Study of Conserved Domain across Divergent Phylogenetic Lineages of Long Neurotoxin from Genus Naja (elapidae Family)

Naja are a toxic genus of Elapids family in the old world. Long neurotoxins are mixtures of peptides, enzymes that interfere with the normal function of the nervous system and potently block α7 homo-oligomeric neuronal AChRs causing paralysis. The aim of the present study is to identify the origin of long neurotoxin, multiple sequences analysis for prediction of structure and function of neurotoxin and observing the conserved pattern of amino acid residues and to construct the phylogenetic tree for organizing evolutionary history of Naja genus from Elapidae family. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Naja genus from the elapids family is most abundant and widely distributed poisonous snake in the world [1]. Elapidae family approximately consists of 300 venomous snakes in 62 genera [2]. The genus Naja consists of currently 26 species of cobra of which 11 inhabit Asia and 15 occur in Africa [3,4]. Some proteins from Elapidae family are potent postsynaptic neurotoxins [5]. The long neurotoxins [6] have high potency and can bind specifically to the nicotinic acetylcholine receptor and block synaptic nerve transmission [7][8][9][10][11][12]. Long neurotoxins of 66-74 amino acids with five disulfide bridges in the central loop of the molecule and having polypeptide chain between residues 65 and 73 that gives rise to a characteristic C-terminal tail [13]. We study the origin and evolution of long neurotoxin from Naja genus by Multiple sequence alignments, phylogenetic analysis that provide the basic information of conserved sequence regions, identifying new members of protein families that derived from a common ancestor [14][15][16].

Sources and sequence information of genus Naja neurotoxins
We have taken ten species of Naja genus from Elapidae family, in which targeted long neurotoxins protein data were used to observe molecular resemble of related protein by phylogenic analysis [17,18].

Multiple sequence alignment of genus Naja neurotoxins
Multiple sequence alignment is conducted by ClustalW [19] that aligns protein sequences of similar Naja genus using a combination of distance matrix and approximate parsimony methods, which is freely available. Multiple sequence alignment analysis shows columns with no gaps are colored in blue or red. The red color indicates highly conserved regions and blue indicates less conserved ones. The Conservation analysis can be used to select a threshold for determining which columns are colored in red. In this study the Conservation Setting is used to select a threshold for determining which columns are colored in red. Numerical setting method is used to study the relative entropy threshold, in bits, that must be met for an alignment column to be displayed in red. A larger number indicates higher degree of conservation. The relative entropy is computed as: ∑i fi log2 (fi/pi), where i is residue type, fi is residue frequency observed in the multiple alignment column, and pi is the background residue frequency. Identity setting used for only columns with one residue type will be colored in red.

Construction of a phylogenetic tree for neurotoxins
Phylogenetic analyses were performed by phylogeny.fr to allow the reconstruction phylogenetic tree of the molecular evolutionary history of various aligned sequences that are useful to align highly evolved gene families clearing evolutionary relationships such as multiple neurotoxins. Trees were obtained by the methods phylogeny.fr. [20,21]. It offers three modes i.e. MUSCLE for multiple alignments, PhyML for tree building, and TreeDyn for tree construction [22][23][24][25][26]. That identify of homologous sequences, construct multiple alignments and phylogenetic tree.

Results and Interpretation
The present study a collection of long neurotoxin protein sequences of Naja genus are used for comparative analysis. Multiple sequence alignment was performed by using ClustalW figure 1 used to identify conserved motifs and to predict functional role in the variable sites as well as conserved sites [24] show the sequence divergence profile of these neurotoxins which demonstrate the sequence enrichment strategy of these sequences for adaptation to different physiological systems. Here we observed that from all sequences of long neurotoxin that Cys(C), Thr(T), Ser(S) (Hydrophilic amino acid) Pro(P), Gly(G), (hydrophobic amino acid), (K)Lys Positive charge, (D)Asp Negative charge which is conserved in all peptides having a common ancestor. That all of these peptides share eight highly conserved cysteines which were involved in the formation of β-strands are almost conserved. Cysteine (C) is conserved in all sequences at 8 sites. Phylogenetic analysis [27][28][29][30] of neurotoxin protein sequences was carried out using phylogeny.fr. That Show the evolutionary relationship among neurotoxins of Naja genus that have common origins but may have common activity. Phylogenetic tree of various neurotoxin and related peptides shows that these neurotoxins are a totally separate entity in terms of phylogenetic tree position. Cladogram [31], Phylogram [32], Radial, Circular trees show the evolutionary relationships amongst various species by means of common ancestors of Naja genus of long neurotoxin (Figures 2-5).

Discussion
Multiple sequence alignment was performed using CLUSTALW figure 1 for ten long neurotoxin proteins of Naja genus. Evolutionary analysis of related proteins is determined by phylogenetic analysis. This showed the evolutionary relationships between different species of Naja which were selected for the study. Study of long neurotoxin protein involves the comparison of homologs sequences that have common origins but may common activity with similar structure. Long neurotoxins might exist in the common ancestor of Naja genus. An alignment will display the following symbols denoting the conservation observed in each column of seventeen Naja genus. '*' indicate that, the residues Cys(C)8, Thr(T)1, Ser(S)1, Pro(P)2, Gly(G)2 , (K)Lys1, (D) Asp1 in the column are identical in all sequences in the alignment is 16.670%. ':' indicate that strongly similar, conserved substitutions have been observed Val,Ile,Tyr, Phe , Lys, Arg,Glu (2), Val, Leu, Ile, Asn and Asp is 6.25%.'. ' indicate that Weakly similar, semi-conserved substitutions are observed Ser Thr,Lys,Asp,Asn,Lys,His,Aln,Val,Pro and Gly(1) is 6.25%. Cysteine (C) that show conserved in all sequences i.e. 8 sites that these entire peptides share highly conserved cysteines involved in the formation of β-strands are almost conserved. Presence of cysteine residues in all neurotoxin of Naja genus indicates their involvement in disulfide bond formation that gives the characteristic secondary structure to these molecules. We also study Phylogenetic analysis by the phylogeny.fr. Cladogram, Phylogram, Radial and Circular tree, were constructed to show the evolutionary relationships amongst various species by way of common ancestors of Naja genus of long neurotoxin (Figure 2-5).  Multiple sequence alignment by ClustalW of various Long Neurotoxins (genus Naja) from Elapidae family. An alignment will display the following symbols denoting the degree of conservation observed in each column. '*' indicate that, the residues in that column are identical in all sequences in the alignment 16 is 16.670%. ':' indicate that strongly similar, conserved substitutions have been observed, 6 is 6.25%. '.' indicate that weakly similar, semi-conserved substitutions are observed 6 is 6.25%.

Conclusion
Ten long neurotoxin proteins from Elapidae family are summarized the identical regions. By using multiple sequences analysis and phylogenetic tree we observe the conserved residues to specify the evolutionary history and analysing sequence structure relationship of long neurotoxin among Naja species. Efficient utilization of Polar, nonpolar, positively and negatively charged amino acids and their distribution in toxin sequence make them a killer element in snake venom. Comparative analyses specify that the long neurotoxin demonstrates how proteins are generated within the nature's testing ground for tailor-made biologic needs. Evolutionary studies of long neurotoxin sequence of Naja genus found the common ancestor of all the Naja species. In future, different long neurotoxin may be converted in laboratories through protein engineering to design synthetic peptide vaccine that have a much positive role.