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Keywords:

  • acrosome reaction;
  • anticoagulant activity;
  • antithrombotic activity;
  • sulfated fucans;
  • sulfated galactans

Abstract

  1. Top of page
  2. Abstract
  3. MARINE SULFATED HOMOPOLYSACCHARIDES: COMPLEX STRUCTURES FROM ALGAE VS. CLEAR STRUCTURES FROM INVERTEBRATES
  4. UNDERSTANDING THE INDUCTION OF THE AR IN SEA-URCHIN SPERM BY SULFATED HOMOPOLYSACCHARIDES
  5. INVERTEBRATE SULFATED HOMOPOLYSACCHARIDES AS ANTICOAGULANT AND ANTITHROMBOTIC REAGENTS: A RELIABLE ALTERNATIVE SOURCE FOR HEPARIN
  6. EXAMPLES OF STRUCTURE–FUNCTION RELATIONSHIP OF THE MARINE SULFATED HOMOPOLYSACCHARIDES ON THE ANTICOAGULANT ACTIVITY AND/OR THE AR
  7. MAIN CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Efforts in both structural and biological studies of sulfated polysaccharides from marine organisms have increased significantly over the last 10 years. Marine invertebrates have been demonstrated to be a source of glycans with particularly well-defined chemical structures, although ordered structural patterns can also be found occasionally in algal sources such as red seaweeds. Clear and regular structural features are essential for a good understanding of the biological activities of these marine homopolysaccharides of which sulfated fucans and sulfated galactans are the most studied. Herein, the main structural features (sugar type, sulfation and glycosylation sites, and orientational binding preferences) of both sulfated fucans and galactans are individually reviewed with regard to their specific contributions to two frequently described biological functions: the acrosome reaction (a physiological event of sea-urchin fertilization), and the anticoagulant and antithrombotic activities (an alternative and highly desirable pharmacological application). © 2009 Wiley Periodicals, Inc. Biopolymers 91: 601–609, 2009.

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com


MARINE SULFATED HOMOPOLYSACCHARIDES: COMPLEX STRUCTURES FROM ALGAE VS. CLEAR STRUCTURES FROM INVERTEBRATES

  1. Top of page
  2. Abstract
  3. MARINE SULFATED HOMOPOLYSACCHARIDES: COMPLEX STRUCTURES FROM ALGAE VS. CLEAR STRUCTURES FROM INVERTEBRATES
  4. UNDERSTANDING THE INDUCTION OF THE AR IN SEA-URCHIN SPERM BY SULFATED HOMOPOLYSACCHARIDES
  5. INVERTEBRATE SULFATED HOMOPOLYSACCHARIDES AS ANTICOAGULANT AND ANTITHROMBOTIC REAGENTS: A RELIABLE ALTERNATIVE SOURCE FOR HEPARIN
  6. EXAMPLES OF STRUCTURE–FUNCTION RELATIONSHIP OF THE MARINE SULFATED HOMOPOLYSACCHARIDES ON THE ANTICOAGULANT ACTIVITY AND/OR THE AR
  7. MAIN CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

The most common marine sulfated homopolysaccharides are the sulfated fucans and sulfated galactans. They are also, after glycosaminoglycans, the most widely studied sulfated polysaccharides. These compounds are essentially isolated from marine organisms such as macroalgae (brown, green, and red), and certain invertebrates like echinoderms (sea cucumber and sea urchins) or tunicates (ascidians). In general, these polymers are mainly composed of fucopyranosyl units exclusively in their α-L-form, or of α-L- and/or α-D- or β-D-galactopyranosyl units. The structures of these strongly anionic macromolecules vary among species, although their main structural features are conserved among phyla. However, they usually show high molecular weights, ≥100 million Da (for a full review of sulfated fucans and galactans, see V. H. Pomin and P. A. Mourão).1

Until now, sulfated fucans have been described only in brown algae (Phaeophyta),1 sea cucumber (Echinodermata, Holothuroidea),2 and sea urchins (Echinodermata, Echinoidea).3 With few exceptions,4–6 most of the structures found in brown algae are highly heterogeneous due to several sulfation and glycosylation sites; no clear evidence of repetitive units, and also the common presence of branching residues in any position. All these heterogeneities often make difficult the complete structural elucidation of the algal sulfated fucans.7 This intricate structural arrangement is directly related to the function of the brown algal sulfated fucans as a structural organizer component of the assembled molecules of the cell wall. This complex structure together with high molecular mass allows the highly complex organization of the cell wall through bonds with different types of molecules: peptides, alginic acid, pectin, cellulose, heteropolysaccharides, among others.8

