ORCID Profile
0000-0002-6100-3251
Current Organisation
National University of Singapore
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Publisher: Springer Science and Business Media LLC
Date: 25-11-2021
DOI: 10.1038/S41467-021-27275-8
Abstract: Despite their limitations, unfractionated heparin (UFH) and bivalirudin remain standard-of-care parenteral anticoagulants for percutaneous coronary intervention (PCI). We discovered novel direct thrombin inhibitors (DTIs) from tick salivary transcriptomes and optimised their pharmacologic activity. The most potent, ultravariegin, inhibits thrombin with a K i of 4.0 pM, 445-fold better than bivalirudin. Unexpectedly, despite their greater antithrombotic effect, variegin/ultravariegin demonstrated less bleeding, achieving a 3-to-7-fold wider therapeutic index in rodent thrombosis and bleeding models. When used in combination with aspirin and ticagrelor in a porcine model, variegin/ultravariegin reduced stent thrombosis compared with antiplatelet therapy alone but achieved a 5-to-7-fold lower bleeding time than UFH/bivalirudin. Moreover, two antibodies screened from a naïve human antibody library effectively reversed the anticoagulant activity of ultravariegin, demonstrating proof-of-principle for antidote reversal. Variegin and ultravariegin are promising translational candidates for next-generation DTIs that may reduce peri-PCI bleeding in the presence of antiplatelet therapy.
Publisher: American Chemical Society (ACS)
Date: 15-10-2015
DOI: 10.1021/ACSCHEMBIO.5B00492
Abstract: Snake venom α-neurotoxins from the three-finger toxin (3FTx) family are competitive antagonists with nanomolar affinity and high selectivity for nicotinic acetylcholine receptors (nAChR). Here, we report the characterization of a new group of competitive nAChR antagonists: Ω-neurotoxins. Although they belong to the 3FTx family, the characteristic functional residues of α-neurotoxins are not conserved. We evaluated the subtype specificity and structure-function relationships of Oh9-1, an Ω-neurotoxin from Ophiophagus hannah venom. Recombinant Oh9-1 showed reversible postsynaptic neurotoxicity in the micromolar range. Experiments with different nAChR subtypes expressed in Xenopus oocytes indicated Oh9-1 is selective for rat muscle type α1β1εδ (adult) and α1β1γδ (fetal) and rat neuronal α3β2 subtypes. However, Oh9-1 showed low or no affinity for other human and rat neuronal subtypes. Twelve in idual alanine-scan mutants encompassing all three loops of Oh9-1 were evaluated for binding to α1β1εδ and α3β2 subtypes. Oh9-1's loop-II residues (M25, F27) were the most critical for interactions and formed the common binding core. Mutations at T23 and F26 caused a significant loss in activity at α1β1εδ receptors but had no effect on the interaction with the α3β2 subtype. Similarly, mutations at loop-II (H7, K22, H30) and -III (K45) of Oh9-1 had a distinctly different impact on its activity with these subtypes. Thus, Oh9-1 interacts with these nAChRs via distinct residues. Unlike α-neurotoxins, the tip of loop-II is not involved. We reveal a novel mode of interaction, where both sides of the β-strand of Oh9-1's loop-II interact with α1β1εδ, but only one side interacts with α3β2. Phylogenetic analysis revealed functional organization of the Ω-neurotoxins independent of α-neurotoxins. Thus, Ω-neurotoxin: Oh9-1 may be a new, structurally distinct class of 3FTxs that, like α-neurotoxins, antagonize nAChRs. However, Oh9-1 binds to the ACh binding pocket via a different set of functional residues.
