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Braz J Med Biol Res 40(1) 2007

A carbohydrate-based mechanism of species recognition

A carbohydrate-based mechanismof species recognition in sea urchinfertilization*

Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho,and Programa de Glicobiologia, Instituto de Bioquímica Médica,Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

P.A.S. Mourão

Abstract

In the present review, we describe a systematic study of the sulfatedpolysaccharides from marine invertebrates, which led to the discoveryof a carbohydrate-based mechanism of sperm-egg recognition duringsea urchin fertilization. We have described unique polymers present inthese organisms, especially sulfated fucose-rich compounds found inthe egg jelly coat of sea urchins. The polysaccharides have simple,linear structures consisting of repeating units of oligosaccharides.They differ among the various species of sea urchins in specificpatterns of sulfation and/or position of the glycosidic linkage withintheir repeating units. These polysaccharides show species specificityin inducing the acrosome reaction in sea urchin sperm, providing aclear-cut example of a signal transduction event regulated by sulfatedpolysaccharides. This distinct carbohydrate-mediated mechanism ofsperm-egg recognition coexists with the bindin-protein system. Possi-bly, the genes involved in the biosynthesis of these sulfated fucans didnot evolve in concordance with evolutionary distance but underwenta dramatic change near the tip of the Strongylocentrotid tree. Overall,we established a direct causal link between the molecular structure ofa sulfated polysaccharide and a cellular physiological event - theinduction of the sperm acrosome reaction in sea urchins. Smallstructural changes modulate an entire system of sperm-egg recogni-tion and species-specific fertilization in sea urchins. We demonstratedthat sulfated polysaccharides - in addition to their known function incell proliferation, development, coagulation, and viral infection -mediate fertilization, and respond to evolutionary mechanisms thatlead to species diversity.

CorrespondenceP.A.S. Mourão

Laboratório de Tecido Conjuntivo

Hospital Universitário

Clementino Fraga Filho, UFRJ

Caixa Postal 68041

21941-590 Rio de Janeiro, RJ

Brasil

Fax: +55-21-2562-2090

E-mail: [email protected]

*This review is dedicated to Prof.

Carl Peter Dietrich, in memoriam.

Research supported by CNPq and

FAPERJ.

Received September 18, 2006

Accepted October 31, 2006

Key words• Acrosome reaction• Fertilization• Sulfated fucans• Glycosaminoglycans• Sulfated galactans• Species diversity• Sperm-egg recognition

Brazilian Journal of Medical and Biological Research (2007) 40: 5-17ISSN 0100-879X Review

A preamble: memories of thebeginning at Prof. C.P. Dietrich’slaboratory

This review is dedicated to the memoryof Prof. C.P. Dietrich, who died on February1, 2005 in São Paulo. I started my scientific

career in 1971 under the supervision of Prof.Dietrich while I was a medical student atEscola Paulista de Medicina (presently Fe-deral University of São Paulo). As I lookback on this period of my life, two majorimpacts related to my involvement with Prof.Dietrich’s group come to mind. The first was

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the conviction he transferred to all his stu-dents that we were involved in internationaland competitive science although workingin a poor and less developed country. Myemphasis on this aspect may surprise mostof us, the Brazilian readers, because theextraordinary development and growth ofscience in our country during the last threedecades. But the general spirit at that timewas that “we do not produce new knowledgebut just repeat what has already been done inthe developed nations”, as some of my friendsand even some professors at Escola Paulistade Medicina used to say. Certainly it was ageneration of Brazilian scientists that helpedplace Brazilian science on its present profes-sional level, and Prof. Dietrich was an im-portant leader of that generation.

