Comparative Biochemistry and Physiology, Part C 168 (2015) 28–38

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Biochemical and pharmacological characterization of three toxic phospholipase A2s from Daboia russelii snake venom J.R. Kumar a,e,⁎, Balapal S. Basavarajappa b,c,d, B.S. Vishwanath a, T. Veerabasappa Gowda a a

Department of studies in Biochemistry University of Mysore, Manasagangothri, Mysore 570006, India Division of Analytical Psychopharmacology, New York State Psychiatric Institute, USA Department of Psychiatry, Orangeburg, NY 10962, USA d Nathan Kline Institute for Psychiatric Research, Orangeburg, NY 10962, USA e Post Graduate Department of Biochemistry, JSS College, Ooty Road, Mysore 570025, India b c

a r t i c l e

i n f o

Article history: Received 3 September 2014 Received in revised form 19 November 2014 Accepted 26 November 2014 Available online 3 December 2014 Keywords: Pre/post synaptic neurotoxin Daboia russelii venom Venom PLA2

a b s t r a c t Three isoenzymes of phospholipase A2 (PLA2), VRV-PL-IIIc, VRV-PL-VII, and VRV-PL-IX were isolated from Daboia russelii snake venom. The venom, upon gel filtration on Sephadex G-75 column, resolved into six peaks (DRG75 I–VI). The VRV-PL-IIIc was purified by subjecting DRG75II to homogeneity by rechromatography in the presence of 8 M urea on Sephadex G-75 column. The other two isoenzymes VRV-PL-VII and VRV-PL-IX were purified by subjecting DRG75III to ion exchange chromatography on CM-Sephadex C-25 column. Mol wt. for the three PLA2s, VRV-PL-IIIc, VRV-PL-VII, and VRV-PL-IX are 13.003 kDa, 13.100 kDa and 12.531 kDa respectively. The VRV-PL-IIIc is not lethal to mice up to 14 mg/kg body weight but it affects blood sinusoids and causes necrosis of the hepatocytes in liver. It causes hemorrhage in kidney and shrinkage of renal corpuscles and renal tubules. The LD50s for VRV-PL-VII and VRV-PL-IX are 7 and 7.5 mg/kg body weight respectively. They induced neurotoxic symptoms similar to VRV-PL-V. All the three PLA2s are anticoagulant and induced varying degree of edema in the foot pads of mice. VRV-PL-V and VRV-PL-VII are shown to act as pre and post synaptic toxins, while VRV-PL-IX acts as presynaptic toxin. This is evident from experiments conducted on cultured hippocampal neurons by patch clamp electrophysiology. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Phospholipase A2s (PLA2) (EC 3.1.1.4) are esterolytic enzymes, which hydrolyse glycerophospholipids at sn-2 position. The PLA2 enzymes are found as both intracellular and extracellular forms. They have crucial roles in various physiological and pathological processes (Dennis et al., 1991). But snake venom PLA2 enzymes exhibit wide variety of pharmacological effects such as pre/post-synaptic neurotoxicity, myotoxicity, cardiotoxicity, pro/antiplatelet activity, edema inducing activity etc. (Ownby et al., 1976). Snake venom is also a source of multimolecular forms of PLA2. The venom of snakes such as Naja naja, Daboia russelii, and Trimersurus flavovirides have been reported to contain multiple forms of PLA2 (Vishwanath et al., 1987; Jayanthi and Gowda, 1988; Bhat and Gowda, 1991). Earlier from our laboratory VRV PL-V (Jayanthi and Gowda, 1988) and VRV PL-VIIIa (Kasturi and Gowda, 1989) two toxic PLA2s were purified and characterized from Abbreviations: PLA2, phospholipase A2; NMDA, N-methyl D-aspartic acid; PC, phosphatidylcholine; TTX, tetrodotoxin; mEPSC, miniature excitatory postsynaptic currents; GABA, gamma amino butyric acid; HEPES, 4-(-2-hydroxyethyl)-1-piperanineethanesulfonic acid; EGTA, ethylene glycol tetra acetic acid; VRV-PL, Vipera russelii venom-phospholipase ⁎ Corresponding author at: Post Graduate Department of Biochemistry, JSS College, Ooty Road, Mysore, India. E-mail address: [email protected] (J.R. Kumar).

http://dx.doi.org/10.1016/j.cbpc.2014.11.005 1532-0456/© 2014 Elsevier Inc. All rights reserved.

