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.     

Plethodontid modulating factor, a hypervariable salamander courtship pheromone in the three-finger protein superfamily

The soluble members of the three-finger protein superfamily all share a relatively simple 'three-finger' structure, yet perform radically different functions. Plethodontid modulating factor (PMF), a pheromone protein produced by the lungless salamander, Plethodon shermani, is a new and unusual member of this group. It affects female receptivity when delivered to the female's nares during courtship. As with other plethodontid pheromone genes, PMF is hyperexpressed in a specialized male mental (chin) gland. Unlike other plethodontid pheromone genes, however, PMF is also expressed at low levels in the skin, liver, intestine and kidneys of both sexes. The PMF sequences obtained from all tissue types were highly variable, with 103 unique haplotypes identified which averaged 35% sequence dissimilarity (range 1-60%) at the protein level. Despite this variation, however, all PMF sequences contained a conserved approximately 20-amino-acid secretion signal sequence and a pattern of eight cysteines that is also found in cytotoxins and short neurotoxins from snake venoms, as well as xenoxins from Xenopus. Although they share a common cysteine pattern, PMF isoforms differ from other three-finger proteins in: (a) amino-acid composition outside of the conserved motif; (b) length of the three distinguishing 'fingers'; (c) net charge at neutral pH. Whereas most three-finger proteins have a net positive charge at pH 7.0, PMF has a high net negative charge at neutral pH (pI range of most PMFs 3.5-4.0). Sequence comparisons suggest that PMF belongs to a distinct multigene subfamily within the three-finger protein superfamily.     

Cloning and characterization of an alpha-neurotoxin-type protein specific for the coral snake Micrurus corallinus

During the cloning of abundant cDNAs expressed in the Micrurus corallinus coral snake venom gland, we cloned an alpha-neurotoxin homologue cDNA (nxh1). Two others isoforms were also cloned (nxh3 and nxh7, respectively). The nxh1 cDNA codes for a potential coral snake toxin with a signal peptide of 21 amino acids plus a predicted mature peptide with 57 amino acids. The deduced protein is highly similar to known toxic three-finger alpha-neurotoxins, with four deduced S-S bridges at the same conserved positions. This is the first cDNA coding for a three-finger related protein described so far for coral snakes. However, the predicted protein does not possess some of the important amino acids for the nicotinic acetylcholine receptor interaction. This protein was expressed in Escherichia coli as a His-tagged protein that allowed the rapid purification of the recombinant protein. This protein was used to generate antibodies which recognized the recombinant protein in Western blot and also a single band present in the M. corallinus venom, but not in the venom of 10 other Micrurus species.     

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.     

Accelerated evolution of small serum proteins (SSPs)-The PSP94 family proteins in a Japanese viper

Five small serum proteins (SSPs) with molecular masses of 6.5-10 kDa were detected in Habu (Trimeresurus flavoviridis) serum; this included two novel proteins SSP-4 and SSP-5. The amino acid sequences of these proteins and of SSP-1, SSP-2, and SSP-3, which were reported previously, were determined on the basis of the nucleotide sequences of their cDNAs. Although these proteins exhibited only limited sequence identity to mammalian prostatic secretory protein of 94 amino acids (PSP94), the topological pattern of disulfide bonds in SSPs was identical to that of the mammalian proteins. SSP-3 and SSP-4 lacked approximately 30 residues at the C-terminal. Each of the full-length cDNAs encoded a mature protein of 62-90 residues and a highly conserved signal peptide. The evolutionary distances between SSPs estimated on the basis of the amino acid changes were significantly greater than those of the synonymous nucleotide substitutions; these finding, together with results from analyses of nonsynonymous to synonymous rates of change (dN/dS) suggest that snake SSPs have endured substantial accelerated adaptive protein evolution. Such accelerated positive selection in SSPs parallels other findings of similar molecular evolution in snake venom proteins and suggests that diversifying selection on both systems may be linked, and that snake SSP genes may have evolved by gene duplication and rapid diversification to facilitate the acquisition of various functions to block venom activity within venomous snakes.     

Structurally conserved alpha-neurotoxin genes encode functionally diverse proteins in the venom of Naja sputatrix

The structure and organization of the genes encoding the long-chain neurotoxins and four other isoforms of weak neurotoxins in the venom of Naja sputatrix are reported. The genes contained three exons interrupted by two introns, a structure similar to other members of the three-finger toxin family. The proteins encoded by these genes, however, show varied affinity towards nicotinic acetylcholine receptors. Phylogenetic analysis of these genes showed that the weak neurotoxin gene is confined to a distinct group. We also observe that specific mutations of the gene provide the diversity in function in these toxins while maintaining a common structural scaffold. This forms the first report where the molecular basis of evolution of postsynaptic neurotoxins from an ancestral gene can be demonstrated using the same species of snake.     