The second impact on my memory comesas Prof. Dietrich participated in a sympo-sium in Argentina in honor of Prof. L. Leloir,who received the Nobel Prize due to hiswork on biosynthesis of carbohydrates. Prof.Dietrich was impressed by the opening lec-ture of Prof. Leloir, when he mentioned thatthe success of a soccer player is to positionhimself properly in the field and then waitfor the ball. If he tries to follow the ball hewill always arrive late. Curiously, this is thesame advice given by Pelé, the famous Bra-zilian soccer player. Prof. Leloir extrapo-lated the philosophy of the soccer game tothe scientific activity of young scientists. Hesuggested that they should direct their workbased mostly on their own results instead offollowing the literature. When a scientificpaper is published, the author is alreadyforward in his experiments, and if we try tofollow his line of work we will tend to arrivelast. Look for new ideas in your own results,advised Prof. Leloir, and create your way inscience with imagination and creativity. Ikept these comments in mind as the “Leloir/Dietrich rule for a successful scientific ca-reer”. This advice is especially relevant forscientists working in laboratories with con-stant lack of funds, with difficulties to access

new reagents and equipment.Once more, this comment is apparently

out of date, when the directions of Brazilianscience follow a “new paradigm”, based onnetworks of research groups concentratedon the investigation of specific topics. Ap-parently, the individuality of a scientist isdiluted and the freedom to delineate his workis limited. But this is not a consensus and the“Leloir/Dietrich rule” is revived through thecomments of several other scientists. Forexample, J.L. Goldstein and M.S. Brown,who received the Nobel Prize for their workon the metabolism of plasma lipoproteins,stated that the fundamental discoveries inmedical science come from creative scien-tists working in individual and low budgetprojects (1). Again, it is the “Leloir/Dietrichrule” in different words!

I believe that an appropriate way to honorthe memory of Prof. Dietrich in this reviewis to describe how we have been followingthe “Leloir/Dietrich rule” and have madefundamental discoveries in our laboratoryon the biological roles of sulfated polysac-charides. In our publications we usually de-scribe the fundamental concepts for our ex-periments but do not mention some of thestrategies behind the work. This review, dedi-cated to Prof. Dietrich, constitutes an oppor-tunity to follow this distinct way of present-ing our experimental results.

Why were we interested in sulfatedpolysaccharides from marineinvertebrates?

During my initial work in Prof. Dietrich’slaboratory I studied glycosaminoglycansfrom different types of cartilages. We re-ported that growth cartilages contain higherproportions of chondroitin 4-sulfate than ar-ticular cartilages, where chondroitin 6-sul-fate is more abundant (2). Furthermore, wedescribed a metabolic error characterized bya deficiency of 6-sulfation of chondroitinsulfate. These patients present pathological

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changes of their articular cartilages but with-out modifications of the growth cartilages(3,4). Overall, these observations indicatethat chondroitin 4-sulfate is related to theossification process while the 6-sulfated iso-mer is important for the structural integrityof the cartilages. Furthermore, the relativeamount of chondroitin 4-sulfate increases intumoral and arthrotic cartilages (5,6), inwhich calcification commonly occurs. Inaddition, phylogenetic studies have shownthat cartilages from bony fish contain morechondroitin 4-sulfate than cartilages fromcartilaginous fish (7). In other studies, Math-ews and Glacov (8) showed that calcifiedskull cartilage contains more chondroitin 4-sulfate than uncalcified skull cartilage. Thenext step, from a phylogenetic point of view,was to look for cartilage-like tissues in in-vertebrates and to investigate the type ofisomeric chondroitin sulfate they contain.

But we then faced a conceptual questionregarding the definition of cartilage. What isin fact cartilage? We found a “definition” forcartilage in a classical book by Mathews (9):“Cartilage is like pornography. It is difficultto define but it can be easily recognized by aspecialist”! However, we preferred a moreformal concept of cartilage-like structures ininvertebrates, which we defined as thosewith properties similar to vertebrate connec-tive tissues, such as a structural function, arelatively high proportion of extracellularmatrix and relatively low proportion of cellsand, finally, a high concentration of proteo-glycan or other acidic polysaccharides. Withthe help of some biology students we wereable to identify two tissues in marine inver-tebrates which fulfilled these characteristics- the tunic of ascidians (Chordata - Tunicata)and the body wall of the sea cucumber (Echi-nodermata - Holothuroidea).

Sulfated L-galactans in the tunic ofascidians

The tunic of ascidians is an external sup-

portive and protective skeleton (10). It con-sists of dense bundles of fibrillar materialembedded in a loose network of fine fibrils,an organization reminiscent of the verte-brate cartilage.