D. russelii pulchella (southern, India). Primary sequence of VRV PL-VIIIa (Gowda et al., 1994) and VRV PL-V has been reported (Vishwanath et al., 1988; Satish, 2004) purified and characterized a multitoxic PLA2, VRV PL-VI, from the D. russelii venom (Northern, India) and has been shown to target pituitary gland. Another PLA2 RVVPF3 was purified and characterized from eastern region D. russelii venom (Chakraborty et al., 2002) exhibiting potent hemolytic activity. The PLA2s may associate with other proteins/peptides to form a complex and induce potent toxicity, for “Reprotoxin” a protein complex from D. russelii venom (Western, India) made up of a PLA2, a protease and a trypsin inhibitor reported (Kumar et al., 2008). The hemorrhagic complex (MCHR) was isolated and characterized from eastern region D. russelii venom (Uma, 1998). It is made up of a PLA2 and two non enzymatic peptides. Apart from this, there is a variation in the composition of acidic and basic PLA2 isoforms in the venoms of Russell's viper from different regions (Prasad et al., 1999). Acidic PLA2s are prominent in northern region D. russelii venom (Vishwanath et al., 1988) and of moderate proportion in the eastern region of India. Southern and western regions contain only basic PLA 2 s and lack acidic PLA2s. The neurotoxic effect of the snake venom is the prominent contributor to the lethal toxicity. Neurotoxicity is brought about either by presynaptic or postsynaptic blockade of the neurotransmission.

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β bungarotoxin (Bon and Saliou, 1983), Crotoxin (Bon et al., 1988), Taipoxin (Fohlman et al., 1976) and Textilotoxin (Pearson et al., 1993) are among the well studied presynaptic neurotoxic PLA2 complexes that have been reported from snake venoms. On the other hand only a few postsynaptic neurotoxic PLA2s are reported from snake venoms. For example Deepa machaiah and Gowda T V (Machiah and Gowda, 2006) have reported the isolation and purification of a postsynaptic PLA2 NN-XIa from N. naja venom (Eastern region, India). Bon and Saliou (1983) reported isolation of Ceruleotoxin from the venom of Bungarus cerulus which blocks post synaptic nerve terminal, But crotoxin is a complex toxin which exhibits both pre and post synaptic neurotoxicity in experimental animals (Hendon and Fraenkel-Conrat, 1971; Eterovic et al., 1975; Fraenkel-conrat, 1983). Several classical methods can explain the effect of toxins on the central nervous system. There are mechanisms underlying the selective inhibition of mEPSCs (miniature excitatory postsynaptic currents) of NMDA (N-methyl D aspartic acid) receptors in neurons. Some reports have revealed the involvement of presynaptic NMDA receptors in physiological functions. Alex et al. (2006) reported the isolation of Conantokin G (Con G) from the venom of Conus geographus and its action on NMDA receptor mediated spontaneous EPSCs in cultured cortical neurons. Although two prominent neurotoxic PLA2 have been reported and characterized from D. russelii venom, the pharmacological activities of these two PLA2 do not completely recapitulate all the toxic effects of the venom such as effects on liver and kidneys. Therefore we set forth to resolve the remaining toxic components of the venom. In the present paper we report the isolation and characterization of three phospholipase A2 from D. russelii venom (Western region). The PLA2, VRV PL-IIIc induces kidney and liver necrosis. The other three PLA2s VRV-PL-VII and VRV-PL-IX including VRV-PL-V inhibited NMDA and nonNMDA mediated spontaneous excitatory neurotransmission in cultured hippocampal neurons.

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separately, and were lyophilized and stored at 4 °C. Protein content for each peak was estimated by the method of Lowry (Lowry et al., 1951). Bovine serum albumin was used as a protein standard. 2.4. Purification of venom PLA2s 2.4.1. VRV PL-IIIc VRV PL-IIIc was purified according to the method of Kumar J R (Kumar et al., 2008). The Sephadex G-75 column (1 × 145 cm) was first equilibrated with 8 M Urea. The three milligrams of DRG-75-II was dissolved in 0.5 ml of 8 M urea and loaded on to the column. Elution was carried out in the same buffer as described earlier. 2.4.2. VRV PL-VII and VRV PL-IX The DRG-75-III peak showing PLA2 activity was loaded (8 mg) on to a CM-Sephadex C-25 cation exchange column (1.5 × 40 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0). PLA2s were resolved by a stepwise gradient elution of phosphate buffers of various molarities and pH. The fractions of 1.5 ml was collected at the rate of 15 ml/h. Protein elution was monitored at 280 nm using a UV–VIS1601 Shimadzu spectrophotometer. Resolved protein peaks were desalted, lyophilized and stored at 4 °C in a refrigerator.

2.4.3. VRV PL-V The most toxic PLA2 found in D. russelii russelii venom, VRV PL-V was purified according to the method described by Shelke (2000). Briefly, whole venom of D. russelii snake venom from southern region India was fractionated on sephadex G-75 column followed by cation exchange chromatography on CM Sephadex C-25 column.

2. Materials and methods

2.5. Phospholipase A2 assay and positional specificity

2.1. Materials and reagents

Phospholipase A2 activity was determined using egg phosphatidylcholine (PC) as substrate according to the method of Bhat and Gowda (1989). The reaction mixture (1 ml) contained 1 μmol of PC in 0.05 M Tris–HCl buffer, pH 7.5, 0.2 ml of diethyl ether, and 40 μmol of Ca2+, mixed well before adding 5 μg of PLA2 peaks and incubated at 37 °C for 60 min with intermittent mixing. The free fatty acid released was extracted as cobalt soap and then the cobalt was complexed with α-nitroso-β-naphthol and estimated by colorimetry method. The phospholipase A2 activity is expressed as nanomoles of free fatty acid released per minute. Positional specificity of PLA2 was determined with [14C] oleate-labeled, autoclaved Escherichia coli (E. coli) cells as substrate according to the method of Vishwanath et al. (1987).

