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Prediction of MHC Class Binding Peptide and High Affinity TAP Binders to Design Synthetic Peptide Vaccine of Long Neurotoxin 3 from <em>Naja naja</em>
ISSN: 1747-0862
Journal of Molecular and Genetic Medicine
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Prediction of MHC Class Binding Peptide and High Affinity TAP Binders to Design Synthetic Peptide Vaccine of Long Neurotoxin 3 from Naja naja

Sherkhane AS, Changbhale SS and Gomase VS*

The Global Open University, Nagaland (TGOUN), India

Corresponding Author:
Gomase VS
The Global Open University
Nagaland (TGOUN), India
Tel: 91-9987770696
E-mail: [email protected]

Received Date: May 27, 2014; Accepted Date: December 26, 2014; Published Date: January 05, 2015

Citation: Sherkhane AS, Changbhale SS, Gomase VS (2015) Prediction of MHC Class Binding Peptide and High Affinity TAP Binders to Design Synthetic Peptide Vaccine of Long Neurotoxin 3 from Naja Naja. J Mol Genet Med 9:150. doi: 10.4172/1747-0862.1000150

Copyright: © 2015 Sherkhane AS, et al. 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(s) and source are credited.

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Abstract

Naja naja is a highly poisonous snake species of cobra in Elapidae family and is commonly found in middle Asia. Long neurotoxin 3 from Naja naja binds to the nicotinic acetylcholine receptors in the postsynaptic membrane, preventing the binding of acetylcholine and blocks excitation of muscles. Antigenic peptides are complex biomolecules that have unique chemical and physical properties resulting of their amino acid composition. In this study, we have predicted the binding affinity of Long neurotoxin 3 from Naja naja having 71 amino acids, which shows 63 nonamers. Peptide fragments of the neurotoxin can be used to select nonamers for use in synthetic peptide vaccine design and to increase the understanding of roles of the immune system in neurotoxin studies. Antigenic peptides of Long neurotoxin 3 from Naja naja are most suitable for synthetic peptide vaccine development because with Small segment' 15-PNGHVCYTKT-24, 26-CDAFCSIRG-34, 36- RVDLGCAATCPTVKTGVDIQCCSTD-60 called the antigenic epitopes is sufficient for eliciting the desired immune response. In this research, we predict of MHC class I and II binding peptide because MHC molecules are cell surface proteins which take active part in immune response, antigenicity, Solvent accessibility, polar and nonpolar residue that are likely exposed on the surface of proteins that are potentially antigenic that allows to design synthetic peptide vaccine.

Keywords

Long neurotoxin 3; Naja naja ; Antigenic peptides; MHCBinders; TapPred; PSSM; SVM; Nonamers

Introduction

Naja naja is one of the most venomous snake species occurs in wild forest and in cultivated areas [1,2]. The genus Naja consists of currently 26 species of cobra of which 11 inhabit Asia and 15 occur in Africa [3,4]. Naja naja ’s venom mainly contains a powerful postsynaptic neurotoxin [5]. Long neurotoxin 3 binds to muscular and neuronal nicotinic acetylcholine receptors and Produces peripheral paralysis by blocking neuromuscular transmission at the postsynaptic site [6-8]. Antigenic peptides from Naja naja are most suitable to design synthetic peptide vaccine because a small segment can generate sufficient immune response. Major histocompatibility complex (MHC) molecules are cell surface proteins that binds to the peptides derived from host or antigenic proteins and present them at the cell surface for recognition by T-cells [9,10]. T cell recognition is a fundamental mechanism of immune system by which the host identifies and responds to foreign antigens [11,12]. There are two types of MHC molecule and are extremely polymorphic [13]. MHC class I molecules present peptides from intracellular proteins that are targeted by proteasome, cleaved them into short peptides of 8-11 amino acids in length. These peptides are bound by the transmembrane peptide transporter (TAP) and translocate them from cytoplasm to endoplasmic reticulum, where they are bound by MHC molecule. The second and the C-terminal position of the peptide are the most important for binding [14,15] and the amino acids at each position contribute a certain binding energy [16]. Whereas, MHC class II molecule present peptides derived from endocytosed extracellular proteins. Identification of MHC-binding peptides and T-cell epitopes helps improve our understanding of specificity of immune responses [17-20].

Methodology

Database searching

The antigenic protein sequence of Long neurotoxin 3 from Naja naja was retrieved from UniProtKB/Swiss-Prot, www.ncbi.nlm.nih.gov [21].