In contrast, the structures of echinoderm-sulfated fucans are much simpler. These glycans are composed of well-defined repetitive units that allow the complete determination of their sulfation patterns, glycosidic linkage types, and anomeric configurations for all residues (see Figure 1). These macromolecules have been isolated from the egg-jelly layer of the sea urchins, and the body wall of sea cucumbers. Different from the random structure of the brown algal sulfated fucans, clear structural patterns are required for invertebrate sulfated fucans due to their specific biological functions. In the case of sea-urchins, they are involved in a very rare case of carbohydrate-induced signal transduction, the acrosome reaction (AR). The characteristic regular structures of each sea urchin species are important to maintain the intra-specificity found in their external fertilization.3

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Figure 1. Chemical structures of the repeating units of the sulfated α-L-fucans from the body wall of the sea-cucumber (A) and from the egg jelly coat of sea-urchins (B–G). The species-specific structures vary in sulfation patterns (exclusively 2- and/or 4-positions), in glycosidic linkages: α(1[RIGHTWARDS ARROW]3) (A–C, E, and G) and α(1[RIGHTWARDS ARROW]4) (D and F), and in number of residues of the repetitive units: tetrasaccharides (A–D), trisaccharides (E-II) and monosaccharides (E-I, F and G), but they are all linear. The structures are the following: (A) Ludwigothurea grisea [[RIGHTWARDS ARROW]3)-α-L-Fucp-2,4(OSOmath image)-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]]n2; (B) Lytechinus variegatus [[RIGHTWARDS ARROW]3)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-4(OSOmath image)-(1[RIGHTWARDS ARROW] 3)-α-L-Fucp-2,4(OSOmath image)-(1[RIGHTWARDS ARROW]]n2; (C) Strongylocentrotus pallidus [[RIGHTWARDS ARROW]3)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-4(OSOmath image)-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-4(OSOmath image)-(1[RIGHTWARDS ARROW]]n9; (D) Arbacia lixula [[RIGHTWARDS ARROW]4)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]4)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]4)-α-L-Fucp-(1[RIGHTWARDS ARROW]4)-α-L-Fucp-(1[RIGHTWARDS ARROW]]n10; (E) Strongylocentrotus purpuratus-I ∼80% [[RIGHTWARDS ARROW]3)-α-L-Fucp-2,4(OSOmath image)-(1[RIGHTWARDS ARROW]]n and ∼20% [[RIGHTWARDS ARROW]3)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]]n and -II [[RIGHTWARDS ARROW]3)-α-L-Fucp-2,4(OSOmath image)-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-4(OSOmath image)-(1[RIGHTWARDS ARROW]3)-α-L-Fucp-4(OSOmath image)-(1[RIGHTWARDS ARROW]]n11; (F) Strongylocentrotus droebachiensis [[RIGHTWARDS ARROW]4)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]]n9; and (G) Strongylocentrotus franciscanus [3)-α-L-Fucp-2(OSOmath image)-(1[RIGHTWARDS ARROW]]n12. All the sulfate groups are highlighted with the grey ellipse.

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The sulfated galactans can also be isolated from the cell wall of green (Clorophyta)13 or of red (Rhodophyta)14, 15 algae. Similarly to the sulfated fucans, the sulfated galactans can be found in the egg jelly coats of a few sea urchin species,10 and the tunics of ascidians (Urochordata, Ascidiacea).16, 17 There is also a single description of a sulfated galactan isolated from a sea grass, a marine angiosperm.18 The sulfated galactans from invertebrates (see Figure 2) and the marine angiosperm show the same structural pattern of simple and well-defined units as found in invertebrate sulfated fucans (see Figure 1). The structures of green algal sulfated galactans are however more heterogeneous, but simpler than brown algal sulfated fucans. The green algal macromolecules are predominantly composed of 3-β-D-Galp, but without a regular or repetitive unit, and with a heterogeneous distribution of sulfation (however, mainly 4- and/or 6-sulfate).1, 13, 20 The red algal sulfated galactans have a very regular backbone, composed always of disaccharide repeating units, but are also highly heterogeneous in their sulfation patterns which vary from species to species (Figure 2D).