Publisher: Wiley
Date: 07-02-2002
DOI: 10.1002/RCM.613
Abstract: Death adders (genus Acanthophis) are unique among elapid snakes in both morphology and venom composition. Despite this genus being among the most ergent of all elapids, the venom has been historically regarded as relatively quite simple. In this study, liquid chromatography/mass spectrometry (LC/MS) analysis has revealed a much greater ersity in venom composition, including the presence of molecules of novel molecular weights that may represent a new class of venom component. Furthermore, significant variation exists between species and populations, which allow for the LC/MS fingerprinting of each species. Mass profiling of Acanthophis venoms clearly demonstrates the effectiveness of this technique which underpins fundamental studies ranging from chemotaxonomy to drug design.
Publisher: Wiley
Date: 31-03-2017
Publisher: Georg Thieme Verlag KG
Date: 2009
DOI: 10.1160/TH09-03-0162
Abstract: Trocarin D is a prothrombin activator from the Tropidechis carinatus venom. It is a functional and structural homologue to mammalian blood coagulation factor Xa.Trocarin D is hypothesised to have evolved from its factor X counterpart (TrFX) through gene duplication and recruitment.The genes of trocarin D and TrFX have significant sequence identities, except for insertions/deletions in their intron 1 and promoter regions. In trocarin D intron 1 region, there are three insertions and two deletions. In trocarin D promoter region, there is a novel 264 bp insertion which has potential cis-elements.This insertion is termed as Venom Recruitment/Switch Element (VERSE) and is hypothesised to account for switching the low-level constitutive expression of factor X in the liver to the high-level inducible expression of trocarin D in the venom gland. To understand the role of VERSE in the trocarin D expression,its cis-elements were characterised by luciferase assays in mammalian cell lines as well as snake venom gland cells. The ability of VERSE to drive luciferase expression is comparable to that of the trocarin D promoter. The predicted cis-elements are important in promoting expression as their mutagenesis resulted in lower luciferase expression.VERSE minimal core promoter and three novel cis-elements (two up-regulatory and one suppressor elements) were identified using deletion/site-directed mutagenesis studies. VERSE is primarily responsible for the increase of trocarin D expression. The insertions/deletions within trocarin D intron 1 need to be characterised for their role in tissue-specific and inducible expression of trocarin D.
Publisher: Elsevier BV
Date: 03-2007
DOI: 10.1016/J.NEUROPHARM.2006.11.002
Abstract: Colubrid snake venoms potentially represent a vast source of novel biological actives and structural motifs owing to their erse phylogeny. The present study describes the identification of rufoxin, a neurotoxin from the venom of Rh hiophis oxyrhynchus (Rufous beaked snake) which is a member of the African colubrid lineage, the psammophiines. Rufoxin (1 microM) displayed reversible post-synaptic neurotoxic activity as evidenced by significant inhibition of indirect twitches and responses to exogenous nicotinic agonists in the chick biventer cervicis nerve-muscle preparation. Rufoxin (0.1-1.0 microM) also caused a rightward parallel shift of cumulative concentration-response curves to carbachol (CCh 0.6-80 microM) without a significant depression of the maximum response, suggestive of classical competitive antagonism at the skeletal muscle nicotinic receptor. Rufoxin lacks NH(2)-terminal sequence homology to previously identified snake venom toxins. This work indicates a wider distribution of neurotoxins across the advanced snake superfamily than previously described.
Publisher: Proceedings of the National Academy of Sciences
Date: 10-01-2022
Abstract: Because dengue viruses are spread by mosquitoes during biting, transmission capacity depends on mosquito-biting behavior. For this reason, it is critical to understand how infection in mosquitoes influences biting. To answer this question, we deployed a multidisciplinary approach including high-resolution, multivariate biting behavior monitoring on mice, in vivo transmission assay, and mathematical modeling. We demonstrated that infected mosquitoes are more attracted to mice and bite more often to get the same amount of blood as uninfected mosquitoes. While the effect of increased attraction to host on transmission capacity is trivial, we showed that increased number of bites results in successive transmission. Eventually, we calculated that the infection-induced behavior changes tripled transmission capacity of mosquitoes.
