Bhargab Kalita

A proteomic analysis of Pakistan Daboia russelii russelii venom and assessment of potency of Indian polyvalent and monovalent antivenom

UNLABELLED To address the dearth of knowledge on the biochemical composition of Pakistan Russells Viper (Daboia russelii russelii) venom (RVV), the venom proteome has been analyzed and several biochemical and pharmacological properties of the venom were investigated. SDS-PAGE (reduced) analysis indicated that proteins/peptides in the molecular mass range of ~56.0-105.0kDa, 31.6-51.0kDa, 15.6-30.0kDa, 9.0-14.2kDa and 5.6-7.2kDa contribute approximately 9.8%, 12.1%, 13.4%, 34.1% and 30.5%, respectively of Pakistan RVV. Proteomics analysis of gel-filtration peaks of RVV resulted in identification of 75 proteins/peptides which belong to 14 distinct snake venom protein families. Phospholipases A2 (32.8%), Kunitz type serine protease inhibitors (28.4%), and snake venom metalloproteases (21.8%) comprised the majority of Pakistan RVV proteins, while 11 additional families accounted for 6.5-0.2%. Occurrence of aminotransferase, endo-β-glycosidase, and disintegrins is reported for the first time in RVV. Several of RVV proteins/peptides share significant sequence homology across Viperidae subfamilies. Pakistan RVV was well recognized by both the polyvalent (PAV) and monovalent (MAV) antivenom manufactured in India; nonetheless, immunological cross-reactivity determined by ELISA and neutralization of pro-coagulant/anticoagulant activity of RVV and its fractions by MAV surpassed that of PAV. BIOLOGICAL SIGNIFICANCE The study establishes the proteome profile of the Pakistan RVV, thereby indicating the presence of diverse proteins and peptides that play a significant role in the pathophysiology of RVV bite. Further, the proteomic findings will contribute to understand the variation in venom composition owing to different geographical location and identification of pharmacologically important proteins in Pakistan RVV.

Proteomic analysis to unravel the complex venom proteome of eastern India Naja naja: Correlation of venom composition with its biochemical and pharmacological properties.

The complex venom proteome of the eastern India (EI) spectacled cobra (Naja naja) was analyzed using tandem mass spectrometry of cation-exchange venom fractions. About 75% of EI N. naja venom proteins were <18kDa and cationic at physiological pH of blood. SDS-PAGE (non-reduced) analysis indicated that in the native state venom proteins either interacted with each-other or self-aggregated resulting in the formation of higher molecular mass complexes. Proteomic analysis revealed that 43 enzymatic and non-enzymatic proteins in EI N. naja venom with a percent composition of about 28.4% and 71.6% respectively were distributed over 15 venom protein families. The three finger toxins (63.8%) and phospholipase A2s (11.4%) were the most abundant families of non-enzymatic and enzymatic proteins, respectively. nanoLC-ESI-MS/MS analysis demonstrated the occurrence of acetylcholinesterase, phosphodiesterase, cholinesterase and snake venom serine proteases in N. naja venom previously not detected by proteomic analysis. ATPase, ADPase, hyaluronidase, TAME, and BAEE-esterase activities were detected by biochemical analysis; however, due to a limitation in the protein database depository they were not identified in EI N. naja venom by proteomic analysis. The proteome composition of EI N. naja venom was well correlated with its in vitro and in vivo pharmacological properties in experimental animals and envenomed human. BIOLOGICAL SIGNIFICANCE Proteomic analysis reveals the complex and diverse protein profile of EI N. naja venom which collectively contributes to the severe pathophysiological manifestation upon cobra envenomation. The study has also aided in comprehending the compositional variation in venom proteins of N. naja within the Indian sub-continent. In addition, this study has also identified several enzymes in EI N. naja venom which were previously uncharacterized by proteomic analysis of Naja venom.

Unraveling the Proteome Composition and Immuno-profiling of Western India Russell’s Viper Venom for In-Depth Understanding of Its Pharmacological Properties, Clinical Manifestations, and Effective Antivenom Treatment

The proteome composition of western India (WI) Russells viper venom (RVV) was correlated with pharmacological properties and pathological manifestations of RV envenomation. Proteins in the 5-19 and 100-110 kDa mass ranges were the most predominate (∼35.1%) and least abundant (∼3.4%) components, respectively, of WI RVV. Non-reduced SDS-PAGE indicated the occurrence of multiple subunits, non-covalent oligomers, self-aggregation, and/or interactions among the RVV proteins. A total of 55 proteins belonging to 13 distinct snake venom families were unambiguously identified by ESI-LC-MS/MS analysis. Phospholipase A2 (32.5%) and Kunitz-type serine protease inhibitors (12.5%) represented the most abundant enzymatic and non-enzymatic proteins, respectively. However, ATPase, ADPase, and hyaluronidase, detected by enzyme assays, were not identified by proteomic analysis owing to limitations in protein database deposition. Several biochemical and pharmacological properties of WI RVV were also investigated. Neurological symptoms exhibited by some RV-bite patients in WI may be correlated to the presence of neurotoxic phospholipase A2 enzymes and Kunitz-type serine protease inhibitor complex in this venom. Monovalent antivenom was found to be better than polyvalent antivenom in immuno-recognition and neutralization of the tested pharmacological properties and enzyme activities of WI RVV; nevertheless, both antivenoms demonstrated poor cross-reactivity and neutralization of pharmacological activities shown by low-molecular-mass proteins (<18 kDa) of this venom.