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Figure 2. Chemical structures of the repeating units of the sulfated galactans from the egg jelly coat of sea-urchin (A), from the tunic of ascidians (B and C), and from red algae (D). The structures are the following: (A) Echinometra lucunter [[RIGHTWARDS ARROW]3)-α-L-Galp-2(SOmath image)-(1[RIGHTWARDS ARROW]]n10; (B) Styela plicata {[RIGHTWARDS ARROW]4)-α-L-Galp-2[[RIGHTWARDS ARROW]1)-α-L-Galp-3(OSOmath image)]-3(OSOmath image)-(1[RIGHTWARDS ARROW]}n17; (C) Herdmania monus [[RIGHTWARDS ARROW]4)-α-L-Galp-3(SOmath image)-(1[RIGHTWARDS ARROW]]n16; and (D) both Botryocladia occidentalis and Gelidium crinale composed of [3)-β-D-Galp-1[RIGHTWARDS ARROW]4-α-Gal-(1[RIGHTWARDS ARROW]]n although with different sulfation contents as showed.19 All the sulfate groups are highlighted with the grey ellipse.

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Although algal homopolysaccharides exhibit potent pharmacological actions, their structural complexities and/or partial characterization do not allow a complete understanding of their biochemical properties. It is usually hard to establish the structure–function relationship for algal polysaccharides, especially sulfated fucans. On the other hand, it is clearer to understand the biological actions of invertebrate polysaccharides because of their well-defined structures. Therefore, the invertebrate homopolysaccharides have been preferentially chosen for subsequent pharmacological studies as potential drug candidates (for more details, see the review of P. A. Mourão).21

In this review, we will individually point out how the main structural features of the polysaccharides (sugar type, sulfation and glycosylation sites, and orientational binding preferences) can specifically account for biological actions. For this purpose, the regular and well-defined marine sulfated homopolysaccharides (sulfated fucans and galactans) from invertebrates and from two species of red algae (Figures 1 and 2) will easily exemplify this structure–biological effect relationship. This will be done through a systematic comparison between the different level of activities of these macromolecules in their two most studied and well-known biological roles: the AR (a natural physiological event of sea urchin fertilization), and the anticoagulant activity (a current highly desirable alternative pharmacological application for thromboembolic occurrences).

UNDERSTANDING THE INDUCTION OF THE AR IN SEA-URCHIN SPERM BY SULFATED HOMOPOLYSACCHARIDES

  1. Top of page
  2. Abstract
  3. MARINE SULFATED HOMOPOLYSACCHARIDES: COMPLEX STRUCTURES FROM ALGAE VS. CLEAR STRUCTURES FROM INVERTEBRATES
  4. UNDERSTANDING THE INDUCTION OF THE AR IN SEA-URCHIN SPERM BY SULFATED HOMOPOLYSACCHARIDES
  5. INVERTEBRATE SULFATED HOMOPOLYSACCHARIDES AS ANTICOAGULANT AND ANTITHROMBOTIC REAGENTS: A RELIABLE ALTERNATIVE SOURCE FOR HEPARIN
  6. EXAMPLES OF STRUCTURE–FUNCTION RELATIONSHIP OF THE MARINE SULFATED HOMOPOLYSACCHARIDES ON THE ANTICOAGULANT ACTIVITY AND/OR THE AR
  7. MAIN CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

In addition to the sulfated homopolysaccharides found in the extracellular matrices of algae, marine angiosperm, ascidians, and sea cucumbers, sulfated fucans and galactans from sea urchins are also localized in the extracellular matrices. In this case they are found in a hydrated, usually transparent jelly layer surrounding the eggs. The sea urchin egg jelly sulfated polysaccharides form a complex extracellular matrix usually containing a single sulfated fucan or sulfated galactan complexed with many unknown proteins of both high and low molecular mass.22 As described below, the egg sulfated homopolysaccharides are intimately involved in gamete recognition.2, 9–12, 23–26

