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The echinoderm lytic systemCalogero Canicattì aa Dipartimento di Biologia , Università di Lecce , Via Prov.le Lecce‐Monteroni, Lecce, I‐73100,ItalyPublished online: 28 Jan 2009.

To cite this article: Calogero Canicattì (1992) The echinoderm lytic system, Bolletino di zoologia, 59:2, 159-166, DOI:10.1080/11250009209386664

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Boll. Zool. 59: 159-166(1992)

The echinoderm lytic system

CALOGERO CANICATTÌDipartimento di Biologia, Università di Lecce,Via Prov.le Lecce-Monteroni, 1-73100 Lecce (Italy)

INTRODUCTION

Lysins are biologically active substances characterizedby their lytic properties against foreign targets. In inver-tebrates, these molecules are important factors involvedin the humoral defence mechanisms (Canicattï, 1990a).

Lytic activity has been evidenced in several groupsboth of acoelomate (Bretting & Renwrantz, 1973; Kamiyaet al., 1985; Norton et al., 1989) and coelomate(Weinheimer et al., 1969, 1970; Day et al., 1972; Bretting& Renwrantz, 1973; Parrinello et al., 1979; Anderson,1980; Parrinello & Rindone, 1981; Cenini, 1983; Tuan &Yoshino, 1984; Tuckova et al., 1986; Canicattï, 1987a;Leippe & Renwrantz, 1988) invertebrates. It depends onmolecules of proteic nature which show a remarkabledegree of resemblance to cytolytic peptides (Bernheimer& Rudy, 1986; Bernheimer, 1990). Generally, the lytic ac-tivity of the invertebrate body fluids is evidenced byusing vertebrate erythrocytes as experimental target. Theresulting lytic effect is therefore referred to as hemolytic.However, other kinds of cells, such as fibroblasts andblood platelets, are lysed by lysins. Bacteria are alsouseful targets to demonstrate killing properties of thelytic system (Roch, 1979; Hültmark et al., 1980; Roch etal., 1981; Anderson & Chain, 1982; Valembois et al.,1986).

The biological properties of invertebrate lyticmolecules appear to depend upon the formation of trans-membrane channels that are formed by aggregation ofmolecules in the membrane bilayer (Bernheimer & Rudy,1986; Roch et al., 1989; Canicattï, 1990a). These obser-vations suggest a resemblance with vertebrate pore-forming proteins (Canicattï, 1990a).

Much attention has been paid to echinodermhemolysins, especially because of their phylogeneticposition. The aim of the present review is to summarizecurrent knowledge on the lytic system of this highlydiversified group.

NATURALLY OCCURRING HEMOLYSIN

ABSTRACT

Echinoderms possess factors responsible for lysis of target cells.Information on their nature, occurrence, physical and chemicalproperties and biological role are summarized. Further results onbinding properties to surface components are discussed, including ahypothetical explanation both for hemolysin-target interaction andregulation.

KEY WORDS: Echinoderms - Hemolysin - Opsonin.

ACKNOWLEDGEMENTS

I am grateful to a number of former and present colleagues whocollaborated in the study of the echinoderm lytic system. They in-clude: V. Arizza, D. Ciulla, E. L. Cooper, G. D'Ancona, E. Farina-Lipari, A. Miglietta, N. Parrinello, D. Rindone, J. Tschopp.

In echinoderms, the coelomic fluid contained in thewide coelomic cavity possesses naturally occurring lyticproperties (Ryoyama, 1973; Parrinello et al., 1979; Ber-theussen, 1983; Canicattï, 1987a, 1989; Leonard et al.,1990) against suitable target cells. The activity dependson molecules of proteinaceous nature unrelated tosaponins (Fig. 1), biologically active, water-soluble mix-ture of at least six glycosides (Nigrelli et al., 1967).

Vertebrate erythrocytes were the main targets usedand, as demonstrated in different species, the specificityof the lytic action was wide, being directed againstalmost all the erythrocyte types tested including mam-malians, reptiles, amphibians and fishes (Ryoyama, 1973;Parrinello et al., 1979; Canicatti, 1987a, 1989). However,in Strongylocentrotus droebachiensis a restrictedspecificity was observed. In this echinoid, in fact, of all

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CH.