Large amounts of sulfated polysaccha-rides were found in ascidian tunics. To oursurprise, we found that this tissue does notcontain glycosaminoglycans, as is the casefor mammal connective tissues, but a uniquesulfated galactan (11). The structure of thiscomplex polysaccharide was determinedusing a vast array of techniques, such asperiodate oxidation, methylation analysis andnuclear magnetic resonance spectroscopy(12-15). This polysaccharide consists mainlyof a carbohydrate core of galactose glyco-sidically linked through position 1→4 andsulfated at position 3. In some species thecentral polysaccharide core is substituted atthe O-2 position by non-sulfated galactoseunits. Even more significant, the sulfatedgalactans from ascidians are entirely consti-tuted of L-galactose, the D-enantiomorphbeing entirely absent (16). This is unusualsince D-galactose is the common constituentof glycoconjugates.

A fucosylated chondroitin sulfate inthe body wall of the sea cucumber

The body wall of the sea cucumber isformed mainly by collagen fibers embeddedin an amorphous matrix. Small irregular mi-crofibrils which resemble the proteoglycansfrom mammalian tissues, form bridges be-tween the collagen fibers (Figure 1A).

Again, in this tissue we did not findglycosaminoglycans with structures similarto those of mammalian tissues. The majorpolysaccharide in the sea cucumber bodywall was denominated fucosylated chon-droitin sulfate. It is composed of a chon-droitin sulfate-like core containing sidechains of sulfated fucose units linked to theglucuronic acid moieties through the O-3position of the acid (Figure 1B). The pres-

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P.A.S. Mourão

sea cucumber makes these polysaccharidesresistant to degradation by D-galactosidases,hyaluronidases and chondroitin sulfate ly-ases, and therefore prevents the digestion ofthe ascidian tunic and the body wall of thesea cucumber by microorganisms present inthe marine environment. Especially note-worthy is also the observation that the poly-saccharides from the marine invertebratesare in general more sulfated than the gly-cosaminoglycans from mammalian connec-tive tissues. Perhaps interactions betweencomponents of the extracellular matrix inthese organisms occur at higher salt concen-trations than in vertebrates and thereforerequire polysaccharides with higher chargedensity.

Our initial comparative biochemicalproject, with a focus on a phylogenetic studyof cartilaginous chondroitin sulfate, led us toan unexpected stage position. We had newsulfated polysaccharides with unique struc-tures extracted from the invertebrate tissues.Of course these findings opened new per-spectives, such as studies on the biosynthe-sis of these unique polysaccharides (20,21),and an interest in their possible effects asmodulators of a variety of events, such ascoagulation (22-25), thrombosis (26-28),proliferation of vascular endothelial andsmooth muscle cells (29), angiogenesis (30),viral infection (31), etc.

Unique sulfated fucans inechinoderms

In addition to the fucosylated chondroitinsulfate, the body wall of the sea cucumbercontains small amounts of a sulfated fucan.The purification of this polysaccharide wasvery laborious. But, once we overcame thischallenge, the structure of the sulfated fucanwas rapidly determined using a combinationof methylation analysis and nuclear mag-netic resonance spectroscopy (32). Againwe were lucky to find a single polysaccha-ride, composed of tetrasaccharide repeating

Figure 1. Electron micrograph ofa section of the body wall of thesea cucumber showing collagenfibrils and the presence of mi-crofibrils (indicated by the arrow)in the ground substance (A). Themajor sulfated polysaccharidesisolated from this tissue are afucosylated chondroitin sulfate(B) and a sulfated fucan (C).See Junqueira et al. (17) for fur-ther details about the ultrastruc-ture of the body wall of the seacucumber.

ence of these unusual sulfated fucosebranches obstructs the access of chondroitinsulfate lyases and hyaluronidases to the chon-droitin sulfate core. However, after partialacid hydrolysis, which removes the sulfatedfucose branches, the polymer is degraded bychondroitin sulfate lyases into 6-sulfated andnon-sulfated disaccharides (18,19).

