Milleporin-1, a new phospholipase A2 active protein from the fire coral Millepora platyphylla nematocysts

Comparative Biochemistry and Physiology, Part C 139 (2004) 267 – 272 www.elsevier.com/locate/cbpc

Milleporin-1, a new phospholipase A2 active protein from the fire coral Millepora platyphylla nematocysts Faisal F.Y. Radwan*, Hosney M. Aboul-Dahab Faculty of Science at Sohag, Department of Zoology, South Valley University, Sohag 82425, Egypt Received 8 October 2004; received in revised form 5 December 2004; accepted 6 December 2004

Abstract Stings of fire corals, potent hydroids common in the Red Sea, are known to cause severe pain and they develop burns and itching that lasts few hours after contact. Nematocyst venom of Millepora platyphylla (Mp-TX) was isolated according to a recent method developed in our laboratory to conduct a previous investigation on the nematocyst toxicity of Millepora dichotoma and M. platyphylla. In this study, Mp-TX was fractionated by using both gel filtration and ion exchange chromatography. Simultaneous biological and biochemical assays were performed to monitor the hemolytic (using washed human red blood cells, RBCs) and phospholipase A2 (using radiolabeled sn-2 C14arachidonyl phosphatidylcholine as a substrate) active venom fractions. The magnitude of both hemolysis and phospholipase A2 activity was found in a fraction rich of proteins of molecular masses ~30,000–34,000 Daltons. The former fraction was purified by ion exchange chromatography, and a major bioactive protein factor (approx. 32,500 Daltons , here named milleporin-1) was recovered. Milleporin-1 enzymatic activity showed a significant contribution to the overall hemolysis of human RBCs. This activity, however, could not be completely inhibited using phospholipid substrates. Melliporin-1 fraction retained about 30% hemolysis, until totally rendered inactive when boiled for 3 min. The overall mechanism of action of milleporin-1 to impact the cellular membrane was discussed; however, it is pending more biochemical and pharmacological future studies. D 2004 Elsevier Inc. All rights reserved. Keywords: Fire corals; Hydrozoa; Millepora; Milleporin-1; Phospholipase A2; Hemolysis; Nematocyst; Red Sea

1. Introduction Fire corals (also called stinging corals) are tropical calcareous hydroids (phylum Cnidaria; order Milleporina), belonging to the genus Millepora. Being hydrozoans rather than anthozoans, Millepora are not classified as true stony corals, although their abundance allows them to be a major contributor to coral reef structure. Millepora platyphylla is a common Red SeaTs fire coral with a plate-like form, a bright yellow-green color, and predominantly occurs on reef crest and in the shallows less than 5 m (Radwan, 2002). Divers often mistake fire corals for seaweed, since * Corresponding author. Present address: National Ocean Service, 219 Fort Johnson Road, Charleston, SC 29412, USA. Tel.: +1 843 762 8637; fax: +1 843 762 8700. E-mail address: [email protected] (F.F.Y. Radwan). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.12.002

accidental contact is very common. Although there were no fatal cases, painful stings and inflammatory effects of Red Sea or Pacific fire corals have been known long ago (Bermet and Ormond, 1981; Vine, 1986; Sagi et al., 1987; Shiomi et al., 1989b). Like other cnidarian venoms, Millepora venoms are contained in nematocysts (Wittle et al., 1974; Shiomi et al., 1989a; Radwan, 2002). These nematocysts contain complex of irritating toxins, which could be injected by sharp threads into contacting preys or human victims, causing burning stings followed by various pathological reactions. Venoms isolated from different cnidarian classes such as hydrozoans, jellyfishes, sea anemones, and corals, contained a complex of cytolytic and cytotoxic proteins. Among these, phospholipases, hemolysins and pore forming peptides are well documented (Klug et al., 1989; Long-Rowe and Burnett, 1994; Grotendorst and Hessinger, 1999; Bloom et

F.F.Y. Radwan, H.M. Aboul-Dahab / Comparative Biochemistry and Physiology, Part C 139 (2004) 267–272

2.1. Collection of fire corals and preparation of nematocyst venom The fire corals M. platyphylla were collected from the Red Sea submerged reef (depths of 0.5–2 m) at a coastal site located 85 km south of Qusier (Egypt). Palm-sized pieces of about two kilograms were cut, transferred into a cooling box, and shipped frozen to the laboratory where they were kept at 20 8C. Nematocysts were collected from the corals and crude venom (Mp-TX) was prepared according to Radwan (2002). Protein determinations were preformed according to the method of Waddell (1956), comparing the absorbancies at 225 and 215 nm [protein (Ag/mL)=(A215 A225)114].

