical oversulfation of Sf SF increases the percentage of AR of Sp sperm, showing that 4-sulfation is important for recognition of SF by Sp sperm. Chemically oversulfated Sp SF-2 is better at inducing the Sf sperm AR, presumably because of increased 2-sulfation. The species, Strongylocentrotus drobachiensis (Sd), has an SF-2 that is exclusively 2-sulfated (like Sf), except the glycosidic linkage in Sd is alpha(1-->4), whereas in Sf it is alpha(1-->3). Sd SF-2 does not induce the AR of Sf sperm, showing the strict requirement for the alpha(1-->3) linkage in recognition between Sf sperm and SF. Egg jelly from Echinometra lucunter (El) contains sulfated galactan (SG) which differs from Sf SF only in that the monosaccharide is L-galactose, not L-fucose. This SG and Sf SF are equally potent in inducing the AR of Sf sperm, showing that modification at C6 of L-fucose is not important for proper recognition between SF and Sf sperm receptors. This system permits study of the structural basis for recognition between sulfated polysaccharide and receptors controlling signal transduction pathways in animal cells.

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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.

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