Indian Journal of Geo-Marine Sciences Vol. 42(6), October 2013, pp. 800-806

Studies on biochemical and biological properties of venom (Turricula javana and Lophiotoma indica)

M Arumugam*, S Giji., S Tamilmozhi, Sunil Kumar & T Balasubramanian Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai-608 502 India [*E-mail: [email protected]] Received 23 March 2012; revised 17 August 2012

Turritoxins were isolated from the venom ducts of snails Turricula javana and Lophiotoma indica collected from Parangipettai waters, south east coast of India. Venom duct yield 1 g/Kg was dissected from both the species. Crude venom exhibited protein concentration of 0.138mg/ml and 6mg/ml respectively. Venom containing toxicity was analyzed by SDS-PAGE and revealed their protein bands with an apparent molecular weight of 25 kDa, 41 kDa and 95 kDa for T. javana and 28 kDa and 66 kDa for L. indica. Many components with molecular mass ranging 29-97kDa exhibited prominent caseinolytic and gelatinolytic activities with prominent hemolytic acitivty upto the dilution of 10-6. The lethal concentration, LC50 was determined in Artemia salina. In addition, the quantitative status of free amino acids was performed through HPTLC. Presence of peptides was revealed by using HPLC, which was indicative of viable biological properties.

[Keywords: Turrids, Marine toxin, HPLC, HPTLC, Peptides, Pharmacology, Venom, Evolution]

Introduction carry out the specific offensive and defensive tasks Marine pharmacology research during 2007–8 needed for their survival5. remained global enterprise, with researchers from 26 Marine mollusks are the largest group of toxic countries, and the United States, contributing to the marine invertebrates that are harmful to humans6. preclinical pharmacology of 197 marine compounds Superfamily , also referred to as the which are part of the preclinical marine toxoglossate snails, includes the families Conidae pharmaceuticals pipeline1. Unexplored marine (Cones), Terebridae (augers) and (Turrids)7, organisms harbors the most biological and chemical the later one being the largest family of deep-sea diversity, it is the vastest resource to discover novel gastropods8. Compared to cones and augers, turrids structures (to date, more than 21855 compounds were are remarkably diverse, and infra specific discovered)2. Drug discovery from marine natural of this group remains uncertain9,10,11. In fact, cones are products has enjoyed a renaissance in the past few hypothesized to have evolved from the turrids12,13. years3. Venomous marine snails are the significant Venomous organisms have developed during components of marine biodiversity, as their venom evolution and are widely spread throughout the are proving to be a promising resource for novel kingdom comprising more than 100,000 biologically active compounds. species distributed RETRACTEDamong all major phyla, such as Extensive Studies describing the chemical and chordates, echinoderms, mollusks, annelids, biological properties of conus venoms have focused nemertines, arthropods and cnidarians, etc. Despite mainly due to the beauty of their shells. Cone snails large number of venomous , the complexity of have evolved towards a rather complex venomous their venoms constitutes only a tiny proportion system, being fruit of 55 million years of evolution. (estimated to represent less than 0.1%) of their venom More than 500-700 Conus species are found to be components have been identified and characterized liable to express 50,000-70,000 distinct conotoxins14. and less than 1% of genetic information is available4. These (‘Conotoxins’ or ‘Conutoxins’), are biologically Animal venoms are highly complex mixtures of active peptides, mostly in the size range of 12-30 biologically active compounds. These biomolecules amino acids15. Consequently, conotoxins encompass a have been tailor-made by millions of years of diverse mixture of pharmacological components, most evolution to endow their possessors with the means to of which are small and structurally constrained 801 ARUMUGAM et al.: STUDIES ON BIOCHEMICAL AND BIOLOGICAL PROPERTIES OF TURRIDS VENOM

