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.     

Dermatan sulfate formation in gastrulae of the sea urchin Clypeaster japonicus

Gastrullation of sea urchin embryos is arrested in sulfate-free sea water. This developmental arrest has been considered to be due to lack of sulfation of glycosaminoglycans in the extracellular matrix of the embryos. In the present study, we characterized a dermatan sulfate type component formed in gastrula-stage embryos of the sea urchin Clypeaster japonicus and examined the effects of sulfate deprivation on the formation. Glycosamino-glycans were prepared from gastrula-stage embryos incubated with [3H]acetate in normal and sulfate-free sea water. Enzymatic analyses indicated that embryos formed a glycosaminoglycan of the dermatan sulfate type which contained an N-acetylgalactosamine-6-sulfate-containing disaccharide as a major unit, plus a minor unidentified component. Under sulfate-free conditions, embryos formed an under-sulfated chondroitin/dermatan sulfate copolymer which mainly consisted of non-sulfate, glucuronic acid-containing (chondroitin) disaccharide units. These results suggest that sulfate deprivation diminishes not only the degree of sulfation but also the formation of L-iduronic acid-containing (dermatan) disaccharide units in dermatan sulfate in sea urchin embryos.     

Highly acidic glycans from sea cucumbers. Isolation and fractionation of fucose-rich sulfated polysaccharides from the body wall of Ludwigothurea grisea

The body wall of the sea cucumber contains high amounts of sulfated glycans, which differ in structure from glycosaminoglycans of animal tissues and also from the fucose-rich sulfated polysaccharides isolated from marine algae and from the jelly coat of sea urchin eggs. In Ludwigothurea grisea, glycans can be separated into three fractions which differ in molecular mass and chemical composition. The fraction containing a high-molecular-mass component has a high proportion of fucose and small amounts of amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction, which represents the major portion of the sea cucumber polysaccharides, contains besides fucose, approximately equimolar proportions of glucuronic acid and amino sugars, and has a sulfate content higher than that in the other two fractions. Both D and L-isomers of fucose are found in these polysaccharides, and the sulfate is linked to the O-3 position of the fucose residues. The attachment position of the sulfate groups to the glucuronic acid units and amino sugars is still undetermined. It is possible that these compounds are involved in maintaining the integrity of the sea cucumber's body wall, in analogy with the role of other macromolecules in the vertebrate connective tissue.     

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.     

Altered fine structures of corneal and skeletal keratan sulfate and chondroitin/dermatan sulfate in macular corneal dystrophy

The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.     

Inhibition of factor X, factor V and prothrombin activation by the bis(lactobionic acid amide) LW10082.

The minimum concentrations of heparin, dermatan sulfate, hirudin, and D-Phe-Pro-ArgCH2Cl required to delay the onset of prothrombin activation in contact-activated plasma also prolong the lag phases associated with both factor X and factor V activation. Heparin and dermatan sulfate prolong the lag phases associated with the activation of the three proteins by catalyzing the inhibition of endogenously generated thrombin. Thrombin usually activates factor V and factor VIII during coagulation. The smallest fragment of heparin able to catalyze thrombin inhibition by antithrombin III is an octadecasaccharide with high affinity for antithrombin III. In contrast, a dermatan sulfate hexasaccharide with high affinity for heparin cofactor II can catalyze thrombin inhibition by heparin cofactor II. A highly sulfated bis(lactobionic acid amide), LW10082 (Mr 2288), which catalyzes thrombin inhibition by heparin cofactor II and has both antithrombotic and anticoagulant activities, has been synthesized. In this study, we determined how the minimum concentration of LW10082 required to delay the onset of intrinsic prothrombin activation achieved this effect. We demonstrate that, like heparin and dermatan sulfate, LW10082 delays the onset of intrinsic prothrombin activation by prolonging the lag phase associated with both factor X and factor V activation. In addition, LW10082 is approximately 25% as effective as heparin and 10 times as effective as dermatan sulfate in its ability to delay the onset of prothrombin activation. The strong anticoagulant action of LW10082 is consistent with previous reports which show that the degree of sulfation is an important parameter for the catalytic effectiveness of sulfated polysaccharides on thrombin inhibition.     