Sephadex G-75 and low-range molecular weight markers were purchased from Sigma Chemicals (St. Louis, MO, USA). [14C] Oleic acid was from Perkin Elmer Life Sciences, Inc., USA. Fatty acid-free bovine serum albumin (BSA) was obtained from PAA Laboratories GmbH, Austria. Scintillation cocktail was obtained from Packard Biosciences BV (The Netherlands). All the other chemicals and reagents were of analytical grade purchased from SRL Chemicals, India. Lyophilized D. russelii snake venom from western India was purchased from the Haffkine Research Institute, Mumbai, India. 2.2. Animals Adult Swiss Wister male mice weighing approximately 21 g (30– 35 days old) were obtained from the central animal facility, University of Mysore. Animal care and handling were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The Institutional Animal Ethics Committee (IAEC) of the University of Mysore approved the protocols for the animal experiments.

2.6. Determination of molecular weights of VRV PL-VII and VRV PL-IX by MS-MALDI The molecular mass of VRV PL-VII and VRV PL-IX was determined by mass spectrometry in Kratos PC-Kompact MALDI-4 in the positive ionization mode (Linear high, Power: 45).

2.7. Determination of LD50 2.3. Gel filtration chromatography and protein estimation The sephadex G-75 column (1 × 145 cm) equilibrated with 0.05 M phosphate buffer (pH 7.0) was loaded with 100 mg of D. russelii venom in 0.5 ml equilibration buffer. Elution was carried out with pre-equilibrated buffer at a flow rate of 15 ml/h and 1.5 ml fractions were collected. Protein elution was monitored at 280 nm using a UV-VIS1601 Shimadzu spectrophotometer. The fractionation showed six discrete peaks (DRG-75 I–VI). Fractions of each peak were pooled

Groups of 10 mice, each mouse weighing 20–24 g, were injected intraperitonially (i.p.) with VRV PL-VII and VRV PL-IX, separately in 250 μl saline at doses of 1.0–10 mg/kg body weight. In case of VRV PL-IIIc dose in between 1 and 14 mg/kg body weight was injected (i.p). The survival time of each animal was recorded for 24 h. The LD50 dose was calculated according to the mathematical scheme of Meier and Theakston (Meier and Theakston, 1986). Animals were also observed constantly for the appearance of signs/symptoms of toxicity.

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2.8. Hemolytic activity Hemolytic activities (indirect/direct) were assayed as described by Boman and Kaletta (1957). Briefly, the substrate for direct lytic activity was prepared by suspending 1 ml of packed fresh human red blood

cells in 9 ml of saline. On the other hand the substrate for indirect hemolytic activity was prepared by suspending 1 ml of packed fresh human RBC and 1 ml of fresh hen egg-yolk in 8 ml of PBS. The suspension (1 ml) was incubated with 10–20 μg of each PLA2 VRV PL-VII and VRV PL-IX fractions for 45 min at 37 °C, and the reaction was stopped

Fig. 1. (A) Gel-permeation chromatography of Daboia russelii venom on a Sephadex G-75 column. Russell's viper venom, dissolved in 0.5 ml of 0.05 M phosphate buffer, pH 7, was loaded on to the column (1 × 145 cm) pre-equilibrated with 0.05 M phosphate buffer, pH 7. The column was eluted with the same buffer and the flow rate was adjusted to 15 ml/h. (B) CM-Sephadex C-25 column chromatography of Peak-III from Sephadex G-75 column. The column of (1.5 × 30 cm) preequilibrated with 0.01 M phosphate buffer (pH 7), was loaded with 8 mg of sephadex G-75 peak-III, dissolved in 3 ml of the equilibration buffer. The column was eluted with 30 ml/h in a stepwise gradient with a phosphate buffer and pH as indicated in the figure. The fractions were collected and protein elution was monitored at 280 nm in a spectrophotometer. RP-HPLC profile for (C) VRV-PL-VII and (D) VRV-PL-IX. VRV-PL-VII and VRV-PL-IX were run on a Vydac C18 column. The column was equilibrated with 0.1% TFA. Elution was carried out with a linear gradient of 70% acetonitrile in 0.1% TFA. (E) SDS PAGE (non reducing) for VRV-PL-VII and VRV-PL-IX. Lane-1 Molecular wt. markers. Lane-2 VRV-PL-VII, Lane-3 VRV-PL-IX. (F) and (G) MS-MALDI: Molecular mass determination of (F) VRV-PL-VII, (G) VRV-PL-IX by MS-MALDI in a Kratos PC-Kompact 4 instrument.

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by addition of 9 ml of ice-cold PBS. The suspension was centrifuged at 2000 rpm for 20 min, and then the released hemoglobin was read at 530 nm.