Fig. 1 - Chemical structure of the echinoderm saponins. A: 22-25epoxyholothurinogenine; B: holothurogenine; C: 12ß-methoxy-7-8-diidro-22-25-epoxyholothurogenine; D: hoioxine.

the erythrocyte types assayed (human, mouse, rabbit andsheep), only rabbit red cells were lysed. Generally thehemolytic titers consistently vary from one erythrocytetype to another. As shown in Figure 2, this different reac:

tivity seems not to depend on the systematic position ofthe different red cells, nor on the structural differencesbetween erythrocytes (presence or absence of nucleus).Rabbit and human cells were the most sensitive targets,whereas sheep erythrocytes were the least reactive. In-dividual variations of the hemolytic activity could bemeasured in echinoid, asteroid and holothuroidcoelomic fluids. As demonstrated by Canicattî (1989),when a total number of 78 Marthasterias glacialis(Asteroidea) coelomic fluids were tested individually,only 38 were reactive towards rabbit erythrocytes. Thereactivity does not depend on sex, nor on age, nor'on theprotein content of the coelomic fluid. It could dependon seasonal parameters (i.e., temperature, photoperiodetc.) or on the biology of the reproductive cycle. At leastfor M. glacialis, the decrease of the number of reactantanimals seems to correspond with the reproductiveperiod: late spring to summer (Tortonese, 1965)-.Biological variability in hemolytic response of thecoelomic fluid was also monitored in another asteroidAsterias forbesi (Leonard et al., 1990). The authorsbelieve that it is a consequence of maintaining theanimals in an artificial environment. This is not true forM. glacialis, nor for P. lividus, since the coelomic fluids;assayed immediately after collection, presented similardegrees of individual variations to those of the specieskept in acquaria.

In addition to hemolytic activity, the echinodermcoelomic fluid exerts also lytic activity against mam-

Hemolytlc titer

frog »naJe» chtok*n rat mou«* g. pVg rabbit ah«*p pig hors* human

t = l A. cra»«l«plnaEE3 P. a*px—um EHUD P. Hvldu« KS3 M. putch*rrlmu«5H3 8. dro*tMK>hl*f£ZD M. glaolall» ESS H. polll

Fig. 2 - Sensitivity of different erythrocytc species to lysis by echinoderm coelomic fluids.

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ECHINODERM HEMOLYSIN 161

malian malignant cells. As demonstrated in Holothuriapolii (Canicatti, unpublished data), the degree of lysiswas influenced by coelomic fluid dilution. Mousemastocytome P815 was the most sensitive target (degreeof lysis of 100+0.2 %). As sensitive as normal rabbiterythrocytes (87.1±1.2%) was human leukemia K562 cellline. The least sensitive target was mouse flbrosarcomaWEHJ 164/14 (65.0*1.0%).

Auto- and allogeneic cells are neither lysed nordamaged. In P. lividus, the incubation of coelomic fluidwith auto- and allogeneic dechorionat'ed eggs did notproduce cytolysis, nor reduction in the ability of the eggsto be fertilized (Canicatti, 1991). These results indicatethat the membrane of «self» cells probably lackshemolysin binding components. On the other hand, it isalso probable that autologous membranes possessprotective components which do not allow any interac-tion between lysins and membrane.

RELATIONSHIP BETWEEN PROTEASE ACTIVITYAND HEMOYLTIC ACTIVITY

Echinoderm coelomic fluid possesses protease activity(Canicatti, 1990b). It depends on thermolabile enzymesinfluenced by pH and temperature, but not affected bycations supplementation in the reaction medium. Theenzymes are produced by coelomocytes which release itin vitro. Inhibitory experiments on protease activitycarried out on H. polii showed that soybean trypsininhibitor and, to a lesser extent, benzamidin PMSF, andTLCK, inhibited both coelomic fluid and coelomocyteenzymes (Canicatti, 1990b). Soybean trypsin inhibitorwas also a strong inhibitor of P. lividus coelomic fluidprotease. Taken together, these results indicate that cell-free coelomic fluid and cell lysate contain enzymes of theserin-protease, type. In H. polii, at least three serin-proteases were identified by affinity labelling with[3H]-DFP. One of these was isolated and characterized.The enzyme, holozyme A, is a 29-kDa protein cleavingN-benzyloxyl-carbonyl-L-lysine thiobenzyl ester sub-strate (Canicatti & Tschopp, 1990). These enzymes are,however, not directly related to hemolytic reactions sin-ce, as demonstrated in P. lividus (Canicatti, 1991),unhemolytic coelomic fluid maintains its proteolytic ac-tivity unchanged. Moreover, coelomic fluid samplescompletely deprived of protease activity, almost entirelymaintained their hemolytic potency. Compared to con-trols (66.1±0.1%), the degree of hemolysis of the STI-treated coelomic fluid ws3 unchanged (64.0±0.1%).