Prediction of antigenicity

Prediction of antigenicity program predicts those segments from neurotoxin protein that are likely to be antigenic by eliciting an antibody response. In this research work antigenic epitopes of Long neurotoxin 3 from Naja naja are determined by using the Hopp and Woods, Welling, Parker, Bepipred, Kolaskar and Tongaonkar antigenicity methods [22-26].

Prediction of MHC binding peptide

The major histocompatibility complex (MHC) peptide binding of Long neurotoxin 3 from Naja naja is predicted using neural networks trained on C terminals of known epitopes. Rankpep predicts peptide binders to MHC-I ligands whose C-terminal end is likely to be the result of proteosomal cleavage using Position Specific Scoring Matrices (PSSMs). Support Vector Machine (SVM) based method for prediction of promiscuous MHC class II binding peptides from protein sequence; SVM has been trained on the binary input of single amino acid sequence [27-36].

Prediction of antigenic peptides by cascade SVM based TAPPred method

In the present study, we predict cascade SVM based several TAP binders which was based on the sequence and the features of amino acids [37]. We found the MHCI binding regions (Table 1), the binding affinity of Long neurotoxin 3 from Naja naja.

Peptide Rank Start Position Sequence Score Predicted Affinity
1 58 STDDCDPFP 8.636 High
2 41 CAATCPTVK 8.618 High
3 30 CSIRGKRVD 8.586 High
4 38 DLGCAATCP 8.549 High
5 61 DCDPFPTRK 8.499 High
6 20 CYTKTWCDA 8.487 High
7 50 TGVDIQCCS 8.385 High
8 57 CSTDDCDPF 8.151 High
9 7 PDITSKDCP 8.071 High
10 40 GCAATCPTV 8.010 High
11 32 IRGKRVDLG 7.958 High
12 36 RVDLGCAAT 7.814 High
13 47 TVKTGVDIQ 7.576 High
14 46 PTVKTGVDI 7.561 High
15 22 TKTWCDAFC 7.380 High
16 48 VKTGVDIQC 7.319 High
17 51 GVDIQCCST 7.318 High
18 60 DDCDPFPTR 7.183 High
19 55 QCCSTDDCD 7.139 High
20 33 RGKRVDLGC 6.855 High
21 44 TCPTVKTGV 6.789 High
22 15 PNGHVCYTK 6.764 High
23 63 DPFPTRKRP 6.707 High
24 16 NGHVCYTKT 6.535 High
25 17 GHVCYTKTW 6.355 High
26 29 FCSIRGKRV 6.308 High
27 11 SKDCPNGHV 6.130 High

Table 1: Cascade SVM based High affinity TAP Binders of Long neurotoxin 3 from Naja naja.

Solvent accessible regions

We also predict solvent accessible regions of proteins having highest probability that a given protein region lies on the surface of a protein Surface Accessibility, backbone or chain flexibility by Emani et al., [38] and Karplus and Schulz [39]. By using different scale we predict the hydrophobic and hydrophilic characteristics of amino acids that are rich in charged and polar residues i.e. Sweet et al., Kyte and Doolittle, Abraham and Leo, Bull and Breese, Guy, Miyazawa, et al., Roseman, Wolfenden et al., Wilson et al., Cowan, Chothia [40-48].

Results and Discussion

Long neurotoxin 3 from Naja naja contain a long residue with 71 amino acids.

IRCFITPDITSKDCPNGHVCYTKTWCDGFCSRRGERVDLGCAA TCPTVKTGVDIQCCSTDDCDPFPTRKRP

Prediction of antigenic peptides

In this study, we found the antigenic determinants by finding the area of greatest local hydrophilicity. The Hopp-Woods scale Hydrophilicity Prediction Result Data found high in position 10-12, 34-37, 64-67 (1.011), 60-61 (1.129) in a protein, assuming that the antigenic determinants would be exposed on the surface of the protein and thus would be located in hydrophilic regions (Figure 1).

molecular-genetic-medicine-Hydrophobicity-plot

Figure 1: Hydrophobicity plot of Hopp and Woods [22] of Long neurotoxin 3 from Naja naja.

Welling antigenicity plot gives value as the log of the quotient between percentage in a sample of known antigenic regions and percentage in average proteins and Prediction Result Data found high in position 20-21 (0.440) (Figure 2).

molecular-genetic-medicine-Long-neurotoxin

Figure 2: Hydrophobicity plot of Welling et al. [23] of Long neurotoxin 3 from Naja naja . Result Data found high in position 37-39(0.38).