2.9. Determination of anticoagulant activity Recalcification time was estimated according to the method of Condrea (Condrea et al., 1981). Briefly, platelet-poor plasma was prepared by diluting whole blood at a ratio of nine parts blood to one part of 0.13 M trisodium citrate. The mixture was centrifuged twice at 2500 g for 15 min. The supernatant obtained is referred to as plateletpoor plasma (PPP). 5–30 μg of VRV PL-VII and VRV PL-IX separately in 0.01 M Tris–HCl, pH 7.4, were added to 300 μl of PPP. The mixture was incubated for 60 s at 37 °C, and the time required for clot formation was recorded (min).

2.10. Histopathology Mice were injected with VRVPL-IIIc 14 mg/kg body weight intraperitoneally. After 24 h after injections, the animals were sacrificed; kidney and liver tissues were rapidly isolated and were fixed in Bouin's fluid for 24 h, washed in distilled water, processed in graded ethanol from 30% to 100%, cleared in chloroform, and embedded in paraffin wax (MP, 58 °C). Sections (5 μm thick) were cut using Spencer-820 microtome, stained with hematoxylin–eosin.

2.11. Whole cell patch clamp electrophysiology 2.11.1. Primary hippocampal neuronal cell culture Hippocampal primary neuronal cultures were prepared from 1-day old C57BL/6 J mouse pups as described previously (Arancio et al., 1995; Di Rosa et al., 2002; Ninan and Arancio, 2004). Cells were dissociated through enzymatic treatment (0.25% trypsin) and subsequent trituration. Hippocampal cells were grown in medium containing 84% Eagle's minimum essential medium (MEM), supplemented with 10% heat-inactivated fetal calf serum, 45 mM glucose, 1% MEM vitamin solution, and 2 mM glutamine. After 24 h this medium was replaced by a medium containing 96.5% neurobasal A, B27-nutrient (2%), heatinactivated fetal calf serum (1%), 0.4 mM glutamine, 0.5 mM kynurenic acid and 6.6 ng/ml 5-fluorodioxyuridine in 16.4 ng/ml uridine to suppress cell division.

2.11.2. Patch clamp recordings Electro physiological studies were carried out on cells of 10– 17 days after plating. Cultured neurons were voltage-clamped with the whole-cell ruptured patch technique throughout the experiment (Arancio et al., 1995; Ninan and Arancio, 2004). The bath solution consisted of (mM) NaCl (119), KCl (5), HEPES (20), CaCl2 (2), MgCl2 (2), glucose (30), glycine (0.001), picrotoxin (0.1), pH 7.3, osmolarity adjusted to 330 mOsm with sucrose. The solution in the whole-cell patch electrode consisted of (mM) K-gluconate (130), KCl (10), MgCl2 (5), EGTA (0.6), HEPES (5), CaCl2 (0.06), Mg-ATP (2), GTP (0.2), leupeptin (0.2), phosphocreatine (20), and creatinephosphokinase (50 U/ml). For the mEPSC experiments, 1 μM tetrodotoxin was also added to the bath to suppress action potentials. Currents were recorded with a Warner amplifier (model PC-501A) (Warner Instruments, Hamden, CT) and filtered at 1 kHz. To eliminate artifacts due to variation of the real properties, the access resistance was monitored for constancy throughout all experiments. The recordings were digitized (Digidata 1322A, Axon Instruments) and analyzed with the mini analysis program (version 4.0) from Synaptosoft, Inc. (Decatur, GA).

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Table 1 Summary of purification of VRV-PL-VII and VRV-PL-IX from the Daboia russelii russelii venom. Step

Fraction

Total proteina (mg)

Enzyme activityb

Sephadex G-75 CM-Sephadex C-25

III peak (DRG75-III) VRV-PL-VII (Peak-II) VRV-PL-IX (Peak-III)

4

1725

431

0.5

566

1132

0.6

350

583

a b c

Specific activityc

Total enzyme activity was estimated in the pooled peaks. nmoles fatty acid released/ml at 37 °C. nmoles fatty acid released/mg/min at 37 °C.

3. Results 3.1. Purification and biochemical characterization of PLA2s D. russelii venom upon gel filtration chromatography on Sephadex G-75 column was resolved into six distinct peaks (Fig. 1A). Peak DRG-75-I showed caseinolytic, peaks DRG-75-II and DRG-75-III exhibited PLA2, and peak DRG-75-IV showed trypsin inhibitor activities. VRV PL-IIIc was purified from DRG-75-II as described before (Kumar et al., 2008). The peak DRG-75-III resolved into three distinct peaks on CM sephadex C-25 column by varying pH and molarities of eluent, phosphate buffer as indicated in the Fig. 1B. The peak II and III exhibited PLA2 activity and hence were numbered as VRV PLVII and VRV PL-IX as per the scheme adopted previously by Vishwanath (Vishwanath et al., 1987). The VRV PL-VII (Peak-II) and VRV PL-IX (Peak-III) accounted for 12.5% and 15% of the protein loaded to the column. The summary of the purification is tabulated in Table 1. The RP-HPLC profiles of VRV PL-VII (Fig. 1C) and VRV PL-IX (Fig. 1D) gave a symmetric sharp peak with a retention time of 42.5 min and 45 min respectively. In Fig. 1E, an illustration of the SDS-PAGE pattern of VRV PL-VII and VRV PL-IX along with reference molecular weight markers is shown. Molecular weights of VRV PL-VII (Fig. 1F) and VRV PL-IX (Fig. 1G) were determined by MS MALDI. They were found to be 13.1 kDa and 12.5 kDa respectively. The VRV PL-VII and VRV PL-IX exhibited indirect hemolytic activity when incubated with egg yolk and freshly isolated RBC, suggesting release of free fatty acid from egg phosphatidylcholine. The specificity of PLA2 activity of VRV PL-IIIc, VRV PL-VII and VRV PL-IX was confirmed by release of radio-labeled fatty acid from E. coli cell membrane containing phospholipids specifically labeled at Sn-2 position with [C14] oleate. Iso-electric points for the PLA2s, VRV PL-VII and VRV PL-IX were 9.5 and N 9.5 respectively (data not shown). Biochemical properties are summarized in the Table 2.