The biological relevance of these un-usual polysaccharides found in marine in-vertebrates is unclear. Possibly, the pres-ence of L-isomers of galactose in the ascid-ian polysaccharides and of sulfated fucosebranches in the chondroitin sulfate from the

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units, in which the 4 residues are 3-linkedfucose units differing only by specific pat-terns of sulfation at the O-2 and O-4 posi-tions (Figure 1C).

Sulfated fucans have been found in otherorganisms, namely the cell walls of marinealgae and the jelly coat of sea urchin eggs(for a review, see Refs. 33 and 34). The firstisolation of sulfated fucans (also known asfucoidan) from marine algae was reportedalmost 90 years ago (35) but studies of theirstructure have led to controversial results.The first evidence showing that sulfatedfucans also occur in sea urchin was pub-lished 58 years ago (36) but no structuralstudy was performed on these compounds.We then decided to undertake a systematicanalysis of the structure of the sulfated fucansfrom echinoderms and marine algae.

Our studies (23,24) and also studies fromother laboratories (37,38) showed that thesulfated fucans from brown algae have com-plex and heterogeneous structures. More re-cent studies have revealed the occurrence ofordered repeat units in the sulfated fucansfrom some species of algae (33,34). Even inthese cases, the presence of highly branchedportions and the complex distributions ofsulfate and acetyl groups highlight the het-erogeneity of algal fucans. In contrast, theechinoderm polysaccharides have simple,ordered structures, which differ in the spe-cific patterns of sulfation and/or position ofthe glycosidic linkages within their repeat-ing units (39-42). Some of these structuresare shown in Figure 2.

Sulfated fucans are species-specificinducers of the acrosome reactionduring sea urchin fertilization

Sulfated fucans from marine brown al-gae and from the body wall of the sea cu-cumber are components of the extracellularmatrix and have a structural function analo-gous to that of the proteoglycans from mam-malian connective tissues. However, in sea

urchins, the sulfated fucans are found on agelatinous layer which surrounds the eggs.This specific localization of the sulfatedfucans suggests that the polysaccharides maybe involved in the fertilization of these in-vertebrates.

A necessary event for successful fertili-zation is the acrosome reaction. When spermapproach the sea urchin egg, the egg jellyinduces the sperm acrosome reaction, whichexposes the protein bindin at the tip of thesperm head. Only then can sperm attach tothe egg and their plasma membrane fuse(43-46).

The acrosome reaction was discoveredin sea urchins, starfish and several othermarine invertebrates by J.C. Dan, workingin Japan in the early 1950s. In 1947 J.C. Danrevisited her home country, the USA, for thefirst time after the difficult years in Japanduring World War II. She took the firstcommercially available phase contrast mi-croscope back to Japan and in the followingyear set up a research program on fertiliza-tion of marine invertebrates. Appreciatingthe great technological advantages of phasecontrast microscopy, she first explored anold problem of sperm entry into eggs. Thenshe concentrated on the changes in the spermtip upon fertilization, for which she alsoapplied electron microscopy, another greattechnological innovation at that time. Shediscovered the biological significance of thechanges, for which she introduced the term“acrosome reaction”, detected through care-ful observation of living spermatozoa underthe phase microscope and intensive exami-nation of spermatozoa, immediately afterfixation in a sea water suspension, with anelectron microscope. The electron micro-scope that she used was a very primitivemodel, although it was one of the latest inthose days - the fourth machine of HitachiElectron Micrograph. For a historical re-view, see Hoshi et al. (47).

Despite extensive studies on the mor-phological and physiological changes that

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P.A.S. Mourão

occur in the spermatozoa during the acro-some reaction in the following four decades,the specific molecule from the egg jellyinvolved in the induction of the acrosomereaction in sea urchins was not identifieduntil 1994, when Keller and Vacquier (48)reported that “purified sulfated fucans hadno significant acrosome reaction-inducingactivity. Instead, acrosome reaction-induc-ing activity was associated only with twoglycoproteins with approximate relative sub-unit masses of 82 and 138 kDa”. Even so, we

decided to test whether sulfated polysaccha-rides from the sea urchin egg jelly induce theacrosome reaction. The well-defined chem-ical structure of these polysaccharides andthe observation that each species possesses apolymer with a different structure (Figure 2)suggested that these sulfated polysaccha-rides may regulate a specific biological eventduring sea urchin fertilization. In fact, whenwe tested purified sulfated polysaccharidesfrom the sea urchin egg jelly with homolo-gous and heterologous sperm from species

