Robin Doley

Protein complexes in snake venom

Snake venom contains mixture of bioactive proteins and polypeptides. Most of these proteins and polypeptides exist as monomers, but some of them form complexes in the venom. These complexes exhibit much higher levels of pharmacological activity compared to individual components and play an important role in pathophysiological effects during envenomation. They are formed through covalent and/or non-covalent interactions. The subunits of the complexes are either identical (homodimers) or dissimilar (heterodimers; in some cases subunits belong to different families of proteins). The formation of complexes, at times, eliminates the non-specific binding and enhances the binding to the target molecule. On several occasions, it also leads to recognition of new targets as protein-protein interaction in complexes exposes the critical amino acid residues buried in the monomers. Here, we describe the structure and function of various protein complexes of snake venoms and their role in snake venom toxicity.

Purification and characterization of an anticoagulant phospholipase A2 from Indian monocled cobra (Naja kaouthia) venom

An anticoagulant, non-toxic phospholipase A(2) was isolated from the venom of Indian monocled cobra (Naja kaouthia) by a combination of ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. This purified protein named NK-PLA(2)-I, had a subunit molecular mass of 13.6 kDa and migrated as a dimer under non-reduced condition in SDS-PAGE. NK-PLA(2)-I was a highly thermostable protein requiring basic pH optima for its catalytic activity and showed preferential hydrolysis of phosphotidylcholine. This protein exhibited higher anticoagulant, indirect hemolysis, liver and heart tissue damaging activity but exerted less toxicity, direct hemolysis, edema and lung tissue damaging activity as compared to whole venom. Treatment of NK-PLA(2)-I with rho-BPB, TPCK, PMSF, antivenom and heating had almost equal effect on PLA(2), and other pharmacological properties except in vitro tissue damaging activity. Current investigation provides a fairly good indication that NK-PLA(2)-I induces various pharmacological effects by mechanisms, which are either dependent or independent of its catalytic activity.

Snake venom: From fieldwork to the clinic

Snake venoms are recognized here as a grossly under‐explored resource in pharmacological prospecting. Discoveries in snake systematics demonstrate that former taxonomic bias in research has led to the neglect of thousands of species of potential medical use. Recent discoveries reveal an unexpectedly vast degree of variation in venom composition among snakes, from different species down to litter mates. The molecular mechanisms underlying this diversity are only beginning to be understood. However, the enormous potential that this resource represents for pharmacological prospecting is clear. New high‐throughput screening systems offer greatly increased speed and efficiency in identifying and extracting therapeutically useful molecules. At the same time a global biodiversity crisis is threatening the very snake populations on which hopes for new venom‐derived medications depend. Biomedical researchers, pharmacologists, clinicians, herpetologists, and conservation biologists must combine their efforts if the full potential of snake venom‐derived medications is to be realized.

Identification of a Novel Family of Snake Venom Proteins Veficolins from Cerberus rynchops Using a Venom Gland Transcriptomics and Proteomics Approach

Cerberus rynchops (dog-faced water snake) belongs to Homalopsidae of Colubroidea (rear-fanged snakes). So far, venom compositions of snakes of the Homalopsidae family are not known. To determine the venom composition of C. rynchops, we have used both transcriptomics and proteomics approaches. The venom gland transcriptome revealed 104 ESTs and the presence of three known snake protein families, namely, metalloprotease, CRISP, and C-type lectin. In addition, we identified two proteins that showed sequence homology to ficolin, a mammalian protein with collagen-like and fibrinogen-like domains. We named them as ryncolin 1 and ryncolin 2 (rynchops ficolin) and this new family of snake venom proteins as veficolins (venom ficolins). On the basis of its structural similarity to ficolin, we speculate that ryncolins may induce platelet aggregation and/or initiate complement activation. To determine the proteome, the whole C. rynchops venom was trypsinized and fractionated by reverse phase HPLC followed by MALDI-MS/MS analysis of the tryptic peptides. Analysis of the tandem mass spectrometric data indicated the presence of all protein families compared to the translated cDNA library. Overall, our combined approach of transcriptomics and proteomics revealed that C. rynchops venom is among the least complex snake venom characterized to date despite the presence of a new family of snake venom proteins.

Purification and biochemical characterization of a thermostable, alkaliphilic, extracellular α-amylase from Bacillus subtilis DM-03, a strain isolated from the traditional fermented food of India

Bacillus subtilis strain DM‐03, which is isolated from starter culture used for the production of alcohol by local Assam tribes, grows optimally at 52–55 °C and secretes a significant amount of α‐amylase at pH 8.0 into the culture media. This α‐amylase, purified by ion‐exchange, gel‐filtration and reverse‐phase HPLC, constitutes 2.9% of the total extracellular protein. This purified enzyme, named Bsamy‐I, has a subunit with molecular mass of 42.8 kDa as determined by SDS/PAGE, and optimum temperature and pH values at 52–55 °C and 9.0 respectively, which makes it ideal for use in the detergent industries. Maximum α‐amylase production is obtained by using soluble starch and NH4Cl as carbon and nitrogen sources respectively. Thermostability of the enzyme is evident from heating the enzyme at 95 °C for 10 min, which results in a loss of 60% of the original enzyme activity. 4‐Bromophenacyl bromide and PMSF at 4 and 1.5 mM concentration respectively completely abolish the enzymic activity, documenting the essential role of histidine and carboxylic residues in the catalytic process.

Venom gland transcriptomics for identifying, cataloging, and characterizing venom proteins in snakes.

Snake venoms are cocktails of protein toxins that play important roles in capture and digestion of prey. Significant qualitative and quantitative variation in snake venom composition has been observed among and within species. Understanding these variations in protein components is instrumental in interpreting clinical symptoms during human envenomation and in searching for novel venom proteins with potential therapeutic applications. In the last decade, transcriptomic analyses of venom glands have helped in understanding the composition of various snake venoms in great detail. Here we review transcriptomic analysis as a powerful tool for understanding venom profile, variation and evolution.