Table 2 Biochemical and pharmacological characterization of VRV-PL-IIIc, VRV-PL-VII, and VRV-PL-IX. Property

VRV-PL-IIIc

VRV-PL-VII VRV-PL-IX

Molecular weight kDa (MALDI) pI LD50 (mg/kg)

13.009 7.3–7.5 Non lethal up to 14 mg/kg body wt. 5 μg

13.100 9.5–10 7

12.531 N11 7.5

3 μg

2 μg

200 ± 0.8 0.3

180 ± 0.5 0.5

Anticoagulant activity Edema A) Edema ratio (5 μg) 160 ± 05 B) Minimum edema dose (MED) (μg) 0.6

Results are given either as mean ± S.D. (n = 5) or as mean of duplicates determination, which differed by not more than 10%. Edema ratio = weight of edematous leg × 100 / weight of normal leg.

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3.2. Pharmacological characterization The PLA2s, VRV PL-VII and VRV PL-IX were lethal to mice at i.p dose 7 mg/kg and 7.5 mg/kg body weight respectively. In contrast VRV PL-IIIc was not lethal to mice up to 14 mg/kg body weight. Mice injected with VRV PL-VII and VRV PL-IX exhibited neurotoxic symptoms such as respiratory distress and paralysis of hind limb. The mice moved with difficulty and were completely immobilized at the time of death. Both the PLA2s induced edema in the mice footpads. The edema ratio was 180 ± 5 and 200 ± 8 respectively. The minimum edema doses for

VRV PL-IIIc, VRV PL-VII and VRV PL-IX were 0.6 μg, 0.3 μg and 0.5 μg respectively. The PLA2s VRV PL-IIIc, VRV PL-VII and VRV PL-IX showed anticoagulant activity. Summary of the pharmacological properties is given in Table 2. The histopathological studies performed on tissues of mice injected with VRV PL-IIIc revealed severe liver necrosis. The enlarged blood sinusoids and necrosis of the hepatocytes, heavy hemorrhage in the hepatic vein and hepatocytes were observed (Fig. 2A). In kidney, shrunken renal corpuscles and renal tubules, hemorrhage in the renal corpuscles and between the renal tubule, and elongation of the epithelial cells were seen (Fig. 2B).

Fig. 2. A. Cross section of liver of control and VRV-PL-IIIc treated mouse. (Hematoxylin and eosin) ×40. 1. Liver section of control mouse showing central vein (arrows) and blood sinusoids (small arrows), and normal hepatocytes. 2. Liver section of the VRV-PL-IIIc treated mouse showing enlarged blood sinusoids (small arrow), and necrosis of hepatocytes. 3. Liver section of the VRV-PL-IIIc treated mouse showing hemorrhage and necrosis of hepatic vein (arrows). 4. Liver section of the VRV-PL-IIIc treated mouse showing heavy hemorrhage in the hepatic vein and necrosis of adjoin cells (arrows). B. Cross section of the kidney of the control and VRV-PL-IIIc treated mouse. (Hematoxylin and eosin) ×40. 1. Kidney showing renal corpuscle (arrows) and renal tubule cells (small arrows) of the control mouse. 2. Kidney section showing hemorrhage in the renal corpuscles (arrows) and necrosis of cells in the treated mouse (small arrows). 4. Kidney section showing hemorrhage in between the renal tubule cells and elongation of epithelial cells and initiation of necrosis (small arrows) in treated mouse.

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Fig. 3. VRV-PL-V inhibits both mEPSC frequency and amplitude in cultured hippocampal neurons. (A) Example of spontaneous mEPSC before (Pre) and after 20 min bath perfusion of VRV-PL-V or saline (control). (B) Average change (mean SEM) in mEPSC amplitude following bath perfusion of VRV-PL-V or saline (control) (n = 6) (C) Average changes in mEPSC frequency following bath perfusion of VRV-PL-V or saline (control) (n = 6). Data were normalized to the average value during the 10 min before VRV-PL-V application (baseline) in each experiment. (D) Average changes (between 15 and 20) in mEPSC amplitude following bath perfusion of VRV-PL-V or saline (control) (n = 6). (E) Average changes (between 15 and 20 min) in mEPSC frequency following bath perfusion of VRV-PL-V or saline (control) (n = 6). (F) Cumulative mEPSC amplitude distribution before and during the bath perfusion of VRV-PL-V or saline (control), in all of the neurons recorded (n = 6). (G) Cumulative inter-event interval distribution before and during the bath perfusion of VRV-PL-V or saline (control) (n = 6).

