THE JOURNAL OF EXPERIMENTAL ZOOLOGY 275444451 (1996) Toxin Compartmentation and Delivery in the Cnidaria: The Nematocyst’s Tubule as a Multiheaded Poisonous b r o w AMIT LOTAN, LENA FISHMAN, AND ELIAHU ZLOTKIN Llepartment of Cell and Animal Biology, Life Sciences Institute, Hebrew IJniversity of Jerusalem, 91904 Jerusalem, Israel ABSTRACT With the aid of dialysis and ion exchange chromatography, a new polypeptide toxin was purified from the tentacles of the Mediterranean jellyfish Rhopilema nomadica. The amino acid sequence of the N-terminal segment of the new toxin revealed that it is a phospholipase A2 ( P U B )toxin closely resembling those previously isolated from reptile and hymenopterous venoms. The occurrence of a PhA2 toxin in the jellyfish tentacles may explain both their local (dermanecrotic) and systemic (cardiac-respiratory) effects upon human envenomation. We used an antibody raised against the above toxin as a probe to explore, for the first time, the site of toxin allocabion in cnidarian nematocysts and its morphological route of delivery. Our immunocytochemical approach revealed that the toxin is stored on the outer (“cytoplasmic”)surface of the inverted tubule folded in the capsule of the resting nematocyst. During discharge the toxin is translocated to the internal surface surrounding the lumen of the everting tubule, and its delivery via extended spirally arrayed barbs is apparently propelled by the high hydrostatic pressure of the capsule. This is a unique example where subcellular translocation and transfer of a polypeptide is driven by mechanical forces. @ 1996 Wiley-Liss, Inc. Cnidaria (hydras,jellyfish, sea anemones, and corals) comprise an early phylum of radially symmetri- cal venomous aquatic animals shown to possess a wide array of neurotoxic (Kem, ’SS), cytolytic, and enzymatic (Walker, ’88)substances. Despite the fact that these toxic substances were isolated substantially from homogenates of entire animals or tentacles, their storage and delivery sites are attributed t o nematocysts (cnidocysts), t h e stinging subcellular organelles common t o all members of the phylum (Mariscal, ’74). The nematocysts are localized in specialized cells (nematocytes or cnidocytes) and consist of a capsule containing a highly folded, eversible tubule. However, no information has so far been obtained on the localization and mode of delivery of nematocyst toxins (Holstein et al., ’94). The present study, while achieving the abovementioned goal, also yields direct proof that cnidarian toxins are localized in the nematocysts (Lotan et al., ,951, and the first chemical characterization of a jellyfish toxin. EXPERIMENTAL PROCEDURES Jellyfish tentacles Rhopilema nomadicc! jellyfish were collected in the Bay of Haifa. Their tentacles were re@ 1996 WILEY-LISS, INC. moved by cutting and stored over dried ice in a deep freezer. Toxicity assays Toxicity of jellyfish tentacle extracts or isolated fractions was assayed by subcutaneous injection into Gambusia affinis fish of 200-300 mg body weight. Lethality, preceded by paralysis, occurred within 5 minutes of injection. Values of lethal doses (LD,,) were determined according to Reed and Muench (’38). Separation methods When needed the frozen tentacles were thawed and centrifuged (l,OOOg,10 min). Supernatant was placed in dialysis bags (cut-off of 10 KDa, Sigma, St. Louis, MO) and dialyzed against 0.01 M ammonium acetate pH 8.2 buffer at 4”C, for removal of salts. The bag contents was separated on an anion exchange (DEAE-Sephadex, Pharmacia, Uppsala, Sweden), column (100 ml), equilibrated and eluted by the above buffer. The fraction obtained at the Received January 15, 1996; revision accepted April 9, 1996. Address reprint requests to E. Zlotkin, Department of Cell and Animal Biology, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, 91904, Israel. LOCALIZATION AND DELIVERY OF A NEMATOCYST TOXIN 445 Tentacles (-70%) 4 Thawing ( 2 5 t ) 4 Centrifugation .Ir Supernatant dialysed Elution volume (nil) 4 Anion exchanger (void volume) 4 Cation exchanger (Monos-HPLC) .Ir Purified toxin (concentrated) Fig. 1. Flow diagram of the purification procedure of a toxin from jellyfish tentacles. Fig. 2. Separation by cation exchange column in an HPLC system. One milligram of protein, obtained by the previous step of DEAE-Sephadex chromatography and concentrated t o a volume of 5 ml, was charged on a Mono-S (Pharmacia, Sweden) column (5 ml) equilibrated with 0.01 M ammonium acetate, pH 8.2 buffer and eluted at a flow rate of 1mumin by a linear gradient of