Snake venom components and their cross-reactivity: a review

Snake venoms are complex mixtures of organic and inorganic compounds, many of which display biological activity. It has been demonstrated that antisera raised against whole venom or a single purified venom protein from one species of snake will react with proteins in the venom of other species. This cross-reactivity between species may have applications in determining snake phylogeny, but recent studies on the variation of venom components within a species make these evolutionary conclusions questionable.     

蛇毒素蛋白毕赤酵母表达进展

蛇毒是许多具有独特生物活性的蛋白质与酶的混合物,在基础科学研究和临床上有重大应用价值,但是通过从蛇毒中分离获取活性组分具有局限性。巴斯德毕赤酵母表达系统是最为常用的真核表达系统之一,其真核加工、折叠、翻译后修饰等能力使得所表达的重组蛋白具有与天然蛋白近似的生物活性,因而该系统在富含二硫键或糖基化的蛇毒素蛋白表达中被广为采用。迄今为止,已经有12个属的25种蛇毒素蛋白(包括蛇毒丝氨酸蛋白酶、金属蛋白酶/去整合素、L-氨基酸氧化 酶、C-型凝集素和神经毒素、血管收缩因子、神经生长因子等家族)在毕赤酵母中获得成功表达,蛇毒富半胱氨酸蛋白、缓激肽增强肽(BPP)等至今尚未见酵母表达的报道。毕赤酵母表达蛇毒素蛋白失败的原因可能在于,有关密码子偏爱性、目的基因转录出的RNA二级结构特征、糖基化程度不均一及糖型差异、所表达毒素对酵母细胞的毒性等方面,并对解决的方法进行了讨论。     

蛇毒抗肿瘤作用研究进展

蛇毒含有多种酶类和毒性蛋白,具有广泛的生物学活性。对蛇毒各种组分的研究,发现蛇毒具有抗血栓,止血,镇痛及抗肿瘤的作用。本就其抗肿瘤方面的研究进展进行了综述,介绍了蛇毒的抑瘤机制,抑瘤活性成分的分离纯化,理化特性及研究方向和应用前景。     

Minor snake venom proteins: Structure,function and potential applications

Snake venoms present a great diversity of pharmacologically active compounds that may be applied as research and biotechnological tools, as well as in drug development and diagnostic tests for certain diseases. The most abundant toxins have been extensively studied in the last decades and some of them have already been used for different purposes. Nevertheless, most of the minor snake venom protein classes remain poorly explored, even presenting potential application in diverse areas. The main difficulty in studying these proteins lies on the impossibility of obtaining sufficient amounts of them for a comprehensive investigation. The advent of more sensitive techniques in the last few years allowed the discovery of new venom components and the in-depth study of some already known minor proteins. This review summarizes information regarding some structural and functional aspects of low abundant snake venom proteins classes, such as growth factors, hyaluronidases, cysteine-rich secretory proteins, nucleases and nucleotidases, cobra venom factors, vespryns, protease inhibitors, antimicrobial peptides, among others. Some potential applications of these molecules are discussed herein in order to encourage researchers to explore the full venom repertoire and to discover new molecules or applications for the already known venom components.     

Viper metalloproteinase (Agkistrodon halys pallas) with antimicrobial activity against multi-drug resistant human pathogens

Metalloproteinases are abundant enzymes in crotalidae and viperidae snake venoms. Snake venom metalloproteinases (SVMPs) comprise a family of zinc-dependent enzymes, which display many different biological activities. A 23.1 kDa protein was isolated from Agkistrodon halys (pallas, Chinese viper) snake venom. The toxin is a single chain polypeptide with a molecular weight of 23146.61 and an N-terminal sequence (MIQVLLVTICLAVFPYQGSSIILES) relatively similar to that of other metalloprotein-like proteases isolated from the snake venoms of the Viperidae family. The antibacterial effect of Agkistrodon halys metalloproteinase (AHM) on Burkholderia pseudomallei (strains TES and KHW), Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacterium) was studied at a concentration 120 microM. Interestingly, we found that the metalloproteinase exhibited antibacterial properties and was more active against S. aureus, P. vulgaris, P. mirabilis and multi-drug resistant B. pseudomallei (strain KHW) bacteria. AHM variants with high bacteriostatic activity (MIC 1.875-60 microM) also tended to be less cytotoxic against U-937 human monocytic cells up to 1 mM concentrations. These results suggest that this metalloprotein exerts its antimicrobial effect by altering membrane packing and inhibiting mechanosensitive targets.     