A necessary event for the sea urchin fertilization is the sperm AR. The sea urchin AR involves the calcium-triggered exocytosis of the acrosome vesicle and the pH-induced polymerization of actin to form the ∼1-μm-long, finger-like, acrosomal process which protrudes from the anterior of the sperm head.27 When sperm approaches the sea urchin egg, the sulfated fucan binds to sperm receptors which are homologs of human polycystin, the protein mutated in autosomal dominant polycystic kidney disease.28 At least two pharmacologically distinct calcium channels open to allow calcium influx from the seawater.29, 30 The internal pH of the sperm also rises about 0.25 pH units due to sodium/proton exchange.31 Both the calcium influx and pH rise are required for AR induction. The AR exposes the protein binding which coats the acrosomal process at the anterior tip of the sperm. The binding attaches the sperm to the EBR1 receptor on the egg surface. Sperm binding mediates both the species-specific attachment of sperm to egg and the fusion of the plasma membranes of the two gametes.32–35 The sequences of bindings are species specific and have been shown to be subjected to positive selection.36 The purified sulfated fucan of egg jelly, devoid of any detectable protein, will by itself induce the sperm AR.22 Induction by the sulfated fucan is potentiated by a polysialic acid containing “sialoglycan” also isolated from egg jelly.37

A preliminary study indicated that the acrosome reaction was induced by sulfated polysaccharides from the sea urchin egg jellies.38 The well-defined chemical structures of the sea urchin egg jelly sulfated fucans, and the observation that each species possesses a polymer with a different structure, strongly suggest that these sulfated polysaccharides are the egg molecules involved in the species-specific induction of the sperm AR.3, 9, 10 Now, it is well-known that cross-species fertilization can be prevented by inhibiting one or more of the five steps that comprise the whole fertilization process: (i) chemotaxis of the sperm to egg-released peptides,39 (ii) induction of the sperm AR by the sulfated fucan (a carbohydrate-based species recognition mechanism) (Figure 3A,a), (iii) binding of the acrosomal process coated with binding to its receptor on the egg surface (a protein-based species recognition mechanism)35 (Figure 3A,b), (iv) penetration of the egg vitelline envelope, and (v) fusion of the plasma membranes of the two gametes.41

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Figure 3. The acrosome reaction. (A) Schematic representation of the two hierarchical steps in sea urchin gamete recognition. (A,a) Sulfated polysaccharide-based species recognition: the sperm acrosome reaction is induced when a sperm with the correct receptor type contacts specific sulfated polysaccharide in the egg jelly coat (red triangles). This reaction exposes the protein binding (blue squares and circles). (A,b) The binding paradigm: the protein binding, coating the outside of the acrosomal process reacts with a matching egg membrane receptor. Data from Biermann et al.25 (B) Confocal images of L. variegatus sperm stained with rhodamine phalloidin and DAPI after incubation with homologous egg jelly (100 μg fucose ml−1). The fluorescent markers are red (phalloidin, for actin), and blue (DAPI, for nucleus). The arrows point to the red rod that represents acrosomal filamentous actin, indicating positive acrosome reaction (R); and the red dot in the sperm head indicating no reaction (NR). Data from Cinelli et al.40

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The synthesis of species-specific structures of the sea urchin sulfated fucans and galactans (Figures 1 and 2) play an important role to induce the AR in a species-specific manner to guarantee the intra-specific egg-sperm recognition. The AR has an intimate relationship with the structural features of the sulfated fucans and galactans. The monosaccharide composition (galactose or fucose), the position of the glycosidic linkage (3- or 4-linked), the sulfation pattern (at 2- or 4-positions), and the number of fucose moieties per repeating unit are all crucial for inducing the sea urchin sperm AR.25, 26

Below we will illustrate how each of these glycosidic structural characteristics can be separately studied on their contribution for the species-specific AR induction in sea urchin sperm. As shown in the example of the AR of Lytechinus variegatus sperm (Figure 3B), the positive AR is detected by fluorescence-labeling of the polymerized actin on the acrosomal process formed at the tip of the reacted sperm head (R at Figure 3B) after the incubation with the inducing sulfated polysaccharide. A weak labeling is noted for the unreacted sperm which show a small tip at the sperm head due to the absence of actin polymerization (NR at Figure 3B). With this procedure, the sulfated fucans (see Figure 1) and sulfated galactans (see Figure 2) were tested by their induction of the AR in homologous as well as heterologous sea urchin sperm from different species. The data obtained from the assays with different types of structure revealed the major structural feature responsible for the AR-induction.

