Ole e 15 and its human counterpart -PPIA- chimeras reveal an heterogeneous IgE response in olive pollen allergic patients

Abstract

Olive pollen is a major cause of immunoglobulin E (IgE)-mediated allergy in Mediterranean countries. It is expected to become a worldwide leading allergenic source because olive cultivation is increasing in many countries. Ole e 15 belongs to the cyclophilin pan-allergen family, which includes highly cross-reactive allergens from non-related plant, animal and mold species. Here, the amino acid differences between Ole e 15 and its weak cross-reactive human homolog PPIA were grafted onto Ole e 15 to assess the contribution of specific surface areas to the IgE-binding. Eight Ole e 15-PPIA chimeras were produced in E. coli, purified and tested with 20 sera from Ole e 15-sensitized patients with olive pollen allergy by ELISA experiments. The contribution of linear epitopes was analyzed using twelve overlapping peptides spanning the entire Ole e 15 sequence. All the patients displayed a diverse reduction of the IgE-reactivity to the chimeras, revealing a highly polyclonal and patient-specific response to Ole e 15. IgE-epitopes are distributed across the entire Ole e 15 surface. Two main surface areas containing relevant conformational epitopes have been characterized. This is the first study to identify important IgE-binding regions on the surface of an allergenic cyclophilin.

Figure 1

Structure comparison between Ole e 15 and human PPIA. (A) Modeled molecular surface of Ole e 15 (left). Light and dark gray colors indicate different and conserved amino acids between Ole e 15 and PPIA, respectively. The isolated surfaces of different (middle) and conserved (right) amino acids are also shown. (B) Modeled molecular surface pairs of Ole e 15 (left) and PPIA (right) for structure comparison. Three patches (Patch 1, 2 and 3) containing most of the different amino acids between both molecules, and a fourth patch (Patch 4) with conserved amino acids were found. All patches are framed. Light and dark gray colors indicate different and conserved amino acids between Ole e 15 and PPIA, respectively. (A,B) All 3D-models were visualized using PyMOL 2.3 (https://pymol.org/2/). (C) Topological diagram showing the secondary elements of the predicted structure of Ole e 15. Patch 1 comprises the amino acid stretch E43 to A59, including the divergent loop; Patch 2 comprises the N-terminal M1 to A26, and the C-terminal Q138 to S172; Patch 3 comprises A76 to G101; and Patch 4 comprises H61 to T75 and M107 to V134. Gray arrow, β-strand; red cylinder, α-helix; blue straight lines, β-turns and random coil structures.

Figure 2

Modeled molecular surface and sequence and comparison between Ole e 15, PPIA and Ole e 15-PPIA chimeras. (A) Front and back (turned 180° on the vertical axis) modeled surfaces of Ole e 15, PPIA and chimeric Ole e 15-PPIA proteins. Grafted amino acids on the chimeras are colored. All 3D-models were visualized using PyMOL 2.3 (https://pymol.org/2/). (B) Amino acid sequence alignment of Ole e 15, PPIA and the chimeras. Amino acid differences between Ole e 15 and PPIA are shaded in gray. Amino acid differences with a change in the SASA value higher than 30 Ų or higher than 10 Ų but with changes in the amino acid charge state are shown in bold. PPIA amino acids grafted on the chimeras are indicated and framed with the same color as represented in (A). (*), fully conserved residues; (:), amino acids with groups of strongly similar properties; (.), amino acids with groups of weakly similar properties.

Figure 3

Biochemical characterization of Ole e 15-PPIA chimeras. (A) Coomassie Blue staining of 1 μg of the purified chimeras after 15% SDS-PAGE under reducing conditions. (B) Analysis of the purification process of Chimeras 1 and 7 by Coomassie Blue staining after 15% SDS-PAGE of fractions obtained from different purification steps. SF, bacterial soluble lysis fraction; NR, not retained proteins; W5, fifth Ni-NTA agarose wash; E, eluted protein (1 μg, not reduced). (C) Circular dichroism spectra of Ole e 15, PPIA and the chimeras. (D) Bar-graph showing percentage values of each secondary structure elements after spectra deconvolution using the CDNN software.

Figure 4

IgE-binding of Ole e 15, PPIA and Ole e 15-PPIA chimeras. (A) Scatter dot-plot representing the IgE-reactivity of the twenty patients to Ole e 15, PPIA and the chimeras assessed by ELISA. Results are shown as OD_492nm values (arbitrary units). Horizontal bars represent the arithmetic mean. Vertical bars represent the standard error of the mean (±SEM, error bars for duplicates). The dashed line represents the cut-off level of IgE-binding. *P < 0.05; ***P < 0.001, ns (not significant). (B) Visualization of the normalized results per patient in OD in (A) by hierarchical clustering analysis performed with MultiExperiment Viewer (MeV). Red, higher IgE levels. Green, low or nor IgE levels.

Figure 5

Immunological characterization of the IgE-response to Ole e 15, PPIA and Ole e 15-PPIA chimeras of two representative olive pollen allergic patients. (A) Bar graphs showing inhibition values of IgE-binding to immobilized Ole e 15 assesed by ELISA after serum preincubation with Ole e 15, PPIA or the Ole e 15-PPIA chimeras. (B) Bar graphs representing the mean OD_492nm (±SD) values obtained by ELISA for the assessment of the IgE-reactivity of serum samples 6 and 11. The dashed line represents the cut-off level of IgE-binding. (C) Two-tailed Spearman correlation analysis at 95% confidence interval between the obtained ELISA OD_492nm values and the IgE-inhibition values at inhibitor concentrations of 0.5 μg/mL. Spearman ρ, Spearman correlation coefficient.