Isolation, purification and biochemical characterization of conotoxin [PDF]

Indian Journal of Biotechnology Vol 8, July 2009, pp 266-271

Isolation, purification and biochemical characterization of conotoxin from Conus figulinus Linnaeus (1758) R Saravanan1*, S Sambasivam1, A Shanmugam1, D Sathish Kumar2, T Tamil Vanan2 and R A Nazeer3 1

Centre of Advanced Study in Marine Biology, Annamalai University, Paraingipettai 608 502, India Department of Biotechnology & 3School of Biotechnology, SRM University, Kattankulathur 603 203, India

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Received 24 July 2008; revised 15 January 2009; accepted 22 March 2009 Cone snails are remarkable for the extent and diversity of gene-encoded peptide neurotoxins that are expressed in their venom apparatus. The protein content of the crude toxin extract of Conus figulinus Linneaus was found to be 1900 µg/mL. The crude extract (dilution up to 10-5) expressed hemolytic activity. The crude extract subjected to gel filtration chromatography yielded 60 fractions; the fractions 7, 12 and 55 showed significant peaks at 280 nm. The fractionated toxin was then characterized by performing SDS-PAGE having the lower peptides ranging from 10 to 43 kDa; two lower peptides below 14 kDa have been identified. The total RNA and purified mRNA were characterized by Agarose gel electrophoresis and for total RNA two prominent bands of 18s and 28s were obtained of which 28s showed double intensity than the other. For mRNA a single band of 6000 base pairs was obtained. Keywords: Conus figulinus,cone snail, chymotrypsin, toxin, trypsin, total RNA, mRNA

Introduction The marine environment is an exceptional reservoir of bioactive natural products, many of which exhibit structural features not found in terrestrial natural products. Research into the pharmacological properties of marine natural products has led to the discovery of many potentially active agents considered worthy of clinical application1. Natural toxins have largely contributed to our understanding regarding the structural and functional characteristics of ion channels and, in this context, much information has been gained about the voltage-sensitive calcium channels by using ω-conotoxin. This toxin is a small peptide present in the venom of the marine snail, C. geographus2. The gastropod genus Conus contains several hundred species. They all possess more or less potent venom, which is used primarily in the capture of prey. According to their feeding habits, the various species can be classified as either piscivorous, molluscivorous or vermivorous. The venom apparatus of some species can also be used for defensive purposes and human fatalities occasionally occur. The isolation and _____________ *Author for correspondeice: Mobile: 09994071533 E-mail: [email protected]

structural elucidation of individual components from these venoms was started at the beginning of the 1980s. So far, major attention has been focused on the mechanism of action of individual toxins from venom of piscivorous Conidae such as C. geographus and C. magus3. Cone snails take advantage of the synergetic effects of different Conus peptides aiming at various targets to capture their preys efficiently. Two distinct phases of their prey are elicited by different sets of Conus peptides (e.g., lightning strike cabal and motor cabal). Such strategy of synergy has been adopted in the treatment of diseases (different kinds of drugs with different pharmacological efficiencies are often under combined administration in clinic)4. Conotoxins exhibit their poisonous effect by blocking specific ion channels of nerve cells. Their channel specificity is quite remarkable. The specificity of conotoxins is due to their disulphidebonding network and specific amino acids in inter cysteine loops. This specificity is one of the attributes that make them valuable diagnostic tools in the characterization of neural pathways, as therapeutic agents, in medicine and potentially as biodegradable toxic agents in agro-veterinary applications5. Many novel Conus active peptides have been characterized not only by chemicals but also by molecular biology

SARAVANAN et al.: CONOTOXINS FROM CONUS FIGULINUS

approaches. Indeed, a great number of novel Conus peptides have been discovered over these years by means of cDNA cloning4. Conotoxins are synthesized by cone shells from mRNA templates derived from toxin genes and expressed in the venom ducts as precursor peptides. There are now numerous gene cloning techniques, which can be used to isolate and characterize the precursor molecules as a preclude for predicting the composition of mature peptides5. As discussed earlier, several of the conotoxins have entered human clinical trials or are in preclinical development stages; some are even commercially available in the market. Hence this paper describes the isolation, purification and biochemical characterization of toxins from C. figulinus. Materials and Methods The samples of Conus figulinus were collected from Mudasalodai landing centre along the Parangipettai coast (Lat. 11° 29′; Long. 79° 46′ E) situated at the South East coast of India, Tamil Nadu. Collection of Venom Duct