We also study Hydrophobicity plot of HPLC/Parker Hydrophilicity Prediction Result Data found 7-PDITSKDDITSKDC-14, 10- TSKDCPNSKDCPNG-17, 55-QCCSTDD-61, 56-CCSTDDC-62 (5.129), 57-CSTDDCD-63 (6.357), 58-STDDCDP-64 (6.457) maximum (Figure 3), BepiPred predicts the location of linear B-cell epitopes Result found that 9-ITSKDCPNG-17, 45-CPTVKT-50 (Figure 4) (Table 2), Kolaskar and Tongaonkar antigenicity methods (Figure 5) Predicted peptides result found i.e. 15-PNGHVCYTKT-24, 26-CDAFCSIRG-34, 36-RVDLGCAATCPTVKTGVDIQCCSTD-60 (Table 3) and the predicted antigenic fragments can bind to MHC molecule is the first bottlenecks in vaccine design.

molecular-genetic-medicine-plot-HPLC

Figure 3: Hydrophobicity plot of HPLC / Parker et al. [24] of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-Bepipred-Linear

Figure 4: Bepipred Linear Epitope Prediction plot showing antibody recognized B-cell epitopes of Long neurotoxin 3 from Naja naja Result found that 9-ITSKDCPNG-17 and 45-CPTVKT-50.

molecular-genetic-medicine-Tongaonkar-antigenicity

Figure 5: Kolaskar and Tongaonkar antigenicity plot for the Long neurotoxin 3 from Naja naja Predicted peptides result found i.e. 15-PNGHVCYTKT-24, 26-CDAFCSIRG-34 and 36-RVDLGCAATCPTVKTGVDIQCCSTD-60 [26].

No. Start Position End Position Peptide Peptide Length
1 9 17 ITSKDCPNG 9
2 45 50 CPTVKT 6

Table 2: Predicted Antigenic epitopes of Long neurotoxin 3 from Naja naja.

No. Start Position End Position Peptide Peptide Length
1 15 24 PNGHVCYTKT 10
2 26 34 CDAFCSIRG 9
3 36 60 RVDLGCAATCPTVKTGVDIQCCSTD 25

Table 3: Predicted Antigenic epitopes of Long neurotoxin 3 from Naja naja.

Solvent accessible regions

We also predict solvent accessible regions in proteins; different measurement was performed for the prediction of antigenic activity, surface region of peptides. Emini et al. [37], (Figure 6) predicts the highest probability i.e. found 31-SIRGKRIRGKRVRGKRVD - 38, 64PFPTRKFPTRKRPTRKRP - 71 (7.808) (maximum), that a given protein region lies on the surface of a protein and are used to identify antigenic determinants on the surface of proteins.

molecular-genetic-medicine-Emini-Surface

Figure 6: Emini Surface Accessibility Prediction plot of Long neurotoxin 3 from Naja naja predicts the highest probability i.e. found 31-SIRGKRIRGKRVRGKRVD-38, 64PFPTRKFPTRKRPTRKRP-71(7.808) (maximum).

Karplus and Schulz (Figure 7) High score is found i.e. found 7- PDITSKD-13 (1.077), 8-DITSKDC-14 (1.084) (maximum), 9- ITSKDCP-15 (1.078). Predict backbone or chain flexibility on the basis of the known temperature B factors of the a-carbons.

molecular-genetic-medicine-Schulz-Flexibility

Figure 7: Karplus and Schulz Flexibility Prediction of Long neurotoxin 3 from Naja naja High score is found i.e. found 7- PDITSKD-13(1.077), 8-DITSKDC-14 (1.084) (maximum), 9- ITSKDCP-15 (1.078).

The hydrophobicity and hydrophilic characteristics of amino acids is determined by using different scales that are rich in charged and polar residues i.e. Sweet et al. [39] hydrophobicity prediction Result Data found high in position 5 (0.352), 21-23, Kyte and Doolittle result high in position 40-42 (1.333), 43-44, Abraham and Leo result high in position 5-7 (0.961), 28-30, Bull and Breese result high in position 12-16,57-60 (0.510), Guy result high in position 10-12, 34-37, 65-67 (0.682), Miyazawa result high in position 5-7 (6.620), 16-2122-27,28-30, 39-42, 52-56, Roseman result high in position 43-44 (0.232), 17-18, 42-43, Wolfenden result high in position 43-44, Wilson et 17-18, 22-23, 27-30 (3.211), Cowan 5-7 (0.727), 28-30, 41-43, Chothia 5-7, 28-30, 40-44 (0.388 (Figures 8-18).

molecular-genetic-medicine-Naja-naja

Figure 8: Hydrophobicity plot of Sweet et al. [39] of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-hydrophobicity-plot

