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.

Background

Widely accepted view related to phylogeny of snakes that they evolved during the era of dinosaurs in the Jurassic period from a family of terrestrial lizards about 200 million years (Myr) ago [1, 2]. Venom, the advanced thesaurus of secretion of venom gland is usually used by snakes in defense and in assault. Within the natural world the venom system of snakes is an example of ultimate sophistication of integrated armory [3]. Natural selection gives the birth of venom which is a naturally engineered lethal admixture of peptides and proteins. The venom helps snakes to affect different prey or victim by exerting action upon different vital system [1]. Snake-bites are one of the serious public health problems in many countries of the world. At global level there are 5 million snake-bites, 2.5 million envenoming and over 125,000 mortality annually [4]. In India the incidence of snake-bites is nearly 200,000 and 35,000- 50,000 people are died every year [5]. The deadly venom contains plethora of polypeptide and non-polypeptide constituents. Cytotoxins and short neurotoxins are the nonenzymatic polypeptides (Molecular weight 5-10 kDA) within in the snake venom [6]. Interestingly the cytotoxins and short neurotoxins are the family members of ‘Three-finger’ protein (TFP) superfamily. The naming of ‘Three-finger’ is for its appearance of three loops (finger like) projected from the core region of the protein. Three finger appearances are maintained by three disulfide bridges within the loops [7]. Cytotoxin exerts their effect upon the target cells by formation of pore within the cell membrane [8]. Short neurotoxins block the neuromuscular transmission by selective binding to muscle nAChR [9]. Other non-toxin family members of Three-finger protein (TFP) superfamily are xenoxin, CD59, Ly-6, Lynx-1 [3, 7]. Xenoxin is a skin secretory protein of Xenopus laevis frog, CD59 is a complement regulatory protein plays a role in complement system in human, mouse and rat [7]. Lynx-1 is a neuronal modulator acts on CNS in mouse [10]. Venom proteins of snakes evolve from the genes of normal body proteins which are responsible for key regulatory processes within the body. These genes are duplicated and selective expression of these duplicated genes facilitates the synthesis of venomous composition of venom gland. In this process the ancestral function is converted into a derived one [3]. The objective of the present study is a comparative compositional, physicochemical characteristics and functional analysis of snake venom toxin proteins and non-toxin proteins of other chordates like hagfish, frog, mouse, rat and human etc. These comparative analyses will help us to understand the occurrence of diversification of different protein sequences in these toxin proteins and nontoxin proteins of other chordates. This also hints the systemlevel adaptability of these three-finger proteins in different physiological milieu. From the applicability view point, the results will provide information necessary for generation of engineered therapeutic proteins from the natural toxins.

Methodology

Amino acid sequences of proteins were obtained from National Centre for Biotechnology Information (http://ncbi/nlm/nih.gov) [11]. SignalP 4.0 server was employed for detection of signal peptide within the amino acid sequences (http://www.cbs.dtu.dk/services/SignalP/) [12]. After processing only main chain of peptides were used for further analysis. Detailed information regarding sequences was mined from Protein Information Resources (PIR) konwlwdgebase and literatures [13]. Protein Information Resources (PIR) is an integrated public bioinformatics resource which helps the genomic and proteomic research. For better understanding a sequence ID code was given to each molecule. Physicochemical characterization including number of amino acids, molecular weight, theoretical isoelectric point (pI), amino acid composition (%) profile, number of positively charged (Arg + Lys) and negatively charged (Asp + Glu) amino acid residues, instability index and Grand Average of Hydropathicity (GRAVY) value were calculated with the help of Expasy ProtParam tool (http://expasy.org/tools/protparam.html).