Publisher: Portland Press Ltd.
Date: 23-10-2020
DOI: 10.1042/BCJ20200529
Abstract: Snake venoms are complex mixtures of enzymes and nonenzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of nonenzymatic proteins. They share a common structure of three β-stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences, they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance the toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as κ-bungarotoxins. This novel dimerization further enhances the ersity in structure and function of 3FTxs.
Publisher: Wiley
Date: 16-03-2016
Publisher: Elsevier BV
Date: 03-2010
Publisher: Wiley
Date: 05-11-2021
DOI: 10.1002/PRO.4212
Abstract: Metallocarboxypeptidases (MCPs) in the mosquito midgut play crucial roles in infection, as well as in mosquito dietary digestion, reproduction, and development. MCPs are also part of the digestive system of plant‐feeding insects, representing key targets for inhibitor development against mosquitoes/mosquito‐borne pathogens or as antifeedant molecules against plant‐feeding insects. Notably, some non‐mosquito insect B‐type MCPs are primarily insensitive to plant protease inhibitors (PPIs) such as the potato carboxypeptidase inhibitor (PCI MW 4 kDa), an inhibitor explored for cancer treatment and insecticide design. Here, we report the crystal structure of Aedes aegypti carboxypeptidase‐B1 (CPBAe1)‐PCI complex and compared the binding with that of PCI‐insensitive CPBs. We show that PCI accommodation is determined by key differences in the active‐site regions of MCPs. In particular, the loop regions α6‐α7 (Leu 242 ‐Ser 250 ) and β8‐α8 (Pro 269 ‐Pro 280 ) of CPBAe1 are replaced by α‐helices in PCI‐insensitive insect Helicoverpa zea CPBHz. These α‐helices protrude into the active‐site pocket of CPBHz, restricting PCI insertion and rendering the enzyme insensitive. We further compared our structure with the only other PCI complex available, bovine CPA1‐PCI. The potency of PCI against CPBAe1 ( K i = 14.7 nM) is marginally less than that of bovine CPA1 ( K i = 5 nM). Structurally, the above loop regions that accommodate PCI binding in CPBAe1 are similar to that of bovine CPA1, although observed changes in proteases residues that interact with PCI could account for the differences in affinity. Our findings suggest that PCI sensitivity is largely dictated by structural interference, which broadens our understanding of carboxypeptidase inhibition as a mosquito population arasite control strategy.
Publisher: Elsevier BV
Date: 05-2002
Publisher: Public Library of Science (PLoS)
Date: 29-10-2012
Publisher: Elsevier BV
Date: 10-2003
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 31-08-2005
Abstract: The Papuan taipan (Oxyuranus scutellatus canni) is widely distributed throughout much of Papua New Guinea. Although neurotoxicity is a major symptom of envenomation, no neurotoxins have been isolated from this venom. Using a series of size exclusion chromatography steps, we report the isolation of cannitoxin, a presynaptic neurotoxin (44,848 Da) that represents approximately 16% of the whole venom. The toxin displayed high phospholipase A2 (PLA2 activity (330 +/- 5 micromol/min/mg) and caused concentration-dependent (11-66 nM) inhibition of indirect (0.2 ms 0.1 Hz supramaximal V) twitches of the chick biventer cervicis nerve-muscle preparation without effecting nicotinic receptor agonists. Prior addition of CSL Taipan antivenom (5 U/ml) or inhibition of phospholipase A2 activity by incubation with 4-bromophenacyl bromide prevented the inhibition of twitches. Cannitoxin is composed of three different subunits, alpha, beta, and gamma, with the possibility of two beta isomers. However, only the alpha subunit displayed in vitro neurotoxic activity of its own. Thus, cannitoxin is similar in structure and pharmacology to taipoxin, which has been isolated from the closely related Australian species O. scutellatus scutellatus (coastal taipan).