In A. forbesi, however, the hemolytic activity appearsto be sensitive to PMSF (Leonard et al., 1990), suggestingthat serin-protease could be an integral component of theasteroid lytic system.

At present, the involvement of serin-protease inechinoderm lytic mechanisms is not clear. At least for P.lividus, we can exclude a direct role in the digestion andconsequent lysis of the red cells. Most likely the protease

acts as regulatory enzyme of the entire process. Thishypothesis is supported by the finding that serin-proteases play a regulatory role in many other biologicalfunctions.

EFFECT OF METAL IONS ON HEMOLYTIC ACTIVITY

The hemolytic activity of the echinoderm coelomicfluids so far studied is, apart from some sea stars, stronglydependent on bivalent ions. In echinoids, as well as inholothuroids, calcium ions were demonstrated to be theonly cations enhancing hemolytic activity. In vitro, to in-creasirjg concentrations of Ca""* (from 5 to 100 mM)there corresponds a respective increase of the degree ofhemolysis (Ryoyama, 1973; Parrinello et al., 1979; Ber-theussen, 1983; Canicatti, 1987a). In many species the ad-dition of physiological concentrations of Ca* * (10 mM)to dialyzed coelomic fluids increases the degree ofhemolysis from 0 to 100% (Table I). Magnesium, whichrepresents a preponderant ion in echinoderm coelomicfluid (about 46.4 mM), cannot substitute calcium.

EDTA and EGTA strongly reduce the hemolyticresponse of echinoid and holothuroid, underlying therole of cations. Presently, the action mechanism ofcalcium is unclear. As suggested by Canicatti (1990a), itcan act as stabilizing agent of the structure of the lyticmolecules, or as mediator for the interaction betweenhemolysin and target. This does not exclude the in-volvement of this cation in mediating hemolysinpolymerization causing membrane damage to the targetcells, as in the more advanced lytic systems (Tschopp,1984; Podack et al., 1985). Ca** and Mg** are notnecessary for the hemolytic activity of Marthasteriasglacialis (Canicatti, 1989) and Asterias forbesi (Leonard

TABLE I - Effect of calcium on echinoderm lytic system.

ECHINOIDEAA. crassispinaS. droebachiensisP. lividus

ASTEROIDEAA. gibbosaE. sepositusM. glacialis

HOLOTHUROIDEAH. poliiH. tubulosaH. impatientH. belleri

Degree

none

2.10.00.3

n.d.0.8

86.6

0.20.10.30.8

of hemolysis (%)

lOmMCa**

40.7100.085.5

4*7.123.987.4

84.584.685.586.9

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162 C. CANICATTÎ

et al., 1990). On the other hand, a prolonged dialysis ofthe sea stars coelomic fluids in EDTA and/or EGTA doesnot reduce their hemolytic potency.

Of interest is the finding that zinc (which, like calcium,is a metal ion of the first transition period of the periodictable) acts as depressor of the hemolytic reaction inholothuroids (Canicattï & Grasso, 1988), asteroids(Leonard et al., 1990), and echinoids (Canicattï, 1991). InP. lividus, very low concentrations (0.1 mM) of the ionsproduce almost 90% of inhibition of the hemolytic ac-tivity of the coelomic fluid (Fig. 3). These properties maymean that the metal is a regulative ion. As suggested byCanicattï & Grasso (1988), Zn** interacting withsulphydryl groups of the hemolysin modifies the proteinstructure leading to inhibition. Alternatively, it wassuggested that the metal interacting with the binding sitemakes the binding between hemolysin and target im-possible. The antagonistic role played by calcium andzinc suggests that these metals could be implicated in theregulation of the hemolytic reaction, respectively ac-tivating and inhibiting the lytic molecules.