Figure 2. Structures of sulfatedpolysaccharides from marine in-vertebrates. The figure shows 9fully characterized structures ofsulfated polysaccharides fromthe egg jelly of sea urchins andthe tunic of ascidians. The spe-cific pattern of sulfation and theposition of the glycosidic linkagevary among sulfated fucans fromdifferent species. The structuresof some α-L-galactans are alsoincluded in the figure. L-galac-tose and L-fucose are closelyrelated sugars, differing only bythe CH2OH and CH3 radicals atposition 5, respectively. In fact,a common designation for L-fu-cose is 6-deoxy-L-galactose.

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that co-habit the same area in Rio de Janeiro,we observed that the sulfated polysaccha-rides are species-specific inducers of theacrosome reaction (Figure 3).

The fact that the sulfated fucans are spe-cies-specific inducers of the acrosome reac-tion in sea urchins was confirmed when thestudy was extended to the species Strongylo-centrotus franciscanus and S. purpuratus,both from the North Pacific Ocean (41,49).These species contain 3-linked fucans, whichdiffer in the proportions of 2- and 4-sulfa-tion. The pattern of sulfation is an importantfeature for recognition of fucans by the sperm.As exemplified in Figure 4, sperm from S.purpuratus were sensitive to homologous 4-sulfated fucan but not to heterologous 2-

sulfated fucan (red symbols in Figure 4). Inthese 3-linked sulfated fucans the 2- and 4-positions are the only ones capable of sulfa-tion. Oversulfation of the sulfated fucan fromS. purpuratus did not change its responsive-ness to homologous sperm, suggesting thatincreased 2-sulfates do not increase or inhib-it the biological activity of this species (indi-cated by blue symbols in Figure 4). How-ever, the sulfated fucan from S. franciscanus,which was ineffective on the S. purpuratussperm, reacted as the homologous fucan af-ter chemical oversulfation because of in-creased 4-sulfation. Thus, the position ofsulfation is crucial for the acrosome reac-tion-inducing activity of the sulfated fucansof these Strongylocentrotus species rather

Figure 3. Structure and effect of sulfated polysaccharides from the sea urchin egg jelly as inducers of the acrosome reaction in sperm of three speciesthat co-exist in the sea harbor of Rio de Janeiro, Brazil. Data modified from Alves et al. (39) (printed with permission).

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than the nonspecific charge density or sul-fate content of the polysaccharides.

Two mechanisms of sperm-eggrecognition in sea urchin fertilization

We demonstrated that sulfated fucansfrom the egg jelly induce the sperm acro-some reaction in a species-specific way.However, we had not investigated the con-tribution of this finding to the final biologi-cal event, that is, the fertilization of the seaurchin eggs. Furthermore, the relationshipbetween the carbohydrate-based mechanismof sperm-egg recognition and the well-knownbindin protein paradigm (43-46) remainedto be clarified.

At this stage we outlined an experimentalapproach to distinguish between the bindinand the carbohydrate-based recognition

mechanisms using three closely related sym-patric Strongylocentrotus species (50). Ini-tially we determined fertilization among thespecies assessed by counting the propor-tions of fertilized eggs (Figure 5A). Forconspecific gametes we observed a high per-centage of successful fertilization, whereasheterospecific sperm fertilized only a smallpercentage of eggs. After pre-reaction of thesperm with conspecific egg jelly, interspe-cific fertilization between S. droebachiensisand S. pallidus increased significantly inboth directions. In contrast, the eggs of S.purpuratus could not be fertilized, even af-ter reacting the heterologous sperm withconspecific egg jelly.

These observations indicated that induc-tion of the acrosome reaction by the egg jellysulfated polysaccharides is the major limita-tion for interspecific fertilization between S.droebachiensis and S. pallidus (carbohy-drate-base species recognition mechanism).In contrast, the major limitation for interspe-cific fertilization between S. purpuratus andthe other two species is a step that followsthe acrosome reaction. Possibly this involvesreaction of the bindin protein, exposed onthe outside of the sperm tip, with the match-ing egg membrane receptor (the protein para-digm of species recognition). These two hi-erarchical steps in sea urchin gamete recog-nition are shown in Figure 6.