Figure 9: Kyte and Doolittle [40] hydrophobicity plot of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-Long-neurotoxin

Figure 10: Abraham and Leo [41] hydrophobicity plot of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-surface-tension

Figure 11: Bull and Breese [42] use surface tension to measure hydrophobicity and also uses negative values to describe the hydrophobicity of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-plot-Miyazawa

Figure 12: Hydrophobicity plot of Miyazawa et al. [43] of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-Naja-naja

Figure 13: Hydrophobicity plot of Roseman [44] of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-plot-Wolfenden

Figure 14: Hydrophobicity plot of Wolfenden et al. [45] of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-Long-neurotoxin

Figure 15: Hydrophobicity plot of Roseman MA [44] of Long neurotoxin 3 from Naja naja.

Figure

Figure 16: Hydrophobicity/HPLC plot of Wilson et al. [46] of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-plot-Cowan

Figure 17: Hydrophobicity/HPLC pH 3.4/ plot of Cowan [47] of Long neurotoxin 3 from Naja naja.

molecular-genetic-medicine-Hydrophobicity-plot

Figure 18: Hydrophobicity plot of Chothia [48] of Long neurotoxin 3 from Naja naja.

Prediction of MHC binding peptide

We found binding of peptides to a number of different alleles using Position Specific Scoring Matrix. Long neurotoxin 3 from Naja naja sequence is 71 residues long, having 63 nonamers. MHC molecules are cell surface proteins, which actively participate in host immune reactions and involvement of MHC-I and MHC-II in response to almost all antigens. We have predicted MHC-I peptide binders of Long neurotoxin 3 from Naja naja was tested with on a set of 4 different alleles i.e. H2-Db (mouse) 8mer with the consensus sequence QNWNCCTI that yields the maximum score i.e. 52.494, H2-Db (mouse) 9mer with the consensus sequence FCIHNCDYM that yields the maximum score i.e. 50.365, H2-Db (mouse) 10mer with the consensus sequence SGYYNFFWCL that yields the maximum score i.e. 58.858, H2-Db (mouse) 11mer with the consensus sequence CGVYNFYYCCY that yields the maximum score i.e. 79.495 (Tables 4-7), and MHC-II peptide binders for I_Ab.p with the consensus sequence YYAPWCNNA that yields the maximum score i.e. 35.632, I_Ad.p with the consensus sequence QMVHAAHAE that yields the maximum score i.e. 53.145 for MHC II allele was tasted.

MHC-I Allele POS. N SEQUENCE C MW (Da) SCORE % OPT.
8mer_H2_Db 41 DLG CAATCPTV KTG 746.89 21.518 40.99 %
8mer_H2_Db 61 STD DCDPFPTR KRP 932.03 4.607 8.78 %
8mer_H2_Db 58 QCC STDDCDPF PTR 880.89 1.654 3.15 %
8mer_H2_Db 2 I RCFITPDI TSK 946.14 0.021 0.04 %

Table 4: Promiscuous MHC ligands, having C-terminal ends are proteosomal cleavage sites, the binding potential (score) of antigenic peptide to the MHC-1 Allele i.e. 8mer_H2_Db.

MHC-I Allele POS. N SEQUENCE C MW (Da) SCORE % OPT.
9mer_H2_Db. 12 ITS KDCPNGHVC YTK 954.08 7.581 15.05 %
9mer_H2_Db. 57 IQC CSTDDCDPF PTR 984.03 7.131 14.16 %
9mer_H2_Db. 13 TSK DCPNGHVCY TKT 989.09 5.283 10.49 %
9mer_H2_Db. 40 VDL GCAATCPTV KTG 803.94 2.756 5.47 %

Table 5: Promiscuous MHC ligands, having C-terminal ends are proteosomal cleavage sites the binding potential (score) of antigenic peptide to the MHC-1 Allele i.e. 9mer_H2_Db.

MHC-I Allele POS. N SEQUENCE C MW (Da) SCORE % OPT.
10mer_H2Db 12 ITS KDCPNGHVCY TKT 1117.26 7.127 12.11 %
10mer_H2Db 60 CST DDCDPFPTRK RP 1175.29 7.065 12.00 %
10mer_H2Db 39 RVD LGCAATCPTV KTG 917.1 1.576 2.68 %

Table 6: Promiscuous MHC ligands, having C-terminal ends are proteosomal cleavage sites the binding potential (score) of antigenic peptide to the MHC-1 Allele i.e. 10mer_H2_Db.