INVERTEBRATE SULFATED HOMOPOLYSACCHARIDES AS ANTICOAGULANT AND ANTITHROMBOTIC REAGENTS: A RELIABLE ALTERNATIVE SOURCE FOR HEPARIN

  1. Top of page
  2. Abstract
  3. MARINE SULFATED HOMOPOLYSACCHARIDES: COMPLEX STRUCTURES FROM ALGAE VS. CLEAR STRUCTURES FROM INVERTEBRATES
  4. UNDERSTANDING THE INDUCTION OF THE AR IN SEA-URCHIN SPERM BY SULFATED HOMOPOLYSACCHARIDES
  5. INVERTEBRATE SULFATED HOMOPOLYSACCHARIDES AS ANTICOAGULANT AND ANTITHROMBOTIC REAGENTS: A RELIABLE ALTERNATIVE SOURCE FOR HEPARIN
  6. EXAMPLES OF STRUCTURE–FUNCTION RELATIONSHIP OF THE MARINE SULFATED HOMOPOLYSACCHARIDES ON THE ANTICOAGULANT ACTIVITY AND/OR THE AR
  7. MAIN CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Unrelated to their natural biological role, the sulfated fucans and sulfated galactans also exhibit potential pharmacological actions in mammalian systems. These include antiviral,42 antimetastatic,43 antiangiogenic,44 antiinflammatory,44, 45 antiadhesive,44, 45 anticoagulant,7, 14, 15, 21, 44, 46, 47 and antithrombotic19, 21, 45, 48 activities. Herein, we will focus on the description of the specific structural features of these sulfated homopolysaccharides only on their anticoagulant and antithrombotic activities due to the pressing need for new antithrombotic drugs as a consequence of the increasing incidence of thromboembolic diseases—cardiovascular diseases are the leading cause of death (30% of total) in the world.

The sulfated fucans and galactans have provided great advantages to be an alternative source for anticoagulant therapies especially because of the massive use of heparin.49 This glycosaminoglycan is well-known to present several limitations due to collateral effects and limited source of material.21 The situation has been further complicated recently because of the alarming discovery that heparin preparations have been contaminated with oversulfated chondroitin sulfate.50 This contaminant induces hypotension associated with kallikrein release when administered by intravenous injection.51

In addition, these new classes of marine sulfated polysaccharides offer a low contamination level of virus and/or prions, because they are exclusively extracted from marine sources. In contrast, contaminated clinical solutions of heparin can occur more easily since this compound is extracted from mammalian sources, porcine intestinal mucosa, or bovine lung.21 In particular, the invertebrate sulfated polysaccharides are promising to be more useful clinical agents than the algal polysaccharides due to their regular and well-defined structures,48 as already mentioned. The invertebrate and red algal macromolecules (Figures 1 and 2) allow a better understanding of their mechanisms of action towards coagulation proteins. On the other hand, the chemical requirements (sulfation and/or glycosylation sites, precise molecular-size, specific structural motif, and influences of branching residues) for the biological effects of the algal polysaccharides are still unknown and hard to be investigated for this specific pharmacological activity (for more details, see Refs.21 and48).

The anticoagulant action of the sulfated polysaccharides resides mainly in the potentiation of the natural inhibitors of the plasma proteases. The plasma proteases include activated factor II (IIa, commonly known as thrombin) and activated factor X (Xa). Inhibitors include the serpins (antithrombin, AT; and heparin cofactor II, HCII). The catalysis of the sulfated polysaccharide can be classified into two distinct mechanisms: the allosteric change on the serpins induced by the sulfated polysaccharides, or the template mechanisms, where the glycan chain of the sulfated polysaccharide can act as a “bridge” that brings together both protease and the serpin.

Next we will describe cases of how each specific structural feature of the marine invertebrate sulfated polysaccharides (and the red algal one) can individually account for the interactions with the serpins and blood proteases, resulting in the blocking of the coagulation process. These particular structural contributions can be properly recognized when we compare together the anticoagulant levels of sulfated polysaccharides that have well-defined structures, whereby they are different, but they share common structural features (Figures 1 and 2). Basically, when only one structural feature is different, the changes of the biological action directly related to the changes of this single structural feature can offer a qualitative result. Thus, this systematic comparison was carried out by two in vitro anticoagulant assays: (i) the aPTT (activated partial thromboplastin time) which measures the general anticoagulant effect where all the plasma proteins are included and (ii) the direct measurement of the inhibition level of factors IIa or Xa by AT and HCII in the presence of the marine sulfated polysaccharides (Table I).