Snake Venom Cardiotoxins-Structure,Dynamics, Function and Folding

Abstract

Snake cardiotoxins are highly basic (pI>10) small molecular weight (～6.5 kDa), all β-sheet proteins. They exhibit a broad spectrum of interesting biological activities. The secondary structural elements in these toxins include antiparallel double and triple stranded β-sheets. The three dimensional structures of these toxins reveal an unique asymmetric distribution of the hydrophobic and hydrophilic amino acids. The 3D structures of closely related snake venom toxins such as neurotoxins and cardiotoxin-like basic proteins (CLBP) fail to show similar pattern(s) in the distribution of polar and nonpolar residues. Recently, many novel biological activities have been reported for cardiotoxins. However, to-date, there is no clear structure-function correlation(s) available for snake venom cardiotoxins. The aim of this comprehensive review is to summarize and critically evaluate the progress in research on the structure, dynamics, function and folding aspects of snake venom cardiotoxins.     

Physicochemical characterization and functional analysis of some snake venom toxin proteins and related non-toxin proteins of other chordates

Snake venom contains a diverse array of proteins and polypeptides. Cytotoxins and short neurotoxins are non-enzymatic polypeptide components of snake venom. The three-dimensional structure of cytotoxin and short neurotoxin resembles a three finger appearance of three-finger protein super family. Different family members of three-finger protein super family are employed in diverse biological functions. In this work we analyzed the cytotoxin, short neurotoxin and related non-toxin proteins of other chordates in terms of functional analysis, amino acid compositional (%) profile, number of amino acids, molecular weight, theoretical isoelectric point (pI), number of positively charged and negatively charged amino acid residues, instability index and grand average of hydropathy with the help of different bioinformatical tools. Among all interesting results, profile of amino acid composition (%) depicts that all sequences contain a conserved cysteine amount but differential amount of different amino acid residues which have a family specific pattern. Involvement in different biological functions is one of the driving forces which contribute the vivid amino acid composition profile of these proteins. Different biological system dependent adaptation gives the birth of enriched bio-molecules. Understanding of physicochemical properties of these proteins will help to generate medicinally important therapeutic molecules for betterment of human lives.     

Predicted structure model of Bungarotoxin from Bungarus fasciatus snake

Snake venoms are cocktails comprising combinations of different proteins, peptides, enzymes and toxins. Snake toxins have diverse characteristics having different molecular configuration, structure and mode of action. Many toxins derived from snake venom have distinct pharmacological activities. Venom from Bungarus fasciatus (commonly known as banded krait) is a species of elapid snake found on the South East Asia and Indian sub-continent, mainly contains neurotoxins. Beta bungartotoxin is the major fraction of Bungarus venom and particularly act pre-synaptically by obstructing neurotransmitter release. This toxin in other snake species functionally forms a heterodimer containing two different subunits (A and B). Dimerization of these two chains is a pre-requisite for the proper functionality of this protein. However, B. fasciatus bungartotoxin contains only B chain and their structural orientation in yet to be resolved. Therefore, it is of interest to describe the predicted structure model of the toxin for functional insights. In this work we analyzed the neurotoxic nature, their alignments, secondary and three dimensional structures, functions, active sites and stability with the help of different bioinformatical tools. A comprehensive analysis of the predicted model provides approaching to the functional interpretation of its molecular action.     

Proteome analysis of snake venom toxins: pharmacological insights

Snake venoms are an extremely rich source of pharmacologically active proteins with a considerable clinical and medical potential. To date, this potential has not been fully explored, mainly because of our incomplete knowledge of the venom proteome and the pharmacological properties of its components, in particular those devoid of enzymatic activity. This review summarizes the latest achievements in the determination of snake venom proteome, based primarily on the development of new strategies and techniques. Detailed knowledge of the venom toxin composition and biological properties of the protein constituents should provide the scaffold for the design of new more effective drugs for the treatment of the hemostatic system and heart disorders, inflammation, cancer and consequences of snake bites, as well as new tools for clinical diagnostic and assays of hemostatic parameters.     

Purification and Functional Characterisation of Rhiminopeptidase A,a Novel Aminopeptidase from the Venom of Bitis gabonica rhinoceros

Background

Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros.

Methodology and Principal Findings

The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped β domain, a mixed α and β catalytic domain, a β-sandwich domain and a C-terminal α helical domain.

Conclusions

This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.     

