In silico identification of IgE-binding epitopes of osmotin protein

Abstract

The identification of B-cell epitopes is an important step to study the antigen- antibody interactions for diagnosis and therapy. The present study aimed to identify B- cell epitopes of osmotin using bioinformatic tools and further modify these regions to study the allergenic property. B-cell epitopes were predicted based on amino acid physicochemical properties. Three single point mutations M1, M2, and M3 and a multiple point mutant (M123) were selected to disrupt the IgE binding. These mutants were cloned, expressed and proteins purified to homogeneity. The IgE binding of the purified proteins was evaluated by ELISA and ELISA inhibition with patients' sera. Three regions of osmotin M1 (57-70 aa), M2 (72-85 aa) and M3 (147-165 aa) were identified as potential antibody recognition sites using in silico tools. The sequence similarity search of the predicted epitopes of osmotin using Structural Database of Allergenic proteins (SDAP) showed similarity with known allergens from tomato, kiwifruit, bell pepper, apple, mountain cedar and cypress. Mutants M1, M2 and M3 showed up to 72%, 60% and 76% reduction, respectively in IgE binding whereas M123 showed up to 90% reduction with patients' sera. The immunoblot of M123 mutant showed 40% reduction in spot density as compared to osmotin. All mutants showed decreased inhibition potency with M123 exhibiting lowest potency of 32% with osmotin positive pooled patients' sera. The three B- cell epitopes of osmotin predicted by in silico method correlated with the experimental approach. The mutant M123 showed a reduction of 90% in IgE binding. The present method may be employed for prediction of B- cell epitopes of allergenic proteins.

Figure 1. Comparative analysis of antigenic index of osmotin epitopes and its mutants by DNAstar protean system.

The region between vertical lines show the three regions mutated by site directed mutagenesis. Mutation in epitope regions shows reduction in antigenic index in DNAstar program.

Figure 2. SDS-PAGE analysis of protein expression in culture.

The uninduced and induced stages of expression of osmotin protein was resolved on 12% gel and stained with Coomassie brilliant blue. Lane M was loaded with Molecular weight marker. Lane U uninduced protein expression. Lane 1, 2, 3 shows expression after induction for 1 h, 2 h, 3 h respectively and lane P represents purified protein.

Figure 3. SDS-PAGE profile of purified osmotin and the three mutants.

(a) The proteins were resolved on 12% gel and stained with Coomassie brilliant blue. (b) SDS-PAGE resolved protein was transferred on to nitrocellulose membrane and probed with osmotin positive patients' pooled sera. Lane M was loaded with Molecular weight marker. Lanes O, M1, M2, M3, M123 were loaded with osmotin and the mutants respectively. (c) Graph showing densitometric scan of spots of the blot.

Figure 4. Circular dichroism analysis.

Far-UV CD analysis of purified osmotin protein and M123 mutant. Results are expressed as mean residue ellipticity (y-axis) at a given wavelength (x-axis).

Figure 5. IgE affinity of osmotin, M1, M2, M3, M123 with food allergic patients' sera (n = 22).

Specific IgE values against the respective proteins in patients' sera by ELISA. Specific IgE values ≥3 times of control were taken as cutoff for the positive results. (─) median value. Statistical difference in IgE binding among osmotin and the mutants is p<0.001 was considered statistically significant.

Figure 6. IgE reactivity of osmotin, M1, M2, M3 and M123.

For IgE inhibition, osmotin positive patients' pooled sera were incubated with 0.1–1000 ng of osmotin protein and mutants as inhibitor. The assay was carried out with purified osmotin protein coated on microtitre plate. Competition of IgE binding to osmotin on the solid phase was quantified by ELISA. The (EC₅₀) value for Osmotin is 4 ng and for M2 is 500 ng.