Two acidic, anticoagulant PLA2 isoenzymes purified from the venom of monocled cobra Naja kaouthia exhibit different potency to inhibit thrombin and factor Xa via phospholipids independent, non-enzymatic mechanism.

Background The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da) and Nk-PLA2β (13282.38 Da) purified from the venom of N. kaouthia. Principal Findings By LC-MS/MS analysis, these PLA2s showed highest similarity (23.5% sequence coverage) with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their “pharmacological target(s)” by uncompetitive mechanism without the requirement of phospholipids/Ca2+. The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation. Conclusion/Significance In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA2α and Nk-PLA2β for the treatment and/or prevention of cardiovascular disorders are proposed.

Proteomics and antivenomics of Echis carinatus carinatus venom: Correlation with pharmacological properties and pathophysiology of envenomation

The proteome composition of Echis carinatus carinatus venom (ECV) from India was studied for the first time by tandem mass spectrometry analysis. A total of 90, 47, and 22 distinct enzymatic and non-enzymatic proteins belonging to 15, 10, and 6 snake venom protein families were identified in ECV by searching the ESI-LC-MS/MS data against non-redundant protein databases of Viperidae (taxid 8689), Echis (taxid 8699) and Echis carinatus (taxid 40353), respectively. However, analysis of MS/MS data against the Transcriptome Shotgun Assembly sequences (87 entries) of conger E. coloratus identified only 14 proteins in ECV. Snake venom metalloproteases and snaclecs, the most abundant enzymatic and non-enzymatic proteins, respectively in ECV account for defibrinogenation and the strong in vitro pro-coagulant activity. Further, glutaminyl cyclase, aspartic protease, aminopeptidase, phospholipase B, vascular endothelial growth factor, and nerve growth factor were reported for the first time in ECV. The proteome composition of ECV was well correlated with its biochemical and pharmacological properties and clinical manifestations observed in Echis envenomed patients. Neutralization of enzymes and pharmacological properties of ECV, and immuno-cross-reactivity studies unequivocally point to the poor recognition of <20 kDa ECV proteins, such as PLA2, subunits of snaclec, and disintegrin by commercial polyvalent antivenom.

Structural and functional characterization of complex formation between two Kunitz-type serine protease inhibitors from Russell's Viper venom

Snake venom Kunitz-type serine protease inhibitors (KSPIs) exhibit various biological functions including anticoagulant activity. This study elucidates the occurrence and subunit stoichiometry of a putative complex formed between two KSPIs (Rusvikunin and Rusvikunin-II) purified from the native Rusvikunin complex of Pakistan Russells Viper (Daboia russelii russelii) venom (RVV). The protein components of the Rusvikunin complex were identified by LC-MS/MS analysis. The non-covalent interaction between two major components of the complex (Rusvikunin and Rusvikunin-II) at 1:2 stoichiometric ratio to form a stable complex was demonstrated by biophysical techniques such as spectrofluorometric, classical gel-filtration, equilibrium gel-filtration, circular dichroism (CD), dynamic light scattering (DLS), RP-HPLC and SDS-PAGE analyses. CD measurement showed that interaction between Rusvikunin and Rusvikunin-II did not change their overall secondary structure; however, the protein complex exhibited enhanced hydrodynamic diameter and anticoagulant activity as compared to the individual components of the complex. This study may lay the foundation for understanding the basis of protein complexes in snake venoms and their role in pathophysiology of snakebite.

A Combination of Podophyllotoxin and Rutin Attenuates Radiation Induced Gastrointestinal Injury by Negatively Regulating NF-κB/p53 Signaling in Lethally Irradiated Mice.

Development of an effective radio protector to minimise radiation-inflicted damages have largely failed owing to inherent toxicity of most of the agents examined so far. This study is centred towards delivering protection to lethally irradiated mice by pre-administration of a safe formulation G-003M (combination of podophyllotoxin and rutin) majorly through regulation of inflammatory and cell death pathways in mice. Single intramuscular dose of G-003M injected 60 min prior to 9 Gy exposure rescued 89% of whole body lethally irradiated C57BL/6J mice. Studies have revealed reduction in radiation induced reactive oxygen species (ROS), nitric oxide (NO) generation, prostaglandin E2 (PGE2) levels and intestinal apoptosis in G-003M pre-treated mice intestine. Restricted nuclear translocation of redox-sensitive Nuclear factor-κB (NF-κB) and subsequent downregulation of cyclo-oxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS; EC 1.14.13.39) and tumor necrosis factor (TNF-α) levels demonstrated the anti-inflammatory effect that G-003M exerts. Support to early hematopoietic recovery was exhibited through G-003M mediated induction of granulocyte colony stimulating factor (G-CSF) and interleukin (IL-6) levels in lethally irradiated mice. Considerable attenuation in radiation induced morphological damage to the intestinal villi, crypts and mucosal layers was observed in G-003M pre-treated mice. Additionally, our formulation did not reduce the sensitivity of tumor tissue to radiation. Altogether, these results suggest that G-003M ameliorates the deleterious effects of radiation exposure by minimising ROS and NO generation and effectively regulating inflammatory and cell death pathways. Mechanism of protection elucidated in the current study demonstrates that G-003M can be used as a safe and effective radio protective agent in radiotherapy for human application.

