In Silico Characterization and Homology Modeling of Histamine Receptors

Abstract

Histamine plays vital role in molecular mechanism of allergic reactions. Therefore, characterization and homology modeling of Histamine receptor is of great importance to design effective vaccines. In this thesis, different methods are applied to analyse biomolecular features of histamine receptors and design best models of these receptors. In addition to this, the study tried to identify potential B cell and T cell epitope based vaccine of an allergen and consequently, emphasized on to develop a B cell prediction tool. Identified four histamine receptors, such as Histamine H1, Histamine H2, Histamine H3 and Histamine H4 have been analysed through ProtParam to extract physiochemical properties and ClastalW algorithm has been applied to identify conserved regions. Motif and Transmembrane regions have been identified through MEME suit and TMHMM servers, respectively. For homology modeling, I-tasser has been used and generated models have been validated through RAMPAGE, ERRAT and PROCHECK. Targeted api m3 allergen then rendered through self-optimized prediction method with alignment for physiochemical feature extraction. NetCTL 1.2 has been applied to identify preliminary T cell epitope candidates and then scrutinized by Stabilized Matrix Base Method, relative to IC50 values. Predicted T cell epitopes have been further analysed for conservancy and population coverage via IEDB tools. B cell epitopes of api m3 allergen have been predicted through, BCPREDS, ABCpred, Bepipred and Bcepred. In addition, classifier based single interface B cell epitope prediction and/or validation tool has been developed through establishing efficient MATLAB algorithms to classify beta turn regions, hydrophilic regions, surface accessible regions and antigenic regions. Lastly, with superimposing graphical representation of these four criteria in a single interface graph plotted to identify B cell epitopes via this tool. Extracted results denotes that, Histamine receptors possess molecular weight around 55.7 KDa, theoretical pI 9.33-9.62, instability index 34.93-47.00, aliphatic index (AI) was above 90 and the receptors were hydrophobic except histamine H1 receptor. Moderately conserved region was found in 75-94 amino acid position. A profound motif has been identified from 84-149 amino acid position for four histamine receptors with significantly lower E-value. It has been identified that, these receptors are seven pass transmembrane protein and a gap between transmembrane helix number five and six was found in each histamine receptor except Histamine H2 receptor, which can be potential drug target candidate. Generated 3D models have been passed through every spheres of validation. Api m3 allergen has been found relative thermostable nature and only 10.46% of the overall secondary structure consisted of beta turn region. Five MHC class I T-cell epitopes were identified and scrutinized and YTEESVSAL found out as the best epitope. For MHC class II T-cell epitopes YPKDPYLYYDFYPLE and GGPLLRIFTKHMLDV have been found as most prominent T-cell epitopes of api m3 allergen. This study also revealed that, GDRIPDEKN and PHVPEYSSS, as the most effective B-cell epitopes of api m3. The proposed tool efficiently identified B cell epitopes and provided result in a single interface. The tool can aid in B cell research and vaccine development. Finally, the suggested potential drug targets can be applied in designing more sustainable antihistamines and relevant drugs in treating allergic diseases. Predicted T-cell and B-cell epitopes of api m3 allergen could help the researchers to test these vaccines further for immunoreactivity applying in vivo analysis. As still there is no report of T-cell and B-cell epitopes of Apis mellifera, this study can be the pioneer in finding effective vaccine against allergens of honeybee. This research also predicted potential B cell epitope regions from an antigenic protein. The most exciting feature of this part of the study is, it presents results of potential B cell epitopes on a single interface, so that, researchers don‘t need to search for every feature (e.g., hydrophilicity, antigenicity, beta turn, surface accessibility etc.) separately. Finally, the study can certainly aid in B cell epitope-based vaccine design research.