Abstract
Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity. So far, no precise information on cross-reactive IgE-epitopes of cyclophilins is available. Here, an 18-kDa IgE-reactive cyclophilin (Rhi o 2) was purified from Rhizopus oryzae, an indoor mold causing allergic sensitization. Based on LC-MS/MS-derived sequences of natural Rhi o 2, the full-length cDNA was cloned, and expressed as recombinant (r) allergen. Purified rRhi o 2 displayed IgE-reactivity and basophil degranulation with sera from all cyclophilin-positive patients. The melting curve of properly folded rRhi o 2 showed partial refolding after heat denaturation. The allergen displayed monomeric functional peptidyl-prolyl cis-trans isomerase (PPIase) activity. In IgE-inhibition assays, rRhi o 2 exhibited extensive cross-reactivity with various other cyclophilins reported as allergens from diverse sources including its homologous human autoantigen. By generating a series of deletion mutants, a conserved 69-residue (Asn81-Asn149) fragment at C terminus of Rhi o 2 was identified as crucial for IgE-recognition and cross-reactivity. Grafting of the Asn81-Asn149 fragment within the primary structure of yeast cyclophilin CPR1 by replacing its homologous sequence resulted in a hybrid molecule with structural folds similar to Rhi o 2. The IgE-reactivity and allergenic activity of the hybrid cyclophilin were greater than that of CPR1. Therefore, the Asn81-Asn149 fragment can be considered as the site of IgE recognition of Rhi o 2. Hence, Rhi o 2 serves as a candidate antigen for the molecular diagnosis of mold allergy, and determination of a major cross-reactive IgE-epitope has clinical potential for the design of next-generation immunotherapeutics against cyclophilin-induced allergies.
Highlights
Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity
ELISA in which 5 different recombinant cyclophilin allergens were separately preincubated with IgG from either anti-Rhi o 2 or anti-TV-3 or anti-⌬Rhi o 2 IgG from rabbit antisera (1:100, v/v) and exposed to sera (1:10, v/v) from cyclophilin-allergic patients (n ϭ 8)
Box-whisker plots with medians and minimum to maximum ranges are shown. *, p Ͻ 0.05 for IgE inhibition by anti-TV-3 versus anti-Rhi o 2 and **, p Ͻ 0.001 for IgE inhibition by anti-⌬Rhi o 2 versus antiRhi o 2 IgG, ␣- refers to anti
Summary
Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity. 10 different cyclophilins have been reported from diverse sources such as carrot, birch pollen, dust mite, and Aspergillus spp Most impressive about these allergens is the presence of a high level of sequence conservation across different taxonomic groups leading to IgE-mediated cross-reactivity [12]. We report a new cyclophilin allergen (molecular mass 18 kDa) from Rhizopus oryzae (mucormycosis mold), which showed frequent IgE sensitization within the mold-allergic population and a broad range of cross-reactivity with other reported cyclophilin allergens. This cyclophilin is designated as Rhi o 2 by WHO/IUIS [18]. We report the possible antigenic determinant of the Rhi o 2 structure through sequence deletion and epitope grafting experiments that may describe the molecular basis of crossreactivity among cyclophilin-sensitive patients
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