peptides that can be synthesized and modified, thus the crude toxins were estimated by comparing increasing their availability and potential utility16. mobility with protein markers such as Phosphorylase There has been much remarkable work on the B (97,400 Da), Bovine Serum Albumin (66,000 Da), characterization of venom of conus snails and Ovalbumin (43,000 Da), Carbonic anhydrase (29,000 comparatively less work on biological and Da), Soyabean trypsin inhibitor (20,000 Da), biochemical characterization of venoms of other two Lysozyme (14,300 Da) by using Gel Documentation toxoglossates (Auger and Turrid snails). system (Lark Fine Technologies, Version 4.0, India). Present study consists a comparative analysis from the venom of two Turrids species. Turrids are the Proteolytic activity of venom duct extracts most diverse groups of marine mollusks comprising Proteolytic activity was assessed by zymography more than 90% of the biodiversity of venomous method using SDS-PAGE23. Electrophoresis was snails17,18,19. Until date, between 5000-10000 species carried using 10% gels containing 2 mg/ml Casein of turrids have been reported20 .These species ranges and Gelatin. After electrophoresis, the gel was rinsed from small to medium size (3-50 mm on average) and in 2.5% (v/v) of Triton X-100 for 1 h in order to they are specialist predators on annelids and other remove SDS and incubated in 20 mM Tris, 0.4 mM mollusks, and occupy all marine habitats from the Calcium Chloride pH 7.4 at 37°C for 16 hrs and tropics to the poles, from Shallow to deep water, and stained with 0.125% Coomassie blue. The proteolysis from hard to soft substrates. Specimens of T. javana activity was detected as colorless bands on the blue and L. indica were collected and biological and gel. biochemical properties from their venom were examined. Hemolytic assay Hemolytic activity was assayed on Chicken Materials and Methods 24 erythrocytes as described earlier . Serial dilution of Specimens of T.javana and L.indica were collected the crude venom was made up to 100 μL in a 96 well from Parangipettai coastal landing centre (Lat.11° v shaped bottom microtitre plates. 100 μL of 1% RBC 28’ N and Long. 79°46′E), South east coast of India was added into all the wells. 1% RBC suspension and and transferred immediately to the laboratory to 100 μL distilled water served as the positive control extract the venom. The venom gland was dissected where 1% RBC suspension and 100 μL normal saline out along their coiled venom duct in a walking cold served as negative control. Then plate was gently storage room (4°C). The collected venom ducts shaken and allowed to stand for 3 hrs at room along their venom gland was homogenized using Tris- temperature. Homogenous red colour suspension buffer (50 mM Tris hydrochloride, 120 mM Sodium indicated the positive reaction due to hemolysis of chloride, 5 mM Potassium chloride, 1 mM Magnesium RBC and negative reaction is indicated by a compact chloride and 2 mM Calcium chloride) in a manual sharply, demarcated disc of regimented cells in the tissue homogenizer. Then the mixture was centrifuged centre bottom of the well. Reciprocal of the highest at 8000 g for 10 minutes at 4ºC. Finally the dilution of the crude extract showing the hemolytic supernatant was collected and lyophilized. Protein pattern was considered as one hemolytic unit (HU). concentration of the crude toxins was determined by the standard method using bovine serum albumin Cytotoxicity bioassay 21 RETRACTED (BSA) . Brine shrimp lethality bioassay was carried out to investigate the cytotoxicity of crude venom. Hatched SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) Brine shrimp eggs were purchased from ornamental SDS-PAGE (12%) was carried out under reducing fish breeding centre (CAS in Marine Biology, conditions according to the standard method22. Annamalai University). Ten nauplii were drawn for Samples were dissolved in reducing buffer (Tris each concentration (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 0.5 M, SDS, glycerol, mercaptoethanol, bromophenol µg/µL) of crude extract containing 100 μL seawater. blue, distilled water) and run at 50 V until the tracking The vials were maintained at 37°C for 24 h under the dye has reached 0.5 cm from the bottom of the gel. light. Surviving larvae were counted. Group of vials After electrophoresis, gels were stained using containing 10 nauplli were filled with100 µL sea Comassie Blue Staining method. Molecular weight of water served control. Survival rate was observed with INDIAN J. MAR. SCI., VOL. 42, NO. 6, OCTOBER 2013 802 the aid of a magnifying glass. This assay was carried version-3). Samples and standards were injected onto out three times in triplicates for each fluid the (10*10 cm) TLC aluminum silica gel plates concentration. The LC50 (mean lethal concentration) (Merck-F254). N-Butanol: acetic acid: water (3:1:1) the results were plotted as % mortality Vs was used as the developing solvent in a 20*10 mm concentration of crude venom extract. twin trough chamber with the saturation up to the distance of 80 mm. Then the plate was allowed to air- Qualitative analysis of free amino acids through dry for 3 minutes. Derivatization was carried out HPTLC using 0.1% Ninhydrin in acetone. Finally, the plate Amino acid analysis venom was performed using was scanned using CAMAG TLC Scanner 3 for the HPTLC system (Camag, Achrom, Mumbai, India detection of amino acids.

High Performance Liquid Chromatography (HPLC) Venom extracts were applied to a Shimadzu reversed-phase C18 analytical column (shim-Pac5µ, 4.6 mm × 215 mm, Phenomonex) and eluted at room temperature at a flow rate of 1 mL/min. HPLC solutions were (A) 0.1% v/v TFA in water and (B) 0.09% v/v TFA in 90%v/v aqueous ACN. The absorbance of elute was monitored at 215 nm.