Fig. 4. VRV-PL-VII inhibits both mEPSC frequency and amplitude in cultured hippocampal neurons.(A) Examples of spontaneous mEPSC before (pre) and after 20 min perfusion of VRV-PL-VII or saline (control). (B) Average change (mean SEM) in mEPSC amplitude following bath perfusion of VRV-PL-VII or saline (control) (n = 6). (C) Average changes in mEPSC frequency following bath perfusion of VRV-PL-VII or saline (control) (n = 6). Data were normalized to the average value during the 10 min before VRV-PL-VII application (baseline) in each experiment. (D) Average changes (between 15 and 20 min) in mEPSC amplitude following bath perfusion of VRV-PL-VII or saline (control) (n = 6). (E) Average changes (between 15 and 20 min) in mEPSC frequency following bath perfusion of VRV-PL-VII or saline (control) (n = 6). (F) cumulative mEPSC amplitude distribution before and during the bath perfusion of VRV-PL-VII or saline (control), in all of the neurons recorded (n = 6). (G) Cumulative inter-event interval distribution before and during the bath perfusion of VRV-PL-VII or saline (control) (n = 6).

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3.3. Patch clamp electro physiology The effect of VRV PL-V, VRV PL-VII and VRV PL-IX on miniature excitatory and inhibitory post-synoptic current (mEPSC) in primary hippocampal neurons. 3.3.1. VRV PL-V The VRV PL-V at 10 μM decreased the frequency of mEPSCs (67.5 ± 6% of baseline at 20 min after VRV PL-V application, P b 0.0001, n = 6), and affected the distribution of their amplitudes; 78.8 ± 3.2% is consistent with both pre-synaptic and post-synaptic site of action. The average baseline mEPSC frequency values were 84 ± 9 min−1 (saline) and 87 ± 7 min− 1 for VRV PL-V and are not significantly different by ANOVA (n = 6). The average baseline amplitude values were 16.9 ± 1.1 pA (saline) and 17.2 ± 1.2 pA (VRV PL-V) and are not significantly different by ANOVA (n = 6) (Fig. 3). 3.3.2. VRV-PL-VII The VRV PL-VII at 10 μM decreased the frequency of mEPSCs (26 ± 1.8% of baseline at 20 min after VRV PL-VII application, P b 0.0001, n = 6), and it affected the distribution of their amplitudes (85 ± 2% of baseline at 20 min after VRV PL-VII, P b 0.0001, n = 6), consistent with both pre-synaptic and post-synaptic site of action. The average baseline mEPSC frequency values were 84 ± 9 min−1 (n = 6) (saline) and 87 ± 7 min−1 (n = 6) (VRV PL-VII), not significantly different by ANOVA. The average baseline amplitude values were 16.9 ± 1.1 pA (n = 6) (saline) and 17.2 ± 1.2 pA (VRV PL-VII), not significantly different by ANOVA (Fig. 4). 3.3.3. VRV-PL-IX The VRV PL-IX 10 μM increased the frequency of mEPSCs (148 ± 3.5% of baseline at 20 min after VRV PL-IX application, P b 0.0001, n = 6), but did not affect the distribution of their amplitudes (97.6 ± 2% of baseline at 20 min after VRV PL-IX toxin, P N 0.5, n = 6), consistent with a purely pre-synaptic site of action. The average baseline mEPSC frequency values were 84 ± 9 min−1 (n = 6) (saline) and 87 ± 7 min−1 (n = 6) (VRV PL-IX), not significantly different by ANOVA. The average baseline amplitude values were 16.9 ± 1.1 pA (n = 6) (saline) and 17.2 ± 1.2 pA (VRV PL-IX), not significantly different by ANOVA (Fig. 5).The VRV PL-V appears to be strong pre and post synoptic neurotoxic PLA2 compared to VRV PL-VII. VRV PL-IX is found to be presynoptic neurotoxic PLA2. 4. Discussion Several isoforms of PLA2 enzymes have been reported from the D. russelii snake venom. The proportions of each of the isoforms of PLA2 vary from region to region in the venom (Jayanthi and Gowda, 1988). The purification of the isoforms of PLA2 often involves multistep procedures (Vishwanath et al., 1987). Isolation and characterization of PLA2s from the southern, northern and eastern regions of India for D. russelii venom have been reported (Vishwanath et al., 1987; Jayanthi et al., 1989; Kasturi and Gowda, 1989). The D. russelii venom from these regions showed a marked difference in the PLA2 content. The PLA2 enzymes constitute the major protein pool of D. russelii pulchella and lack acidic isoform. The venom from India's northern region contains a moderate amount of basic PLA2 enzymes and high acidic PLA2 enzymes (Jayanthi and Gowda, 1988). In eastern regional D. russelii russleii venom contains moderate amounts of both acidic and basic isoforms of PLA2 (Prasad et al., 1999). In contrast we found, the presence of moderate basic PLA2 enzyme isoforms and absence of acidic PLA2 enzymes from D. russelii venom in the western region. In this study three new basic PLA2 iso-forms are purified from the western regional D. russelii venom. The PLA2s VRV PL-VII and VRV PL-IX were purified to homogeneity by subjecting DRG75-III to ion exchange chromatography on CM Sephadex C-25. The elution profiles indicated

