Purification and characterization of two hemolysins from Stichodactyla helianthus

Toxicon 39 (2001) 187±194

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Puri®cation and characterization of two hemolysins from Stichodactyla helianthus MarõÂ a Eliana Lanio a, Vivian Morera b,*, Carlos Alvarez a, Mayra Tejuca a, Teresita GoÂmez a, Fabiola Pazos a, Vladimir Besada b, Diana MartõÂ nez a, Vivian Huerta b, GabrõÁ el PadroÂn b, MarõÂ a de los Angeles ChaÂvez a a

Department of Biochemistry, Biology Faculty, University of Havana, Calle 25 No 455, Plaza, P.O. Box 10400, Havana, Cuba Physical-Chemistry Division, Protein Structure Department, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba

b

Received 8 February 1999; accepted 21 January 2000

Abstract Two hemolysins, Sticholysin I (St I) and Sticholysin II (St II) were puri®ed from the sea anemone Stichodactyla helianthus combining gel ®ltration and ion exchange chromatography. The amino acid composition of both cytolysins was determined revealing a high proportion of glycine, lysine, tyrosine and non-polar amino acids (alanine, leucine and valine). Cysteine was not found in either polypeptide. Molecular masses of St I and St II were 19401 and 19290 Da, respectively. N-terminal sequence analysis of St I and St II showed a high homology between them suggesting they are isoforms of the same cytolysin. Compared with other sea anemone cytolysins, St I and St II contain a 22 amino acid insertion fragment also present in Eq T II/Tn C and probably in CaT I and Hm T and absent in C III, the major hemolysin previously reported in this anemone. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Hemolysin; Cytolysin; Sea anemone; Stichodactyla helianthus

1. Introduction Several polypeptides isolated from sea anemones are bioactive substances probably synthesized by the animal for use in feeding or defense against predators. Among them, a number of hemolytic factors have been puri®ed to homogeneity and analyzed from many di€erent species of anemones but relatively few of

* Corresponding author: Fax: +1-53-7-336008. E-mail addresses: [email protected] (M.E.MarõÂ a E. Lanio), [email protected] (V. Morera).

them are well understood in terms of their lytic mechanism (Turk, 1991). Cytolysins are water-soluble polypeptides exhibiting the unique property of inserting and accommodating spontaneously into membranes. Due to their lytic capacity and the possibility to address them to speci®c tissues, cytolysins have been evaluated as promising anti-tumor agents (Avila et al., 1988; Pederzolli et al., 1995). Cardiostimulatory and anticoagulant properties have also been reported for some of them. (Cline et al., 1995; DõÂ az et al., 1992; Galletis and Norton, 1990). Characterization of these proteins revealed that most of them are single-chain basic polypeptides of approximately 20 kDa that

0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 0 ) 0 0 1 0 6 - 9

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Nomenclature AjH-2

Hemolysin 2 from Anthopleura japonica CaT I and Ca T II Caritoxins from Actinia cari C I, II, III and IV isoforms of the cytolysin from Stichodactyla helianthus ESI - MS electrospray ionization mass spectrometry

increase ion membrane permeability by forming a channel complex resulting from the association of toxin monomers. A singular characteristic of these anemone cytolysins is that their hemolytic e€ects can be prevented by pre-incubation with sphingomyelin (Belmonte et al., 1993; Macek, 1992; Michaels, 1979; Varanda and Finkelstein, 1980). Stichodactyla helianthus is a sea anemone relatively abundant along Cuban sea coasts. Devlin (1974) ®rst reported the presence of a toxic polypeptide causing lysis of red blood cells in this anemone. Since then much work has been done to purify and characterize the molecular entities of this anemone. The laboratories of Mebs in Germany (Mebs et al., 1992) and Bernheimer (Bernheimer and Rudy, 1986) and Kem in USA (Kem, 1988; Kem and Dunn, 1988) have carried out most of this work. In this paper, we employed a very similar puri®cation procedure previously reported by Kem and Dunn (1988), which allows isolating, with an adequated degree of purity, of two hemolytic polypeptides: Sticholysin I and II (St I and St II) from the Caribbean sea anemone Stichodactyla helianthus. The Nterminal sequence of Sticholysin II was similar to the sequence previously reported by Blumenthal and Kem (1983) for Sh Cytolysin III, except that it additionally contains a 22 amino acid peptide segment (position 30±51) similar to what has been reported for Tenebrosins and Equinatoxins. This ®nding eliminates the only reported di€erence between Sticholysin II and Sh C III and allows the conclusion that they are one and the same molecule.