Countering effects of a combination of podophyllotoxin, podophyllotoxin β-D-glucoside and rutin hydrate in minimizing radiation induced chromosomal damage, ROS and apoptosis in human blood lymphocytes

The present study was conceptualized with the aim of developing a safe radioprotector for human application against radiation induced toxicity. For this study, a formulation (G-002M) prepared by combining three active principles isolated from rhizomes of Podophyllum hexandrum, was evaluated for its potential to protect genomic DNA of human blood cells exposed to different doses of radiation (5,7&10Gy). Blood samples were pretreated (-1hr to exposure) with G-002M. Parameters of Premature Chromosome Condensation (PCC) assay like PCC-index, PCC-rings and PCC-fragments were used to estimate radiation induced chromosomal aberrations. Radiation (7Gy) induce ROS generation and its modulation by G-002M was determined by flow-cytometry and fluorescent microscopy while apoptosis (0,2,24&48 hr) was analyzed using TUNEL assay. Effect on spindle organization in G2/M arrested cells by all the three compounds individually was studied using immunofluorescence microscopy. Irradiation caused dose dependent linear increase in PCC-rings and fragments, while decline in PCC index. G-002M pretreatment significantly decreased these chromosomal aberrations at all the radiation doses and assisted cell survival as indicated by increased PCC index compared with radiation only group. Significant decrease in radiation induced intracellular ROS (45 ± 3%) and apoptosis (49.9%) was also exhibited by the formulation. On podophyllotoxin treatment, most of the cells have shown blocked spindles however, depicted normal arrangement. G-002M also demonstrated a highly significant Dose Modifying Factor or DMF (PCC-rings: 2.27 and PCC-fragments: 1.60). Present study based on many parameters along with DMF study, strongly suggests that G-002M is an effective formulation with a potential to minimize chromosomal damage even at very high radiation doses.

Proteomic Analysis and Immuno-Profiling of Eastern India Russell’s Viper (Daboia russelii) Venom: Correlation between RVV Composition and Clinical Manifestations Post RV Bite

The proteomes of Russells viper venom (RVV) from Burdwan (RVV B) and Nadia (RVV N), the two districts of West Bengal, eastern India (EI), were investigated by gel-filtration chromatography (GFC) followed by tandem mass spectrometry of tryptic fragments of the fractions. A total of 73 and 69 proteins belonging to 15 snake venom protein families were identified in RVV B and RVV N, respectively, by MS/MS search against Viperidae (taxid 8689) protein entries of the nonredundant NCBI database. The minor differences in venom composition of both the EI RV were established unequivocally by their biochemical and pharmacological properties and by SDS-PAGE, gel filtration chromatography, and LC-MS/MS analyses. The composition of EI RVVs was well correlated with published reports on the pathophysiology of RV-envenomed patients from this part of the country. Venom-antivenom cross-reactivity determined by ELISA, Western blotting, and antivenomics approaches demonstrated poor recognition of low molecular mass (<20 kDa) RVV proteins by commercial polyvalent antivenoms, which was substantiated by neutralization of RVV enzymes by antivenom.

Quantitative proteomic analysis and antivenom study revealing that neurotoxic phospholipase A 2 enzymes, the major toxin class of Russell's viper venom from southern India, shows the least immuno-recognition and neutralization by commercial polyvalent antivenom

The proteome composition of Russells viper venom (RVV) from southern India (SI) was investigated by 1D-SDS-PAGE of venom followed by tandem mass spectrometry analysis of protein bands. A total of 66 proteins belonging to 14 snake venom protein families were identified by LC-MS/MS analysis against Viperidae (taxid 8689) protein entries from the non-redundant NCBI database. Phospholipase A2 (43.25%) and snaclec (14.57%) represented the most abundant enzymatic and non-enzymatic proteins, respectively. SI RVV was characterized as containing a higher quantity of PLA2 and a lower amount of Kunitz-type serine protease inhibitors, in comparison to RVV from other regions of the Indian subcontinent. The enzymatic activities, pharmacological properties, and clinical manifestations of RV envenomation in SI were well correlated with its proteome composition; however, ATPase, ADPase, and hyaluronidase enzymes were not identified by LC-MS/MS analysis, owing to paucity of the existing database. Neurological symptoms exhibited by RV-bite patients in SI were correlated to the presence of abundant neurotoxic phospholipase A2 enzymes (15.66%) in SI RVV. Neutralization studies, immunological cross-reactivity, and antivenomics studies unequivocally demonstrated the poor recognition and lowest neutralization of PLA2 enzymes by commercial polyvalent antivenom, which is a major concern for the treatment of RV-envenomed patients in SI.