The animals were aseptically transferred into a sterile room for dissection. The shell was broken with the help of a hammer and the venom duct was removed. The removed venom ducts were stored in 25% ethanol and frozen at –80°C for further study6.

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et al method9. The elution buffer was 50 mM Tris-HCl and the volumetric flow rate was 0.33 mL/min. Every 4 mL of the effluent obtained from gel filtration was collected in a fraction. SDS-PAGE analysis of proteins was carried out using 12.5% polyacrylamide gel at 120 V for 90 min. Solutions for preparing 12.5% resolving gel included H2O (6.4 mL), 30% acrylamide mix (8.3 mL), 1.5 M Tris (pH 8.8, 5 mL), 10% SDS (0.2 mL), 10% ammonium persulfate (0.1 mL), and N,N,N,N’tetramethylethylenediamine (0.008 mL). The gel separation was performed based on a suggested protocol of Anderluh et al10. Hemolytic Activity

The hemolytic activity of the fractionated toxin was studied using the method of Lin et al11. RBC (1 mL of 1%) was taken in 12 tubes and serially diluted. The tubes were then incubated at 37°C for 30 min. For positive control 1 mL of RBC and 0.5 mL of distilled water was added to achieve 100% lysis. For negative control, 1 mL of RBC and 0.5 mL of PBS was added. After 30 min the tubes were centrifuged at 2000 rpm for 5 min. The hemoglobin released was estimated by reading the absorbance of the supernatant at 420 nm. Biochemical Characterization Trypsin and Chymotrypsin Assays

The protein content of crude toxin was estimated by the method of Lowry et al8 using BSA as a standard.

Trypsin and chymotrypsin inhibitory assays were carried out following the method of Yakoby and Raskin12. Trypsin, with a specific activity of 50,500 U/mL, and α-chymotrypsin with a specific activity of 51 U/mg proteins, were purchased from Sigma-Aldrich. Trypsin was used at a dilution of 3-5 U/mL, and chymotrypsin was used at 3-5 mU/mL. Dilutions were made with 50 mM citric acid buffer at pH 3 and 5 for trypsin and chymotrypsin, respectively, except for the last dilution (to 0.5 U/mL or 0.5 mU/mL), which was made in 50 mM Tris-HCl (pH 8.0). All the experiments were started after enzyme-dilutions. One unit of chymotrypsin hydrolyzed 1.0 µmol of N-benzoyl-L-tyrosine ethyl ester (BTEE) per min at pH 7.8 at 25°C. One unit of trypsin hydrolyzed 1.0 µmol of N-α-benzoyl-Larginine ethyl ester (BAEE) per min at pH 7.6 at 25°C.

Purification and SDS-PAGE Analysis

Isolation and Estimation of Total RNA Content

The crude toxin was purified on a 5×90 cm column of Sephadex G-25 (Sigma) adopting Cruz

The total RNA was isolated using Chomoczynkski and Sacchi method13 and studied through agarose gel

Isolation of Crude Extract

For the isolation of crude venom from C. fugilinus, homogenization buffer containing 50 mM Tris hydrochloride, 120 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium chloride and 2 mM calcium chloride was used. Two fresh venom ducts were homogenized with 2 mL of buffer in a manual tissue homogenizer and sonicated three times for 50 sec/cycle (10 sec on, 20 sec off). During sonication the vessel was cooled with an ice bath. The mixture was centrifuged at 17,200 rpm for 10 min at 4°C. The supernatant (considered to be crude extract) was retained and stored at –20°C for further use7. Protein Estimation