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the presence of PLA2 in peaks located differently compared to the elution profile of VRV PL-VI and VRV PL-VIIIa. The identities of these isoforms are supported by pI values (VRV PL-VII-9.5–10, VRV PL-IX- N10). The catalytic specificity of PLA2 isoforms was confirmed when they specifically released radioactive fatty acid from autoclaved E. coli cells labeled with phospholipids at the sn-2 position with [14C] oleate. The homogeneity of these PLA2s was confirmed by RP-HPLC and SDS-PAGE. The molecular weights of VRV PL-VII and VRV PL-IX were found to be 13.1 kDa and 12.53 kDa respectively. The molecular weights of these PLA2s are close to the previously reported values from D. russelii venom PLA2s (Jayanthi and Gowda, 1988; Kasturi and Gowda, 1989). The PLA2 VRV PL-IIIc is non toxic, while VRV PL-VII and VRV PL-IX are toxic to mice at 7 and 7.5 mg/kg body wt. LD50 dose respectively; hence they can be grouped into low toxicity PLA2s (LD50 N 1 mg/kg) (Rosenberg, 1988). It is evident from the data that apparently there is no correlation between enzyme activity and lethal toxicity (Rosenberg, 1988). It is well documented that several PLA2 isoforms from Russell's viper venom tend to target the vital organs in experimental animals as well as victims of snake bites (Vishwanath et al., 1988). For example VRV PL-VI was shown to target the kidney. Renal failure leading to death in victims of Russell's viper bite has been reported by Tin-Nu-Swe (Tin Nu et al., 1993), though they survive the early effect of envenomation. The PLA2 VRV PL-V from Russell's viper venom is most toxic (1.8 kg/body wt.) and attacks presynaptic site (Kasturi and Gowda, 1989). Uma and Veerabasappa Gowda (2000) have reported that VRV PL-VIIIa injures lung leading to hemorrhage. Similarly VRV PL-IIIc is associated with other factors leading to atrophy of reproductive organs in experimental mice (Kumar et al., 2008). In rural India, it has been observed that some of the victims of Russell's viper bite suffer from necrosis of the kidney and also known to cause impotency. The necrosis of the kidney is characterized by the shrinkage and hemorrhage in the renal corpuscles. Apart from affecting the kidney this PLA2 also damages the liver as shown by enlarged blood sinusoids, necrosis and hemorrhage in hepatic veins. Similar effect was also induced by VRV PL-VI a PLA2 from the northern region D. russelii venom. In addition, it also induced hemorrhage in the endocrine glands such as thyroid and pituitary (Vishwanath et al., 1988). The patch clamp electrophysiology technique has been extensively used to determine the site of action of both pre and postsynaptic neurotoxins (Capogna et al., 1996). Spontaneous neurotransmitter release (mEPSCs) was induced by quantum transmitters, which were released from presynaptic membrane (Neher and Sakaba, 2001). In the present study we have made an attempt to demonstrate the site of action of VRV PL-V, VRV PL-VII and VRV PL-IX in cultured hippocampal neurons. Gippert (Geppert et al., 1998) demonstrated the effect of presynaptic toxicity using mEPSCs. The mEPSCs showed sensitivity against three PLA2 isoforms studied. Both VRV PL-V and VRV PL-VII markedly decreased the frequency of spontaneous release of neurotransmitter from neuronal cells as measured by mEPSCs. Simultaneously, the decrease in amplitude was also affected by both PLA2s; it is similar to the action of opiods on hippocampal neurons (Liao et al., 2007). This action was mediated by both presynaptic and postsynaptic sites, because VRV PL-VII and VRV PL-V decreased the distribution of mEPSC amplitudes. mEPSC frequency modulation occurs almost immediately for VRV PL-V, while a delayed modulation was observed for VRV PL-VII. On the other hand results of change in distribution of amplitude were observed differently for the two PLA2 isoforms. Change in amplitude for VRV PL-V is delayed (5 min) compared to VRV PL-VII (immediately). These observations taken together suggest that VRV PL-VII and VRV PL-V decrease the neurotransmitter release at presynaptic neuron by modulating the proteins underlying the vesicular release machinery, such as decreased glutamate releasing sites, release rates and release probabilities as well as modulation of postsynaptic receptors. Kasturi and Gowda (1989) have reported that VRV PL-V is a strong presynaptic neurotoxin. In