Identification and characterization of a taxon-specific three-finger toxin from the venom of the Green Vinesnake (Oxybelis fulgidus; family Colubridae)

Snake venoms contain a variety of protein and peptide toxins, and the three-finger toxins (3FTxs) are among the best characterized family of venom proteins. The compact nature and highly conserved molecular fold of 3FTxs, together with their abundance in many venoms, has contributed to their utility in structure-function studies. Although many target the nicotinic acetylcholine receptor of vertebrate skeletal muscle, often binding with nanomolar K_ds, several non-conventional 3FTxs show pronounced taxon-specific neurotoxic effects. Here we describe the purification and characterization of fulgimotoxin, a monomeric 3FTx from the venom of Oxybelis fulgidus, a neotropical rear-fanged snake. Fulgimotoxin retains the canonical 5 disulfides of the non-conventional 3FTxs and is highly neurotoxic to lizards; however, mice are unaffected, demonstrating that this toxin is taxon-specific in its effects. Analysis of structural features of fulgimotoxin and other colubrid venom 3FTxs indicate the presence of a “colubrid toxin motif” (CYTLY) and a second conserved segment (WAVK) found in Boiga and Oxybelis taxon-specific 3FTxs, both in loop II. Because specific residues in loop II conventional α-neurotoxic 3FTxs are intimately associated with receptor binding, we hypothesize that this loop, with its highly conserved substitutions, confers taxon-specific neurotoxicity. These findings underscore the importance of rear-fanged snake venoms for understanding the evolution of toxin molecules and demonstrate that even among well-characterized toxin families, novel structural and functional motifs may be found.     

Unique gene organization of colubrid three-finger toxins: complete cDNA and gene sequences of denmotoxin, a bird-specific toxin from colubrid snake Boiga dendrophila (Mangrove Catsnake)

Denmotoxin is a colubrid three-finger toxin isolated from the venom of Boiga dendrophila, which exhibits bird-specific neurotoxicity. We have sequenced the full-length cDNA and the gene encoding the precursor of denmotoxin. This is the first glimpse of genomic organization of a colubrid three-finger toxin. Denmotoxin cDNA shows low similarity to elapid three-finger toxins, except for the conserved signal peptide region. The open reading frame of denmotoxin possesses an additional fragment encoding a part of the putative signal peptide followed by an extra long N-terminus. The exon/intron organization of denmotoxin is also different from elapid three-finger toxin genes. The denmotoxin gene contains four exons and three introns, while elapid genes share virtually identical gene organization consisting of three exons and two introns. It appears that Elapidae snakes have lost the extra second exon after the divergence of the snake families.     

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因的克隆及分析

从广西产眼镜王蛇（Ophiophagus hannah）毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBank登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。     

Identification of novel serum proteins in a Japanese viper: homologs of mammalian PSP94

Three small serum proteins (SSP-1, -2, and -3), with molecular masses of 6.5-10kDa, were isolated from Habu (Trimeresurus flavoviridis) serum, and the amino acid sequences were determined by protein and cDNA analysis. Despite only limited sequence identity to any mammalian prostatic secretory protein of 94 amino acids (PSP94), all of the Cys residues in these SSPs were well conserved. SSPs are the first PSP94 family proteins to be identified in reptiles. SSP-1 and -3 weakly inhibited the proteolytic activity of a snake venom metalloproteinase. On the other hand, SSP-2 formed a tight complex with triflin, a snake venom-derived Ca(2+) channel blocker that suppresses the smooth muscle contraction. This suggests a role for SSP-2 in the self defense system of venomous snakes.     

Identification and Structural Characterization of a New Three-Finger Toxin Hemachatoxin from Hemachatus haemachatus Venom

Snake venoms are rich sources of biologically active proteins and polypeptides. Three-finger toxins are non-enzymatic proteins present in elapid (cobras, kraits, mambas and sea snakes) and colubrid venoms. These proteins contain four conserved disulfide bonds in the core to maintain the three-finger folds. Although all three-finger toxins have similar fold, their biological activities are different. A new three-finger toxin (hemachatoxin) was isolated from Hemachatus haemachatus (Ringhals cobra) venom. Its amino acid sequence was elucidated, and crystal structure was determined at 2.43 Å resolution. The overall fold is similar to other three-finger toxins. The structure and sequence analysis revealed that the fold is maintained by four highly conserved disulfide bonds. It exhibited highest similarity to particularly P-type cardiotoxins that are known to associate and perturb the membrane surface with their lipid binding sites. Also, the increased B value of hemachotoxin loop II suggests that loop II is flexible and may remain flexible until its interaction with membrane phospholipids. Based on the analysis, we predict hemachatoxin to be cardiotoxic/cytotoxic and our future experiments will be directed to characterize the activity of hemachatoxin.     

