Sequence analysis and phylogenetic study of some toxin proteins of snakes and related non-toxin proteins of chordates

Snakes are equipped with their venomic armory to tackle different prey and predators in adverse natural world. The venomic composition of snakes is a mix of biologically active proteins and polypeptides. Among different components snake venom cytotoxins and short neurotoxin are non-enzymatic polypeptide candidates with in the venom. These two components structurally resembled to three-finger protein superfamily specific scaffold. Different non-toxin family members of three-finger protein superfamily are involved in different biological roles. In the present study we analyzed the snake venom cytotoxins, short neurotoxins and related non-toxin proteins of different chordates in terms of amino acid sequence level diversification profile, polarity profile of amino acid sequences, conserved pattern of amino acids and phylogenetic relationship of these toxin and nontoxin protein sequences. Sequence alignment analysis demonstrates the polarity specific molecular enrichment strategy for better system adaptivity. Occurrence of amino acid substitution is high in number in toxin sequences. In non-toxin body proteins there are less amino acid substitutions. With the help of conserved residues these proteins maintain the three-finger protein scaffold. Due to system specific adaptation toxin and non-toxin proteins exhibit a varied type of amino acid residue distribution in sequence stretch. Understanding of Natural invention scheme (recruitment of venom proteins from normal body proteins) may help us to develop futuristic engineered bio-molecules with remedial properties.     

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.     

Snake venoms. The amino-acid sequence of protein S2C4 from Dendroaspis jamesoni kaimosae (Jameson's mamba) venom.

A major component (S2C4) was purified from Jameson's mamba by gel filtration on Sephadex G-50 and ion-exchange chromatography on CM-cellulose. Protein S2C4 comprises 62 amino acid residues including 8 half-cystine residues. The complete amino acid sequence of the protein has been established. The sequence and the invariant amino acid residues of protein S2C4 resemble a short neurotoxin, a long neurotoxin, a cytotoxin and an angusticeps type protein. However, the position of its four disulphide bridges differs from those encountered in a short neurotoxin or a cytotoxin. Mixtures of protein S2C4 and angusticeps type proteins revealed a marked synergistic effect, in that their toxicity in combination was greater than the sum of their individual toxicities.     

Snake venom toxins. The amino-acid sequences of three toxins (9B, 11 and 12A) from Hemachatus haemachatus (Ringhals) venom.

Three toxins (9B, 11 and 12A) were purified from the venom of Hemachtus haemachatus as described previously. Whereas toxin 11 and 12A comprise 61 amino acid residues, toxin 9B contains 63 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have elucidated. The properties of the toxins were compared with those of the cytotoxin group. The toxicities, the sequences and some of the invariant residues of toxin 11 and 12A resemble the corresponding properties of the cytotoxin group. However their immunochemical properties indicate that they are distinct from both the cytotoxin and neurotoxin groups. The sequence of toxin 9B shows that it is related to the cytotoxins, but its toxicity is much lower than those encountered among members of this group.     

平颏海蛇3种短链神经毒素的基因克隆与功能比较

通过构建平颏海蛇毒腺cDNA文库得到 3个编码短链神经毒素的cDNA ,sn12、sn36和sn16 0 ,它们编码6 0个氨基酸的成熟肽 ,氨基酸序列比较表明三者之间只存在第 46位氨基酸残基的差异 ,分别为Pro4 6、His4 6和Arg4 6。在大肠杆菌中重组表达以上 3种同源序列 ,重组神经毒素SN12、SN36和SN16 0对昆明小鼠 (i.p .)的半致死剂量 (LD50 )分别是 0 .0 95 6mg/kg、0 .346 7mg/kg和 0 .2 192mg/kg ;在对昆明小鼠 (i.p .)的醋酸扭体反应镇痛实验中 ,SN12和SN16 0表现出相似的效果 ,而SN36则存在明显的差异。平颏海蛇短链神经毒素SN12、SN36和SN16 0之间只存在第 46位氨基酸残基的差异 ,在生物活性上却显示出明显的区别 ,因而推测第 46位氨基酸与短链神经毒素对烟碱型乙酰胆碱受体结合功能相关     

Toxic components of the venom of the Central Asian scorpion, Orthochirus scrobiculosus

Four polypeptide neurotoxins, possessing paralytic activity for mice, were isolated from the venom of the Central Asian black scorpion Orthochirus scrobiculosus. All these toxins, Os-1 - Os-4, were shown to be homogeneous by disc-electrophoresis and N-terminal group analyses. The amino acid composition of the toxins was determined, methionine residues being found in toxin Os-1. The neurotoxin Os-3 was subjected to tryptic and chymotryptic hydrolyses and its total amino acid sequence was established. It was shown that neurotoxin Os-3 consists of 67 amino acid residues with four intramolecular disulfide bonds.     