Publisher: Springer Science and Business Media LLC
Date: 13-05-2016
DOI: 10.1038/SREP25935
Abstract: Anticoagulant therapy is used for the prevention and treatment of thromboembolic disorders. Blood coagulation is initiated by the interaction of factor VIIa (FVIIa) with membrane-bound tissue factor (TF) to form the extrinsic tenase complex which activates FX to FXa. Thus, it is an important target for the development of novel anticoagulants. Here, we report the isolation and characterization of a novel anticoagulant ringhalexin from the venom of Hemachatus haemachatus (African Ringhals Cobra). Amino acid sequence of the protein indicates that it belongs to the three-finger toxin family and exhibits 94% identity to an uncharacterized Neurotoxin-like protein NTL2 from Naja atra . Ringhalexin inhibited FX activation by extrinsic tenase complex with an IC 50 of 123.8 ± 9.54 nM. It is a mixed-type inhibitor with the kinetic constants, Ki and Ki’ of 84.25 ± 3.53 nM and 152.5 ± 11.32 nM, respectively. Ringhalexin also exhibits a weak, irreversible neurotoxicity on chick biventer cervicis muscle preparations. Subsequently, the three-dimensional structure of ringhalexin was determined at 2.95 Å resolution. This study for the first time reports the structure of an anticoagulant three-finger toxin. Thus, ringhalexin is a potent inhibitor of the FX activation by extrinsic tenase complex and a weak, irreversible neurotoxin.
Publisher: Elsevier BV
Date: 03-2003
DOI: 10.1016/S0041-0101(02)00388-4
Abstract: Non-conventional toxins constitute a poorly characterized class of three-finger toxins isolated exclusively from Elapidae venoms. These toxins are monomers of 62-68 amino acid residues and contain five disulfide bridges. However, unlike alpha/kappa-neurotoxins and kappa-neurotoxins which have the fifth disulfide bridge in their middle loop (loop II), the fifth disulfide bridge in non-conventional toxins is located in loop I (N-terminus loop). Overall, non-conventional toxins share approximately 28-42% identity with other three-finger toxins including alpha-neurotoxins, alpha/kappa-neurotoxins and kappa-neurotoxins. Recent structural studies have revealed that non-conventional toxins also display the typical three-finger motif. Non-conventional toxins are typically characterized by a lower order of toxicity (LD(50) approximately 5-80 mg/kg) in contrast to prototype alpha-neurotoxins (LD(50) approximately 0.04-0.3 mg/kg) and hence they are also referred to as 'weak toxins'. Further, it is generally assumed that non-conventional toxins target muscle (alpha(2)beta gamma delta) receptors with low affinities several orders of magnitude lower than alpha-neurotoxins and alpha/kappa-neurotoxins. However, it is now known that some non-conventional toxins also antagonize neuronal alpha 7 nicotinic acetylcholine receptors. Hence, non-conventional toxins are not a functionally homogeneous group and other, yet unknown, molecular targets for this class of snake venom toxins may exist. Non-conventional toxins may therefore be a useful source of ligands with novel biological activity targeting the plethora of neuronal nicotinic receptors as well as other physiological processes.