To investigate whether failure of thesperm acrosome reaction, induced by eggjelly sulfated polysaccharides, is in fact themajor limitation of interspecific fertilizationbetween S. droebachiensis and S. pallidus,we determined the proportion of sperm thatundergoes the acrosome reaction in responseto egg jelly sulfated polysaccharide (Figure5B). S. droebachiensis sperm responded ex-clusively to the conspecific polysaccharide,whereas S. pallidus and S. purpuratus cross-reacted. S. droebachiensis contains a sulfatedfucan with a structure unique among the otherStrongylocentroid species examined, since itcontains 4-linked instead of 3-linked fucose

Figure 4. Structures and effect of sulfated fucans from the egg jelly of two Strongylocentro-tus species as inducers of the acrosome reaction on homologous and heterologous sperm.These species contain 3-linked fucans, which differ in the proportion of 2- and 4-sulfation(represented in red). Chemical oversulfation of these polysaccharides adds additionalsulfate esters to the polysaccharides at the 2- and 4-positions (represented in blue), theonly ones capable of sulfation. Note that oversulfation of sulfated fucan conferred acro-some reaction activity to heterologous sperm but did not increase the activity of homolo-gous ones. Data modified from Hirohashi et al. (49) (printed with permission from Elsevier).

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Figure 5. Experimental approach usedto distinguish between the bindin andthe carbohydrate-based mechanismsof sperm-egg recognition in sea urchinfertilization. A, Successful fertilizationamong three Strongylocentrotus spe-cies using sperm diluted in sea water(A-1) and pre-reacted with conspecificegg jelly (A-2). B, Structures of sul-fated fucans isolated from the egg jel-lies of sea urchins and their effects asinducers of the acrosome reaction.The sulfated fucans were used at aconcentration of 100 µg hexose/mL.Note that this is a higher concentrationof sulfated polysaccharide than thoseused in the experiments in Figure 4.Egg jellies of S. purpuratus containtwo isoforms of sulfated fucans shownin the panel. Data modified fromBiermann et al. (50) (printed with per-mission from Blackwell Publishing).See text for further details.

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terizes a carbohydrate-based species recog-nition mechanism. There are no other sig-nificant barriers to interspecific fertilizationbetween these two species. Other speciespairs in the same genus acrosome react non-specifically to egg jelly polysaccharide butexhibit species-specific sperm binding dueto the bindin protein (the protein paradigmof species recognition) (Figure 6).

Sulfated fucans: another avenue forspeciation in sea urchins?

Phylogenetic relationships, the times ofdivergence of sea urchin species and a sum-mary of the structure of the polysaccharidesfrom their egg jellies are shown in Figure 7.These observations indicate that the genesinvolved in the biosynthesis of the sulfatedfucans did not evolve in concordance withevolutionary distance but underwent a dra-matic change near the tip of the Strongylo-centrotid tree driven by natural selection.Possibly, the acrosome reaction specificitymay have played a direct role in the separa-

Figure 6. Schematic representa-tion of the two hierarchical stepsin sea urchin gamete recogni-tion. A, Carbohydrate-basedspecies recognition: the spermacrosome reaction is inducedwhen a sperm with the correctreceptor type contacts specificsulfated polysaccharide in theegg jelly coat (red triangles).This reaction exposes the bindinprotein (shown in blue). B, Theprotein paradigm: the bindin pro-tein, coating the outside of thesperm tip, reacts with a match-ing egg membrane receptor. AR= acrosome reaction. Reprintedfrom Biermann et al. (50) (withpermission from Blackwell Pub-lishing).