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle alpha(1)betagammadelta-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 A by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snake's diet.     

Isolation of a Neurotoxin (α-colubritoxin) from a Nonvenomous Colubrid: Evidence for Early Origin of Venom in Snakes

The evolution of venom in advanced snakes has been a focus of long-standing interest. Here we provide the first complete amino acid sequence of a colubrid toxin, which we have called -colubritoxin, isolated from the Asian ratsnake Coelognathus radiatus (formerly known as Elaphe radiata), an archetypal nonvenomous snake as sold in pet stores. This potent postsynaptic neurotoxin displays readily reversible, competitive antagonism at the nicotinic receptor. The toxin is homologous with, and phylogenetically rooted within, the three-finger toxins, previously thought unique to elapids, suggesting that this toxin family was recruited into the chemical arsenal of advanced snakes early in their evolutionary history. LC-MS analysis of venoms from most other advanced snake lineages revealed the widespread presence of components of the same molecular weight class, suggesting the ubiquity of three-finger toxins across advanced snakes, with the exclusion of Viperidae. These results support the role of venom as a key evolutionary innovation in the early diversification of advanced snakes and provide evidence that forces a fundamental rethink of the very concept of nonvenomous snake.     

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.     

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assembling an arsenal: origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences

We analyzed the origin and evolution of snake venom toxin families represented in both viperid and elapid snakes by means of phylogenetic analysis of the amino acid sequences of the toxins and related nonvenom proteins. Out of eight toxin families analyzed, five provided clear evidence of recruitment into the snake venom proteome before the diversification of the advanced snakes (Kunitz-type protease inhibitors, CRISP toxins, galactose-binding lectins, M12B peptidases, nerve growth factor toxins), and one was equivocal (cystatin toxins). In two others (phospholipase A(2) and natriuretic toxins), the nonmonophyly of venom toxins demonstrates that presence of these proteins in elapids and viperids results from independent recruitment events. The ANP/BNP natriuretic toxins are likely to be basal, whereas the CNP/BPP toxins are Viperidae only. Similarly, the lectins were recruited twice. In contrast to the basal recruitment of the galactose-binding lectins, the C-type lectins were shown to be Viperidae only, with the alpha-chains and beta-chains resulting from an early duplication event. These results provide strong additional evidence that venom evolved once, at the base of the advanced snake radiation, rather than multiple times in different lineages, with these toxins also present in the venoms of the "colubrid" snake families. Moreover, they provide a first insight into the composition of the earliest ophidian venoms and point the way toward a research program that could elucidate the functional context of the evolution of the snake venom proteome.     

Identification and purification of vitamin K-dependent proteins and peptides with monoclonal antibodies specific for gamma -carboxyglutamyl (Gla) residues

Novel monoclonal antibodies that specifically recognize gamma-carboxyglutamyl (Gla) residues in proteins and peptides have been produced. As demonstrated by Western blot and time-resolved immunofluorescence assays the antibodies are pan-specific for most or all of the Gla-containing proteins tested (factors VII, IX, and X, prothrombin, protein C, protein S, growth arrest-specific protein 6, bone Gla protein, conantokin G from a cone snail, and factor Xa-like proteins from snake venom). Only the Gla-containing light chain of the two-chain proteins was bound. Decarboxylation destroyed the epitope(s) on prothrombin fragment 1, and Ca(2+) strongly inhibited binding to prothrombin. In Western blot, immunofluorescence, and surface plasmon resonance assays the antibodies bound peptides conjugated to bovine serum albumin that contained either a single Gla or a tandem pair of Gla residues. Binding was maintained when the sequence surrounding the Gla residue(s) was altered. Replacement of Gla with glutamic acid resulted in a complete loss of the epitope. The utility of the antibodies was demonstrated in immunochemical methods for detecting Gla-containing proteins and in the immunopurification of a factor Xa-like protein from tiger snake venom. The amino acid sequences of the Gla domain and portions of the heavy chain of the snake protein were determined.