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Fig. 5. VRV-PL-IX enhanced mEPSC frequency in cultured hippocampal neurons. (A) Examples of spontaneous mEPSC before (pre) and after 20 min bath perfusion of VRV-PL-IX or saline (control). (B) Average change (mean SEM) in mEPSC amplitude following bath perfusion of VRV-PL-IX or saline (control) (n = 6). (C) Average changes in mEPSC frequency following bath perfusion of VRV-PL-IX or saline (control) (n = 6). Data were normalized to the average value during the 10 min before VRV-PL-IX application (baseline) in each experiment. (D) Average changes (between 15 and 20 min) in mEPSC amplitude following bath perfusion of VRV-PL-IX or saline (control) (n = 6). (E) Average changes (between 15 and 20 min) in mEPSC frequency following bath perfusion of VRV-PL-IX or saline (control) (n = 6). (F) Cumulative mEPSC amplitude distribution before and during the bath perfusion of VRV-PL-IX or saline (control), in all of the neurons recorded (n = 6). (G) Cumulative inter-event interval distribution before and during the bath perfusion of VRV-PL-IX or saline (control) (n = 6).

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contrast to VRV PL-V and VRV PL-VII, the VRV PL-IX increased mEPSC frequency without modulating distribution of amplitude, obviously suggesting it to be a presynaptic neurotoxin. Similarly, α-LTx, a component of black widow spider venom, increased the mEPSCs frequency without affecting the amplitude in hippocampal slice culture (Capogna et al., 1996). α-LTx toxin binds to a specific receptor that is exclusively associated with axon terminal plasma membranes (Geppert et al., 1998) in all brain regions (Malgaroli et al., 1989). Putative observations suggest that VRV PL-IX appears to be presynaptic neurotoxin in contrast to the action of VRV PL-V and VRV PL-VII, increasing the neurotransmitter release of presynaptic neuron by modulating the proteins underlying the vesicular release machinery, such as increased glutamate releasing sites, release rates and release probabilities. Similarly, the β-bungarotoxin, a potent presynaptic neurotoxin that exerted potent toxic effect on cultured cerebellar granular neurons (CGNs) via activation of NMDA receptors and/or L-type calcium channels leading to the degranulation of [Ca2+] homeostasis and induction of neuronal death (Tseng and Lin-Shiau, 2003) affecting mEPSC frequency but not amplitude, mediated by ά-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA)/kainite preferring glutamate receptors, was enhanced by focal application of low concentrations of (b 0.5 nM) of α-latrotoxin (α-LTx) in hippocampal slice culture. These observations are consistent with the selective presynaptic action of this toxin (Capogna et al., 1996). Later it was shown that α-LTx enhances spontaneous neurotransmitter release by binding to presynaptic receptor protein (Geppert et al., 1998). There are several PLA2 isoforms from the venoms shown to act as presynaptic neurotoxins, for example: Ammodytoxin (Ritonja and Gubensek, 1985), Notexin (Halpert and Eaker, 1976) and Agkistrodoxin (Tang et al., 1998). Similarly there are reports on complex toxins containing PLA2 as one of the subunits; for example Textilotoxin (Pearson et al., 1993), Taipoxin (Fohlman et al., 1976), and Cannitoxin (Kuruppu et al., 2005) act as presynaptic neurotoxins. On the contrary there are only a few reports on the postsynaptically active PLA2s from snake venoms, for example: NNXIa from N. naja (Machiah and Gowda, 2006), hostoxin-1 (Tan et al., 2006), and Ceruleotoxin (Bon and Saliou, 1983). Only few reports are available on toxins from the venom acting as both pre and postsynaptic toxins, for example: Crotoxin (Hendon and Fraenkel-Conrat, 1971; Eterovic et al., 1975; Fraenkel-conrat, 1983). 5. Conclusion We are reporting isolation, purification and characterization of three PLA2 isoforms of which VRV-PL-IIIc is a component of “Reprotoxin” a multi subunit complex toxin, which is by itself nonlethal in experimental mice, while VRV PL-VII and VRV-PL-IX were lethal to mice. Both VRV-PL-V and VRV-PL-VII exhibit pre and postsynaptic neurotoxicity, while VRV-PL-IX is presynaptic neurotoxic PLA2. Acknowledgment The authors thank to Dr Gopal K Marathe, Department of Biochemistry, University of Mysore, Mysore, and Dr Satish S, University of Pennsylvania, K C Ponnappa, Thermoscietific, Bangalore for valuable suggestion and critical comments. TVG and JRK thank UGC, New Delhi, Government of India Grant No. (42-652/2013), for financial support. References Alex, A.B., Baucum, A.J., Wilcox, K.S., 2006. Effect of Conantokin G on NMDA receptormediated spontaneous EPSCs in cultured cortical neurons. J. Neurophysiol. 96, 1084–1092. Arancio, O., Kandel, E.R., Hawkins, R.D., 1995. Activity-dependent long-term enhancement of transmitter release by presynaptic 3′,5′-cyclic GMP in cultured hippocampal neurons. Nature 376, 74–80.

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