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.     

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.     

Inhibition of autonomous human keratinocyte proliferation and amphiregulin mitogenic activity by sulfated polysaccharides

Summary We previously demonstrated that human keratinocyte cultures proliferate in the absence of polypeptide growth factors (autonomous growth) and that this autonomous growth is blocked by interaction of heparin with a human keratinocyte-derived autocrine factor (KAF) which we identified as amphiregulin (AR). In the present study, we demonstrate that sulfated polysaccharides other than heparin (low and high molecular weight dextran sulfates) also inhibit the AR-mediated autonomous proliferation of human keratinocytes. Furthermore, sulfated polysaccharides such as high and low molecular weight dextran sulfates, heparan sulfate and, to a lesser extent, chondroitin sulfates B and C were also shown to be inhibitors of human keratinocyte-derived AR (k-d AR)-stimulated DNA synthesis in quiescent murine AKR-2B cell cultures. Our results demonstrate that sulfation of polysaccharides is required for AR inhibitory activity, and that several sulfated polysaccharides (other than heparin) can act as inhibitors of AR-mediated autonomous proliferation in human epidermal keratinocytes and as inhibitors of k-d AR-mediated mitogenic activity in AKR-2B cells.     

Structure and anticoagulant activity of sulfated fucans. Comparison between the regular, repetitive, and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae

Sulfated fucans are among the most widely studied of all the sulfated polysaccharides of non-mammalian origin that exhibit biological activities in mammalian systems. Examples of these polysaccharides extracted from echinoderms have simple structures, composed of oligosaccharide repeating units within which the residues differ by specific patterns of sulfation among different species. In contrast the algal fucans may have some regular repeating structure but are clearly more heterogeneous when compared with the echinoderm fucans. The structures of the sulfated fucans from brown algae also vary from species to species. We compared the anticoagulant activity of the regular and repetitive fucans from echinoderms with that of the more heterogeneous fucans from three species of brown algae. Our results indicate that different structural features determine not only the anticoagulant potency of the sulfated fucans but also the mechanism by which they exert this activity. Thus, the branched fucans from brown algae are direct inhibitors of thrombin, whereas the linear fucans from echinoderms require the presence of antithrombin or heparin cofactor II for inhibition of thrombin, as reported for mammalian glycosaminoglycans. The linear sulfated fucans from echinoderms have an anticoagulant action resembling that of mammalian dermatan sulfate and a modest action through antithrombin. A single difference of one sulfate ester per tetrasaccharide repeating unit modifies the anticoagulant activity of the polysaccharide markedly. Possibly the spatial arrangements of sulfate esters in the repeating tetrasaccharide unit of the echinoderm fucan mimics the site in dermatan sulfate with high affinity for heparin cofactor II.     

Separation,purification, anticoagulant activity and preliminary structural characterization of two sulfated polysaccharides from sea cucumber Acaudina molpadioidea and Holothuria nobilis

In this study, we isolated two fucosylated polysaccharide sulfates (ACP and HOP) from sea cucumber Acaudina molpadioidea and Holothuria nobilis, with an average molecular weight of 90.8 and 135.8 kDa, respectively. We investigated and compared their anticoagulant activities through anticoagulant assay. Our data showed that both polysaccharides possessed good anticoagulant activity, but HOP's activity was higher than that of ACP. Due to the different anticoagulant activities of ACP and HOP, we compared the preliminary structural characterizations of these two sulfated polysaccharides, and found that both ACP and HOP consisted of β-d-glucuronic acid, β-d-N-acetyl-galactosamine, α-l-fucose and sulfate groups. ACP and HOP had almost identical ratios of glucuronic acid, N-acetyl-galactosamine and fucose. However, the sulfate contents and sulfation patterns of fucose residues of ACP and HOP were obviously different. There were 4-O-sulfated fucose, 3,4-O-disulfated fucose and 2,4-O-disulfated fucose in ACP, but only 3-O-sulfated fucose and 2,4-O-disulfated fucose were present in HOP. Therefore, their distinct anticoagulant activities might be due to the different sulfate contents and sulfation patterns of their fucose residues.     