Mp-TX (5 mg protein, 3 mL volume) was applied to a maintained Sephacryl S200 column (dimensions 1.660 cm) and eluted by 0.05 M phosphate buffer, pH 7 at 4 8C at flow rate 1.5 mL/min. The absorbance was read at 280 nm and individual fractions were collected per 1 min through 60 min run. The operation was repeated 10 times. Post-gel filtration fractions matched between runs were combined, concentrated and screened for hemolytic activity. Accordingly, the fractions which possessed the highest hemolytic activity (hemolytic peak includes fractions from 44 to 52, see Fig. 1A) were pooled, concentrated and re-chromatographed on a CM-32 cellulose column (2.245 cm), equilibrated with 0.01 M ammonium acetate buffer, pH 6.0. The column was eluted with NaCl in a gradient of concentration (0–0.5 M) in the same buffer at the flow rate of 0.8 mL/min. Fractions (2 mL each) were collected, whereas, fractions (13 through 17, see Fig. 1B), composing the major protein peak, were pooled, dialyzed for 4 h against distilled water (Biocompare dialysis tubing, 5kDa) and concentrated using a Millipore Microcon columns (YM-10). The former fraction was used for the following assays and here was named milleporin-1.

A

20 mAbs at 280 nm 15

mAbs at 545 nm

10 5 0 1

10

19

28

37

46

55

Fraction (min)

B

25

0.5 mAbs at 280nm M NaCl

20 15

0.25 10

M NaCl

2. Materials and methods

2.2. Gel filtration and ion exchange chromatography

Absorbance

al., 2001; Torres et al., 2001; Radwan and Burnett, 2001; Radwan et al., 2000, 2001, 2002; Anderluh and Macek, 2002). Some of those peptides have been extensively characterized (Nagai et al., 2000, 2002; Talvinen and Nevalainen, 2002; Malovrh et al., 2003; Oshiro et al., 2004). Both of the difficulty to separate the milleporan polyps from skeletal fragments and concurrent contamination of nematocyst harvest, caused by cellular algal symbionts, have hampered the study of the nematocyst toxins of stinging corals in general. Nonetheless, a simple dissolution method of coral exoskeleton, resulting in dissolving of its calcareous chalices and releasing of animal tissues, was recently developed (Radwan, 2002) and used in this study. Venom of M. platyphylla nematocysts was found lethal to mice (LD50=0.25 Ag/g mouse) and exhibited potent hemolytic, dermonecrotic and vasopermeable activities (Radwan, 2002). The hemolytic activity correlated with mice toxicity, and could be blocked using various lipid membrane compounds such as phosphatidylcholine, phosphatidylserine or dihydrocholesterol-suggesting a possible involvement of phospholipase activity. Also, the venoms, which previously extracted from the same Pacific species (Shiomi et al., 1989a), have shown lethal, hemolytic and capillary permeability-increasing activities as well as positive acid phosphatase and phospholipase A activities. The Red Sea fire corals showed greater lethal and hemolytic potencies than the Pacific ones—a result possibly because of using different techniques for nematocyst isolation and venom preparation. Nonetheless, both previous reports have accused a major protein factor of a molecular mass ranging from 31.5 to 33 kDa (post-gel filtration), for most of lethal and hemolytic venom potentials showed by fire corals. In this study, we tried to purify and partially characterize that protein factor (named here as milleporin-1), as a first step to further the understanding of its mode of action, which in turn should permit the development of more effective remedies against fire coral stings.