PHYSICO-CHEMICAL PROPERTIES

The echinoderm hemolysins express homogeneousphysico-chemical properties (Table II). Usually thehemolytic reaction of the coelomic fluids greatly in-creases in the temperature range 20° C to 37° C, mostprobably because at these temperatures the in vitrostability of the lytic principle is maximal.

Also pH influences the hemolytic activity of coelomicfluid preparations. It seems that the degree of hemolysisis proportional to the pH value of the medium, although

O .001 .002 .00« .012 .026 .05 .1 2. .6

Concentrations (mM)

Fig. 3 - Depressive effect of Zn* * on the hemolytic activity of P.lividus coelomic fluid.

the highest degree of hemolysis is recorded at alkalinepH (about pH 8.0). In addition, the active principle is alsopH stable to some extent.

Temperatures exceeding 50° C generally destroy thehemolytic activity of the coelomic fluids. However, insome species, a very heat-labile (37° C) lytic principle(Bertheussen, 1983), as well as a thermo-stable one(Canicatti, 1989) could exist. Thermo-stable and thermo-labile hemolysins cooccur in H. polii after antigenicstimulation (Canicattï & Párrinello, 1985)..

The course of hemolysis, followed for several concen-trations of coelomic fluid preparations, indicates that thedegree of hemolysis depends on concentration. Theresulting curve has a sigmoidal appearance (Ryoyama,1973; Canicattï & Parrinello, 1985; Canicattï, 1987a,1989), suggesting that a multiple binding of a singlemolecular species or, alternatively, a synergic effect of

TABLE II - Physico-chemical properties of tbe hemolytic activity of ecbinoderfn coelomic ßuid.

Optimal reactiontemperature

Optimal pH pH stability Thermal inactivation Ref.

ECH1NOIDEAA. crassispinaP. depressusH. pulcberrimusS. droebachiensisP. lividus

HOLOTHUROIDEAH. poliiH. tubulosaH. impatient

ASTEROIDEAM. glacialisA. forbesi

37°37°37°

37°

37°37°37°

20°25°

CC

cc

ccc

cc

8-6.3 4-9

4-9

6-9

3-11

45-56° C

37°45-46

>50°50°50°

C° C

CCC

thermostable56° C

11123

455

67

Ref. = reference: 1: Ryoyama, 1973; 2: Bertheussen, 1983; 3: Canicattï, 1987a; 4: Parrinello et al., 1979; 5: Canicattï, unpublished data;6: Canicattî, 1989; 7: Leonard et al., 1990.

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ECHINODERM HEMOLYSIN 163

more components, could be responsible for the mem-brane damage of the target cell.

LYTIC COMPONENTS AND LYSIN-PRODUCER CELLS

The main difficulty encountered in the purification ofechinoderm hemolysins was the loss of biological ac-tivity during the isolating procedure. In H. polii, by usinga Chromatographie method (Gel filtration on BiogelA5m), the coelomic fluid was resolved in three majorpeaks, of which only the third one was lytic (Canicattî &Parrinello, 1983). Results in identifying H. poliihemolysins can be obtained when coelomocyte lysate isused instead of coelomic fluid. With an overlaytechnique, two lytic bands with different electrophoreticmobilities could be located by their biological activity ina polyacrylamide gel slab (Canicatti & Ciulla, 1988). Thecomponents were identified as hemolysin 1 (Hel) andhemolysin 2 (He2). Hel is the calcium dependent, heat-labile cellular component, He2 the calcium-independent,heat-stable one (Canicattî & Ciulla,.1987). The antiseraproduced against these lytic bands labelled in im-munoblot an 80-kDa component, which was retained tocorrespond to the molecular weight of the lytic com-ponents (Canicatti & Ciulla, 1988).

The nature of the cells producing hemolysins has beeninvestigated in H. polii by using a sodium metrizoategradient to separate homogeneous cell populations(Canicattî et al., 1988). From this study it was demon-strated that two hemolysin producing amoebocytepopulations occur in sea cucumber (Fig. 4). Theamoebocytes of population 1 are responsible for theproduction of Hel, whereas those of population 2produce He2. The H. polii amoebocytes are large cells(12-21 urn) with a roundish or ovoid central nucleus(Canicattî et al., 1989), which assume a great functionalimportance in the internal defence system of the seacucumber (Canicattî & D'Ancona, 1989).