Biochemical and molecular modeling analysis of the ability of two p-aminobenzamidine-based sorbents to selectively purify serine proteases (fibrinogenases) from snake venoms

Snake venoms contain several trypsin-like enzymes with equivalent physicochemical characteristics and similar inhibition profiles. These are rather difficult to separate by classical purification procedures and therefore constitute a good model for affinity chromatography analysis. Some of these trypsin homologues present fibrinogenase activity, mimicking one or more features of the central mammalian coagulation enzyme, thrombin. It was previously demonstrated that a number of amidine derivatives are able to interact specifically with some of these serine proteases. To understand the enzyme-sorbent interactions we have investigated the ability of two commercially available benzamidine affinity matrices to purify thrombin-like serine proteases (TLSP) with similar biological properties from two snake venoms (Bothrops jararacussu and Lachesis muta rhombeata). Curiously, each sorbent retained a single but distinct TLSP from each venom with high yield. Molecular modeling analysis suggested that hydrophobic interactions within a specific region on the surface of these enzymes could be generated to explain this exquisite specificity. In addition, it was demonstrated that a specific tandem alignment of the two benzamidine sorbents enables the purification of three other enzymes from B. jararacussu venom.     

Characterization of hyaluronidase isolated from Agkistrodon contortrix contortrix (Southern Copperhead) venom

Snake venoms are a rich source of enzymes including many hydrolytic enzymes. Some enzymes such as phospholipase A2, proteolytic enzymes, and phosphodiesterases are well characterized. However many enzymes, such as the glycosidase, hyaluronidase, have not been studied extensively. Here we describe the characterization of snake venom hyaluronidase. In order to determine which venom was the best source for isolation of the enzyme, the hyaluronidase activity of 19 venoms from Elapidae, Viperidae, and Crotalidae snakes was determined. Since Agkistrodon contortrix contortrix venom showed the highest activity, this venom was used for purification of hyaluronidase. Molecular weight was determined by matrix-assisted laser desorption ionization mass spectroscopy and was found to be 59,290 Da. The molecular weight value as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 61,000 Da. Substrate specificity studies indicated that the snake venom enzyme was specific only for hyaluronan and did not hydrolyze similar polysaccharides of chondroitin, chondroitin sulfate A (chondroitin 4-sulfate), chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin 6-sulfate), chondroitin sulfate D, chondroitin sulfate E, or heparin. The enzyme is an endo-glycosidase without exo-glycosidase activity, as it did not hydrolyze p-nitrophenyl-beta-D-glucuronide or p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The main hydrolysis products from hyaluronan were hexa- and tetrasaccharides with N-acetylglucosamine at the reducing terminal. The cleavage point is at the beta1,4-glycosidic linkage and not at the beta1,3-glycosidic linkage. Thus, snake venom hyaluronidase is an endo-beta-N-acetylhexosaminidase specific for hyaluronan.     

Plant natural products active against snake bite--the molecular approach

The article surveys the substances identified in plants reputed to neutralize the effects of snake venoms. Protective activity of many of them against the lethal action of the venom of the jararaca (Bothrops jararaca) snake was confirmed by biological assays. It was shown that all belong to chemical classes capable of interacting with macromolecular targets--receptors and enzymes. In a few cases it has been shown that exogenous natural micromolecules can mimic the biological activity of endogenous macromolecules. From the evidence presented, it can be inferred that micromolecules which neutralize the action of snake venoms mechanistically replace endogenous antitoxic serum proteins with venom neutralizing capacity such as produced by some animals.     

Self-proteolysis regulation in the Bothrops jararaca venom: the metallopeptidases and their intrinsic peptidic inhibitor

Snake venom proteome variation is a well-documented phenomenon, whereas peptidome variation is still relatively unknown. We used a biological approach to explore the inhibitory activities present in the whole venom of Bothrops jararaca that prevents the venom self-proteolysis and/or digestion of the glandular tissue. Although snake venom metallopeptidases have long been known from the biochemical up to the clinical point of view, the mechanisms by which these enzymes are regulated in the reptile's venom gland remain fairly unknown. We have successfully demonstrated that there are three synergistic weak inhibitory mechanisms that are present in the crude venom that are able to abolish the metallopeptidase activity in situ, namely: (i) citrate calcium chelation; (ii) acidic pH and; (iii) enzymatic competitive inhibition by the tripeptide Pyroglutamyl-lysyl-tryptophan. Taken together, these three factors become a strong set-up that inhibits the crude venom metallopeptidase activity as well as a purified metallopeptidase from this same venom. However, this inhibition can be totally reverted by dilution into an optimal pH solution, such as the blood.     

The diversity of bioactive proteins in Australian snake venoms

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.     