Absorbance

268

5 0

0 1

5

9

13

17

Fraction number Fig. 1. Chromatography of Mp-TX on Sephacryl S200 column. The column was equilibrated with 0.05 M phosphate buffer, pH 7 at 4 8C. Fractions (1.5 mL each) were eluted at flow rate of 1.5 mL/min and screened for hemolytic activity (Fig. 1A). Thereafter, the fractions composing the hemolytic peak (44–52, indicated by a bar) were pooled and re-chromatographed on CM-32 cellulose column (Fig. 1B). The column was maintained with 0.01 M ammonium acetate, pH 6, at 4 8C and fractions were eluted at 0.8 mL/min with a gradient concentration of 0–0.5 M NaCl. Fractions 13– 17 (indicated by a bar) were pooled and processed as milleporin-1 fraction.

F.F.Y. Radwan, H.M. Aboul-Dahab / Comparative Biochemistry and Physiology, Part C 139 (2004) 267–272

2.3. Electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out, according to Laemmli (1970). Samples of 5–10 Ag/well were run in 7.5–10% discontinuous gels at 200 V for 45 min with Tris-glycine as a running buffer at 4 8C. Protein bands were visualized with silver staining (Wray et al., 1981). Molecular masses were determined using Bio-Rad Broad-range standards (Cat #161-0321). 2.4. Bioassays 2.4.1. Phospholipase activity assay Phosphatidylcholine (1-palmitoyl, 2-arachidonyl [arachidonyl-1-14C], 1.85 GBq/mmol (BioTrend, Germany) was prepared in the form of vesicles to access enzyme activity as described by Reylonds et al. (1991). A stock solution of substrate was prepared by adding four parts of organic solvent (toluene/ethanol; 1:1) to one part of radioactive phosphatidylcholine (0.05 ACi/AL). 5 AL of the substrate were dispensed into each microcentrifuge tube. Solvents were removed under N2 and 80 AL of 0.1 M Tris buffer was added to each tube. The PLA2 assays were initiated by adding enzyme source to microcentrifuge tubes containing substrate vesicles, and vortexing the tubes for 15 s. In assay, doses ranged from 20– 200 Ag protein (Mp-TX or milleporin-1) were incubated (in duplicates) in 250 AL total volume for 30 min at 28 8C. Meanwhile, negative control tests were processed; however, positive standard curve was established using phospholipase A2 honey bee venom standards (P9279, Sigma-Aldrich). The reactions were terminated by adding extraction solvent (600 AL chloroform/methanol; 2:1) acidified with 100 AL 1.0 N HCl. Arachidonic acid (20 nM in 10 AL chloroform) was added to each tube as a carrier and free fatty acid standard. Each tube was vortexed for 15 s and centrifuged at 11,750g for 2 min. The lower organic phase was transferred to another microcentrifuge tube and two more extractions steps followed with 500 AL chloroform. The free fatty acid fraction was transferred into liquid scintillation vials. The radioactivity was estimated by adding Ecolite scintillation medium then counting on Liquid Scintillation Analyzer. The PLA2 reactive equivalents per mg toxin were estimated based on the standard curve analysis. 2.4.2. Hemolysis assay The hemolytic effects of M. platyphylla venom and fractions were monitored as described by Klug et al. (1989). Briefly; red blood cells (RBCs) from freshly drawn human blood were washed several times in a buffer containing 0.12 mM NaCl, 2 mM EDTA, 25 mM Na-phosphate (pH 7.2). Aliquots of 1 mL of a 0.5% RBCs suspension in 0.9% NaCl, were incubated for overnight (18 h) at 4 8C with different doses [ranging from 10 to 200 Ag protein/mL (n=3)] of crude venom or fractions. The incubation was stopped by