Role of accelerated segment switch in exons to alter targeting (ASSET) in the molecular evolution of snake venom proteins

BackgroundSnake venom toxins evolve more rapidly than other proteins through accelerated changes in the protein coding regions. Previously we have shown that accelerated segment switch in exons to alter targeting (ASSET) might play an important role in its functional evolution of viperid three-finger toxins. In this phenomenon, short sequences in exons are radically changed to unrelated sequences and hence affect the folding and functional properties of the toxins.ResultsHere we analyzed other snake venom protein families to elucidate the role of ASSET in their functional evolution. ASSET appears to be involved in the functional evolution of three-finger toxins to a greater extent than in several other venom protein families. ASSET leads to replacement of some of the critical amino acid residues that affect the biological function in three-finger toxins as well as change the conformation of the loop that is involved in binding to specific target sites.ConclusionASSET could lead to novel functions in snake venom proteins. Among snake venom serine proteases, ASSET contributes to changes in three surface segments. One of these segments near the substrate binding region is known to affect substrate specificity, and its exchange may have significant implications for differences in isoform catalytic activity on specific target protein substrates. ASSET therefore plays an important role in functional diversification of snake venom proteins, in addition to accelerated point mutations in the protein coding regions. Accelerated point mutations lead to fine-tuning of target specificity, whereas ASSET leads to large-scale replacement of multiple functionally important residues, resulting in change or gain of functions.

Transcriptomic analysis of the venom gland of the red-headed krait ( Bungarus flaviceps ) using expressed sequence tags

BackgroundThe Red-headed krait (Bungarus flaviceps, Squamata: Serpentes: Elapidae) is a medically important venomous snake that inhabits South-East Asia. Although the venoms of most species of the snake genus Bungarus have been well characterized, a detailed compositional analysis of B. flaviceps is currently lacking.ResultsHere, we have sequenced 845 expressed sequence tags (ESTs) from the venom gland of a B. flaviceps. Of the transcripts, 74.8% were putative toxins; 20.6% were cellular; and 4.6% were unknown. The main venom protein families identified were three-finger toxins (3FTxs), Kunitz-type serine protease inhibitors (including chain B of β-bungarotoxin), phospholipase A2 (including chain A of β-bungarotoxin), natriuretic peptide (NP), CRISPs, and C-type lectin.ConclusionThe 3FTxs were found to be the major component of the venom (39%). We found eight groups of unique 3FTxs and most of them were different from the well-characterized 3FTxs. We found three groups of Kunitz-type serine protease inhibitors (SPIs); one group was comparable to the classical SPIs and the other two groups to chain B of β-bungarotoxins (with or without the extra cysteine) based on sequence identity. The latter group may be functional equivalents of dendrotoxins in Bungarus venoms. The natriuretic peptide (NP) found is the first NP for any Asian elapid, and distantly related to Australian elapid NPs. Our study identifies several unique toxins in B. flaviceps venom, which may help in understanding the evolution of venom toxins and the pathophysiological symptoms induced after envenomation.

Isolation of a snake venom phospholipase A2 (PLA2) inhibitor (AIPLAI) from leaves of Azadirachta indica (Neem): Mechanism of PLA2 inhibition by AIPLAI in vitro condition

A compound (AIPLAI (Azadirachta indica PLA(2) inhibitor)) purified from the methanolic leaf extract of A. indica (Neem) inhibits the cobra and Russells viper venoms (RVVs) phospholipase A(2) enzymes in a dose-dependent manner. Inhibition of catalytic and tested pharmacological properties of cobra venom (Naja naja and Naja kaouthia) PLA(2) enzymes by AIPLAI is significantly higher (P<0.05) compared to the inhibition of PLA(2) enzymes of crude RVV (Daboia russelli) when tested under the same condition. Kinetic study reveals that in in vitro condition, AIPLAI inhibits the purified N. kaouthia PLA(2) enzymes in a non-competitive manner. The AIPLAI is quite stable at room temperature. The present study shows that AIPLAI holds good promise for the development of novel anti-snake venom drug in future.

Unusual accelerated rate of deletions and insertions in toxin genes in the venom glands of the pygmy copperhead (Austrelaps labialis) from kangaroo island

BackgroundToxin profiling helps in cataloguing the toxin present in the venom as well as in searching for novel toxins. The former helps in understanding potential pharmacological profile of the venom and evolution of toxins, while the latter contributes to understanding of novel mechanisms of toxicity and provide new research tools or prototypes of therapeutic agents.ResultsThe pygmy copperhead (Austrelaps labialis) is one of the less studied species. In this present study, an attempt has been made to describe the toxin profile of A. labialis from Kangaroo Island using the cDNA library of its venom glands. We sequenced 658 clones which represent the common families of toxin genes present in snake venom. They include (a) putative long-chain and short-chain neurotoxins, (b) phospholipase A2, (c) Kunitz-type protease inhibitor, (d) CRISPs, (e) C-type lectins and (f) Metalloproteases. In addition, we have also identified a novel protein with two Kunitz-type domains in tandem similar to bikunin.ConclusionInterestingly, the cDNA library reveals that most of the toxin families (17 out of 43 toxin genes; ~40%) have truncated transcripts due to insertion or deletion of nucleotides. These truncated products might not be functionally active proteins. However, cellular trancripts from the same venom glands are not affected. This unusual higher rate of deletion and insertion of nucleotide in toxin genes may be responsible for the lower toxicity of A. labialis venom of Kangroo Island and have significant effect on evolution of toxin genes.