2. Materials and methods 2.1. Puri®cation procedure Specimens of the anemone Stichodactyla helianthus were collected along the coast of Havana City. Total extracts were obtained by mincing and homogenizing the whole body of the anemone (GoÂmez et al., 1986). Typically, 500 mg of crude extract was applied to a

Eq T II Hm T IEF Tn C

Equinatoxin II from Actinia equina L cytolysin from Heteractis magni®ca Isoelectric focusing Tenebrosin C from Actinia tenebrosa

Sephadex G-50 (Medium) column (dimensions 4  100 cm; Pharmacia-LKB, Sweden) equilibrated with 0.02 M sodium acetate bu€er, pH 5 and eluted with the same bu€er at a ¯ow rate of 36 ml hÿ1. The second peak which showed the highest hemolytic activity was chromatographed on a CM-52 cellulose column (dimensions 1:5  30 cm; Pharmacia-LKB, Sweden) equilibrated with 0.1 M sodium acetate bu€er, pH 5. Elution was carried out with an ionic strength linear gradient (0.1±0.4 M) of the same bu€er at a ¯ow rate of 23 ml hÿ1. The fractions corresponding to each peak were concentrated and dia®ltrated with distilled water using an Amicon ultra®ltration device equipped with a membrane whose cut-o€ was 1000 Da. Protein concentration was determined as previously described (Lowry et al., 1951) in the presence of 0.1% sodium deoxycholate. Absorption coecients e0:1% , 1 cm at 280 nm was determined from dried samples of known weight in a Cary 219 spectrophotometer (USA).

2.2. Biological activity assays 2.2.1. Determination of hemolytic activity (HA) Erythrocyte suspensions were prepared using fresh human red blood cells, washed and resuspended in physiological bu€er solution (Tris bu€ered saline, TBS: 0.145 M NaCl, 0.01 M Tris±HCl, pH 7.4). The concentration of the standard cellular suspension was adjusted by addition of TBS in such a way that, when complete lysis of 1 ml of the suspension was achieved after addition of 14 ml of 0.1% sodium carbonate, the absorbance at 540 nm, measured in a 1 cm cuvette, was 0.7. This value corresponds to an approximate cellular concentration of 1  108 cells mlÿ1. For the determination of the HA, a few microlitres of the protein solution were added to 1 ml of the standard erythrocyte suspension. After protein addition, the erythrocyte suspension was incubated for 30 min at room temperature. Elapsed this time, the samples were centrifuged at 300  g for 15 min and the absorbance of the supernatant was measured at 540 nm against a control of hemolysis without cytolysin.

M.E. Lanio et al. / Toxicon 39 (2001) 187±194

2.2.2. Evaluation of phospholipase activity Phospholipase activity was determined by a colorimetric procedure previously described using egg phosphatidyl choline as substrate (Lobo de Araujo and Radvanyi, 1987).

2.3. Preliminary physical±chemical characterization The purity of proteins was checked by SDSPAGE according to Laemmli (1970) using a 17.5% polyacrylamide gel. Samples (20 mg) were dissolved in Laemmli bu€er (10% SDS, 0.02 M 2-mercaptoethanol, pH 6.8) and heated at 1008C for 10 min. Before molecular mass and amino acid analysis were performed, St I and St II were submitted to a last puri®cation step by HPLC (Pharmacia-LKB, Sweden) on a reversed phase column RP-C8 …4:6  250 mm, J.T. Baker, USA). Elution was performed with an acetonitrile gradient from 15% to 60% in water containing 0.1% tri¯uoroacetic acid with a ¯ow rate of 0.8 ml minÿ1 at 378C. Protein peaks were detected by absorbance measurements at 226 nm. Molecular masses were determined by ESI-MS in a JEOL JMS-HX110/110A spectrometer (JEOL, Japan) equipped with an ESI double focusing interface. The injection ¯ow and the acceleration voltage employed were 1 ml minÿ1 and 7 kV, respectively. Samples were dissolved in 10 mL methanol:water (1:1 v/v) containing 2% acetic acid. Amino acid analysis was performed after hydrolysis of the pure protein according to Allen's method (Allen, 1989) using an automatic analyzer Alpha Plus 4151 (Pharmacia-LKB, Sweden). Fluorescence and absorption spectra of the proteins in 0.01 M Tris±HCl, pH 7.5 at 378C were recorded from 310 to 440 nm, after excitation at 295 nm, using a Perkin-Elmer MPF-44B spectro¯uorimeter (PerkinElmer, USA) and from 250 to 350 nm employing a Cary 219 spectrophotometer, respectively. N-terminal amino acid sequence analysis was performed by automated Edman degradation of the protein using a dual phase sequencer (model 810/ 816, Knauer, Germany) equipped with an on-line phenylthiohydantoin amino acid analyzer. Isoelectric focusing was carried out on the PhastSystem2 (Pharmacia-LKB, Sweden) using the optimized method for IEF with PhastGel IEF 3-9 and a high pI calibration kit (pH 5±10.5). Proteins were visualized according to the Pharmacia-LKB Owners Manual using the PhastGel2 silver kit.