Composition and degree of sulfation of glycosaminoglycans from tissues of different animal species: heterogeneity and tissue specificity of heparan sulfates

The composition and degree of sulfation of glycosaminoglycans (GAG) in proteoglycans from various animal tissues were studied. It was shown that sulfated GAG contain chondroitin sulfates AC and B as well as heparan sulfates. The bulk of GAG in the majority of tissues under study is represented by 2-3 types of heparan sulfate molecules differing in the degree of sulfation. According to the degree of sulfation heparan sulfates from all tissues studied can be classified into three groups. Homologous tissues of various animal species are characterized by a similar composition and the degree of sulfation The data obtained are discussed in terms of the feasible role of proteoheparan sulfates in specific cell-to-cell interactions.     

Sulfated glycosaminoglycans (GAG) in the developing mouse brain : Quantitative aspects on the metabolism of total and individual sulfated GAG in vivo

Sulfation and desulfation of total glycosaminoglycans (GAG) as well as of chondroitin sulfates (A + C), dermatan sulfate, and heparan sulfate were quantified in the developing cerebrum and cerebellum of mice by labeling with [35S]sulfate combined with chases started 24 hr after [35S]sulfate injection. In both the developing cerebrum and cerebellum, the rate of biosynthesis of total sulfated GAG was highest shortly after birth (2 days), decreased sharply thereafter, and reached a plateau after 14 days. The biosynthetic activities of chondroitin sulfates and heparan sulfate decreased sharply up to 14 days and retained constant levels afterward. By contrast, the rates of biosynthesis of dermatan sulfate increased up to 14 days. The biodegradation rates of total sulfated GAG as well as of chondroitin sulfates, heparan sulfate, and dermatan sulfate were strongly correlated with the corresponding rates of biosynthesis during the first 2 postnatal weeks. Total and individual sulfated GAG showed high degradation rates resulting in half-life times of a few hours up to 1 1/2 days. Thus sulfated GAG are synthesized in excess and the actual net content seems to be co-regulated to a high degree by lysosomal degradation. In both brain parts, a proportional increase of the sulfated GAG content vs the total GAG content from 40% at birth to 90% at 28 days was observed. Since during development heparan sulfate and dermatan sulfate manifested a relative increase in their daily net synthesis besides a decrease of chondroitin sulfates, a developmental increase of the sulfate groups linked to GAG is evidenced. This molecular differentiation resulting in microenvironmental changes may be of high functional significance.     

Specific inhibition of binding of antistasin and [A103,106,108] antistasin 93-119 to sulfatide (Gal(3-SO4)beta 1-1Cer) by glycosaminoglycans.

Leech-derived antistasin is a potent anticoagulant and antimetastatic protein that binds sulfatide (Gal(3-SO4)beta 1-1Cer) and sulfated polysaccharides. In this study, the synthetic fragment [A103,106,108] antistasin 93-119, which corresponds to the carboxyl terminus, showed specific and saturable binding to sulfatide. Binding was competitively blocked by glycosaminoglycans (GAGs) in the order: dextran sulfate 5000 congruent to dextran sulfate 500,000 greater than heparin greater than dermatan sulfate much greater than chondroitin sulfates A and C. This rank order of inhibitory potency was identical to that observed with whole antistasin. We suggest that residues 93-119 of antistasin represent a critical domain for binding GAGs and sulfated glycolipids.     

Lymphocytes express a diverse array of specific receptors for sulfated polysaccharides

Lymphocyte receptors for sulfated polysaccharides were detected in two ways, namely, by the ability of lymphocytes to form rosettes with sheep red blood cells (SRBC) coupled with one of fourteen different sulfated polysaccharides, and by the ability of cholate extracts of lymphocytes to hemagglutinate the same sulfated polysaccharide-coupled SRBC. It was found that murine lymphocytes lacked receptors for a number of glycosaminoglycans, such as hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate, but reacted strongly with heparin, arteparon, and a number of sulfated polysaccharides of plant and bacterial origin. In each case receptor activity was demonstrated by rosetting and by the ability of lymphocyte lysates to strongly agglutinate sulfated polysaccharide-coupled SRBC. The receptors exhibited a high degree of diversity as evidenced by (a) only subpopulations of lymphocytes, particularly splenic B cells, expressing receptors for some of the sulfated polysaccharides and (b) hemagglutination-inhibition analyses revealing numerous subsets of receptors with different binding specificities. Receptor diversity was further highlighted by a 48% difference in the hemagglutination-inhibiton results between thymus and spleen. It is proposed that these receptors are involved in cell-cell communication and lymphocyte homing and recirculation. The likely target structures for the receptors in vivo are the heparan sulfates, a ubiquitous and structurally diverse family of sulfated glycosaminoglycans.     

