The echinoderm lytic system
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The echinoderm lytic systemCalogero Canicatt aa Dipartimento di Biologia , Universit di Lecce , Via Prov.le LecceMonteroni, Lecce, I73100,ItalyPublished online: 28 Jan 2009.
To cite this article: Calogero Canicatt (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 CANICATTDipartimento di Biologia, Universit di Lecce,Via Prov.le Lecce-Monteroni, 1-73100 Lecce (Italy)
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 (Canicatt, 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; Canicatt, 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; Hltmark 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; Canicatt, 1990a). These obser-vations suggest a resemblance with vertebrate pore-forming proteins (Canicatt, 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
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.
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; Canicatt, 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
160 C. CANICATT
Fig. 1 - Chemical structure of the echinoderm saponins. A: 22-25epoxyholothurinogenine; B: holothurogenine; C: 12-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 Canicatt (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-
frog naJe chtok*n rat mou* g. pVg rabbit ah*p pig hors* human
t = l A. cralplnaEE3 P. a*pxum EHUD P. Hvldu KS3 M. putch*rrlmu5H3 8. dro*tMK>hl*fZD M. glaolall ESS H. polll
Fig. 2 - Sensitivity of different erythrocytc species to lysis by echinoderm coelomic fluids.
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 (87.11.2%) 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 (66.10.1%), the degree of hemolysis of the STI-treated coelomic fluid ws3 unchanged (64.00.1%).
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
of hemolysis (%)
162 C. CANICATT
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 (Canicatt & Grasso, 1988), asteroids(Leonard et al., 1990), and echinoids (Canicatt, 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 byCanicatt & 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.
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
Fig. 3 - Depressive effect of Zn* * on the hemolytic a...