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In Silico Prediction and Threading Based Epitope Mapping of Ompa-Like Outer Membrane Leptospiral Lipoprotein Loa22

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In Silico Prediction and Threading Based Epitope Mapping of Ompa-Like Outer Membrane Leptospiral Lipoprotein Loa22 International Journal Of Scientific & Engineering Research, Volume 6, Issue 3, March-2015 477 ISSN 2229-5518 In silico prediction and threading based epitope mapping of OmpA-like outer membrane Leptospiral Lipoprotein Loa22 Angela Asir Ramasamy Victor1$, Sunil Abraham1$, Jebasingh Tennyson1, Nityananda Pradhan2* $ Authors equally contributed to this paper Abstract — Loa22 is OmpA-like outer membrane protein from Leptospira interrogans characterized in the C-terminus domain which play an important role in the infection and immunological responses of leptospirosis. [1,2]. Phylogenetic tree was constructed for Loa22 by comparing it with Lipoproteins (LipL 71, 45, 41, 31, 32, 21 and 48) and with outer membrane proteins (OmpL 1, 47, 54, 36, 37). The comprising lineages of Loa 22 have largely varying rates of evolution. In the present study the physicochemical properties of Loa22 were assessed. The structure of Loa22 was predicted by threading method in RaptorX server and the structure was energy minimized and validated by using SAVES server. The stereochemical quality and the protein backbone conformations were done by Ramachandran Plot analysis. Four sequential and conformational B cell epitopes of Loa22 were found in Ellipro server and mapped to find the sequential epitopes. The same B cell linear epitopes was also predicted by ABC Pred, BepiPred 1.0 server. All these epitopes were found to be in higher values. The results of the present study will help to elucidate the function of Loa22 and epitope-based vaccine development for Leptospirosis. Key words: Leptospira, Lipoprotein, OmpA, outer membrane protein, Loa22, Phylogenetic analysis, B-cell epitope, List of abbreviations: SAVES: Structural Analysis and Verification Server; MEGA: Molecular Evolutionary Genetic Analysis; PI: Protrusion Index; LBP: Local Bootstrap values; ACC: Auto Cross Covariance NEFF: Number of Effective sequence homologs. 1. Introduction the southern states of India like Kerala, Tamil Nadu and Loa22 an OmpA like Outer membrane protein from Leptospira certain parts of Andhra Pradesh [12-14]. interrogans characterized by C-terminus domain invokes leptospiral pathogenesis. In other bacteria, OmpA acts as a Leptospira genus is sub-classified into 18 genomospecies multifunctional protein involved in cell adhesion, tissue that included both saprophytic and pathogenic species [7, 15]. invasion [3, 4], immune evasion [5], and induction of the Based on serological methods, approximately 300 serovars immune response [6]. Leptospirosis is one of the most have been identified; of which more than 200 are pathogenic widespread zoonotic diseases in the world which is caused by [7, 16, 17]. The availability of genome sequence data for the pathogenic bacteria Leptospira [7]. It is also known as Weil’s different Leptospira strains drives the discovery of new syndrome, and the clinical manifestations of leptospirosis diagnostic tools and vaccines for Leptospirosis [18]. The major occur when humans acquire the pathogen Leptospira from problem associated with Leptospirosis is the diagnosis of the animals via skin or gastrointestinal contact with water, food or disease, as it shows multiple symptoms; very often it is been soil. Clinical symptoms of leptospirosis include high fever, confused with other diseases. bleeding, and renal failure. The major target of Leptospira in the A number of leptospiral outer membrane proteins (OMPs) kidney is the renal proximal tubular cells, and the pre- and lipoproteins have been characterized including OmpL1 treatment with Leptospira outer membrane proteins, (OMPs) [19], LipL41 [20], LipL36 [21], LipL32 [22], LipL21 [23], LipL46 which leads to tubule interstitial nephritis and acute renal [24], LenA [25], Loa22 [23] and Omp52 [19]. However their malfunction. [8] Mortality ranges from 10-15% with Weil’s performance in diagnostic assays for acute leptospirosis or as disease to 70% in the case of pulmonary hemorrhage vaccine candidate have been problematic [19, 25]. Loa22 is syndrome (SPHS) [9-11]. Leptospirosis is endemic in most of highly conserved among pathogenic Leptospira species; Loa22 is essential for virulence of L. interrogans in animal model and represents the first genetically defined virulence factor in 1 School of Biological Sciences, Madurai Kamaraj University, Madurai-625021, Tamil Nadu, India. Leptospira species. More recent studies have revealed that 2 Senior Professor & Head, Department of Psychopharmacology, National Institute Loa22 may play an important role in the infection and of Mental Health and Neuro Sciences (NIMHANS), Hosur Road, Bangalore- immunological responses of Leptospirosis. Some outer 560029, Karnataka, India. * Corresponding author membrane proteins of Leptospiral lipoproteins LipL32 (also Email: [email protected] vaccineTelephone Number:candidates +91-0 80have- 26995108. been problematic [19, 25]. Loa22 is highly conserved among pathogenic Leptospira species; Loa2 is IJSER © 2015 http://www.ijser.org International Journal Of Scientific & Engineering Research, Volume 6, Issue 3, March-2015 478 ISSN 2229-5518 known as Hap1), LipL41 and the porin OmpL1, have been from the species of L. interrogans serogroup found to be protective immunogens that are conserved among Icterohaemorrhagiae serovar copenhageni (strain Fiocruz L1- various pathogenic leptospires. [26, 27]. 