Results The amount of protein present in T. javana and L. indica was estimated as 0.138 and 6 mg/mL respectively. Protein patterns of crude toxins were determined under reducing conditions. The electrophoretic profile of T. javana exhibited 3 components with their molecular weight approximately at 25 kDa, 41 kDa and 95 kDa. In contrast to T. javana the venom of L. indica exhibited two exclusive bands with the molecular mass ranging Fig. 1  Proteins of T. javana and L. indica venom extract separated by SDS-PAGE 28 kDa and 66 kDa respectively (Fig. 1).

RETRACTED

Fig. 2  Shows the Caseinolytic and Gelatinolytic activities of T. javana and L .indica venom 803 ARUMUGAM et al.: STUDIES ON BIOCHEMICAL AND BIOLOGICAL PROPERTIES OF TURRIDS VENOM

Table 1  Shows the Rf values of amino acids and venom

S.No. Amino Acid Rf 1. Glycine 0.27 2. Alanine 0.29 3. Valine 0.42 4. Lysine 0.07 5. Serine 0.84 6. Proline 0.71 7. Leucine 0.85 8. Isoleucine 0.29 9. Trytophan 0.82 10. Histidine 0.28 11. Tyrosine 0.74 12. Methionine 0.59 13. Threonine 0.49 Fig. 3  Shows the hemolytic activity of T. javana and L. indica venom 14. Glutamic acid 0.33 15. Arginine 0.75 16. Phenylalanine 0.27 17. Cysteine 0.02 18. Aspartic acid 0.75 19. Aspargine 0.35 20. Glutamine 0.47 21. T. javana 0.07, 0.85, 0.28, 0.59, 0.49, 0.47, 0.74, 0.71 22. L. indica 0.42, 0.29, 0.49, 0.27, 0.74, 0.75, 0.47, 0.71

Therefore, L. indica had more potent cytotoxic compounds than T. javana. The refractive index of respective amino acids is reported in the Table 1. Glycine, Leucine, Histidine, Methionine, Threonine, Glutamine, Tyrosine and Proline were observed in T. javana whereas Isoleucine, Threonine, Phenylalanine, Tyrosine, Fig. 4  Shows the cytotoxicity of the crude venom Arginine, Glutamine, Proline and Valine were Proteolytic activities present in T. javana and observed in L. indica. Proline, Glutamine, Threonine L. indica venom extracts was detected using SDS- and Tyrosine are the amino acids common for both PAGE. Venom extracts of both T. javana and T. javana and L. indica. L. indica catalyzed the hydrolysis of the protein Four different peaks were detected in T. javana at substrates (Casein and gelatin). Venom extract had the retention time 3.611, 6.778, 7.658 and 42.670. At large array of both caseinolytic and gelatinolytic the same time three different peaks were detected in proteases. Several components with molecular mass L. indica at the retention time 1.556, 6.576 and ranging between 97 to 29 kDa exhibited Caseinolytic 42.857. Both T. javana and L.indica recorded the and Gelatinolytic activitiesRETRACTED (Fig. 2). Crude extracts major peak at the retention time around 6.778 and induced moderate level of hemolysis on chicken 6.576 indicating similar biochemical characteristics blood. The hemolytic effect was observed up to a may exist among the species of Turridae family dilution of 10-6 for both T. javana and L. indica (Fig. 3). (Figs 5&6).

Lethal Assay Discussion Both the extracts showed moderate toxicity against In any habitat there is a competition for resources, brine shrimp nauplii. Moderate LC50 value indicated and every ecosystem on earth supporting life contains 25 higher toxicity and higher LC50 values are suggestive poisonous or venomous organisms . Among them, of lower toxicity (Fig. 4). LC50 was calculated as Conoidea superfamily, comprised of cone snails, 150 µg/mL for T. javana and 50 µg/mL for L. indica. terebrids, and turrids, is an exceptionally promising INDIAN J. MAR. SCI., VOL. 42, NO. 6, OCTOBER 2013 804