Anticoagulant proteins from snake venoms: structure, function and mechanism

Over the last several decades, research on snake venom toxins has provided not only new tools to decipher molecular details of various physiological processes, but also inspiration to design and develop a number of therapeutic agents. Blood circulation, particularly thrombosis and haemostasis, is one of the major targets of several snake venom proteins. Among them, anticoagulant proteins have contributed to our understanding of molecular mechanisms of blood coagulation and have provided potential new leads for the development of drugs to treat or to prevent unwanted clot formation. Some of these anticoagulants exhibit various enzymatic activities whereas others do not. They interfere in normal blood coagulation by different mechanisms. Although significant progress has been made in understanding the structure-function relationships and the mechanisms of some of these anticoagulants, there are still a number of questions to be answered as more new anticoagulants are being discovered. Such studies contribute to our fight against unwanted clot formation, which leads to death and debilitation in cardiac arrest and stroke in patients with cardiovascular and cerebrovascular diseases, arteriosclerosis and hypertension. This review describes the details of the structure, mechanism and structure-function relationships of anticoagulant proteins from snake venoms.     

S1 subsite in snake venom thrombin-like enzymes: can S1 subsite lipophilicity be used to sort binding affinities of trypsin-like enzymes to small-molecule inhibitors?

Thrombin-like enzymes isolated from snake venoms comprise a group of serine proteinases responsible for many important coagulation disorders in the envenomed victims. Besides, these proteinases have great biotechnological interest as antithrombotic agents and as diagnostic tools. However, in spite of the recent overflow of snake venom thrombin-like enzymes (SVTLEs) on protein sequence databases, there is a lack of three-dimensional (3D) structural information on this family. Without such 3D structures available many aspects of the biological function and biochemical properties of these enzymes still remain obscure. Therefore, we have gone through a series of computational techniques, which enabled us to identify the set of residues involved in molecular recognition of inhibitors bound to the S1 subsite of snake venom thrombin-like enzymes (SVTLEs) and ultimately conclude that nonpolar (van der Waals) intermolecular interactions and ligand's hydrophobicity are the most important factors affecting binding affinities to the S1 subsite of a SVTLE isolated from the venom of Lachesis muta muta (Lmm-TLE). Consequently, we have proposed that S1 subsite lipophilicity may be used to sort binding affinities of trypsin-like enzymes to small molecules by showing that the inhibitory potency of several S1-directed compounds follows subsite lipophilicity among Lmm-TLE and other three homologous proteases. Noteworthy, in the course of our analyses we determined that thrombin's S1 subsite should, in fact, be considered less lipophilic than that of trypsin if we account for the presence of the sodium-controlled water channel communicating with the S1 subsite in the coagulant enzyme.     

A comparative in silico characterization of functional and physicochemical properties of 3FTx (three finger toxin) proteins from four venomous snakes

Snake venom is an abundant resource of diverse pharmacologically bioactive proteins and peptides and a good natural source of drug lead compounds and used as important research tools in the field of toxicology, pharmacology and neuroscience. Three finger toxins (3FTx) is an important super-family of snake venom proteins which has a conserved three finger like appearance in three dimensional structures. Members of 3FTx family show a wide array of pharmacological effects by targeting different receptors and ion channels with high specificity and many of them are being investigated as potential drug target. Therefore, with a vision to verdict a new edge and attempt we determined the amino acid compositional (%) profile, physiochemical properties, secondary structural and functional analysis and phylogenetic relationship of three finger toxins present in four different elapid snake species namely, Naja naja, Astrotia stokesii, Hydrophis cyanocintus and Pelamis platura using different bioinformatics tools. From the outcome of the current studies, it will be possible to know about a range of biological functions which are responsible mainly for the glowing amino acid composition profile of these proteins. Amino acid composition (%) profile although represents differential amount of different amino acid residues which encompasses a family precise model but all the protein sequence have a conserved amount of cysteine. The analysis of physicochemical properties can be used as a basic approach to contribute in developing rational drug through protein engineering and understanding different physiological function which will be beneficial for the welfare of human being.     

Molecular mechanism analysis of Gloydius shedaoensis venom gloshedobin interaction with inhibitors by homology modeling

Snake venom thrombin-like enzymes (SVTLEs) are widely applied in the treatment of thrombotic diseases, however, the molecular mechanism of its inhibition by synthetic and natural proteinaceous inhibitors is not yet understood. Here we investigated effects of protease inhibitors including phenylmethylsulfonil fluoride (PMSF), benzamidine (BMD) and its derivates on the activity of recombinant gloshedobin, a SVTLE from the snake Gloydius shedaoensis. The molecular inhibition mechanism was postulated by separately docking inhibitors into three-dimensional model of gloshedobin using protein C activator from Agkistrodon contortrix contortrix venom (ACC-C, which bear 78% identity with gloshedobin) as template. The analysis indicated that the strongest inhibitor, PMSF, was via a covalent bond with the catalytic Ser195, while other inhibitors showing weaker inhibitory activity were via hydrogen bond with Ser195 or non-catalytic residues.