Publisher: MDPI AG
Date: 12-07-2022
DOI: 10.3390/BIOMEDICINES10071679
Abstract: Activated factor XI (FXIa) is an important antithrombotic drug target. Clinical and pre-clinical data have demonstrated that its inhibition attenuates thrombosis with minimal risk of excessive bleeding. We isolated Fasxiator from the venom of banded krait Bungarus fasciatus and subsequently engineered FasxiatorN17R,L19E, with improved affinity (Ki = 0.9 nM) and selectivity towards FXIa. Here, we assess the in vivo efficacy and bleeding risk of rFasxiatorN17R, L19E in pre-clinical animal models. Rats injected intravenously (i.v.) with bolus rFasxiatorN17R, L19E showed the specific in vivo attenuation of the intrinsic coagulation pathway, lasting for at least 60 min. We performed the in vivo dose-ranging experiments for rFasxiatorN17R, L19E as follows: FeCl3-induced carotid artery occlusion in rats (arterial thrombosis) inferior vena cava ligation in mice (venous thrombosis) tail bleeding time in both rats and mice (bleeding risk). Head-to-head comparisons were made using therapeutic dosages of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) for arterial and venous thrombosis, respectively. In the arterial thrombosis model, 2 mg/kg i.v. rFasxiatorN17R,L19E achieved a similar antithrombotic efficacy to that of UFH, with -fold lower bleeding time. In the venous thrombosis model, the 10 mg/kg subcutaneous (s.c.) injection of rFasxiatorN17R,L19E achieved similar efficacy and bleeding levels to those of LMWH enoxaparin. Overall, rFasxiatorN17R,L19E represents a promising molecule for the development of FXIa-targeting anticoagulants.
Publisher: Wiley
Date: 10-04-2001
DOI: 10.1016/S0014-5793(01)02342-0
Abstract: Alpha-toxins from scorpion venoms prolong the action potential of excitable cells by blocking sodium channel inactivation. We have purified bukatoxin, an alpha-toxin from scorpion (Buthus martensi Karsch) venom, to homogeneity. Bukatoxin produced marked relaxant responses in the carbachol-precontracted rat anococcygeus muscle (ACM), which were mediated through the L-arginine-nitric oxide synthase-nitric oxide pathway, consequent to a neuronal release of nitric oxide. Based on the presence of proline residues in the flanking segments of protein-protein interaction sites, we predicted the site between (52)PP(56) to be the potential interaction site of bukatoxin. A homology model of bukatoxin indicated the presence of this site on the surface. Buka11, a synthetic peptide designed based on this predicted site, produced a concentration-dependent nitric oxide-mediated relaxant response in ACM. Using alanine-substituted peptides, we have shown the importance (53)DKV(55) flanked by proline residues in the functional site of bukatoxin.
Publisher: Wiley
Date: 04-04-2019
DOI: 10.1002/PRO.3605
Publisher: Wiley
Date: 02-08-2002
DOI: 10.1046/J.1440-1681.2002.03726.X
Abstract: 1. The autonomic effects of venoms and toxins from several species of scorpions, including the Indian red scorpion Mesobuthus tamulus, the Chinese scorpion Buthus martensi Karsch and the Israeli scorpion Leiurus quinquestriatus quinquestriatus, all belonging to Buthidae, and the Asian black scorpions Heterometrus longimanus and Heterometrus spinifer, belonging to Scorpionidae, are reviewed. 2. The effects of the venoms of M. tamulus and L. q. quinquestriatus on noradrenergic and nitrergic transmission in the rat isolated anococcygeus muscle revealed that both venoms mediated their pharmacological effects via a prejunctional mechanism involving the activation of voltage-sensitive sodium channels with consequent release of neurotransmitters that mediate target organ responses, similar to the effects mediated by other alpha-scorpion toxins. 3. Two new toxins, Makatoxin I and Bukatoxin, were purified to homogeneity from the venom of B. martensi Karsch. Determination of their complete amino acid sequences confirmed that both toxins belonged to the class of alpha-scorpion toxins. The effects of both toxins on noradrenergic and nitrergic transmission in the rat anococcygeus muscle provided firm evidence that their pharmacological actions also closely resembled those mediated by other alpha-scorpion toxins on neuronal voltage-sensitive sodium channels. 4. The venoms of H. longimanus and H. spinifer were found to have high concentrations of noradrenaline (1.8 +/- 0.3 mmol/L) and relatively high concentrations of acetylcholine (79.8 +/- 1.7 micromol/L) together with noradrenaline (146.7 +/- 19.8 micromol/L), respectively, which can account for their potent direct cholinergic and noradrenergic agonist actions in the rat anococcygeus muscle. 5. Our studies confirmed that the rat anococcygeus muscle is an excellent nerve-smooth muscle preparation for investigating the effects of bioactive agents on noradrenergic and nitrergic transmission, as well as the direct agonist actions of these agents on post-synaptic alpha-adrenoceptors and M3 muscarinic cholinoceptors. Although many studies, including our own, have documented that scorpion venoms and toxins mediate their primary effects via a prejunctional mechanism that leads to the marked release of various autonomic neurotransmitters, our studies have shown that there are exceptions to this generally accepted phenomenon. In particular, we have provided firm evidence to show that the venoms from H. longimanus and H. spinifer do not have such a prejunctional site of action but, instead, the venoms mediate their autonomic effects through direct agonist actions on post-junctional muscarinic M3 cholinoceptors and alpha-adrenoceptors.
