Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

ORIGINAL PAPER

Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

Karthikayalu S^{I, II, III}; Rama V^II; Kirubagaran R^I; Venkatesan R^I

^INational Institute of Ocean Technology, Chennai, Tamil Nadu, India

^IIDepartment of Industrial Biotechnology, Dr. M.G.R. University, Maduravoyal, Chennai, Tamil Nadu, India

^IIIAxioGen Biotech Private Limited, Pondicherry, India

^{Correspondence to}

ABSTRACT

It is well established that sea anemones comprise a rich source of cytolytic toxins. The present study reports the isolation and characterization of a cytolysin obtained from the sea anemone Heteractis magnifica collected in the Andaman Islands of the Indian Ocean. The crude extract was screened for hemolytic activity by a blood agar plate method and a 6-mm zone of clearance was observed after incubation. The hemolytic property of the crude extract, tested by the microtiter plate method, revealed positive results at concentrations as low as 120 ng/mL. Furthermore, it was favored by alkaline pH and was stable up to 60°C. On the other hand, the hemolytic effect was abolished by the addition of human serum. Purification steps involved ammonium sulfate precipitation and subsequent desalting by dialysis, followed by anion- and cation-exchange chromatographies. The purified fractions displayed the presence of a 19-kDa cytolysin when analyzed by SDS-PAGE. The conserved region of the cytolysin (with 303 bp) was amplified by RT-PCR and was sequenced. The sequence showed maximum homology (97%) with the already reported cytolysins from other sea anemone species.

Key words:Heteractis magnifica, marine toxin, hemolysin, cytolysin.

INTRODUCTION

The cnidarians comprise species of mostly marine aquatic invertebrates that constitute the phylum Cnidaria represented by sea anemones, corals, sea fans, sea pens, jellyfish and zooanthids, which are known to secrete a variety of cytolytic toxins mainly for defensive purposes (1-5). Four major groups of these cytolysins are found in cnidarians (5, 6). Among them, the 19-kDa pore-forming cytolysins are the most predominant (5-15). In previous studies, the sea anemone Heteractis magnifica was subjected to the analysis of different cytolysins including magnificalysins Hmg I, Hmg II and Hmg III (14-18). However, Hmg III is the most potent and active cytolysin, whose molecular mass has been determined as 19 kDa (14-15). The N-terminal amino acid sequences of Hmg III also exhibited homology with other sea anemone hemolysins such as equinatoxins from Actinia equina, tenebrosin-C from Actinia tenebrosa and cytolysin III from Stichodactyla helianthus (19-21).

Besides being known as a pore-forming cytolysin, Hmg III has not been further investigated on its physiological characteristics regarding the cytolytic activity. Hence, the present study has been undertaken on the isolation, physiological characterization and purification of the cytolysin Hmg III from the sea anemone Heteractis magnifica employing a human erythrocyte model. Furthermore, the sequence verification has also been performed on this Indian sea anemone species and compared with a Singaporean species of which the sequence has been already known. The sequence comparison among cytolysins of other cnidarians - such as Stichodactyla helianthus, Heteractis crispa, Actinia equina and Oulactis orientalis - has also been performed by a phylogenetic analysis for establishing a genetic relationship.

MATERIALS AND METHODS

Collection of the Sea Anemone Heteractis magnifica

The sea anemone Heteractis magnifica - identified by Fautin and Allen (22) - was collected from Andaman Islands, India, at a depth of 5 m by scuba diving.

Preparation of the Toxin

In the laboratory, the live animal was induced through osmotic thermal stress to eject the epithelial mucus that contains the toxins (8). The cnidarian toxins are stored in the cnida, a capsule within specialized cells that contains a barbed, threadlike tube that delivers a paralyzing sting when propelled into attackers and prey. Approximately 2 kg of anemone was added to 1 L of warm distilled water (40 to 45°C) for 15 minutes and subject to intermittent stirring to minimize tissue damage. After 15 minutes, the anemone was removed and the solution was filtered. The filtrate was stored in 100-mL aliquots in liquid nitrogen for transportation. Subsequently, the extract was removed from the nitrogen and stored at -70°C until analysis. When required, the aliquots were thawed, concentrated by lyophilization and reconstituted in phosphate buffered saline at pH 7.4.

Protein Assay

Protein concentration was determined by the Lowry et al. method using bovine serum albumin (BSA) as standard (23). The protein concentration during purification studies was measured from the absorbance at 280 nm. One hundred microliters of purified fractions were read at 280 nm using SpectraMax® microplate reader (Molecular Devices, USA).

Hemolytic Activity on Human Blood Agar Plate

Human blood agar plate was prepared by adding 5 mL of human blood to 95 mL of sterile nutrient agar and pouring the mixture immediately on petri plates. After solidification, wells were cut on the agar plate using a corkscrew borer (8 mm diameter). Wells were loaded with 50 μL (1 mg/mL) of protein samples. The plates were observed for hemolysis after overnight incubation at room temperature.

Hemolytic Assay

Hemolytic activity of the toxin was measured quantitatively in terms of attenuance on human red blood cells at room temperature using a microplate reader. Freshly collected human blood with heparin was centrifuged to remove the buffy coat. Afterward, the obtained erythrocytes (RBC) were washed three times in 0.85% saline and stored at 4°C. Toxins at determined concentrations were added in the first well to erythrocyte buffer (140 mM NaCl, 10 mM Tris-HCl, pH 7.4) and then subjected to two-fold serial dilution. RBC (100 μL; D₆₃₀ = 0.5) in erythrocyte buffer were added to the toxins, and hemolysis was monitored by measuring attenuance at 630 nm for 20 minutes at room temperature. The final volume in all wells was 200 μL. The percentage of hemolysis was determined at the end of the assay using the following equation of Maček et al. (24):

in which D_obs is the measured attenuance in the well after 20 minutes and D_max is the maximal attenuance by distilled water and D_min is the minimal attenuance by erythrocyte buffer.

Characterization of the Hemolytic Extract

The crude hemolytic extract was characterized with respect to thermal and pH stability, freeze-thaw stability and treatment with human serum.

Effect of temperature and freeze-thaw process

In order to study the stability of the extract under different temperatures, it was exposed to different heat treatments: 25°C (room temperature), 60°C for one hour, 80°C for one hour, 100°C for one hour and 121°C and 15 psi by autoclaving for 15 minutes. The extract was also frozen at -80°C and then quickly thawed to 37°C. The freeze-thaw procedure was repeated three times and the hemolytic activity was assessed.

Effect of pH

The buffer pH of the extract was 9 and was adjusted to 3, 4, 5, 6, 7 and 8 with hydrochloric acid (HCl), then, incubated for one hour at room temperature and assayed.

Effect of human serum

Human serum was added to the extract at a final concentration of 1% and incubated at room temperature for one hour. The mixtures were assessed for hemolytic activity against human RBC, with serum-free extract as control.

Ammonium Sulfate Precipitation

The crude extract was treated with ammonium sulfate (Himedia Laboratories, India) to 20, 40, 60, 80 and 100% saturation according to the Rosenberg table (25). The mixtures were stirred for 30 minutes at 4°C and centrifuged afterward at 10000 x g for ten minutes at 4°C. The precipitates were resuspended in 10 mM sodium phosphate buffer (pH 7.2) and desalted in a dialyzing tube with a cutoff of 12 to 14 kDa (Himedia Laboratories, India) against 10 mM sodium phosphate buffer at pH 7.2.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS- PAGE)

Protein samples were analyzed by SDS-PAGE using 5% stacking gel and 12% resolving gels (26). Samples were denatured by boiling in loading buffer containing SDS and β-mercaptoethanol before they were placed on the gel. Thirty micrograms of crude protein and 10 μg of purified fractions were placed on the gel and underwent electrophoresis. Following electrophoresis at 15 mA for four hours, gels were stained with Coomassie brilliant blue R-250 (Himedia Laboratories, India) and destained in a solution of 50% (v/v) methanol and 10% (v/v) acetic acid.

Purification of Hemolytic Proteins by Ion-Exchange Chromatography

The dialyzed fractions were purified by ion-exchange chromatography. Anion-exchange chromatography was performed using DEAE-cellulose column (1 x 5 cm) at a flow rate of 1 mL/minute (25). The column-stabilizing buffer was 10 mM sodium phosphate buffer, pH 7.0. Two-milliliter (10 mg/mL) of the crude protein extract in stabilizing buffer was placed on the column. Elution of the bound proteins was performed using a linear gradient of sodium chloride (0.1 M, 0.25 M, 0.5 M, 1 M and 1.25 M). Protein concentrations of the collected fractions were measured by absorbance at 280 nm using a microplate reader and fractions showing hemolytic activity were pooled together, lyophilized and dialyzed against 10 mM phosphate buffer overnight at 4°C. The dialyzed fractions were subjected to cation-exchange chromatography using CM-cellulose column and a similar method previously described in this study.

Isolation of RNA

Total RNA was isolated from the tentacles of the sea anemone H. magnifica using TRIzol® reagent according to the manufacturer's protocol (ABgene, UK). The quality of the RNA preparation was determined by electrophoresis in agarose gel (1.2%) and the RNA was employed for reverse transcription reaction.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

One microgram of H. magnifica RNA was used in the RT-PCR performed with Reverse-iT® One-step RT-PCR kit (ABgene, UK) in a total reaction volume of 20 μL. Gene-specific primers (HMg2F 5P-ATTGATGGTGCAAGTCTGGG-3P, HMg2R 5P-GTTGCTGTACCAGTTATAGTC-3P) were utilized for the amplification reaction. Primer sequences were obtained according to Wang et. al (14). Other components of the RT-PCR reaction were 2x RT-PCR master mix (Thermoprime plus DNA polymerase, reaction buffer, 0.2 mM dNTP mix and 1.5 mM MgCl₂ - ABgene, UK) and 50 U/μL of Reverse-iT® RTase blend (ABgene, UK). The mixture was incubated for one hour at 42°C and inactivated by heating at 95°C for five minutes. PCR was performed by denaturation at 95°C for 20 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds and final extension at 72°C for five minutes. The PCR product was electrophoresed on 1.2% agarose gel. The PCR band was excised from the agarose gel and eluted and purified using a gel elution kit (Eppendorf, Germany) according to the manufacturer's protocol. The purified PCR product was sequenced.

RESULTS

Hemolytic Assay

The hemolytic activity of the crude extract is shown in the Figure 1. A 6-mm diameter clear zone of lysis was observed around the wells in human blood agar plates when 50 mg of crude extract was added after incubation (Figure 1). The hemolysis induced by hemolysins in red blood cells suspension was concentration-dependent (Figure 2). The crude extract revealed 50% hemolysis at a concentration of ~120 ng/mL by microtiter plate assay (Figure 2).

Physiological Properties

Hemolytic activity peaked at room temperature (25°C) and was partially reduced at 60°C. No activity was observed when the extract was subject to more than 80°C for one hour (Figure 3 - A). Frequent freeze-thaw processes of the stored samples at -80°C affected considerably the hemolytic activity (Figure 3 - A). The pI of this toxin ranges from 9.0 to 10.0. Therefore, its activity in in vitro conditions has also been assumed to occur at this high pH level. Hence, hemolysis was favored in an alkaline pH (Figure 3 - B) whereas activity was reduced in acidic medium. On the contrary, addition of 1% human serum to the extract resulted in neutralization of the hemolytic activity (Figure 3 - C).

Precipitation of Hemolytic Toxin

Table 1 shows the relative distribution of the hemolytic activity of H. magnifica crude extract, precipitated by ammonium sulfate. Crude extract proteins were precipitated in 20%, 40%, 60%, 80% and 100% fractions. All the fractions obtained during salting out process showed hemolytic activity, which indicates that the toxin was precipitated in all fractions (Table 1). However, proteins separated by 60 to 80% ammonium sulfate precipitation were found to be more active. SDS-PAGE analysis of ammonium sulfate precipitated fractions possessing cytolytic activity revealed the presence of a protein band with a molecular weight of 19 kDa (Figure 4).

Toxin Purification by Ion-Exchange Chromatography

The fractionation of the ammonium sulfate precipitated crude extract of H. magnifica is shown in Figure 5 - A, after purification by ion-exchange chromatography. Six major peaks, called A, B, C, D, E and F, were obtained by DEAE-cellulose column chromatography. Peak A fractions were found to exhibit hemolytic activity. These fractions were pooled, lyophilized and dialyzed against 10 mM phosphate buffer overnight at 4°C. Dialyzed fractions were lyophilized and purified by carboxymethyl-cellulose (CM) cation-exchange chromatography. Subsequently, six major peaks - called A1, A2, A3, A4, A5 and A6 - were obtained by CM-cellulose column (Figure 5 - B) and the fractions under peak A6 showed hemolytic activity. Then, fractions corresponding to peak A6 were pooled together, lyophilized and analyzed by SDS-PAGE.

SDS-PAGE

SDS-PAGE analysis of the active fractions revealed two bands of approximately 14 and 19-20 kDa respectively (Figure 6). The result reveals the presence of the 19 kDa pore-forming cytolysin in the purified fractions.

RT-PCR

One hundred micrograms of RNA were extracted from 100 mg of tissue. One microgram of RNA was reverse-transcribed and amplified by RT-PCR. Using PCR primers, HMg2F and HMg2R, a 303 bp PCR product coding for the conserved region of the cytolysin was obtained (Figure 7) and purified. The purified PCR product was sequenced (accession n. AB254368) and analyzed by BLASTn (Figure 8), BLASTx (Figure 9) and Clustal W (Figure 10).

DISCUSSION

The present study demonstrated the hemolytic activity of a cytolysin isolated from the sea anemone H. magnifica. The isolated toxin required alkaline pH (7 to 9) and a determined temperature (25°C) for its optimal activity. Additionally, this study included cDNA amplification which enabled comparison of the conserved region of this cytolysin with others.

The purified active protein from ion-exchange chromatography was separeted into two bands by SDS-PAGE, one with ~14 kDa and the other with ~19 kDa. The nucleotide sequence of H. magnifica cytolysin was compared with the Hmg III cytolysin cDNA from the same species of Singaporean waters. Surprisingly, it showed variation in seven positions (273, 315, 354, 377, 441, 433 and 456) of the cDNA bases together with corresponding differences in three amino acids (glutamine is replaced by lysine in 55^th position, arginine by threonine in 113^th position and asparagine by histidine in 132^nd position) in their deduced amino acid sequences.

The cytolysin sequence from the sea anemone H. magnifica was aligned with the reported cytolysins of other cnidarians. The multiple sequence alignment revealed that H. magnifica cytolysin shares more than 80% homology with the reported cytolysins from other sea anemones.

BLAST analysis of both this cytolysin and the cytolysin of the same Singaporean species revealed 97% (Figure 8) nucleotide homology and 96% homology with deduced protein sequence (Figure 9). Furthermore, it showed up to 95% nucleotide homology with sticholysin II and 94% with sticholysin I from the sea anemone Stichodactyla helianthus and 94% with actinoporin RTX-A from Heteractis crispa. Based on this homology study, a phylogenetic tree was constructed (Figure 10), which indicated a close genetic relationship among H. magnifica cytolysin, sticholysin and actinoporin RTX-A, rather than connection with equinatoxin.

In the present study, the cytolytic effect has been assessed using human erythrocytes. Interestingly, human serum possesses a total inhibitory effect on the cytolytic activity when added to the incubated erythrocytes. Further studies are necessary on the cytolytic effect of cnidarian cytolysins on other marine invertebrate, as the body fluid of the latter lacks RBC or molecules of human serum nature.

Received: May 16, 2009

Accepted: February 9, 2010

Abstract published online: February 12, 2010

Full paper published online: May 30, 2010

Conflicts of interest: There is no conflict.

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Publication Dates

• Publication in this collection
  07 June 2010
• Date of issue
  2010

History

• Accepted
  12 Feb 2010
• Received
  16 May 2009