sodium chloride molarity. The various fractions were collected according to their elution pattern, concentrated by Centricon columns (Amicon) and assayed for their toxicity to fish. The marked fraction (arrow) which possessed the highest toxicity (LDs0 = 0.6 pg/lOO mg b.w.), revealed a single band on SDS-PAGE and was assessed for homogenicity by isoelectric focusing and amino acid analysis (data not shown). Protein content of venom fractions was determined by the procedure of Lowry et al. ('51) with bovine serum albumin (BSA) as the standard. Purified peptides were quantsed by amino acid analysis. zilber et al. ('91), and the modified peptides were desalted on a Vydac C18 column in 0.1% TFA acid with a linear gradient of 0 4 0 % acetonitrile/Z-propanol 1:l. Desalted peptides were lyophilized, resuspended in phosphate-buffered saline, pH 7.4, and adsorbed onto polyvinylidene difluoride (PVDF) membranes (Speicher, '89). Amino acid sequence analysis was performed by automated Edman degradation with an Applied Biosystems (Foster City, CA) 475A gas-phase protein sequencing system at the Bletterman Laboratory of Macromolecular Research (Faculty of Medicine, Hebrew University). The chromatography system was calibrated prior to each analysis with phenylthiohydatoin (PTH) amino acid standards. Each sequence was confirmed in at least two separate determinations utilizing different batches of peptide. Amino acid analysis Anti bodies void volume was separated by a Mono-S column (5 ml, Pharmacia) in an HPLC (Spectra-Physics, San Jose, CA) system eluted by a gradient of molarity of NaCl (Figs. 1 and 2). Electrophoresis Electrophoretical techniques included SDSPAGE and analytical isoelectric focusing (see Fig. 4 and Lester et al., '82). Protein determination Preimmune serum was collected from rabbits Analysis of amino acid composition, after acid hydrolysis and 9-fluorenylmethoxycabonyl chloride and assayed by dot blot against a mixture of pro(FMOC-Cl) derivatization, was performed on a teins extracted from tentacles, yielding a clear Merck-Hitachi reverse-phase HPLC system, ac- negative response. The above rabbits were immucording t o Gooderham ('83).The system was cali- nized according to Vaitukaitis ('81). Purified toxin brated prior to each analysis with FMOC-amino (90-180 pg) were suspended in the solution of complete adjuvant in 0.1% SDS to a final volume of 2 acid standards. ml and injected subcutaneously at 15 points. Blood Peptide sequencing was collected after two applications. The serum Purified toxins were reduced and alkylated with was examined for antibodies by immunoblots (see 4-vinylpyridine as previously described by Fain- Fig. 4). ~,In order to obtain a toxin-snecific antibodv ~ ~~ . I A. LOTAN ET AL. 446 the rabbit serum was incubated with nitrocellu- the thawed tentacles, when examined microscopilose blot strips containing purified toxin. The an- cally, was shown to include mainly discharged tibodies which reacted with the blot were eluted nematocysts (data not shown). Toxicity was thereand assayed against a variety of protein bands fore attributed to substances originating from nemaobtained from jellyfish tentacles. The antibodies tocysts. The suspension containing the discharged were shown to react exclusively with the toxin nematocysts was centrifuged (l,OOOg, 10 min) and (Fig. 4). the supernatant was dialyzed and separated by a DEAE-cellulose anion exchange column (data not Immunocytochemistry shown). The eluate collected from the void volume Proteins extracted from jellyfish tentacles of the anion exchange column was separated on a were separated on a Bio-Rad (Richmond, CA) Mono-S HPLC cation exchange column (Fig. 2). minigel apparatus a n d transferred to nitrocelAll the fractions eluted from the Mono-S collulose membranes overlayed with the toxin-spe- umn (Fig. 2) were concentrated and desalted by cific antibodies detected w i t h the a id of a centricon filters (Amicon, Danvers, MA) and were BM-chemiluminescence Western Blotting Kit (Rab- shown to be toxic to fish. The last fraction (Fig. 2, bit) (Boehringer-Mannhcim, Indianapolis, IN). The marked fraction) revealed only one band on SDSaffinity-purified antibodies were used for toxin de- PAGE (Fig. 4A), was toxic to fish by injection (LD50 tection by immunoblots (Fig. 4) and light (Fig. 5), = 0.6 Fg/100 mg body weight), and was therefore examined for purity. as well as electron microscopy (Fig. 6). For light microscopy, tentacles were sliced with a Toxin characterization Jung CM 3000 (Leica, 13Uffal0, NY)Microtome to The single component