Amino acid sequence of a short chain neurotoxin from the venom of banded krait (Bungarus fasciatus)

The amino acid sequence of a short chain neurotoxin obtained from Bungarus fasciatus venom consists of 64 amino acid residues: Arg-Ile-Cys-Leu-Asn-Gln-Gln-Gln-Ser- Thr-Pro-Glu-Asp-Gln-Pro-Thr-Asn-Gly-Gln-Cys-Tyr-Ile-Lys-Thr-Asp-Cys-Gln- Asn-Lys - Thr-Trp-Asn-Thr-His-Arg-Gly-Ser-Arg-Thr-Asp-Arg-Gly-Cys-Gly-Cys-Pro-Lys- Val-Lys - Pro-Gly-Ile-Asn-Leu-Arg-Cys-Cys-Lys-Thr-Asp-Lys-Cys-Asn-Glu. The above result was obtained primarily from the amino acid analyses and sequencing of tryptic peptides accompanied with the necessary analyses and sequencing of the chymotryptic and lysyl endopeptidic peptides for alignment.     

Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins

Summary. Thermophilic proteins show substantially higher intrinsic thermal stability than their mesophilic counterparts. Amino acid composition is believed to alter the intrinsic stability of proteins. Several investigations and mutagenesis experiment have been carried out to understand the amino acid composition for the thermostability of proteins. This review presents some generalized features of amino acid composition found in thermophilic proteins, including an increase in residue hydrophobicity, a decrease in uncharged polar residues, an increase in charged residues, an increase in aromatic residues, certain amino acid coupling patterns and amino acid preferences for thermophilic proteins. The differences of amino acids composition between thermophilic and mesophilic proteins are related to some properties of amino acids. These features provide guidelines for engineering mesophilic protein to thermophilic protein. Authors’ addresses: Yuan-Jiang Pan, Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Zhejiang University Road 38, Hangzhou 310027, China; Wei-Fen Li, Microbiology Division, College of Animal Science, Zhejiang University, Hangzhou 310029, China     

Amino acid composition analysis of human secondary transport proteins and implications for reliable membrane topology prediction

Secondary transporters in humans are a large group of proteins that transport a wide range of ions, metals, organic and inorganic solutes involved in energy transduction, control of membrane potential and osmotic balance, metabolic processes and in the absorption or efflux of drugs and xenobiotics. They are also emerging as important targets for development of new drugs and as target sites for drug delivery to specific organs or tissues. We have performed amino acid composition (AAC) and phylogenetic analyses and membrane topology predictions for 336 human secondary transport proteins and used the results to confirm protein classification and to look for trends and correlations with structural domains and specific substrates and/or function. Some proteins showed statistically high contents of individual amino acids or of groups of amino acids with similar physicochemical properties. One recurring trend was a correlation between high contents of charged and/or polar residues with misleading results in predictions of membrane topology, which was especially prevalent in Mitochondrial Carrier family proteins. We demonstrate how charged or polar residues located in the middle of transmembrane helices can interfere with their identification by membrane topology tools resulting in missed helices in the prediction. Comparison of AAC in the human proteins with that in 235 secondary transport proteins from Escherichia coli revealed similar overall trends along with differences in average contents for some individual amino acids and groups of similar amino acids that are presumed to result from a greater number of functions and complexity in the higher organism.     

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.     

Amino acid sequence of cytotoxin IIa isolated from the venom of the Indian cobra (Naja naja)

A cytotoxic basic polypeptide, designated as cytotoxin IIa, was purified to homogeneous state from the venom of the Indian cobra (Naja naja) by a combination of gel filtration on Sephadex G-50, CM-cellulose chromatography, and fast protein liquid chromatography. Cytotoxin IIa is a single polypeptide consisting of 60 amino acid residues with four intramolecular disulfide linkages. The toxin showed high cytotoxicity toward Yoshida sarcoma and ascites hepatoma cells as did cytotoxins I and II isolated from the same venom. Analysis of the amino acid sequence revealed that cytotoxin I, IIa, and II are highly homologous in their primary structures and that cytotoxin IIa differs from cytotoxin I only in having Phe 25 and Val 52 in place of Tyr 25 and Glu 52 residues.     

Topogenic signals in integral membrane proteins

Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane-assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner-membrane proteins, but also for eukaryotic.proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive-inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information.     

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.     