269

centrifuging the samples for 5 min (700g) and the absorbance of the supernatant was measured at 545 nm against a control of hemolysis without venom. The lytic potency of tested venoms was expressed as % hemolysis relative to % hemoglobin released by toxins compared to the total hemoglobin present after a maximal lysis of similar amount of erythrocytes (induced by adding 0.5% SDS). One hemolytic unit (HU) was expressed as a dose caused 50% hemolysis per mg protein of venom. 2.4.3. Inhibition test of phospholipase A2 and/or hemolytic activities of melliporin-1 Phosphatidylcholine (PC) (soybean l-a-phosphatidylcholine, Sigma) was sonicated as a stock solution (5 mg/ mL) in a physiological saline (0.9% NaCl) and applied at concentrations ranged from 40 to 300 Ag to microcentrifuge tubes containing 75 Ag of milleporin-1, incubated for 30 min in a total volume 250 AL, before being added to previously washed RBCs (see Section 2.4.2) to make a total volume of 1 mL/test (Long-Rowe and Burnett, 1994). Meanwhile, concentrations from 10 to 75 Ag/mL of milleporin-1 were incubated directly with RBCs. Other tubes which contain the incubated 300 Ag PC/75 Ag milleporin-1 mixture or only 75 Ag of milleporin-1 in 250 AL saline, were boiled (in a water bath for 3 min) shortly before being added to the blood cells. Incubation with blood was for 18 h at 4 8C. All tests were done in triplicates and blank saline were used for negative control tests. Reaction was stopped and % hemolysis was estimated as mentioned in Section 2.4.2.

3. Results and discussion Fire corals are a known cause of discomfort to those who used to undersea recreational activities. They cause painful stings with burning and itching that last for hours after contact (Addy, 1991). Several techniques have been tried to isolate, purify and test the toxic contents of fire corals (Wittle et al., 1971, 1974; Wittle and Wheeler, 1974; Shiomi et al., 1989a,b). Most of those techniques adopted extraction of toxic substances from materials that shaved off coral surface rather than extracting pure nematocysts—suggesting that extra-nematocyst structures could be also possible sources of active components. However, a simple dissolution method used to dissolve coral exoskeleton and release of animal tissues was recently developed (Radwan, 2002) and applied in this study. This technique promoted the ability to obtain better recovery of intact nematocysts, ready for further venom preparations, and lower contamination with extra-nematocyst materials such as coral debris or algal symbionts (to less than 10%). Nematocyst venom of M. platyphylla (Mp-TX), a potent fire coral hydroids common in the Red Sea, was fractionated using Sephacryl S200 column, meanwhile, a screen for the hemolytic fractions of toxin was carried out using human

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F.F.Y. Radwan, H.M. Aboul-Dahab / Comparative Biochemistry and Physiology, Part C 139 (2004) 267–272 Table 1 Estimation of specific hemolytic and phospholipase activities of M. platyphylla toxins Hemolysis (HU/mg)a (meanFSD)

Toxin

Crude M. platyphylla toxin Mp-TX Milleporin-1

Phospholipase A2 equivalents/mgb (meanFSD)

359F26

27F5

2364F121

423.5F14

a

RBCs (Fig. 1A). Three prominent peaks were resolved. The magnitude of hemolytic activity has been found in the back of the third peak—a toxin fraction rich of proteins of molecular masses of ~30,000–34,000 Daltons (Fig. 2). This fraction was subjected to purification by ion exchange CM32 cellulose column, hence, its major component (here named as milleporin-1) was separated (Fig. 1B). The presence of PLA2-like activity was confirmed in both crude Mp-TX and milleporin-1 purified preparations of nematocyst venom (Fig. 3). Purified milleporin-1 toxin fraction showed greater recovery of phospholipase A2 as well as about 13 times higher hemolysis than crude toxin Mp-TX (Table 1). In fact, red blood cells were completely lysed when incubated with milleporin-1 at dose about 75 Ag/ mL (Fig. 4A). At similar dose, however, milleporin-1 hemolytic activity was greatly inhibited when being incubated with a phosphatidylcholine as PLA2 substrate

Mp-TX Milleporin-1 Control

2000

A 100 75 50 25 0 Control

10

20

40

60

70

75

75 boiled

[Milleporin-1] µg/ml RBCs

B % Hemolysis .

3000

(Fig. 4B). Although, milleporin-1 retained about 30% hemolysis-assuming that a cytolytic part of this fraction is non-enzymatic and greatly thermal unstable (see Fig. 4A

% Hemolysis .