Table I. Anticoagulant Activities of Marine Invertebrate and Algal Sulfated Fucans and Sulfated Galactans Measured by APTTa and by IC50 for Thrombin (IIa) and Factor Xa Inhibition in the Presence of Antithrombin (AT) or Heparin Cofactor II (HCII)21, 48
PolysaccharideSourceStructure (Figure)APTT (IU mg−1)IC50 (μg ml−1)
IIa/ATIIa/HCIIXa/AT
  • a

    The activity is expressed as international U mg−1 using a parallel standard curve based on the International Heparin Standard (193 U mg−1).

3-Linked sulfated α-L-fucansS. purpuratus (I)1E-I760.30.32
S. purpuratus (II)1E-II100.92ND
S. pallidus1C18>500>500>500
L. variegatus1B3>500>500>500
S. franciscanus1G∼2>500>500250
L. grisea1A<1>500>500>500
4-Linked sulfated α-L-fucansS. droebachiensis1F<1NDNDND
A. lixula1D∼2150150>500
Sulfated α-L-galactansE. lucunter2A203620
H. monus2C∼2>500>500>500
S. plicata2B<1>500>500>500
Algal sulfated galactans15, 19B. occidentalis2D930.021.12.5
G. crinale650.02251.5

EXAMPLES OF STRUCTURE–FUNCTION RELATIONSHIP OF THE MARINE SULFATED HOMOPOLYSACCHARIDES ON THE ANTICOAGULANT ACTIVITY AND/OR THE AR

  1. Top of page
  2. Abstract
  3. MARINE SULFATED HOMOPOLYSACCHARIDES: COMPLEX STRUCTURES FROM ALGAE VS. CLEAR STRUCTURES FROM INVERTEBRATES
  4. UNDERSTANDING THE INDUCTION OF THE AR IN SEA-URCHIN SPERM BY SULFATED HOMOPOLYSACCHARIDES
  5. INVERTEBRATE SULFATED HOMOPOLYSACCHARIDES AS ANTICOAGULANT AND ANTITHROMBOTIC REAGENTS: A RELIABLE ALTERNATIVE SOURCE FOR HEPARIN
  6. EXAMPLES OF STRUCTURE–FUNCTION RELATIONSHIP OF THE MARINE SULFATED HOMOPOLYSACCHARIDES ON THE ANTICOAGULANT ACTIVITY AND/OR THE AR
  7. MAIN CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

A Sugar-Type-Dependent Case

When we compare all the structures in Figures 1 and 2, we can trace structural similarities and differences for the sulfated fucans and galactans. Both the sulfated fucan from Strongylocentrotus franciscanus (Figure 1G), and the sulfated galactan from Echinometra lucunter (Figure 2A) present the same sulfation pattern (exclusive and entirely 2-sulfated), the same anomeric configuration (α-form), the same glycosidic linkage position (1[RIGHTWARDS ARROW]3), and the same molecular mass (∼100 kDa), however, they differ exclusively by their sugar types (fucose or galactose, respectively).47 Interestingly, this single structural difference is itself enough to promote great changes in the anticoagulant properties of these homopolysaccharides. The 2-sulfated α-galactan from E. lucunter exhibits a significant anticoagulant activity (aPTT of 20 IU mg−1, although almost 10-fold less than UFH, Table I). The specific anticoagulant assay with the purified proteases revealed that this galactan enhances both IIa and factor Xa inhibition by AT or HCII (Table I). On the other hand, the 2-sulfated α-fucan from S. franciscanus has its anticoagulant effect exclusively based on the catalysis of AT inhibition over factor Xa, however 12.5-fold less active than the sulfated galactan. This single effect over Xa/AT system explains the much lower activity of the compound from S. franciscanus (aPTT of ∼2 IU mg−1, 100-fold less active than UFH), since the anti-Xa activity has relatively minor influence on the aPTT. This is an illustrative example of a sugar-type-dependent biological effect of polysaccharides.

