Abstract
Background/ObjectiveEpitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH) allergen from Curvularia lunata using in-silico methods and immunoassay.MethodB cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients' sera.ResultThe homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1–P6) and four T cell (P7–P10) epitopes were predicted by a combination of methods. Peptide P2 (epitope P2) showed E(X)2GGP(X)3KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively) followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding.ConclusionFour B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic diseases.
Highlights
Allergic diseases have multi-factorial aetiology induced by interplay of allergens and immune system components
Template Selection The C. lunata alcohol dehydrogenase (ADH) (CADH) sequence was searched for homology in the Protein Data Bank (PDB)
The suitability of selected model was checked by BioInfoBank Metaserver which returned a 3D Jury score (Jscore) of 279.71
Summary
Allergic diseases have multi-factorial aetiology induced by interplay of allergens (usually proteins) and immune system components. The allergens derived from pollens, insects, animals and fungi have been implicated in allergy. They trigger activation of B and T cells, cross-linking of effector cell bound IgE and release of inflammatory mediators inducing allergic response [1]. The molecular determinants present on allergens are involved in determining the type and intensity of the immune response. Identification of IgE binding and activation of CD4+ helper T cells in response to recognition of allergen epitopes on MHC class II molecules play a crucial role in development of the immune response [2]. The incidence of mold allergy ranges from 6 to 24% in the general population, up to 44% among atopics and 80%
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