Fig. 2. SDS-PAGE of crude Mp-TX (1), post-Sephacryl S200 hemolytic peak (2) and milleporin-1 fraction purified with CM-32 cellulose column (3). Protein bands were visualized with silver staining (Wray et al., 1981). Molecular masses were determined using Bio-Rad Broad-range standards marker (4) (Cat #161-0321).

One hemolytic unit (HU) was expressed as a dose caused 50% hemolysis per mg protein of venom. b Phospholipase A2 equivalents were calculated using a standard curve established with phospholipase A2 of purified honey bee venom. One PLA2 equivalent equal to the amount of protein required to cause the release of 50% free fatty acid.

100 75 50 25 0

CPM

0/75 40/75 80/75 100/75 150/75 200/75 300/75 300/75 boiled

[PC] / [ Milleporin-1] µg/ml RBCs 1000

20

0µ

g

g 0µ 15

µg 80

µg 40

µg 20

Co

ntr ol

0

TOXIN Fig. 3. Phospholipase A2 activity of Mp-TX and milleporin-1 toxin fraction. Increasing doses of toxins were applied to radiolabeled sn-2 C14arachidonyl phosphatidylcholine substrate prepared as described in Section 2.4.1. Free fatty acid fraction was extracted and analyzed (as counts/min, CPM) using liquid scintillation counter (bars indicate FSD).

Fig. 4. (A) Hemolytic activity of milleporin-1 toxin fraction. Increasing doses of milleporin-1 were incubated with washed human RBCs for 18 h. Hemolysis occurred without toxin was used as control. Another test contain 75 Ag toxin was boiled in 250 AL saline for 3 min before being mixed with blood cells. Tests were carried out in triplicates and % hemolysis was estimated as described in Section 2.4.2 (bars indicateFSD). (B) Inhibition of milleporin-1 hemolytic activity using phosphatidylcholine (PC) as a phospholipids substrate. Increasing concentrations of PC were incubated with constant dose of milleporin-1 (75 ug, a dose that caused a maximal hemolysis to RBCs) for 30 min at room temperature. The mixture was applied to RBCs to make 1 mL total volume. Another test, containing the maximal inhibiting dose, was boiled in 250 Al saline for 3 min before being mixed with blood cells. Tests were carried out in triplicates and % hemolysis was estimated as described in Sections 2.4.2 and 2.4.3 (bars indicateFSD).

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and B). These findings suggest that the underlying mechanism of fire coral nematocyst venom-induced hemolysis is highly due to a direct enzymatic action of venom phospholipase A2 on membrane phospholipids, and indirect lysis facilitated by a possible binding with the non enzymatic part of toxin. The mechanism of hemolysis induced by nematocyst venom and the role of phospholipase A and the direct lytic factors has been proposed (Hessinger and Lenhoff, 1976). It was documented that amphiphilic structure of some cytolytic toxins, separated from nematocyst venoms separated from jellyfish and sea anemones, was account for their potent hemolytic activity (Grotendorst and Hessinger, 1999; Nagai et al., 2000; Malovrh et al., 2003). These bco-factorsQ allow the formation of pores in susceptible membranes and play a role as precursors of more complicated cytolytic and cytotoxic events. Interestingly, the better known direct lytic venoms such as cobra and bee venoms contain a second lytic protein in addition to their PLA2. Milleporin-1, which showed a tight link to its non-enzymatic factor, suggests a similar synergistic action to harm the integrity of the cell membrane. Yet, it is unclear whether specific receptors, or some other mechanism, may play a role in addition to a simple lipid binding and formation of pores. However, future biochemical and pharmacological studies on milleporin-1 would give details of its structure and biomedical properties.

Acknowledgements The authors wish to thank Dr. J.W. Burnett, University of Maryland Baltimore, USA for his valuable thoughts and support of this work and Mr. Refaat Galal (deceased in 2003), South Valley University (SVU) Electron Microscope Unit, Faculty of Science at Sohag, for technical assistance in nematocyst investigations. Thanks are also due to Ms. Hanan Nagdi for typing the manuscript. This study was supported by SVU Scientific Research Funds from the Ministry of Higher Education (Egypt) (FY2002/2003).

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