Fig. 5  Shows the HPLC profiling of T. javana and L. indica

Fig. 6  Shows the HPLC profiling of T. javana and L. indica RETRACTED20 group for the discovery of natural peptide toxins . profiling of peptide toxin for drug discovery. Venom toxins disturb the activity of critical enzymes, L. indica and T. javana upon SDS-PAGE revealed receptors, or ion channels, thus disarranging the sharp protein bands under reduced conditions central and peripheral nervous systems, the signifying the presence of peptides on their toxin. cardiovascular and the neuromuscular systems, blood Three molecular weight peptide bands representing coagulation and homeostasis. On the other hand, due 25 kDa, 41 kDa and 95 kDa were detected for to their high degree of target specificity, venom toxins T. Javana, whereas 2 peptides with the molecular have been increasingly used as pharmacological tools weight of 28 kDa and 66 kDa were detected in and as prototypes for drug development25. Hence, the L. indica. It is estimated that between 500 to 700 present study biochemical approach was carried on conus species exist, each possessing 50-200 two turrid species to come up with the comparative conopeptides on their venom26. In the case of turrids 805 ARUMUGAM et al.: STUDIES ON BIOCHEMICAL AND BIOLOGICAL PROPERTIES OF TURRIDS VENOM

only two to three peptides were documented. above principle, toxin induced membrane Previously, reported the protein content from 5 to permeabilization is detected by Hemolytic assay (loss 25% in the C. textile, C. aulicus, C.tulipa, of hemoglobin from red blood cells). Hemolytic effect C. geographus, C. marmoreus and also suggested that was observed up to a dilution of 10-6 for both these high levels of protein could be responsible for T. javana and L. indica. Thus Hemolytic activity their potent content nature27. Similarly, the protein indicated the presence of cytolytic activity by the content was estimated as 1.9 mg/ml in the crude toxin venom through pore-formation onto biological of C. figulinus28. Accordingly, the higher protein membranes33. Haemolytic activity was recorded for content was observed in L. indica, than that of the gastropod Conus figulinus toxin Upto 10-5 T. javana. Thus L. indica indicated the higher toxic dilutions28, which is very close to our turrid toxin content when compared to T. javana. potency. Earlier the HU (50) of jelly fish Venoms are usually a cocktail of proteins in which R. esculentum full venom (RFV) against chicken protease occupied a special position. Profile of erythrocytes was recorded at 3.40 µg/mL34. enzymatic degradation was similar in distinct The interesting trait is higher protein content was substrates, indicating the presence of proteases with observed in L. indica, when compared to that of broad substrate specificity. In both the turrids, clear T. javana. But both of the samples showed more or areas in the gel indicated regions of enzyme activity. less similar toxicity against blood erythrocytes cells Prominent fact we observed here is, the peptide and brine shrimps. Venom represents a huge and accounting for protease ranges in the similar essentially unexplored treasure of bioactive molecular weight i.e., 25 and 28 kDa. Proteolysis components; hence to sort out this, the crude sample activity of toxin samples on casein and gelatin were was analyzed in reverse phase HPLC, where a major assayed due to the probable involvement of proteases peak at the retention time of 6.778 and 6.576 were in the instability of biological activities. These results recorded. The major peaks may denote the presence suggest that, such proteases could contribute to of protease peptide in the turri toxin. Besides this degradation of proteins and components present in the some minor peaks accounting for the presence of extracellular matrix, for diffusion factors or they can nucleosides, CHO, lipids and biogenic amines were be directly involved in the degradation of proteins. also recorded. Earlier a significant proteolytic activity towards casein had been reported in the venom extracted from The HPTLC results revealed the presence of amino the anterior regions of venom ducts of same conidae acids like threonine tyrosine, glutamine and proline. family29. Previously four jellyfish venoms Besides that lysine, leucine, histidine, methionine demonstrated a huge variation in their proteolytic were present only in T. javana whereas valine, activities in quantitative and qualitative manner isoleucine, phenylalanine, arginine were exclusively depending on the species35. present only in L. Indica. There have been relatively

The brine shrimp assay is considered as a reliable only few studies on this turrid groups. Holding a indicator for the preliminary assessment of toxicity30. unique position among toxic groups, further efforts The Brine shrimp test (BST) represented a rapid, will highlight more about their biological properties inexpensive and simple bioassay for testing toxin of biomedical impact. lethality which in most cases correlates with cytotoxic and anti-tumour propertiesRETRACTED31. Percentage of lethality Conclusion was found to be directly proportional to the Present study is a comprehensive report of the concentration of the toxin. Our samples showed 50% isolation and characterization of Turrid venoms. mortality at 50 μg/mL and 150 μg/mL. LC50 values Within this group the classical family Turridae less than 250 μg/mL may considered significantly 32 broadly defined, more than two hundred genera had active and had the potential for further investigation . been described, this turritoxins may also exhibits Ultimately our samples too would have biopotency similar properties as extensively demonstrated for the for further scrutiny. conotoxins so as to act on ion channels, receptors are Most commonly used physical definition of life is transporters. Therefore, this information will open a based on the principle that an intact impermeable cell new field of investigation to understand more detailed membrane is essential for life. In assay based on the pharmacology of the turrid venoms. INDIAN J. MAR. SCI., VOL. 42, NO. 6, OCTOBER 2013 806

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