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.     

The heterogeneity of dermatan sulfate and heparan sulfate in rat liver and a shift in the glycosaminoglycan contents in carbon tetrachloride-damaged liver

The glycosaminoglycan of rat liver can be separated into five distinct fractions; a hyaluronic acid franction, a heparan sulfate fraction with a molar ratio of sulfate to hexosamine (S/HexN) around 0.7, a heparan sulfate fraction with a S/HexN ratio around 1.4, a dermatan sulfate fraction with a S/HexN ratio near unity, and a dermatan sulfate fraction with a S/HexN ratio around 1.3.Enzymatic analysis of the two dermatan sulfate fractions indicates that they differ significantly in that the high sulfated fraction contains relatively more N-acetylgalactosamine 4,6-bissulfate units (about 26% of the total hexosamine). In experimental injury produced by carbon tetrachloride, the low sulfated fraction increases as much as 9-fold on a dry weight basis, bearing no linear relationship to the amount of the high sulfated fraction which increases only 2-fold. A significant shift is also observed in the levels of the two heparan sulfate fractions. In this case, however, the high sulfated fraction shows a much more pronounced increase than does the low sulfated fraction. On the basis of these observations, it is suggested that for each of the dermatan sulfate and heparan sulfate classes are at least two pools, distinguished by sulfation degree and perhaps by turnover rate and physiological function.     

Inhibition of thrombin by sulfated polysaccharides isolated from green algae

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

Structure of the sulfated alpha-L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus: patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition.

The egg jelly coats of sea urchins contains sulfated polysaccharides responsible for inducing the sperm acrosome reaction which is an obligatory event for sperm binding to, and fusion with, the egg. Here, we extend our study to the sea urchin Strongylocentrotus franciscanus. The egg jelly of this species contains a homofucan composed of 2- O -sulfated, 3-linked units which is the simplest structure ever reported for a sulfated fucan. This polysaccharide was compared with other sulfated alpha-L-fucans as inducers of acrosome reaction in conspecific and heterospecific sperm. Although all these fucans are linear polymers composed of 3-linked alpha-L-fucopyranosyl units, they differ in the proportions of 2-O- and 4-O-sulfation. The reactivity of the sperm of each species is more sensitive to the egg jelly sulfated fucan found in their own species. The reactivity of the sperm does not correlate with the charge density of the fucan, but with the proportion of 2-O- and 4-O-sulfation. The pattern of sulfation may be an important feature for recognition of fucans by the sperm receptor contributing to the species-specificity of fertilization.     

Polysaccharides sulfated at the time of gastrulation in embryos of the sea urchin Clypeaster japonicus

Based on the fact that the development of sea urchin embryos is arrested at the blastula stage in sulfate-free sea water (SFSW), we attempted in the present study to elucidate the nature of sulfated polysaccharides (PSs) which appear at the time of gastrulation in embryos of the sea urchin Clypeaster japonicus. Electrophoretic analysis of PSs prepared from embryos at different developmental stages revealed that three kinds of PSs (3A, 3B, 3C) appear de novo at the gastrula stage, and that these PSs are not found in embryos at the hatching blastula stage, nor are they found in permanent blastula reared in SFSW. These, three PSs were mostly of extracellular matrix origin. Among them, 3C was identified as dermatan sulfate on the basis of its electrophoretic mobility and sensitivity to enzymatic digestion. 3A and 3B remained to be identified. Further, a plausible precursor of 3C, which was sulfated under normal conditions, was detected as 6D in the embryos reared in SFSW. Autoradiographic analysis using [35S]sulfate revealed that these three PSs, accounted for more than 90% of [35S]sulfate incorporated into the acid PS fraction during gastrulation.