Publisher: Wiley
Date: 03-03-2015
Abstract: Natriuretic peptides (NP) play important roles in human cardiac physiology through their guanylyl cyclase receptors NPR-A and NPR-B. Described herein is a bifunctional O-glycosylated natriuretic peptide, TcNPa, from Tropidechis carinatus venom and it unusually targets both NPR-A and NPR-B. Characterization using specific glycosidases and ETD-MS identified the glycan as galactosyl-β(1-3)-N-acetylgalactosamine (Gal-GalNAc) and was α-linked to the C-terminal threonine residue. TcNPa contains the characteristic NP 17-membered disulfide ring with conserved phenylalanine and arginine residues. Both glycosylated and nonglycosylated forms were synthesized by Fmoc solid-phase peptide synthesis and NMR analysis identified an α-helix within the disulfide ring containing the putative pharmacophore for NPR-A. Surprisingly, both forms activated NPR-A and NPR-B and were relatively resistant towards proteolytic degradation in plasma. This work will underpin the future development of bifunctional NP peptide mimetics.
Publisher: MDPI AG
Date: 02-10-2020
Abstract: Venomous snakes are important subjects of study in evolution, ecology, and biomedicine. Many venomous snakes have alpha-neurotoxins (α-neurotoxins) in their venom. These toxins bind the alpha-1 nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction, causing paralysis and asphyxia. Several venomous snakes and their predators have evolved resistance to α-neurotoxins. The resistance is conferred by steric hindrance from N-glycosylated asparagines at amino acids 187 or 189, by an arginine at position 187 that has been hypothesized to either electrostatically repulse positively charged neurotoxins or sterically interfere with α-neurotoxin binding, or proline replacements at positions 194 or 197 of the nAChR ligand-binding domain to inhibit α-neurotoxin binding through structural changes in the receptor. Here, we analyzed this domain in 148 vertebrate species, and assessed its amino acid sequences for resistance-associated mutations. Of these sequences, 89 were sequenced de novo. We find widespread convergent evolution of the N-glycosylation form of resistance in several taxa including venomous snakes and their lizard prey, but not in the snake-eating birds studied. We also document new lineages with the arginine form of inhibition. Using an in vivo assay in four species, we provide further evidence that N-glycosylation mutations reduce the toxicity of cobra venom. The nAChR is of crucial importance for normal neuromuscular function and is highly conserved throughout the vertebrates as a result. Our research shows that the evolution of α-neurotoxins in snakes may well have prompted arms races and mutations to this ancient receptor across a wide range of sympatric vertebrates. These findings underscore the inter-connectedness of the biosphere and the ripple effects that one adaption can have across global ecosystems.
Publisher: Wiley
Date: 17-05-2011
DOI: 10.1111/J.1742-4658.2011.08115.X
Abstract: Snake venoms are cocktails of enzymes and non-enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l-amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A(2) . Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists. Here, we review the structures of these enzymes and describe their structure-based mechanisms of catalysis and inhibition. Some of the enzymes exist as protein complexes in the venom. Thus we also discuss the functional role of non-enzymatic subunits and the pharmacological effects of such protein complexes. The structures of inhibitor-enzyme complexes provide ideal platforms for the design of potent inhibitors which are useful in the development of prototypes and lead compounds with potential therapeutic applications.