The production of lytic molecules by circulating cellshas also been demonstrated in the sea star M. glacialis(Canicattî, 1989). In this asteroid, the extract obtained bysonication of coelomocytes was lytic against rabbiterythrocytes. As for coelomic fluid, the lytic principlewas calcium-independent and thermo-stable. However,we do not know which of the nine different cell typesdescribed in Asteroidea (Kanungo, 1984) are hemolysin-producer coelomocytes in M. glacialis.

HEMOLYSIN BINDING TO SURFACE COMPONENTSAND MEMBRANE DAMAGE

Not much is known about interactions betweenhemolysins and membrane integrated molecules of targetcells. Since the surface of bacteria, fungi, or cells ischaracterized by determinant carbohydrates which arepart of polysaccharides or glycoproteins or glycolipids,

D«gre« of lytla (%)

pop2

Fig. 4 - Hemolytic activity in cell lysate preparations from Na-metrizpate separated H. polit amoebocyte populations.

experiments were carried out to demonstrate an in-volvement of saccharide molecules of foreign targets onthe hemolysin attachment. However, apart from S.droebanchiensis in which D-glucosamine andD-galactosamine exerted inhibitory activity onhemolysin-rabbit erythrocyte interaction (Bertheussen,1983), sugars seem not to be effective in producinghemolysia inhibition. In other echinoderms, lipidsdisplayed inhibitory activity (Fig. 5). In H. polii,sphingomyelin is a strong inhibitor of the hemolyticreaction (Canicattî et al., 1987) suggesting a specific in-

100Degree of lysis (%)

8 0 -

8 0 -

4 0 -

2 0 -

H. poltl

S 3 M. flUiclalU

EM) P. llvldu*

Con Sph Ph Cho

Fig. 5 - Inhibitory effect of lipids on echinoderm lytic system. Con:control; Sph: sphyngomyelin; Ph: phosphatidyl-inositol andphosphatidyl-ethanolamine; Cho: cholesterol.

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164 C. CANICATTÎ

volvement of this integral membrane component in thelysis of the target cells. Sphingomyelin is also an inhibitorof the M. glacialis hemolysin (Çanicattî, 1989). In P.lividus, neither sugars nor sphingomyelin inhibited thehemolytic activity of the coelomic fluid (Canicattï,1987a); however, cholesterol, phosphatidyl-inositol, andphosphatidyl-ethanolamine were the most effective(Canicattï, 1991). Relatively less effective (>10%) werethe inhibitions produced by phosphatidylcholine,phosphatidylserine and phosphatidylglicerol.

As demonstrated in H. polii, the interaction betweenlipid and hemolysin was not of the enzymatic type;moreover, the coelomic fluid did not possesssphingomyelinase activity (Canicattï et al., 1987).

From these studies it is conceivable that thehemolyticreaction is mediated by a rapid binding of the moleculesto membrane targets through sugars or lipids, with con-sequent lysis of the cell (Canicattï, 1988).

The cytolytic effect is expressed through the formationof holes with diameters between 5 and 25 nm. In H.polii, the first observations of erythrocyte membraneslysed by coelomic fluid revealed irregular lesions whichwere apparently not surrounded by structured rings(Canicatti, 1987b) characteristic for more complex lyticsystems (complement and perforins) and for the ringsproduced by bacterial and cnidarian toxins. A moredetailed analysis carried out with coelomocyte •hemolysins evidenced the presence of structured lesionswith an average diameter of 10 nm, which is charac-teristic for all the pore-forming proteins studied so far.Similar results have recently been obtained in P. lividus(Canicattï, 1991).

When analyzed in SDS-PAGE after lysis, the iodinatedproteins of H. polii coelomic fluid associated to rabbiterythrocyte membranes showed a pattern constitutedby a high m.w. protein which does not penetrate into a10% gel, by a band of about 27 kDa between the majorcomponents, and a feeble band of 31 kDa between theminor components. In reduced conditions, the highm.w. band disappears and is substituted by two highlyradioactive bands of about 80.5 and 64 kDa. The 31-kDaband presents a higher intensity, whereas the 27-kDaband is lost, probably because of the reduction effect(Canicattï, 1987b). The fact that in this pattern there is ahigh m.w. protein which is resolved in components of80.5 and 64 kDa, leads one to think that, analogously towhat happens for C9 of the vertebrate complement(Tschopp, 1984) or perforins (Podack et al., 1985), thelytic molecules could polymerize on the target cellmembranes. Through these processes the rings obser-ved under electron microscope in negative stainingcould be constituted. A priori, these considerationsseem to be conceivable. However, we must recognizethat the reaction between hemolysin and foreign surfacecould be more complex and include not yet knownelements.