derived from the Mono-S sections of 8 ,um. The sections were attached to glass slides with polylysine, and incubated with the af- marked fraction (Fig. 2) was shown t o possess a n finity-purified antitoxin antibodies, followed by Mr of 17,000 by SDS-PAGE separation (Fig. 3). goat anti-rabbit fluorescein isothiocyanate-labeled Isoelectric focusing (data not shown) indicated that the single component is a basic protein of a antibodies (Jackson, Inc., Bar Harbor, ME). For electron microscopy, nematocysts were fixed PI around pH 9.0. The component induced rapid with form and glutaraldehyde (1,4%, respectively) lethality in Gambusza fish (1 pg/lOO mg body in cacodylate buffer 0.1 M, pH 7.4, for 24 h at 4°C weight within 1min). The above toxicity was comand 45 min at room temlperature. The immunogold pletely abolished by: (1)incubation in 10%enzyme technique was employed for the postembedding to substrate ratio of pronase solution for 30 min method (Castel et al., '93). Grids were stained with at 25"C, (2) lyophilization and (3) immersion in a boiling water bath for 5 min, thus indicating that uranyl acetate and lead citrate. the toxin is a polypeptide. The amino acid sequence of a n N-terminal segment of 32 amino acRESULTS ids of the new toxin is presented in Figure 3 and Toxinpurification compared to two PhA2 toxins previously isolated The procedure of toxin purification is schemati- from the venoms of a lizard (Holoderma, Gila moncally presented i n Figure 1. The supernatant of ster) and the honey bee (Shipolini et al., '74; Sosa Lizard G I PGTLWC MG GA I PGTLWC A N NA Honeybee Fig. 3. The sequence of the first 32 amino acids from the N-terminal end of the jellyfish toxin is presented and compared to PhA2 toxins derived from honey bee and lizard venoms (Shipolini el; al., 1974; Sosa et al., 1986). LOCALIZATION AND DELIVERY OF A “EMATOCYST TOXIN et al., ’86). The above comparison (Fig. 3) suggests that the present toxin may be defined as PhA2 like toxin. The obvious similarity in the amino acid sequences of about 60% residue identity among toxins derived from venoms of animals belonging t o three different phyla is striking and emphasizes the importance and priority of PhA2 toxins in toxinology (Rosenberg, ’90) (see also Discussion). Toxin in the jellyfish tissues 447 o-isorhiza (Mariscal, ’7411 and revealed the same pattern of toxin localization. In the discharged nematocysts the immunocytochemical staining occurred all along the everted tubule (Fig. 5A). The allocation of the toxin on the tubule revealed a helical shape (Fig. 5C), which parallels the helical arrangement of the barbs (Fig. 5D) (Tardent, ’88). The latter observation has raised the possibility that the hollow barbs (Hessinger and Ford, ‘88) may function as a device for toxin delivev. This essential aspect was further studied by the high resolution immunogold technique and electron microscopy (Fig. 6). The occurrence and localization of the toxin in the jellyfish organs might further indicate its involvement in the prey capture system. Proteins exToxin localization by electron tracted from the jellyfish bell and its tentacles were microscopy (EM) separated electrophoreticaly, blotted and reacted The precise distribution of the toxin in both restwith the toxin-specific antibody. As shown in Figing and discharged nematocysts was studied by ure 4, the toxin is expressed in the tentacles, but is the immunogold EM method (Caste1 et al., ’93) completely absent from the bell tissues which, in and results are presented in Figure 6, indicating contrast to tentacles, are devoid of nematocysts. (I)the occurrence of the toxin in association with Toxin localization by light microscopy (LM) the tubular structures and its absence from the As shown in Figure 5, LM immunocytochemi- capsular lumen and walls (Fig. 6A,B); (2) the excal staining of cryostat-sliced nematocytes reveals ternal (on the “cytoplasmic”surface) allocation of highly specific localization of the toxin, limited to the toxin on the surface of the inverted-undisthe tubular segments of the nematocysts (Fig. charged tubule (Fig. 6C,D); and (3) occurrence of 5A,B,C). The capsular lumen and wall were de- higher concentrations of toxin at the bases of the void of immunofluorescent staining in the resting extended barbs, and their close vicinity in the and discharged nematocysts (Fig. 5A,B). Three everted-discharged tubule (Figs. 6E, 7). The above data (Figs. 5 and 6) allow visualmorphologically distinct types of discharged nemaization of t h e morphological route of toxin tocysts