Publisher: International Union of Crystallography (IUCr)
Date: 21-07-2007
Publisher: International Union of Crystallography (IUCr)
Date: 28-09-2002
DOI: 10.1107/S0907444902011022
Abstract: Bucain is a three-finger toxin, structurally homologous to snake-venom muscarinic toxins, from the venom of the Malayan krait Bungarus candidus. These proteins have molecular masses of approximately 6000-8000 Da and encompass the potent curaremimetic neurotoxins which confer lethality to Elapidae and Hydrophidae venoms. Bucain was crystallized in two crystal forms by the hanging-drop vapour-diffusion technique in 0.1 M sodium citrate pH 5.6, 15% PEG 4000 and 0.15 M ammonium acetate. Form I crystals belong to the monoclinic system space group C2, with unit-cell parameters a = 93.73, b = 49.02, c = 74.09 A, beta = 111.32 degrees, and diffract to a nominal resolution of 1.61 A. Form II crystals also belong to the space group C2, with unit-cell parameters a = 165.04, b = 49.44, c = 127.60 A, beta = 125.55 degrees, and diffract to a nominal resolution of 2.78 A. The self-rotation function indicates the presence of four and eight molecules in the crystallographic asymmetric unit of the form I and form II crystals, respectively. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated.
Publisher: Proceedings of the National Academy of Sciences
Date: 02-12-2013
Abstract: Snake venoms are toxic protein cocktails used for prey capture. To investigate the evolution of these complex biological weapon systems, we sequenced the genome of a venomous snake, the king cobra, and assessed the composition of venom gland expressed genes, small RNAs, and secreted venom proteins. We show that regulatory components of the venom secretory system may have evolved from a pancreatic origin and that venom toxin genes were co-opted by distinct genomic mechanisms. After co-option, toxin genes important for prey capture have massively expanded by gene duplication and evolved under positive selection, resulting in protein neofunctionalization. This erse and dramatic venom-related genomic response seemingly occurs in response to a coevolutionary arms race between venomous snakes and their prey.
Publisher: Wiley
Date: 29-11-2021
DOI: 10.1002/PRO.4245
Abstract: Kazal‐type protease inhibitor specificity is believed to be determined by sequence of the reactive‐site loop that make most, if not all, contacts with the serine protease. Here, we determined the complex crystal structure of Aedes aegypti trypsin inhibitor (AaTI) with μ‐plasmin, and compared its reactivities with other Kazal‐type inhibitors, infestin‐1 and infestin‐4. We show that the shortened 99‐loop of plasmin creates an S2 pocket, which is filled by phenylalanine at the P2 position of the reactive‐site loop of infestin‐4. In contrast, AaTI and infestin‐1 retain a proline at P2, rendering the S2 pocket unfilled, which leads to lower plasmin inhibitions. Furthermore, the protein scaffold of AaTI is unstable, due to an elongated Cys‐V to Cys‐VI region leading to a less compact hydrophobic core. Chimeric study shows that the stability of the scaffold can be modified by swapping of this Cys‐V to Cys‐VI region between AaTI and infestin‐4. The scaffold instability causes steric clashing of the bulky P2 residue, leading to significantly reduced inhibition of plasmin by AaTI or infestin‐4 chimera. Our findings suggest that surface loops of protease and scaffold stability of Kazal‐type inhibitor are both necessary for specific protease inhibition, in addition to reactive site loop sequence. PDB ID code: 7E50 .
Publisher: Wiley
Date: 06-2003
Publisher: MDPI AG
Date: 14-05-2020
Abstract: A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography–mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.
No related grants have been discovered for Manjunatha Kini.