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3. Results 3.1. Puri®cation of St I and St II from Stichodactyla helianthus The puri®cation procedure described leads to the isolation of two resolved proteins having hemolytic activity: St I and St II. The ®rst chromatographic separation on a Sephadex G-50 column rendered ®ve resolved peaks (Fig. 1(A)). Most of the hemolytic activity coincided with the second absorbance peak, which also showed very low phospholipase A activity. Peak I (the void volume peak) contained phospholipase A activity (5 U mgÿ1) and relatively low hemolytic activity …HC50  100 mg mlÿ1) (Pazos et al., 1996). On the other hand, peak III exhibits proteinase inhibitory activity whose main component has been puri®ed, sequenced and thoroughly evaluated (Delfõ n et al., 1994, 1996). Peak III, IV and V contain neurotoxins; some of them have been puri®ed to homogeneity and functionally analyzed (CastanÄeda et al., 1995; Kem et al., 1989). The results obtained in the gel ®ltration chromatographic step are similar to those reported by Kem and Dunn (1988), using the toxin-enriched exudate instead of whole animal homogenate. The hemolytic fractions (peak II) from two Sephadex G-50 column runs were applied onto a CM-52 cellulose column yielding two well resolved peaks St I and St II, eluting at 0.22 and 0.27 M sodium acetate, respectively (Fig. 1(B)). This chromatographic pro®le was di€erent to what obtained by Kem and Dunn (1988) using non-denaturing concentration of urea during elution. The minor peaks observed in the pro®le (Fig. 1(B)) did not exhibit a signi®cant hemolytic activity. St I and St II showed a high hemolytic activity …HC50  30±45 ng mlÿ1 where HC50 is the protein concentration necessary to lyse 50% of the cells) and very low phospholipase activity (010ÿ3U mgÿ1 of protein). SDS-PAGE of both fractions showed a single band for each one of apparent molecular mass of about 20 kDa, suggesting the homogeneity of both fractions (Fig. 2(A)). The sequence of steps summarized in Table 1 seems to be adequate for the puri®cation of St I and St II as the main hemolytic components present in the total extract of Stichodactyla helianthus. The low yield obtained (estimated on protein grounds) is compensated by the high purity obtained for both cytolysins (36 and 50 puri®cation factor, Table 1). From the total extract to the gel ®ltration chromatographic step, there is an apparent increase in the total hemolytic units (data not shown). This increase could be explained by the dissociation of hemolysin-inhibitor complexes that takes place when the total extract is submitted to gel ®ltration chromatography. The assessment of protein purity was ®nally per-

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Fig. 1. Puri®cation of Stichodactyla helianthus hemolysins. A: Chromatographic separation of Stichodactyla helianthus total extract on Sephadex G-50. 500 mg of total extract were applied on the column …4  100 cm) equilibrated and developed with 0.02 M sodium acetate bu€er, pH 5. Fractions of 3 ml were collected at a ¯ow rate of 36 ml hÿ1and monitored at 280 nm. B: Ion exchange chromatography of the two Stichodactyla helianthus cytolysins. The second peak of the gel ®ltration step (Fig. 1(A)) showing the highest hemolytic activity was pooled, dia®ltrated and chromatographed (90 mg) on a CM cellulose 52 column …1:5  30 cm) equilibrated with 0.1 M sodium acetate bu€er, pH 5. Elution was carried out with an ionic strength linear gradient made from 200 ml each of 0.1 and 0.4 M of the same bu€er. Fractions of 3 ml were collected at a ¯ow rate of 23 ml hÿ1.