MHC-I Allele POS. N SEQUENCE C MW (Da) SCORE % OPT.
11mer_H2Db 11 DIT SKDCPNGHVCY TKT 1204.34 14.334 18.03 %
11mer_H2Db 22 VCY TKTWCDGFCSR RGE 1262.45 -3.336 -4.20 %

Table 7: Promiscuous MHC ligands, having C-terminal ends are proteosomal cleavage sites the binding potential (score) of antigenic peptide to the MHC-1 Allele i.e. 11mer_H2_Db.

Alleles highlighted in red represent predicted binders (Tables 8 and 9). Here RANKPEP report PSSM-specific binding threshold and is obtained by scoring all the antigenic peptide sequences included in the alignment from which a profile is derived, and is defined as the score value that includes 85% of the peptides within the set. Peptides whose score is above the binding threshold that are highlighted here and peptides produced by the cleavage prediction model are also highlighted here. We also use a cascade SVM based TAPPred method which found 27 High affinity TAP Transporter peptide regions which represents predicted TAP binders residues which occur at N and C termini from Long neurotoxin 3 from Naja naja (Table 1). TAP is an important transporter that transports antigenic peptides from cytosol to ER. TAP binds and translocate selective antigenic peptides for binding to specific MHC molecules. The efficiency of TAP-mediated translocation of antigenic peptides is directly proportional to its TAP binding affinity. Thus, by understanding the nature of peptides, that bind to TAP with high affinity, is important steps in endogenous antigen processing. In this study, we found the MHCI and MHCII binding regions. T cell immune responses are derived by antigenic epitopes hence their identification is important for design synthetic peptide vaccine. Therefore, the prediction of peptide binding to MHCI molecules by appropriate processing of antigen peptides occurs by their binding to the relevant MHC molecules. Because, the C-terminus of MHCI-restricted epitopes results from cleavage by the proteasome and thus, proteasome specificity is important for determining T-cell epitopes. Consequently, RANKPEP focus on the prediction of conserved epitopes. C-terminus of MHCI-restricted peptides is generated by the proteasome, and thus RANKPEP also determines whether the C-terminus of the predicted MHCI-peptide binders is the result of proteasomal cleavage and these sequences are highlighted in the output results.

MHC-II Allele POS. N SEQUENCE C MW(Da) SCORE %OPT.
MHC-II I_Ab 25 TKT WCDAFCSIR GKR 1059.27 17.73 49.76 %
MHC-II I_Ab 40 VDL GCAATCPTV KTG 803.94 14.967 42.00 %
MHC-II I_Ab 21 HVC YTKTWCDAF CSI 1093.25 12.459 34.97 %
MHC-II I_Ab 41 DLG CAATCPTVK TGV 875.06 10.95 30.73 %
MHC-II I_Ab 29 CDA FCSIRGKRV DLG 1047.29 10.823 30.37 %
MHC-II I_Ab 38 KRV DLGCAATCP TVK 831.96 10.5 29.47 %
MHC-II I_Ab 4 IRC FITPDITSK DCP 1003.16 10.03 28.15 %

Table 8: Prediction of MHCII ligands all rows highlighted represent predicted binders to the MHC-II Allele i.e. MHC-II I_Ab.

MHC-II Allele POS. N SEQUENCE C MW(Da) SCORE % OPT.
MHC-II I_Ad 39 RVD LGCAATCPT VKT 817.97 7.417 13.96 %
MHC-II I_Ad 39 RVD LGCAATCPT VKT 817.97 7.417 13.96 %

Table 9: Prediction of MHCII ligands all rows highlighted represent predicted binders to the MHC-II Allele i.e. MHC-II I Ad.

Prediction Result Data found 7-PDITSKDDITSKDC-14, 10- TSKDCPNSKDCPNG-17, 55 QCCSTDD-61, 56-CCSTDDC-62 (5.129), 57-CSTDDCD-63 (6.357) and 58-STDDCDP 64 (6.457) (maximum).

Conclusion

From the above result and discussion it is concluded that the ability of RANKPEP to predict MHC binding peptides, and thereby potential T-cell epitopes, Antigenic peptides should be located in solvent accessible regions and contain both hydrophobic and hydrophilic residues. High peaks in the surface accessibility plot predict regions that have a higher chance of producing antibodies that can bind to native protein. This means the increase in affinity of MHC binding peptides may result in enhancement of immunogenicity of Long neurotoxin 3 of Naja naja and are helpful in the designing of synthetic peptide vaccine. This approach can help reduce the time and cost of experimentation for determining functional properties and helps to minimize the number of validation experiments.

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