were observed [heterotrichous isorhiza, translocation and release as it is schematically heterotrichous microbasic eurytele, holotrichous presented in Figure 7. In the resting nematocysts toxin is localized in the crypts (invaginations) of the outer surface of the twisted, folded, and inverted tubule, the lumen of which is occupied by A B C D the internalized barbs. During discharge and tubule eversion toxin is translocated from the outer to the inner surface of the everted and extended tubule while aggregating near the bases and within the hollow barbs in the process of release (Figs. 6 and 7). DISCUSSION Fig. 4. Analyses of the new toxin purified from the R. nomadica jellyfish. A, SDS-PAGE electrophoresis of 2 pg of toxin. B,C,D, immunoblots: Separation of 10 pg of protein extracted from the fishing tentacles (b), as above from bell tissues (C), and 0.1 pg of purified toxin (D). The experimental approach of the present study comprised two steps, the first aimed at the isolation and purification of an, as yet unknown, polypeptide toxin from a jellyfish, and the second aimed at employing the newly characterized toxin as a probe to study its localization, translocation, and delivery in the nematocyst. The choice of the jellyfish as an experimental animal was directed not only by the preexisting information on its phenology, distribution, and 448 A. LOTAN ET AT.,. Fig. 5. Resting (B) and dlischarged (A,C,D) hematocysts stained by an immunofluorescent technique. D: Scanning electron microscopy of the discharged extended tubule. Notice that the fluorescent staining reveals the toxin on the tubule (A,B,C) appearing in the form of helically arranged dots (C) parallel to the helical arrangement of the barbs on the extended tubule (D). Arrowhead points to unstained capsule of the discharged nematocysts. Bars: in A, C, 2 pm; in B,D, 1pm. availability (Lotan et al., ’92, ’94), but also by its public health hazard and the absence of any clear, specific information on the chemistry and pharmacological essence of its toxic substances (Carrie, ’94). The failure of previous efforts (Endean et al., ’93) to isolate and purify jellyfish toxins follows from the extreme lability of these compounds t o standard treatments such as lyophilization. In our approach we bypassed this difficulty through the use of separation methods which avoid lyophilization, such as dialysis and ion exchange column chromatography. Surprisingly, the resulting toxin belongs t o one of the most common categories of toxic substances found in reptile and artliropod venoms, namely a PhA2 like toxin identified by its primary structure (Fig. 3). The effect of PhA2 toxins is based on two separate mechanisms of intoxication. The first comprises a synergic interaction with membrane-active amphipathic venom polypeptides or “direct lytic factors” in disruption of biological membranes and lysis of various cells (Rosenberg, ’90). The second mecha- nism includes a more specific neurotoxic action in the form of presynaptic toxins that affect neurotransmitter release. Furthermore, phospholipase toxins have been shown to serve as potential models of clinical disorders such as epilepsy (convulsant action), hemolytic disorders, coagulation disorders, and inflammation (increased levels of arachidonic acid) (Rosenberg, ’90). With this background the occurrence of PhA2-like toxins in jellyfish tentacles and nematocytes may explain both the local (lytic, necrotic), as well as systemic (cardiac and respiratory), effects (Carrie, ’94) of jellyfish envenomation. Our main concern, however, was focused on the localization, translocation, and morphological route of delivery of the toxin by the nematocyst system. This aspect was studied substantially by immunocytochemistry using light and electron microscopy. The discharge of the nematocysts is driven by the capsule’s high internal hydrostatic pressure of 15 megapascals (approximately 150 atmospheres), which causes the eversion of the internally folded tubule (Holstein et al., ’94) and LOCALIZATION AND DELIVERY OF A NEMATOCYST TOXIN Fig. 6. Tmmunogold electron microscopy of resting nematocyst and discharged tubule. A: Cross-section through intact nematocysts containing profiles of the folded tubule (TI, which reveal the immunogold grains. Examination by higher magnification (data not shown) indicates the absence of toxin in capsule’s lumen and wall. B: Insert from A revealing immunogold grains located exclusively on the folded inverted tubule C: Cross-section of inverted (resting) tubule. Immunogold grains can be seen