Discussion

In the present study, snake venom toxins (cytotoxins and short neurotoxin of naja annulifera and naja naja) and related nontoxin proteins of other chordates were analyzed with the help of bioinformatical tools Table 1 (see supplementary material). The analysis of amino acid composition of each sequence depicts that conservation of cysteine amino acid took place in different molecules in different organisms Table 2 (see supplementary material). Cytotoxins, short neurotoxins and related non-toxin proteins are similar in their cysteine profile but substantially different in composition of other amino acids. Cysteine profile is conserved because it is responsible for disulphide bridging which is crucial for maintenance of internal core structure of three-finger proteins [7]. Positively charged lysine amino acid is present in very high percentage in cytotoxin, short neurotoxin and in xenoxins. Lysine with the help of ionic bonds interacts with other charged biomolecules of cells, increasing the reactivity of the protein. Lethality of cytotoxins is facilitated by an invariant lysine residue of these (cytotoxins) peptides [14]. Short neurotoxin binding to nAChR is governed by positively charged amino acid lysine [15, 16]. Arginine is also present in high amount in short neurotoxin which is another positively charged amino acid, is responsible for the receptor binding mechanism. Both Lysine and Arginine and their adequate presence help cytotoxin and short neurotoxin to become an effective lethal bio-molecule. Additionally short neurotoxins also manage negatively charged amino acid efficiently than cytotoxins. Negatively charged aspartic acid and glutamic acid assisted proper attachment to membrane receptor [17]. Very high amount of negatively charged amino acid is also present in other three-finger proteins like Plethodontid modulating factor (PMF) and Lymphocyte antigen 6H (Ly6H) molecules of different organisms. In PMF the high amount of negativity is contributed by the presence of Aspartic acid and Glutamic acid residues in the sequence.

Three -finger proteins function mainly by binding to other proteins. The PMF also follows that direction by binding to positively charged female receptors for pheromone attachment [18]. More negativity of PMF by presence of negatively charged amino acid accelerates the binding mechanism of pheromone to a receptor in very expeditiously way. Other non-toxin protein of chordates contains a balanced proportion of positively and negatively charged amino acids. It is because these proteins play different key regulatory cellular processes within the internal physiological system (cellular communication system, complement system and nervous system). Large perturbation in amino acid composition affects the system in a detrimental path, although they evolved efficiently for better adaptation to system [19]. Family members of a particular family of threefinger proteins present in different species show same conservation of amino acid composition profile (e.g., Lynx-1). Moderate deviations were also evidenced in complement system proteins (CD59). Involvement in different biological functions is one of the driving forces which contribute the vivid amino acid composition profile of these proteins.

Table 3 (see supplementary material) furnishes details of the physicochemical characterization, which shows that the minimum amino acid residue containing protein is snake venom cytotoxins and maximum amino acid residue containing protein is Lynx-1, a neuromodulator. Computation of Isoelectric point (theoretical pI) and molecular weight (Mw) of an amino acid sequence is worthy because these data dictate the approximate area of a 2D-gel where a protein of interest may be detected. The cytotoxins and short neurotoxins are highly basic in nature (pI 8.69 - 9.48) where as other related protein molecules are acidic or basic. PMF is one of the chief acidic molecules with a pI range of 3.74 to 3.96. Instability index shows that Xenoxins, HLMP1, HEP21 and Lynx-1 are stable in nature (instability index <40). The relative volume of a protein occupied by its aliphatic side chains is termed as Aliphatic index (AI). Aliphatic index plays role in protein thermal stability. With a high Aliphatic index proteins are more thermally stable. Aliphatic amino acids also are hydrophobic in nature. The aliphatic index of cytotoxins in the range of 66.5 to 84.33 indicated that these proteins are thermally stable as well as they contain high amount of hydrophobic amino acids. Copresence of hydrophobic and polar (charged) residues within cytotoxins generates amphipathic nature of cytotoxins. For biological membrane perturbation this is an important criterion for a molecule. Short neurotoxin ranges an aliphatic index of 30.33 to 54.26. All different family of three-finger proteins exhibit family specific aliphatic index profile. All proteins included in this study are hydrophilic (negative GRAVY value), whereas exceptionally SLURP1 and SLURP2 are slightly hydrophobic in nature. Short neurotoxin with GRAVY value of -1.213 is the most potent hydrophilic molecule.

Conclusion

In the post-genomic era not only the generation of data but also proper assimilation of knowledge from these data is a significant deed. Development of different computational resources for exploration of biological data thrusts the discoveries of new insights into the different areas of biological sciences. Comparative physicochemical characterization of proteins from its sequence of a protein superfamily portrays the family specific molecular compositional strategy for improve system adaptability. The present study on snake venom toxin proteins and non-toxin body proteins help to understand what kind of compositional biasness and differences plays role for adaptation to different biological systems namely venom system, pheromone system, complement system and cellular communication system. Notably the exploitation of a protein scaffold which is involved in diverse biological function, used in snakes as venom architecture describes the uniqueness of process of evolution. Physicochemical characterization of these proteins describes within the Laboratory of Nature how proteins are engineered for customized biological needs. This in turn assists to generate therapeutic molecules of medicinal importance.