M.E. Lanio et al. / Toxicon 39 (2001) 187±194

191

Fig. 2. Assessment of hemolysins purity by SDS-PAGE and reversed-phase HPLC. (a) SDS-PAGE analysis of St I and St II. Lanes A) molecular weight markers (67 kDa bovine albumin, 45 kDa egg albumin, 25 kDa chymotrypsinogen, 18 kDa a-interferon, 14 kDa lysozime), B) St I C) St II. (b) Puri®ed hemolysins St I and St II chromatographed on reversed phase HPLC. Chromatographic conditions: Baker RP-C8 column (300 nm pore size, 5 mm octylsilica packed into a cartridge 4:6  250 mm); gradient: from 15% to 60% in A, where solvent A was 0.1% (by vol.) aqueous TFA and solvent B was acetonitrile containing 0.05% (by vol.) aqueous TFA. Flow rate was 0.8 ml minÿ1; column temperature was 37oC.

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Table 1 Characteristics of the main hemolytic fractions obtained during the puri®cation of the cytolysins from Stichodactyla helianthus Fractions

Protein (mg)

HU/mg protein (10ÿ3)a

Yield (%)

Puri®cation factor

Total extract Peak II St I St II

570 42 7.5 15

0.61 13.9 21.7 30

100 7.4 1.3 2.6

1 23 36 50

a HU Ð Hemolytic activity units. One HU is the of amount of protein that dissolved in 1 ml of the test erythrocyte suspension caused 50% lysis after 30 min incubation at 378C.

formed on a reversed phase HPLC (Fig. 2(B)). The chromatograms showed one major component at 56% and 60% of acetonitrile for St I and St II, respectively, apparently contaminated one with the other. 3.2. Some molecular characteristics of both cytolysins The amino acid composition of St I and St II are shown in Table 2. As for the rest of the sea anemone cytolysins (Turk, 1991) both proteins contain a high content of glycine and of non-polar amino acids, the highest values found were for alanine, leucine and valine. Aromatic amino acids are relatively abundant in St I and St II and tyrosine is the best represented of them. Among the basic amino acids, lysine is the most abundant. No cystine was found in hydrolysates of native proteins and no cysteic acid following performic acid oxidation. It means that Stichodactyla helianthus

Table 2 Amino acid compositions of proteins St I and St II. Values are given for 24-h total acid hydrolysis. The experimental values are residues molÿ1. AA Ð amino acid, EV (a) Ð average experimental value, SD Ð standard deviation St I AA B T S Z G A V M I L Y F H K R

EV (a) 17.1 8.9 10.0 10.4 20.3 12.8 10.1 5.3 8.3 11.9 13.5 4.5 0.3 10.8 7.5

St II SD 0.00 0.07 0.14 0.14 0.28 0.14 0.71 0.14 0.07 0.14 0.35 0.21 0.00 0.35 0.14

EV (a) 16.6 9.9 8.9 10.4 19.4 14.8 10.1 4.4 8.3 12.9 12.3 5.4 1.3 11.2 7.9

SD 0.28 0.14 0.64 0.14 0.78 0.14 0.00 0.64 0.00 0.07 0.64 0.00 0.14 0.35 0.35

hemolysins lack either cystine or cysteine and therefore there are no disul®de bridges involved in the covalent structure of these proteins. St I and St II display very similar absorption and ¯uorescence spectra. The absorbance spectra for both proteins were characterized by a maximum at 278 nm and the presence of a broad absorption band (270±290 nm) typical of polypeptides containing aromatic amino acids. Absorption coecients e0:1% , 1 cm at 280 nm of both proteins were 2:1320:37 for St I and 1:8720:21 for St II. The maximum of the emission band after excitation at 295 nm was located at 335 nm for both St I and St II suggesting a relative exposition of tryptophan to the aqueous environment. The average masses determined by ESI-MS were 19401210 Da and 1929027 Da for St I and St II, respectively. Isoelectric focusing indicated that both proteins are basic, exhibiting pIs equal or higher than 9 (data not shown). A highly basic pI (10.5) has also been reported for Eq T II, a cytolysin puri®ed from the Mediterranean anemone Actinia equina (Macek and Lebez, 1988). The N-terminal sequence analysis on the intact proteins permitted to establish the N-terminal sequence up to the 58th and 64th residues for St I and St II, respectively. Although the sequences are di€erent, indicating that St I and St II are not the same molecular entity, it is clear that they correspond to isoforms of the same hemolysin, since there is a very high sequence homology (93%) (Fig. 3). The comparison of the Nterminal sequence of St I with that of St II revealed four substitutions, three of them non-conservative and one conservative.