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electrophoresis. 100 mg of venom bulb was homogenized in 1.5 mL of denaturing solution. 1 mL of phenol and 200 µL of freshly prepared chloroformisoamyl alcohol (49:1) was added to the homogenized sample and mixed thoroughly and then incubated in ice for 15 min. The mixture was centrifuged at 10,000 rpm for 20 min at 4°C. Upper aqueous phase was transferred carefully to another tube. RNA precipitate obtained by adding 1 mL of 100% isopropanol was incubated at –20°C for 30 min. After incubation the sample was again centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was discarded and the pellet was resuspended in 0.3 mL of denaturing solution and then adding 0.3 mL of 100% isopropanol to precipitate the RNA. After centrifuging at 10,000 rpm for 20 min the supernatant was discarded. The RNA pellet was resuspended in 75% ethanol and incubated at room temperature (RT) for 10-15 min to dissolve residual amounts of guanidine. After centrifuging at 10,000 rpm for 20 min at 4°C the supernatant was discarded. Finally the pellet of RNA was dissolved in 50-100 µL of stabilized formamide. The total RNA isolated was stored in –80°C for further use. The RNA was estimated by the method of Lin et al11 using orcinol reagent.

bromophenol blue reached 2 cm from the bottom of the gel. Results The protein content of the crude toxin extract was found to be 1900 µg/mL. Partial Purification of Toxin

Out of the 60 fractions, 3 fractions showed maximum absorbance at 280 nm. The hemolytic activity of all the 3 fractions was detected up to 10-5 dilution against human RBC. The toxins in these peaks were named conotoxins 7, 12 and 55 in the order of elution. The toxic fractions obtained by gel filtration were mostly occupied by these 3 conotoxins. Purification by gel filtration chromatography and the hemolytic activity has been shown in Figs 1 and 2, respectively. Biochemical Characterization

In SDS-PAGE, the conotoxins afforded 3 bands (Fig. 3), of which two lower peptides below 14 kDa have been identified. The purified conotoxins showed a complete inhibition of the trypsin and chymotrypsin enzyme at absorbance 252 nm as shown in Figs 4 and 5, respectively.

mRNA Purification from Total RNA

For the purification of mRNA from total RNA, method of Anderluh et al10 was followed. The isolated total RNA was added to the vials of oligo dT25, which was mixed well by tapping gently. The volume was made upto 200 µL by adding DEPC water and it was incubated at 65°C for 5 min with intermittent mixing. 20 µL of 5 M NaCl was added and incubated at 37°C for 10 min. Then the sample was centrifuged at 10,000 rpm for 10 min at RT and the supernatant was removed carefully without disturbing the pellet. The pellet was washed with 200 µL of wash buffer and suspended by tapping. Then it was centrifuged at 10,000 rpm for 10 min and the supernatant was removed without disturbing the pellet. This step was repeated twice. The washed pellet was suspended gently in 25-50 µL of TE buffer and incubated at 65°C for 5 min. The suspension was then transferred onto the spin column using micropipette. The column was placed into a sterile 1.5 mL Eppendorf vial. The column containing the suspension was then centrifuged at 5000 rpm for 5 min at RT to recover purified mRNA which was stored at –80°C for further use. The agarose gel (0.8%) was run at 50 mV and the power supply was switched off when the

Fig. 1—Chromatographic purification of the toxin isolated from C. figulinus.

Fig. 2—Hemolytic activity

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In addition to purification of conotoxins, baseline work has been carried out to isolate total RNA from the venom gland of C. figulinus and purification of mRNA from the same in order to know the gene responsible for secreting the conotoxins. The results for total RNA yielded two prominent bands of 18S and 28S, of which 28S showed (Fig. 6) double intensity than the other. The concentration of RNA present in the isolated sample of C. figulinus was found to be 850 µl/mL. For mRNA a single band of 6000 bp was obtained (Fig. 7). After mRNA purification from the isolated total RNA sample from the venom gland of C. figulinus, these can be used as