The Sulfation Site-Dependent Cases

Based on this same systematic comparison, the sulfated galactans from the two red algal Botryocladia occidentalis and Gelidium crinale exhibit identical backbone, same chain size but slight differences in their sulfation patterns (Figure 2D). As a consequence of these differences the two sulfated galactans differ in their anticoagulant and venous antithrombotic activities as described by Fonseca et al.19 Sulfated galactan from G. crinale exhibits pro-coagulant and pro-thrombotic effects in low doses (up to 1.0 mg kg−1 body weight), but in high doses (>1.0 mg kg−1) this polysaccharide inhibits both venous and arterial thrombosis in rats. In contrast, sulfated galactan from B. occidentalis is a very potent anticoagulant and antithrombotic compound in low doses (up to 0.5 mg kg−1 body weight), inhibiting venous experimental thrombosis, but these effects are reverted in high doses. Conversely, arterial thrombosis is only inhibited at high doses (>1.0 mg kg−1) of the polysaccharide from B. occidentalis. These results indicate that slight differences in the proportions and/or distribution of sulfated residues along the galactan chain may be critical for the interaction between proteases, inhibitors, and activators of the coagulation system, resulting in a distinct pattern in anti- and pro-coagulant activities and in the antithrombotic action. As summarized in Table I, these structural differences account for 30% of difference in the anticoagulant activity (aPTT) of these algal macromolecules, especially on the catalytic effect of the sulfated polysaccharide on HCII-mediated anti-IIA activity.

Indeed, the structural requirement for the interaction of these polysaccharides with coagulation cofactors and their target proteases and inhibitors are stereospecific.46 The site of sulfation has a major impact on the activity. This can be illustrated by the fact that 2,4-di-sulfated units have an amplifying effect on the AT-mediated anticoagulant activity in the series of 3-linked α-L-fucans (Figure 1, Table I). Specific sulfation sites are required for the interaction with plasma serine-protease inhibitors. Notably, the occurrence of the 4-sulfated unit content in the 3-linked α-L-fucans: L. variegatus (a single 4-sulfated unit/tetrasaccharide—Figure 1B), S. pallidus (a double 4-sulfated unit/tetrasaccharide—Figure 1C), and S. purpuratus, isotype II (a double 4-sulfated unit/trisaccharide—Figure 1E-II), is the structural motif required to enhance the inhibition of IIa by HCII. In contrast, the presence of 2-sulfated residues seems to have a deleterious effect on HCII-mediated anti-IIA activity of the polysaccharide.21

The sulfation site-dependent effect is also observed in the comparative studies of the AR of the species S. franciscanus and S. purpuratus, both from the North Pacific Ocean.12, 24 These species contain 3-linked α-fucans, which differ in the proportions of 2- and 4-sulfation (Figures 1G and 1E). The pattern of sulfation is an important feature for recognition of fucans by the sperm. Sperm from S. purpuratus were sensitive to homologous type-II predominantly 4-sulfated fucan (Figure 1E-II), but not to heterologous 2-sulfated fucan (Figure 1G). In these 3-linked α-sulfated fucans the 2- and 4-positions are the only ones capable of sulfation. Oversulfation of the sulfated fucan from S. purpuratus did not change its responsiveness to homologous sperm, suggesting that increased 2-sulfates do not increase or inhibit the biological activity of this species.3, 26 However, the sulfated fucan from S. franciscanus, which was ineffective on the S. purpuratus sperm, reacted as the homologous fucan after chemical oversulfation because of increased 4-sulfation. Thus, the position of sulfation is crucial for the acrosome reaction-inducing activity of the sulfated fucans of these two Strongylocentrotid species rather than the nonspecific charge density or sulfate content of the polysaccharides.

The Glycosidic Linkage-Dependent Cases

The sea urchin species S. droebachiensis expresses an entirely 2-sulfated α-L-fucan, identical to the sulfated homopolysaccharide from the egg jelly of S. franciscanus (Figure 1G), except for the glycosidic linkage at the 4-position (Figure 1F) instead of the 3-position. The sulfated fucan from S. droebachiensis does not induce the AR on the sperm from S. franciscanus, showing a strict requirement for the (1[RIGHTWARDS ARROW]3) linkage glycosidic bond in the recognition between the S. franciscanus sperm and the 2-sulfated fucans. The small structural change of the glycosylation position is itself enough to block the inter-specific fertilization of these species.24

Another example of AR mainly regulated by glycosidic linkage type comes from the observation of the cross-AR between the species S. purpuratus and S. pallidus. These species express α(1[RIGHTWARDS ARROW]3) linked sulfated fucans, although with different sulfation patterns (Figures 1C and 1E). However, both sulfated fucans were able to induce the AR in heterologous sperm. In this case, other structural modifications (such as the sulfation pattern) do not impair the induction of interspecific AR.3 Both cases described above are fine and rare examples of a glycosidic linkage position-dependent biological action of carbohydrates in the glycobiology field.3, 24