130) and Loa22 from L. interrogans serovar Grippotyphosa (Table. 3) were retrieved from protein knowledgebase Considering the potential importance of the outer (UniProt KB) (http://www.uniprot.org/uniprot/). The retrieved membrane protein Loa22 as an epitope-based vaccine sequences were subjected to Multiple Sequence Alignment candidate, we examined the evolutionary relationship of Loa22 with ClustalW algorithm, implemented in Molecular with several outer membrane and Lipoproteins, which showed Evolutionary Genetic Analysis (MEGA 5.2.2) comprising lineages. Given the potential of the Loa22 proteins (http://www.megasoftware.net) by using distance matrix and as diagnostic antigens and vaccine candidates, the was trimmed to consensus. Maximum Likelihood (ML) trees evolutionary relationship of Loa22 was examined, followed by were constructed with 1000 bootstraps at uniform divergence protein threading and mapping of B-cell epitopes from the rates with the distance ‘p’ as the evolutionary model and with conserved region of the alignment to develop a vaccine a data subset to use with missing data treatment [28]. Posterior candidate for Leptospirosis. probability and the conserved regions among the closely related sequences were aligned with MEGA 5.2.2. (Figure 1). 2. Methodology Maximum likelihood method selects the most suitable substitution model. The purpose of the molecular 2.1. Phylogenetic analysis phylogenetic tree was to estimate the relationships among the species represented by the sequences and to understand the A total of 7 sequences of Lipoproteins such as LipL 71, LipL relationships among the sequences themselves without regard 45, LipL 41, LipL 31, LipL 32, LipL 21 and LipL 48 (Table. 1) to the host species. and 5 outer membrane proteins such as Omp L 1, Omp L 47, Omp L 54, Omp L 36, Omp L 37 (Table. 2) IJSER © 2015 http://www.ijser.org International Journal Of Scientific & Engineering Research, Volume 6, Issue 3, March-2015 479 ISSN 2229-5518 Phobious signal predictor [39]. SOSUI [40] was used to characterize whether the protein is soluble or transmembrane in nature. Interproscan [41] analysis of the query protein for domain study showed that it has a match of outer membrane protein, OmpA / MotB, C- terminal (IPR006665) of Omp A like superfamily. A collection of protein fingerprints by PRINTS [42] was used to study the tentative functional assignments for the selected query protein. Since there is no any structure in the form of X- Figure 1: Diagram showing the phylogenetic Ray crystallographic data from PDB all these above comparision of the Putative Lipoprotein Loa 22 works were done only with the primary sequence which has a predicted OmpA domain based on information which was available from Uniprot for sequence identity Loa22. 2.2. Physicochemical parameters 2.3. Threading based-Modelling, Energy Physicochemical analysis including molecular weight, minimization and Validation of the three theoretical pI, amino acid composition, instability index, dimensional protein aliphatic index, and grand average of hydropathicity The retrieved Loa 22 sequence (Uniprot Id: M4QAZ7) (GRAVY) of Loa22 was analysed by using ProtParam tool was submitted into the RaptorX server [29] (http://web.expasy.org/ protparam/). [34]. Prediction of (http://raptorx.uchicago.edu/) to derive the 3-D subcellular localisation of the protein was carried out by structure. The modelled protein structure was viewed CELLOv.2.5 [35].The secondary structure of the protein by using Swiss-PdbViewer (http://www.expasy. was studied by using PSIPRED server [36] BLAST [38] org/spdbv/) and the individual residues were from NCBI was used to compare the query sequence with collected using 100 cycles of steepest descent the database sequence and its homologues. Conserved algorithm carried out in GROMOS96 [30] until the domains [38] were also analysed from CDD of BLAST. side chain interactions in the vicinity is readjusted Signal peptide regions were also predicted by using and brings up lower potential energy and becomes more stable. IJSER © 2015 http://www.ijser.org International Journal Of Scientific & Engineering Research, Volume 6, Issue 3, March-2015 480 ISSN 2229-5518 The
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