Figure 7. Phylogenetic relation-ship and divergence times ofsome sea urchin species and asummary of the structure of thepolysaccharides from their eggjelly. Possibly, the carbohydrate-mediated mechanism of egg-sperm recognition played a di-rect role in the separation of S.droebachiensis from S. pallidus.The bindin mechanism mayhave functioned as an isolationmechanism on the earlier sepa-ration of the joint lineage from S.purpuratus. Myr = million yearsof evolutionary divergence. Datamodified from Biermann et al.(50) (printed with permissionfrom Blackwell Publishing).

units. This sulfated fucan specifically trig-gered the acrosome reaction only in spermfrom its own species. Sperm from S. pallidusand S. purpuratus responded to conspecificand heterospecific 3-linked but not to 4-linkedsulfated fucans, independent of their sulfationpatterns at the O-2 and O-4 positions.

Overall, we have shown that sulfatedcarbohydrate from egg jelly induces theacrosome reaction species specifically in S.droebachiensis and S. pallidus. This charac-

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tion of these species. There is evidence thatS. droebachiensis and S. pallidus separatedfrom S. purpuratus before their divergencefrom each other. The bindin mechanism mayhave functioned as an isolation mechanismin the earlier separation of the joint lineagefrom S. purpuratus. A renewed and laterspeciation event originated the species S.droebachiensis and S. pallidus due to theegg jelly sulfated fucans and sperm incom-patibility (carbohydrate-based mechanism).

Surprisingly the egg jelly of the sea ur-chin S. droebachiensis contains a 4-linkedsulfated fucan clearly distinct from those ofthe phylogenetically related species but simi-lar to that from A. lixula - a species thatdiverged approximately 200 millions yearsago. This fact characterizes an event of con-vergent evolution - identical sulfated fucansin species that diverged millions of yearsago. The occurrence of the same fucan inthese two species is not relevant for theircross-fertilization because the populationsof A. lixula and S. droebachiensis do notoverlap geographically. Nevertheless, thisobservation suggests that the gene for thebiosynthesis of 4-linked fucan may havebeen retained during the evolution of S.droebachiensis, but remained repressed ornon-expressed until a period of strong natu-ral selection. This observation reminds us ofa general concept regarding evolution, stat-ing that “the past of an organism not onlydetermines its future but also gives an enor-mous reserve for a rapid modification, basedon little genetic changes” (51).

Conclusions and challenges

Our studies on the structural characteriza-tion of sulfated polysaccharides from marineinvertebrates led us to discover unique poly-mers in these organisms, especially sulfatedfucose-rich compounds found in the egg jellyof sea urchins. These polysaccharides havesimple, linear structures composed of repeat-ing oligosaccharide units. They differ among

the various species of sea urchins in terms ofspecific patterns of sulfation, the position ofthe glycosidic linkage within their repeatingunits and in the type of constituent monosac-charide. These polysaccharides show speciesspecificity in inducing the acrosome reactionin sea urchins, providing a clear-cut exampleof a biological transduction event regulated bysulfated polysaccharide. This distinct carbo-hydrate-mediated mechanism for cell-cell rec-ognition during fertilization co-exists with thebindin-protein system. Our results may lead toan overall view of carbohydrate-mediated fer-tilization in sea urchins and indicate how theegg jelly polysaccharide evolved and led tospecies diversity.

The major challenge at this stage is theidentification of the metabolic pathways in-volved in the biosynthesis of the egg jellypolysaccharides. This is not only a fascinat-ing challenge for carbohydrate researchers,but may also help to define the genetic basisfor the proposed carbohydrate mechanismof species recognition.

Finally, we hope this review is convinc-ing in the sense that we were able to obtainnew results of general biological interestfollowing the “Leloir/Dietrich advice”. Themotive to follow this route was to look al-ways for inspiration in our own results. Wehope this is a proper way to honor the memoryof Prof. Carl Peter Dietrich.

Acknowledgments

The contributions to this work by A.P.Alves, A.C.E.S. Vilela-Silva and A.P. Va-lente (Universidade Federal do Rio de Janei-ro), B. Mulloy (National Institute for Bio-logical Standards and Control), V.D.Vacquier (Scripps Institution of Oceanogra-phy) and C.H. Biermannn (Friday HarborLaboratories, University of Washington) areduly acknowledged. We are grateful to Dr.Maria Christina Mello (Universidade Fede-ral do Rio de Janeiro) for revising the manu-script.

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