Purification, characterization, and cDNA cloning of a new fibrinogenlytic venom protein, Agkisacutacin, from Agkistrodon acutus venom

Agkisacutacin is a new fibrinogenlytic protein from Agkistrodon acutus venom. It consists of two heterologous subunits linked by an intersubunit disulfide bond. The cDNAs encoding the two chains of Agkisacutacin were cloned from a lambdagt11 cDNA library of the snake venom gland and sequenced, including the leader peptides (23/23 amino acid residues) and mature subunits (129/123 amino acid residues). It is structurally related to the family of IX/X-binding protein (IX/X-bp)-like proteins and shows high similarity (alpha-70%/beta-64%) to habu IX/X-bp from Trimeresurus flavoridis, but displays distinct biological activity with direct action on fibrinogen.     

Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria

One of the well-known observations of proteins from thermophilic bacteria is the bias of the amino acid composition in which charged residues are present in large numbers, and polar residues are scarce. On the other hand, it has been reported that the molecular surfaces of proteins are adapted to their subcellular locations, in terms of the amino acid composition. Thus, it would be reasonable to expect that the differences in the amino acid compositions between proteins of thermophilic and mesophilic bacteria would be much greater on the protein surface than in the interior. We performed systematic comparisons between proteins from thermophilic bacteria and mesophilic bacteria, in terms of the amino acid composition of the protein surface and the interior, as well as the entire amino acid chains, by using sequence information from the genome projects. The biased amino acid composition of thermophilic proteins was confirmed, and the differences from those of mesophilic proteins were most obvious in the compositions of the protein surface. In contrast to the surface composition, the interior composition was not distinctive between the thermophilic and mesophilic proteins. The frequency of the amino acid pairs that are closely located in the space was also analyzed to show the same trend of the single amino acid compositions. Interestingly, extracellular proteins from mesophilic bacteria showed an inverse trend against thermophilic proteins (i.e. a reduced number of charged residues and rich in polar residues). Nuclear proteins from eukaryotes, which are known to be abundant in positive charges, showed different compositions as a whole from the thermophiles. These results suggest that the bias of the amino acid composition of thermophilic proteins is due to the residues on the protein surfaces, which may be constrained by the extreme environment.     

Insights from the analysis of conserved motifs and permitted amino acid exchanges in the human, the fly and the worm GPCR clusters

G-protein coupled receptors (GPCRs) belong to biologically important and functionally diverse and largest super family of membrane proteins. GPCRs retain a characteristic membrane topology of seven alpha helices with three intracellular, three extracellular loops and flanking N' and C' terminal residues. Subtle differences do exist in the helix boundaries (TM-domain), loop lengths, sequence features such as conserved motifs, and substituting amino acid patterns and their physiochemical properties amongst these sequences (clusters) at intra-genomic and inter-genomic level (please re-phrase into 2 statements for clarity). In the current study, we employ prediction of helix boundaries and scores derived from amino acid substitution exchange matrices to identify the conserved amino acid residues (motifs) as consensus in aligned set of homologous GPCR sequences. Co-clustered GPCRs from human and other genomes, organized as 32 clusters, were employed to study the amino acid conservation patterns and species-specific or cluster-specific motifs. Critical analysis on sequence composition and properties provide clues to connect functional relevance within and across genome for vast practical applications such as design of mutations and understanding of disease-causing genetic abnormalities.     

The amino acid sequence and position of the free thiol group of a short-chain neurotoxin from common-death-adder (Acanthophis antarcticus) venom.

The amino acid sequence of a short-chain neurotoxin Acanthophis antarcticus c (toxin Aa c) from the venom of an Australian elapid snake, the common death adder (Acanthophis antarcticus, subfamily Acanthophiinae) was elucidated. Toxin Aa c is composed of 62 amino acid residues, including eight half-cystine residues and a cysteine residue. The amino acid sequence of toxin Aa c is homologous with those of other short-chain neurotoxins found in snakes of the family Elapidae, especially with those from snakes of the subfamily Hydrophiinae. The single cysteine residue was located in position 4. Toxin Aa c has a lethal dose (LD50) of 0.08 micrograms/g body weight of mouse on intramuscular injection.     

重组虎纹捕鸟蛛毒素—I在巴氏毕赤酵母中的表达及纯化

虎纹捕鸟蛛毒素I是从虎纹捕鸟蛛粗毒中分离纯化,具有镇痛活性的肽类神经毒素。对巴氏毕赤酵母生产的重组HWTX－I进行多步纯化,首先将分泌到培养上清的rHWTX-I进行90％饱和度的（NH4）2SO4沉淀,再用截留分子量3kD的滤膜脱盐,再用CM阳离子交换层析分离,最后用C18反相层析脱盐纯化,真空干燥后得到的rHWTX-I经Tricine SDS-PAGE,质谱鉴定,氨基酸组成分析,N－端序列测定后活性鉴定,证明已获得高纯度的重组HWTX－I,摇瓶表达量约为80mg/L,约占总分泌量的23．6％,并对摇瓶发酵条件进行了优化,为利用基因工程方法生产HWTX－I的规模化生产及临床应用提供了证据。     

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.