4. Discussion The puri®cation procedure presented in this paper leads to the obtainment of two hemolysins: St I and St II from the Caribbean sea anemone Stichodactyla helianthus. The results obtained in the ion exchange chromatographic step on CM-52 cellulose (Fig. 1(B)) are di€erent from those previously reported by Kem

M.E. Lanio et al. / Toxicon 39 (2001) 187±194

and Dunn (1988) working with an analogous fraction from Stichodactyla helianthus. The two additional peaks obtained in that work are probably the result of pre-treating the original sample with 4 M urea that solubilized two additional proteins not found as a result of the procedure here developed. Peaks corresponding to C II and C III (Kem and Dunn, 1988) are very similar in shape and relative absorbances to the two hemolytic proteins puri®ed in this work (St I and St II, respectively). A common feature of many cytolysins puri®ed from sea anemones, including St I and St II (Table 2), is the absence of cysteine and the presence of high proportion of non polar amino acids (Macek, 1992). According to this, St I and St II can be classi®ed into the group of sea anemone cytolysins comprising Eq T II, Tn C, Ca T II, C III, AjH-2, Parasitoxin and their isotoxins, all of them having very similar amino acid composition (Turk, 1991). Amino terminal sequencing of the puri®ed protein St II exhibits exactly the same amino acids per cycle, up to the 29th, as has already been reported for C III (Blumenthal and Kem, 1983). In contrast, from cycle 30 to 51 the amino acids detected did not match at all with those of C III and coincided again after cycle 51 to 64 (Fig. 3). The comparison St II amino-terminal sequence with those of Eq T II puri®ed from the Mediterranean anemone Actinia equina (Belmonte et al., 1994) or Tn C puri®ed from the Australian anemone Actinia tenebrosa (Simpson et al., 1990) (Fig. 3), revealed that these two polypeptides also contain the 22 amino acid insertion fragment absent in the original C III (Blumenthal and Kem, 1983) and present in St II. Comparing the sequence of the insert present in St II with those present in Eq T II or Tn C, only three substitutions can

193

be observed: 1 conservative and 2 non conservative substitutions, thus between St II insertion fragment and those of Eq T II and Tn C there exist 19 identical amino acids. Therefore, we can regard this insert as a high similarity fragment (86%). The comparison of the St II insert with the relatively small fragment sequenced of Ca T I from Actinia cari (Sencic and Macek, 1990) showed complete identity and slight di€erences with the portion of Hm T from Heteractis magni®ca (Mebs et al., 1992) suggesting that these two proteins might contain the 22 amino acid insertion fragment present in St II, Tn C and Eq T II and absent in the original C III (Fig. 3). The molecular size of C III was recently shown by ESI-MS to be of 19303230 Da; apparently a tryptic fragment corresponding to the missing sequence near the N-terminal was lost during HPLC puri®cation (Kem, personal communication). Even though St I and C I show complete homology in the small-analyzed N-terminal fragment (Fig. 3), it was impossible to suggest that they could be the same molecular entity. Considering their di€erences in solubility and relative proportions in the chromatographic pro®le (Fig. 1(B); Kem and Dunn, 1988) and lacking of additional sequence information it is impossible to conclude the exact correspondence among St I and the isoform C I. The high homology found between the N-terminal of St II and C III primary sequences and the re-evaluation of C-III molecular size (Kem, personal communication) indicate that C III and St II are the same protein. These results de®nitively solve the apparent contradiction that existed among proteins exhibiting similar functional characteristics but di€erent amino acid sequences (C III di€erent from Eq T II and Tn C).

Fig. 3. Alignment of N-terminal amino acid sequences of sea anemone cytolysins. Asterisks denote identical amino acid residues. X indicates unidenti®ed amino acids. Eq T II Ð Equinatoxin II from Actinia equina (Pederzolli et al., 1995). Tn C Ð Tenebrosin C from Actinia tenebrosa (Sencic and Macek, 1990). CaT I Ð Caritoxin I from Actinia cari (Simpson et al., 1990). Hm T Ð cytolysin from Heteractis magni®ca (Mebs et al., 1992). C I, C II, C III and C IV Ð isoforms of cytolysin from Stichodactyla helianthus (Kem and Dunn, 1988). St I and St II Ð Sticholysin I and II, isoforms of the cytolysin from Stichodactyla helianthus puri®ed in this work.

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