An Orientational Binding Preference-Dependent Case

The interaction energies obtained from molecular dynamic (MD) simulations were statistically equivalent for both 2-sulfated α-L-galactan from E. lucunter (Figure 2A) and 2-sulfated α-L-fucan from S. franciscanus (Figure 1H),52 as it could be expected based on the structural similarities of these two compounds, especially conformations in solution. However, they revealed markedly different interactions for the binding with the complex AT/IIa explained by their extremely different orientations on the AT binding site (∼90°, Figures 4C and 4D). Such difference in orientation upon binding to AT explains the lack of activity of fucan under the same experimental conditions and fully supports the expected bridging mechanism in the activity of galactan, as previously described.53 Thus, both compounds are capable of interacting with AT, as indicated by our theoretical results and on experimental data showing that both polysaccharides are retained on an AT-affinity column.53 However, only the complex formed by the sulfated galactan and the AT (Figure 4D) allows a further interaction with IIa, analogous to the ternary complex between heparin, AT, and IIa (Figure 4A), supporting the potentiation of this protease inactivation by AT in the presence of the sulfated galactan. This is an example of how the orientational constraints in solution can influence the spatial preferences of the sulfated polysaccharides for their binding affinities with target proteins.52

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Figure 4. Structures of the complexes between different polysaccharides and AT. (A) ternary complex between AT, thrombin and a heparin derivative (PDB ID 1TB6); (B) AT bounded to the synthetic pentasaccharide (PDB ID 1E03); (C) final structure from a 5 ns MD of AT complexed to a sulfated fucan decasaccharide with pyranose rings in 1C4 form; (D) final structure from a 5 ns MD of AT complexed to a sulfated galactan decasaccharide. For (B)–(D) two orientations of the complexes are presented. Data from Becker et al.52

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MAIN CONCLUSIONS

  1. Top of page
  2. Abstract
  3. MARINE SULFATED HOMOPOLYSACCHARIDES: COMPLEX STRUCTURES FROM ALGAE VS. CLEAR STRUCTURES FROM INVERTEBRATES
  4. UNDERSTANDING THE INDUCTION OF THE AR IN SEA-URCHIN SPERM BY SULFATED HOMOPOLYSACCHARIDES
  5. INVERTEBRATE SULFATED HOMOPOLYSACCHARIDES AS ANTICOAGULANT AND ANTITHROMBOTIC REAGENTS: A RELIABLE ALTERNATIVE SOURCE FOR HEPARIN
  6. EXAMPLES OF STRUCTURE–FUNCTION RELATIONSHIP OF THE MARINE SULFATED HOMOPOLYSACCHARIDES ON THE ANTICOAGULANT ACTIVITY AND/OR THE AR
  7. MAIN CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

The example cases in this review showed clearly how the structural features of the marine homopolysaccharides (sulfated fucans and galactans) can be distinctly evaluated for interactions with the coagulation proteins (acting as promising anticoagulant or antithrombotic reagents) as well as with the specific receptor at the sea urchin sperm which triggers the AR (a very rare case of pure carbohydrate-based signaling transduction). It was observed that the carbohydrate–protein complexes are stereospecific and not a mere consequence of charge density of the sulfated polysaccharide through electrostatic interactions. It is important to notice that these structural characteristics do not account independently and/or exclusively for these actions. Obviously, the active or inactive conformations of these homopolysaccharides arise from the presence of all structural features together which comprise the “final code” required for cell signaling, recognition, and interaction with other molecules. However, specific structural features of the carbohydrates can indeed preponderantly regulate the biological processes, as described above.

REFERENCES

  1. Top of page
  2. Abstract
  3. MARINE SULFATED HOMOPOLYSACCHARIDES: COMPLEX STRUCTURES FROM ALGAE VS. CLEAR STRUCTURES FROM INVERTEBRATES
  4. UNDERSTANDING THE INDUCTION OF THE AR IN SEA-URCHIN SPERM BY SULFATED HOMOPOLYSACCHARIDES
  5. INVERTEBRATE SULFATED HOMOPOLYSACCHARIDES AS ANTICOAGULANT AND ANTITHROMBOTIC REAGENTS: A RELIABLE ALTERNATIVE SOURCE FOR HEPARIN
  6. EXAMPLES OF STRUCTURE–FUNCTION RELATIONSHIP OF THE MARINE SULFATED HOMOPOLYSACCHARIDES ON THE ANTICOAGULANT ACTIVITY AND/OR THE AR
  7. MAIN CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES