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,1
,1
* Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore;
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore;
Temasek Life Sciences Laboratory, National University of Singapore, Singapore; and
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA
1Correspondence: Protein Science Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, Science Dr. 4, Singapore 117543. E-mail: dbskinim{at}nus.edu.sg (R.M.K.); Plant Morphogenesis Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, Science Dr. 4, Singapore 117543. E-mail: dbskumar{at}nus.edu.sg (P.P.K.)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: heart rate • bradycardia • adrenergic receptors • GPCR ligands • beta-blocker peptide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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, 2)
. This arsenal of pharmacologically active molecules has been utilized in the past for obtaining several therapeutic agents and lead compounds like bradykinin-potentiating peptides as the inhibitor of angiotensin-converting enzyme (3)
, eptifibatide, and tirofiban to inhibit platelet aggregation (4
5
6
7
8
9)
and ancrod to reduce blood fibrinogen levels (10)
. Snake venom toxins have also found extensive application as research tools. For example, 
-bungarotoxin has been used to characterize the fundamental mechanisms involved in neuromuscular transmission (11)
.
Proteins from snake venoms fall under two categories, namely enzymatic and nonenzymatic proteins. Among the nonenzymatic proteins, three-finger toxin (3FTX) family is the most abundant and well-characterized family. All members of this family share a similar fold consisting of three finger-like loops made of ß-sheet structure, projecting from a globular core and stabilized by four or five intramolecular disulfide linkages giving rise to the compact "ß-cross" motif (12
13
14
15
16)
. Despite the similar structural fold, different 3FTXs have diverse molecular targets in the prey. For example, short- and long-chain -neurotoxins target 1 nicotinic acetylcholine receptors (nAChRs), long-chain -neurotoxins target 7 nAChR, 
-bungarotoxins target 3 and 4 nAChRs (15)
, muscarinic toxins target muscarinic AChRs (17)
, fasciculin targets acetylcholinesterase (18)
, calciseptine and FS2 toxin target L-type calcium channel (19)
, dendroaspin targets integrin IIbß3 (20)
, cardiotoxins target phospholipids and glycospingolipids (21)
, hemextin AB complex targets coagulation factor VIIa (22)
, and cardiotoxin A5 targets integrin vß3 (23)
. However, there are a number of "orphan groups" of 3FTXs whose molecular target in the prey and hence their functional roles in the snake venom are not yet known (24)
.
In an effort to exploit this rich miscellany, we constructed a partial cDNA library using mRNA extracted from Ophiophagus hannah venom gland tissue and identified five new genes coding for proteins belonging to the 3FTX family. We report the identification, purification, and characterization of one of these novel proteins, ß-cardiotoxin from the venom of O. hannah. This protein differs from classical cardiotoxins in its structure as well as function. Here we report the interaction of this novel protein with the ß-adrenergic receptor (AR) system causing a marked reduction in heart rates in whole animals as well as isolated perfused rat hearts. Thus, we named this novel member of the 3FTX family as ß-cardiotoxin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Lyophilized O. hannah venom was obtained from D. MacRae (Bali, Indonesia). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), except the following: reagents for Edman degradation N-terminal sequencing (Applied Biosystems, Foster City, CA, USA), and acetonitrile (Merck KGaA, Darmstadt, Germany). Superdex 30 Hiload (16/60) column and Jupiter C18 (5 µ, 300 Å, 10x250 mm) were purchased from Amersham Biosciences (Uppsala, Sweden) and Phenomenex (Torrance, CA, USA), respectively. Water was purified using a MilliQ system (Millipore, Billerica, MA, USA). All chemical reagents used were of the highest purity available.
Animals
Swiss albino male mice (
20 g) were used for the in vivo toxicity study. Male Sprague-Dawley rats were used for electrocardiogram (ECG) monitoring (3–4 wk old, 80–100 g), and the Langendorff isolated perfused heart experiments (8 wk old, 250 g). Animals were acquired from the university Laboratory Animal Center and were acclimatized to the Animal Holding Unit surroundings for at least 3 days before the experiments. Animals were kept under standard conditions with food and water available ad libitum. All animal experiments were conducted according to the protocol (776/05a) approved by the Institutional Animal Care and Use Committee of the National University of Singapore.
Isolation of total RNA
Total RNA was isolated from O. hannah venom gland using RNeasy Mini kit as described earlier (25)
. For each extraction, 30 mg venom gland tissue were first pulverized in liquid nitrogen using a cooled mortar and pestle and further homogenized for 20 to 30 s using a Heidolph DIAX600 homogenizer (Schwabach, Germany) in the presence of 600 µl RLT lysis buffer containing guanidine thiocyanate (provided in the RNeasy Mini kit). The integrity of the RNA extracted was examined by denaturing agarose gel electrophoresis.
cDNA library construction
The 5'-RACE ready cDNA was obtained using a SMART RACE cDNA amplification kit from Clontech. The BD SMART IIA oligonucleotide and an oligo (dT) primer supplied with the kit were used for this purpose. The double stranded cDNA for cloning was obtained using the long-UP and oligo (dT) primers provided in the kit, cloned into pGEMT-easy vector, and introduced into competent E. coli DH5 cells by heat shock transformation.
Sequencing of cDNA clones
Transformed clones were selected on a plate containing IPTG and X-gal by blue-white selection. Plasmids were extracted from overnight cultures using QIAprep Miniprep kit, digested using EcoRI, and fractionated on a 1% agarose gel to confirm the presence of the insert. Plasmids having the inserts were sequenced separately using T7 and SP6 primers, and the full-length sequences were assembled and analyzed.
Purification of the protein
O. hannah crude venom (100 mg in 1 ml of MilliQ water) was loaded onto a Superdex 30 (16/60) gel filtration column that was equilibrated with 50 mM Tris-HCl buffer (pH 7.4) and eluted with the same buffer using an AKTA purifier system (Amersham Biosciences, Uppsala, Sweden). The eluted samples were pooled into eight fractions. These were further subfractionated by reverse phase-high performance liquid chromatography (RP-HPLC) using a Jupiter C18 (5 µ, 300 Å, 10 mmx250 mm) column that was equilibrated with 0.1% trifluoroacetic acid and the proteins eluted with a linear gradient of 80% acetonitrile in 0.1% trifluoroacetic acid. The fractions were collected and directly injected into an API-300 liquid chromatography/tandem mass spectrometry system (PerkinElmer Life Sciences, Wellesley, MA, USA) for mass determination. Fractions showing the expected molecular mass were pooled and lyophilized.
Electrospray ionization mass spectrometry
The mass and homogeneity of the novel protein were determined by electrospray ionization mass spectrometry (ESI-MS) using an API-300 liquid chromatography/tandem mass spectrometry system (PerkinElmer Life Sciences). RP-HPLC fractions were directly used for the analysis. Ion spray, orifice, and ring voltages were set at 4600, 50, and 350 V, respectively. Nitrogen was used as the nebulizer and curtain gas. A Shimadzu LC-10AD pump was used for solvent delivery (40% acetonitrile in 0.1% formic acid) at a flow rate of 40 µl/min. BioMultiview software (PerkinElmer Life Sciences) was used to analyze and deconvolute the raw mass data.
N-terminal sequencing
N-terminal sequencing of the native protein was performed by automated Edman degradation using a PerkinElmer Applied Biosystems 494 pulsed-liquid phase protein sequencer (Procise) with an on-line 785A phenlythiohydantion (PTH)-derivative analyzer. The PTH-derivatized amino acids were sequentially identified by mapping the respective separation profiles with the standard chromatogram.
Circular dichroism (CD) spectroscopy
Far-UV CD spectra (260–190 nm) were recorded using a Jasco J-810 spectropolarimeter (Jasco Corporation, Tokyo, Japan). All measurements were carried out at room temperature using a 0.1 cm path length capped cuvette. The instrument optics and cuvette chamber were continuously flushed with 30 l of nitrogen/min before and during the recording of the spectra to provide an oxygen-free environment. The spectra were recorded using a scanning speed of 50 nm/min, a resolution of 0.1 nm and a bandwidth of 1 nm. A total of three scans was recorded and averaged for each spectrum, and the baseline was subtracted. All samples were dissolved in MilliQ water.
Methods for protein administration
The protein was dissolved in 200 µl of 0.9% NaCl (Braun, Malaysia) and administered through the intraperitoneal route for the in vivo toxicity study in mice. Intratail vein administration was done in the electrocardiogram (ECG) monitoring study in rats. The various protein doses were dissolved in 200 µl of 0.9% NaCl solution and injected into the tail vein using a 27 G x [
]" needle (Becton-Dickinson, Franklin Lakes, NJ, USA) held at an angle of 10° to the tail. The infusion was made very slowly and at a steady rate.
In vivo toxicity study
After purification of the protein from snake venom, intraperitoneal doses of 1, 10, and 100 mg/kg were administered to healthy male mice (3 animals per group), and the symptoms were observed and recorded; 0.9% NaCl was injected as control. Postmortem examinations were conducted on the animals after their death.
Anticoagulant activity
The anticoagulant activity of the purified protein was tested by two coagulation tests using a BBL fibrometer. Blood plasma was collected from healthy human volunteers.
Prothrombin time
The prothrombin times were measured as described by Banerjee et al (22)
; 100 µl of 50 mM Tris-HCl buffer (pH 7.4), 100 µl of plasma, and 50 µl of the protein dissolved in the assay buffer were preincubated for 2 min at 37°C. Clotting was initiated by the addition of 150 µl of thromboplastin with calcium reagent (Sigma-Aldrich).
Recalcification time
The recalcification times were measured according to the method of Banerjee et al. (22)
; 100 µl of 50 mM Tris-HCl buffer (pH 7.4), 100 µl of plasma, and 50 µl of the protein dissolved in the assay buffer were preincubated for 2 min at 37°C. Clotting was initiated by the addition of 50 µl of 50 mM CaCl2.
Hemolytic assay
Hemolytic activity was assayed as described earlier (26)
. Blood from healthy human volunteers was collected into a solution of 3.8% sodium citrate (9:1, v/v) and centrifuged for 15 min at 3000 rpm at room temperature. The plasma was discarded, and red blood cells (RBCs) were washed three times with 0.9% NaCl. RBCs (5 µl) were added to 0.95 ml of 0.9% NaCl containing various concentrations of CM18 or ß-cardiotoxin. For negative control, no protein was added. The tubes were incubated at 37°C for 1 h. RBC were removed by centrifugation at 3000 rpm for 15 min at room temperature, and the absorbance of the supernatant was read at 418 nm and 540 nm.
ECG monitoring and heart rate determination
For ECG measurements, Sprague-Dawley rats were anesthetized with an intraperitoneal injection of ketamine + xylazine at a dose of 75 + 10 mg/kg. Needle probes (29-gauge, MLA1204) were inserted into the front and back left paw pads of each rat, and signals were captured using the Animal BioAmp differential amplifier (ML136). Signals were digitized using an eight-channel Powerlab 8SP (ML785), and recordings were displayed with Chart 5 software (ADInstruments, Castle Hill, NSW, Australia). All probes and equipment for ECG measurements were obtained from ADInstruments (Castle Hill, NSW, Australia). The normal heart rates of the animals were in the range of 470 to 500 beats per minute (BPM). Continuous recording was made from 5 min before an intratail vein injection of various doses of the protein until 20 min after the injection. The same volume of 0.9% NaCl was administered to the control group. The heart rate at different time points before and after the injection of the protein was estimated using the Chart 5 software.
Isolated perfused heart
The isolated perfused hearts were prepared according to the method described by Cerra et al. (27)
. Rats were anesthetized with an intraperitoneal injection of ketamine + xylazine at a dose of 75 + 10 mg/kg; 2 ml of 50 I.U. heparin (David Bull Laboratories, Warwick, UK) were injected intraperitoneally 15 min before the death of the animal. The heart was excised rapidly and placed in oxygenated Krebs-Henseleit (KH) solution (in mM: 118.0 NaCl, 4.5 KCl, 1.4 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 1.4 CaCl2, and 11 glucose; pH 7.4) before the aorta was cannulated on the Langendorff apparatus. Nonrecirculating mode of retrograde perfusion with KH solution was carried out at a constant flow (6–7 ml/min) at 37°C. The buffer was continuously bubbled with 95% O2 and 5% CO2. The hearts were allowed to equilibrate for 30 min, and in study groups the buffer flow was switched to a second reservoir that contained 5 µM of the protein dissolved in KH solution; plain KH solution was passed for a further 15 min in the control group.
Measurement of isovolumetric cardiac performance
A water-filled latex balloon was attached to a pressure transducer and inserted through the mitral valve into the left ventricle (LV) through an incision in the left atrium. The pressure transducer was connected to a Powerlab (ADInstruments, Castle Hill, NSW, Australia) data recording system. The following indices of cardiac performance were measured and averaged from 10 beats for each condition, and premature contractions were excluded from the analysis: heart rate (HR, in beats/min) and left ventricle diastolic end pressure (LVDEP, in mmHg), which is an index of contractile activity.
Competitive binding assay
Cloned human ß-adrenergic receptor subtypes 1 and 2 (ß1 and ß2) produced in Sf9 cells were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA, USA). The radioligand (–)-[3H]CGP-12177 was purchased from GE Healthcare (Buckinghamshire, UK). The competitive binding assay was carried out using the method described by Lewis et al. (28)
, and a few recommendations by the receptor manufacturers (PerkinElmer Life and Analytical Sciences) were also incorporated. For the competitive binding studies, we used 0.15 nM (–)-[3H]CGP-12177, ß1 or ß2 and ß-cardiotoxin ranging from 5 nM to 10 µM in 75 mM Tris-HCl buffer containing 12.5 mM MgCl2 and 2 mM EDTA, pH 7.4. Total reaction volume was 1050 µl. Nonspecific binding was determined to be 2–3% for ß1 and 1–2% for ß2 by the inclusion of 2 µM (S)-(–)-propranolol hydrochloride. After 60 min incubation at room temperature, the reaction mixtures were filtered through Whatman GF/C glass microfibre filters (Maidstone, England) presoaked in ice-cold wash buffer (50 mM Tris-HCl buffer, pH 7.4). The filters were washed nine times with 500 µl (each time) of ice-cold wash buffer. A Beckman LS3801 liquid scintillation counter was used to measure the radioactivity retained on the washed filters.
The binding affinity Ki was calculated from the IC50 using the equation of Cheng and Prusoff (29)
,
Ki = IC50 ÷ {1 + ([Radioligand]/Kd)}
where, the IC50 (concentration of the inhibitor that displaces 50% of bound ligand) values were determined by plotting the percent specific binding in the y-axis vs. log [molar concentration of protein used] in the x-axis, Kd is the binding affinity of the radioligand to the receptor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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). Two nontoxin genes belonging to the ribosomal complex were also identified [AY354199 (protein accession #AAR10441) and AY357074]. Several other sequences (19%) that were obtained did not show any match with available sequences from the database (see Supplemental Fig. S1).
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The cDNA AY354198 encodes for a protein with a molecular weight of 7012.42 Da, which is closely related to proteins reported as precursors of cardiotoxins (CTXs) (ABB83631, ABB83632, ABB83633, ABB83634, and ABB83635) all identified from O. hannah venom glands (Fig. 1A
). These sequences are highly similar to each other and differ from each other only at two to five sites out of which the substitutions K21R, V23I, and L54V are conserved changes (Fig. 1A
). Despite being classified as CTXs, the biological properties of none of these proteins have been characterized. Interestingly, they showed only 55–65% sequence identity with conventional CTXs and CTX-like basic proteins (Fig. 1B
) and even poorer sequence identity with neurotoxins (data not shown). Furthermore, most of the structural differences between AY354198 and conventional CTXs were found in the loop regions (see Supplemental Fig. 2A), which play an important role in the interaction of 3FTXs with various target proteins (13)
. Therefore, we expected this protein to interact with a distinct protein target and to exhibit different pharmacological effects than classical CTXs and hence examined the biological properties of this protein. As shown below, this protein indeed shows distinct biological properties compared to other snake venom CTXs, and hence, it belongs to a new class of 3FTXs. Unlike conventional CTXs, it acts as a beta-blocker and decreases the heart rate (see below) and hence was named as ß-cardiotoxin.
Isolation and purification of ß-cardiotoxin
Our initial liquid chromatography/mass spectrometry (LC/MS) studies of O. hannah venom (30)
showed that it contains a protein with the molecular weight of 7013.80 ± 1.27 Da indicating the presence of AY354198 protein in the venom. The novel protein was purified by following the calculated mass of the protein while fractionating the crude venom of O. hannah using chromatographic techniques. We used a two-step chromatography approach, where the first step comprised of separating the venom components based on their sizes into eight peaks using gel filtration chromatography (Fig. 2
A). Subsequently, each peak was fractionated by RP-HPLC using a Jupiter C18 semipreparative column (for example, peak 5; Fig. 2B
). Each RP-HPLC fraction was then subjected to ESI-MS analysis to determine the mass and hence the tentative identification of the protein (data not shown). The ESI-MS of fraction 5d (Fig. 2B
) showed three peaks with mass/charge ratios ranging from +4 to +6 charges (data not shown), and the final reconstructed spectrum showed a molecular weight of 7012.43 ± 0.91 Da similar to the expected mass (Fig. 2C
). The ESI-MS spectrum also revealed the purity of the protein (Fig. 2C
). N-terminal Edman degradation sequencing of the first 30 residues further confirmed the identity of the protein.
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Far-UV CD spectroscopy
The secondary structural elements present in conventional cardiotoxins and ß-cardiotoxin were analyzed using CD spectroscopy. Nanixain, a CTX isolated from the venom of Naja nigricollis (Dugar et al., unpublished observations), showed a small minimum at 215–220 nm and an intense maximum at 190–195 nm and CM18, a CTX from the venom of Naja atra, showed a maximum at 220–225 nm, a small minimum at 210–212 nm and an intense maximum at 190–195 nm (Fig. 3
). Interestingly, ß-cardiotoxin showed intense minimum at 212–215 nm and small maximum at 198 nm and differed significantly from the CD spectra of the other two conventional CTXs (Fig. 3)
.
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In vivo toxicity study
Three groups of male Swiss albino mice (20 g) were used in the toxicity studies for ß-cardiotoxin. Each group of three mice received one dose (1, 10, or 100 mg/kg) of ß-cardiotoxin, and the ensuing effects were closely monitored. The protein was not lethal up to 10 mg/kg dosage. At 100 mg/kg, the mice showed symptoms of labored breathing, impaired locomotion, lack of response to external stimuli, and death occurred after 30 min. Postmortem examinations did not reveal any hemorrhage or visual damage to internal organs. To another group, a single dose (2.5 mg/kg) of -bungarotoxin, a potent neurotoxin isolated from the venom of Bungarus candidus was injected. These animals exhibited severe paralysis of the hind limbs and they died in 10 min after the administration. To the last group, 0.9% NaCl was administered as a negative control and no symptoms developed until the animals were ultimately killed at the end of the study.
Anticoagulant effects
The effects of ß-cardiotoxin (final concentration of 50 µM protein per assay) on the extrinsic and intrinsic pathways of blood coagulation were assessed using the prothrombin time and recalcification time, respectively. ß-Cardiotoxin did not cause any significant changes in the clotting time compared to the control in both assays (Fig. 4
A). Thus, we conclude that ß-cardiotoxin does not affect blood coagulation.
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Hemolytic activity
Washed human erythrocytes were treated with ß-cardiotoxin at concentrations ranging from 1 to 100 µg/ml (final concentration per assay) to determine the hemolytic activity. The protein did not show any significant hemolytic activity, as the absorbance values of the highest doses were comparable to that of the negative control (Fig. 4B
). In contrast, CTX CM18 showed significant hemolytic activity (Fig. 4B
).
Cardiac effects of ß-cardiotoxin
The in vivo activity of the protein was determined by administering the protein into anesthetized male rats and monitoring the changes induced in the ECG patterns. For the control animals, the carrier alone (0.9% NaCl) was injected and there was no change in the heart rate after injection (Fig. 5
A). The administration of CTX CM18, as expected, increased the heart rate and induced a positive chronotropic effect (Fig. 5B
). In contrast, there was decrease in the heart rate as indicated by an increase in the distance between successive QRS complexes on administration of ß-cardiotoxin, suggesting a negative chronotropic effect that might cause bradycardia (Fig. 5C
). Thus, a conventional CTX increases the heart rate, while ß-cardiotoxin decreases the heart rate (Fig. 5D
). This decrease in the heart rate induced by ß-cardiotoxin is dose dependent (Fig. 5E
). Thus, ß-cardiotoxin induces negative chronotropism in the heart rate in rats unlike conventional CTXs.
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Isolated perfused heart studies
The direct effects of ß-cardiotoxin on cardiac tissue was determined using the Langendorff isolated perfused rat hearts. There were no changes in any of the cardiac parameters in the control group (Fig. 6
A), whereas, ß-cardiotoxin induced a negative chronotropic effect (Fig. 6B
). ß-Cardiotoxin at 5 µM caused a marked reduction in the heart rate (Fig. 6C
) without any significant change in the contractility as indicated by the LVDEP (Fig. 6D
). Thus, the decrease in the heart rate induced by ß-cardiotoxin in rats is most probably due to its direct action on the cardiac muscles.
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Interaction of ß-cardiotoxin with human ß-adrenergic receptors (ß-ARs)
ß-ARs are expressed abundantly in cardiomyocytes, and the adrenergic signaling cascade is responsible for the control of heart rate (31)
. Therefore, we hypothesized that the change in heart rate observed in anesthetized rats and isolated perfused rat hearts could be due to interaction of ß-cardiotoxin with ß-ARs, and hence we performed radioligand binding assays. At first, the nonspecific binding of radioligand (–)-[3H]CGP-12177 to the receptor preparations was defined using 2 µM (S)-(–)-propranolol hydrochloride. The radioligand showed only 2 - 3% non-specific binding to ß1-AR preparation and 1–2% nonspecific binding to ß2-AR preparation. (S)-(–)-propranolol hydrochloride was also used as a positive control in the study, and IC50 values for the displacement of bound radioligand to ß1-AR and ß2-AR were determined as 3.5 and 0.5 nM, respectively (Fig. 7
). The Ki of (S)-(–)-propranolol hydrochloride to ß1-AR and ß2-AR were calculated as 1.9 nM and 0.2 nM, respectively. These values are in good agreement with the Ki values given by the manufacturer (2.6 and 0.2 nM for ß1-AR and ß2-A, respectively; see Supplemental Fig. S3). ß-Cardiotoxin showed a dose-dependent displacement of the radioligand (–)-[3H]CGP-12177. IC50 values for the inhibition of ligand binding to ß1-AR and ß2-AR were determined as 10 and 5 µM, respectively (Fig. 7A, B
). The Ki for ß-cardiotoxin binding to ß1 and ß2 ARs were calculated as 5.3 and 2.3 µM, respectively. Thus, ß-cardiotoxin induces a negative chronotropic effect on the heart rate by binding to ß1-AR in cardiomyocytes. Its interaction with ß2-AR in the bronchi may induce some respiratory symptoms because of bronco-constriction. This exogenous protein interacts with ß-ARs and hence was named as ß-cardiotoxin.
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DISCUSSION |
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71.3 million American adults suffer from one or more forms of CVD (32)
. CVD causes nearly one in every three deaths and was the number one killer in the USA as it was the cause for 37.3% deaths in 2003. The economic impact of CVD is huge, and it is estimated that the combined direct and indirect costs of treating CVDs in 2006 would be $403.1 billion (32)
. Beta-blockers, which are antagonists of ß-ARs, are the drugs of choice in the treatment of CVDs. Most patients (93%) with myocardial infarction are given beta-blockers on arrival at hospitals. They are also prescribed after discharge as a chronic therapy (32)
. In addition, beta-blockers are used in the treatment of many pathological conditions afflicting the heart and vasculature, such as ventricular arrhythmias, heart failure, digitalis intoxication, and fetal tachycardia. Owing to the presence of ß-ARs in various noncardiac tissues, beta-blockers have also been used to treat conditions like, migraine, essential tremor, situational anxiety, alcohol withdrawal, hyperparathyroidism, glaucoma, portal hypertension, and gastrointestinal bleeding (33)
. Most of the beta-blockers in clinical use currently are small molecules belonging to the aryloxypropanolamine class (34)
. Although they are widely used, physicians have encountered several problems associated with the currently available beta-blockers. All beta-blockers are available as racemic mixtures of L- and D-enantiomers, where only the L-enantiomer exerts beta-blockade while the D-enantiomer, apart from being inert, may have adverse side effects. For example, in a clinical trial using the D-enantiomer of the widely used beta-blocker sotalol, it was reported that the mortality increased by 65% compared to placebo (35)
. Many beta-blockers exhibit varying levels of lipophilicity and so have the ability to cross membrane barriers and reach the central nervous system, causing adverse effects like hallucinations and insomnia (34)
. Some beta-blockers are shown to increase insulin resistance and raise the risk of diabetes (36)
. Thus, new beta-blockers with high specificity and low side effects are being actively pursued.
Isolation of a novel protein from O. hannah venom
On screening, the cDNA library from the venom gland tissue of O. hannah, we identified five new 3FTXs. One of these 3FTXs showed only 55% sequence identity with conventional CTXs isolated from Naja sp. (Fig. 1B
). Subsequently, two other reports (37
, 38)
have described the sequencing of the same toxin and also some closely related isoforms from cDNA libraries (Fig. 1A
and Supplemental Fig. S2B), which shows the usefulness of this approach for identification of novel low abundant proteins that have eluded detection by conventional approaches. Here we have described a two-step chromatographic approach used for the isolation of this novel protein (Fig. 2)
.
Identification of unique secondary structural conformation of ß-cardiotoxin
CTXs from different cobra venoms have been classified into two distinct structural subclasses based on their CD spectra (39)
. Almost all CTXs studied so far fall clearly into either of the two classes. The ß-cardiotoxin spectrum on the other hand, neither shows the positive maximum at 220–225 nm shown by all group 2 CTXs, nor has the intense maximum at 190–195 nm like all group 1 CTXs (Fig. 3)
. The secondary structural conformation of ß-cardiotoxin is unique compared to both group 1 CTX nanixain and group 2 CTX CM18 (Fig. 3)
, and hence, it differs from both classes of CTXs. Thus, ß-cardiotoxin has unique secondary structural elements compared to all other CTXs.
Conventional CTXs have a few well-conserved residues apart from the eight Cys residues, which are thought to play an important role in maintaining structural integrity of the three-finger fold. Among them are Tyr 22 and Tyr 51, which have been shown by chemical modification to play vital structural and functional (especially Tyr 22) roles in CTXs (40)
. Replacement of Tyr 22 led to changes in interaction of ß-sheet regions between loops I and II and replacement of Tyr 51 led to structural perturbation in the globular core region of the molecule, the overall effect being a destabilized structural core and highly perturbed dynamics in the 3-stranded ß-sheet region (41)
. Unlike conventional CTXs, in ß-cardiotoxin there are Val residues in both positions 23 and 53 (homologous to positions 22 and 51 of CTXs; Fig. 1B
). These changes could possibly explain the observed unique secondary structural features of ß-cardiotoxin. It would be interesting to see the effects of these two replacements on the tertiary structure of the protein.
ß-Cardiotoxin belongs to a new class of 3FTXs
3FTXs constitute 50% of the weight of most elapid and hydrophid venoms and are the leading cause of death and morbidity as they are highly lethal (42)
. As mentioned in the Introduction, despite the similarity in overall protein fold, they target different receptors, ion channels, or proteins to exhibit various pharmacological effects. As shown here, ß-cardiotoxin exhibits unique biological effects compared to any of the 3FTXs known. Although its amino acid sequence shows similarity to CTXs (55% identity), it is structurally and functionally distinct from conventional CTXs. ß-Cardiotoxin was nonlethal up to a dose of 10 mg/kg, in contrast to CTXs, which are highly lethal proteins with LD50 values in the range of 1 to 2 mg/kg (1)
. Unlike CTXs, which show potent hemolytic activity (43
44
45)
, ß-cardiotoxin failed to show hemolytic activity on washed human erythrocytes (Fig. 4B
). Further, unlike CTXs that cause an increase in heart rate when injected into anesthetized rats (Fig. 5B, D
; ref. 46
), ß-cardiotoxin caused a dose-dependent decrease in heart rate indicated by the prolongation of successive QRS complexes in the ECG recordings (Fig. 5. C-E
). We also found that it can act directly on the cardiac tissue causing a marked reduction in heart rate (negative chronotropism) in Langendorff preparations of perfused rat hearts without affecting the contractility (inotropism) (Fig. 6B-D
). Many beta-blockers like esmolol exert a direct inhibitory effect on membrane Ca2+ channels apart from acting as beta-blocking agents. This inhibition of membrane currents would lead to pronounced negative inotropism leading to adverse complications like severe reduction of blood pressure (47)
. Peptides engineered from ß-cardiotoxin would be useful therapeutic prototypes as they lack intrinsic negative inotropism like conventional beta-blockers. We have shown that the above mentioned pharmacological effects are due to its direct binding to ß1- and ß2-ARs. The Ki values indicate that it has higher affinity for ß2-AR compared to ß1-AR (5.3 and 2.3 µM for ß1- and ß2-ARs, respectively). The small molecule beta-blockers in current clinical use have strong affinities to the two receptors (in nanomolar ranges) compared to ß-cardiotoxin that binds at low micromolar ranges. This may be caused by steric hindrance due to the larger molecular size of the protein compared to the small molecule drugs, and the affinities and specificity could be increased further by protein engineering approaches like mutagenesis and peptide minimization. ß-Cardiotoxin belongs to a new class of 3FTXs with a unique molecular target in the prey. This finding further broadens the array of molecular targets identified for various 3FTXs. The ß-AR blocking ß-cardiotoxin along with the 1-AR blocking conopeptide from cone snail venom (28)
form a novel class of exogenous peptide adrenergic-blocking agents that may have immense applications in developing novel research tools and therapeutic agents.
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ACKNOWLEDGMENTS |
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Received for publication April 8, 2007. Accepted for publication May 17, 2007.
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