Fig. 3—SDS-PAGE

Fig. 6—Agarose gel electrophoresis of total RNA

Fig. 4—Trypsin inhibition assay

Fig. 5—Chymotrypsin inhibitory assay

Fig. 7—Agarose gel electrophoresis of mRNA

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a template for the synthesis of cDNA. This can be further cloned and expressed so that a cDNA library of entire species could be obtained. Discussion In this study, three toxins (conotoxin 1, 2 and 3) with hemolytic activity were partially purified from the salivary gland of C. figulinus by gel filtration on Sephadex G-25. The biochemical properties of the conotoxins are found to be very similar to one another. They are all monomeric simple proteins with molecular weight lying between 10-43 kDa. This is in agreement with the results of Shiomi et al14, that the partially purified echotoxins extracted from Monoplex echo, has molecular mass 7 kDa by gel filtration on Sephadex G-75 column. Conotoxins cause in vitro hemolysis in human blood. Some hemolytic toxins such as sea anemone toxins15 and melittin from bee venom16, which form a channel in the lipid membrane by assembling of several molecules, are also known to increase their α-helical structure content upon binding to the lipid membrane. Similar to these toxins, conotoxins probably form a channel in erythrocyte membrane resulting in the release of hemoglobin. The purity of the toxins was demonstrated by slab gel analysis and further supported by pharmacological data. Their molecular weight was estimated to be 24 and 25.5 kDa, respectively by SDS-PAGE and their protein nature was suggested by the complete loss of activity after incubation with trypsin or after boiling17. Partially purified eburnetoxin and tessulatoxincontaining fractions were found to contain two major proteins18. This is similar to our results on the C. fugilinus toxin. Whereas Kobayashi et al18 isolated the proteins (eburnetoxin and tessulatoxin) with the highest molecular weight, but were unsuccessful in isolating the low molecular weight proteins. In this context, it may be noticed that the SDS-PAGE profiles of both the proteins are almost identical for the 3 toxins. Provided that there is some structural relationship between the proteins with the lowest molecular weight, it appears that the technique which was adopted by Kobayashi et al for their further purification was not suitable; i.e. the pI of these proteins (above 9.5 for the toxin from C. distans) was too high to allow their purification on an electrofocusing column with a pH gradient from 3.5 to 9.5. Thus, the conotoxins may serve as a good repertoire for studying the structure-function relationship of conotoxins. Two new classes of

conopeptides inhibit the a1-adrenoceptor and noradrenaline. A modern development in pharmacology which has attracted considerable attention is the use of combination drug therapy, particularly for more intractable health problems such as AIDS or incurable tumours. The cone snails appear to have anticipated the development of pharmacological combination strategies by over 40 million years. The peptides which contribute to excitotoxic shock (the lightningstrike cabal) as well as the peptides that disrupt neuromuscular transmission (the motor cabal) comprise, in effect, a highly sophisticated application of a combination drug strategy in a natural system19. Although the molecular mechanisms that lead to rapid interspecific divergence are not understood, the phenomenon has become better defined. Conus peptides are initially translated as larger preporpeptide precursors; a mature Conus peptide of 20 amino acids is generally processed from a 70 to 80 amino acid precursor, with a single non-repeated copy of the toxin encoded at the C-terminal end20. In conclusion, an attempt was made to isolate different peptides of conotoxins from C. figulinus. The concentration of protein in the crude extract was found to be 1900 µg/mL. Two lower peptides below 14 kDa have been identified in SDS-PAGE. Activity studies were performed with the purified toxin containing proteolytic peptides, which inhibit the trypsin and chymotrypsin enzymes. Haemolytic assay carried out with the purified toxin suggests that these peptides also act as cytolytic agents. So there is a need for further purification of conotoxins, using ultra modern techniques such as 2D-gel electrophoresis, HPLC, FPLC, which may throw light on the individual characterization of peptides. Structural elucidation of the peptides can also be done by 2D-NMR, X-ray crystallography, etc. All the above explained techniques may lead to the development of wonderful new peptide drug in future from Conus species. Acknowledgement The authors are thankful to the authorities of SRM and Annamalai Universities for the basic facilities provided to carry out this work. One of the authors (RS) is thankful to Indian Council of Medical Research for the financial support.

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