BIOLOGICAL ROLE

Vertebrate erythrocytes, which are not replaceable,membrane models in the study of lytic systems, certainlydo not represent natural targets for echinoderms.However, we can state that the same mechanisms areused against the whole range of foreign agents, such asbacteria, fungi, parasites, or modified self cells.

In echinoids, as well as in holothuroids, hemolysinsseem to assume a mainly opsonic role. In S. droebachien-sis, rabbit erythrocytes treated with high concentrationsof coelomic fluid in which agglutinin was inhibitedproduced a strong opsonic activity. The echinoidphagocytic cells, in fact, phagocytize more avidly op-sonized red cells. Since this accelerated phagocytosis isabsent when coelomic fluid inhibited with aminosugarsis used, involvement of the sea urchin hemolysins issuspected (Bertheussen, 1983). Also in the sea cucumber,hemolysins appear to be involved as opsonic elements(Canicatti & Parrinello, 1985). In these organisms the in-jection of large quantities of sheep red cells into thecoelomic cavity induces a strong decrease of the lytic ac-tivity of the coelomic fluid at day 1 from the injection.The decrease is most probably due to hemolysin- in-volvement in erythrocyte clearance.

Of interest is the finding that hemolysin occurs inechinoderm epidermal secretion. As demonstrated in M.glacialis, the watery mucous liquid produced by theepidermal secretroy cells exerts lytic activity against rab-bit, erythrocytes (Canicattï & D'Ancona, 1990). Sincemucus assumes, among other activities, an importantdiscouraging function against potential pathogens, it ispossible that hemolysin contitutes one of the molecularbarriers against host exploitation.

More recent interest has been focused on the role ofhemolysins in the cytotoxicity mediated by echinodermcoelomocytes.

In invertebrates, spontaneous killing cell-mediatedcytotoxicity is a widely occurring event (Boiledieu &Valembois, 1977; Decker et al., 1981; Koyama &Watanabe, 1982; Scofield et at., 1982; Buscema & Van deVyver, 1985; Söderhäll et al., 1985; Mukai & Shimoda,1986), most probably involved in intraspecific orallogeneic histoincompatibility.

In echinoderms, amoebocytes recognize allogeneicand xenogeneic cell combination in vitro (Bertheussen,1979; Decker et al., 1981). The reaction is contact-dependent, bilateral (Bertheussen, 1979), and as demon-strated in Pisaster gigantus, inhibited by sugars (Deckeret al., 1981). Apparently, cell-surface glycoproteins ontarget cells could be responsible for the specificy of thereaction, but the mechanisms by which effector cellscarry out the killing of the targets remains unknown.Perhaps it is significant that amoebocytes, which arehemolysin producer cells (Canicattï, 1990a), are able tokill the target cells.

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ECHINODERM HEMOLYSIN 165

EVOLUTION

From the small selection of echinoderm hemolysinsdiscussed in this article, it is apparent that there are stillconsiderable areas of ignorance about these molecules.An intriguing problem remains the phylogeneticalrelationship between echinoderm and vertebrate (com-plement and perform) lytic proteins. A certain degree offunctional similarity between the two lytic systems couldimplicate a common evolutionary ancestor, although it isalso conceivable that echinoderms and vertebrates haveindependently developed proteins (convergentevolution) with similar killing mechanisms. For manyAuthors, the echinoderm hemolytic activity may be en-visaged as complement-like, with the attractive idea thatit could represent a primitive alternative pathway (Day etal., 1972; Bertheussen, 1983, 1984; Leonard etal., 1990).Recently, it was suggested that there exists a relationshipbetween vertebrate pore-forming proteins (C9 and per-forin) and H. polii hemolysin (Canicatti, 1988). This im-plicates a different level of phylogenetic relationship be-tween vertebrate and invertebrate lytic systems(Canicatti, 1990a). However, since no structural genecoding information is available at this moment, theproblem is still open to debate.

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