only on the outer surface (“cytoplasmic” surface) of 449 the tubule wall (arrows) while the internalized barbs (B) occupy the tubule’s lumen. D: Insert form C revealing immunogold grains along the external, “cytoplasmic” surface of the tubules’ wall. E: Cross-section of the discharged everted tubule revealing the externally directed extended barbs and the immunogold grains localized on the inner surface of the tubule wall. Notice that grains accumulate a t the bases of the barbs and occur within their cavities (arrows and Fig. 4). a, tubule membrane; B, barbs; T, tubule. Bars: in A, 0.8 pm; B, 0.1 pm, C,D,E, 0.5 pm. 450 A. LOTAN ET AL. Fig. 7. Schematic presentation of toxin compartmentation and delivery in the nematocyst system. The central part reveals the resting tubule who,se lumen is filled with barbs. The toxin, represented by dots, is located on the external side (“cytoplasmic”surface) of the tubule’s membrane (Fig. 6D). During the discharge toxin is translocated into the tubule’s lumen while the barbs emerge and extend. Toxin is delivered from the everted-extended tubule through the hollow barbs. enables its penetration into the tissues of potential prey or an opponent. The latter phenomenon is reminiscent of the penetration of plant leaves by pathogenic fungi using enormous turgor pressures (Howard et al., ’91). In cnidarian nematocysts high speed cinematography revealed that tubule eversion occurs within 3 ysec at accelerations of up t o 40,OOOg (Fig. 2B) and therefore comprises one of the fastest events in biology. However, the ultrastructural and mechanical data do not explain toxin allocation and mode of delivery. Furthermore, even the notion that nematocysts serve as the venom source has not been unequivocally established. At least four different hypotheses/models have been proposed to explain venom release (Thomason, ’91;Tardent, ’88). These models were based on the assumption that the toxins are stored in the lumen of either the capsule or the tubule and are delivered through either the tip or the surface of the tubule during eversion. Venom production and delivery systems in metazoans are formed of epithelial secretory glands, collecting ducts, sharp, hollow, rigid stinging devices, and accompanying apparati of muscles and motor, as well as sensory, nerves (Edstrom, ’92). This multiple organ system is analogous t o a syringe-injection apparatus. The present study clarifies how a subcellular organelle such as the nematocyst is able to fulfill the role required of an entire organ system in other phyla. Our immunocytochemical approach suggests that the cnidarian venom is stored on the outer surface of the folded, undischarged tubule and is delivered upon its discharge (eversionextension) through the spirally arranged array of the extended hollow barbs, thus resembling a multiheaded poisonous arrow (Fig. 7). Our data do not support the various proposed models (Thomason, ’91; Tardent, ’88) of nematocyst toxin delivery, which were proposed solely on the basis of morphological information. Our data, however, support the hypothesis of Hessinger and Ford (’88)that in the nematocysts of the man-ofwar hydromedusa, the barbs are hollow and are occupied by electron-dense granules supposedly containing venom protein, “thereby making each barb a tiny hypodermic syringe for delivery of the nematocyst toxins.” We assume that the hydrostatic pressure raised in the dischargeable capsule causing the tubule eversion (Holstein e t al., ’94; Tardent, ’88) also supplies the appropriate propulsion for toxin delivery through the barb system. This notion is supported by a most recent study revealing that the fiber-like structure of the capsule’s inner wall provides the tensile strength necessary t o withstand the capsule’s high osmotic pressure (15 megapascals), which continues along the tubule wall (Holstein et al., ’94). Thus, the nematocyst delivery system supplies a unique example for subcellular protein translocation based on simple mechanical forces. In summary, the cnidarian subcellular nematocyst fulfills the role of the entire venom apparatus of advanced organisms (Lotan et al., ’95). However, from a mechanistic point of view, the nematocyst system is not strictly a syringe apparatus, but rather an ammunition device where the LOCALIZATION AND DELIVERY OF A NEMATOCYST TOXIN capsule functions as the cartridge case employed for propelling the toxin-loaded tubule, which serves as the shell or the warhead of the system. LITERATURE CITED Carrie, B. 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