Molecular characterization of a fungal cyclophilin allergen Rhi o 2 and elucidation of antigenic determinants responsible for IgE–cross-reactivity

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.

Analysis of allergen components and identification of bioactivity of HSP70 in pollen of Populus deltoides

“…Plant-derived allergens mainly belong to disease-related protein 10 (DRP-10), thomas protein-like protein (TLP), Non-specific lipid transfer proteins (nsLTPs), expansion proteins, calcium binding proteins and profilin protein families [ 26 , 27 ]. These proteins are called pan-allergen in specialized terms such as profilin, because the same family of proteins has a common antigenic determinant, and they can cause a wide range of cross-reactions [ 28 ]. Proteins of the same family share a common domain and are relatively conservative in structure, which caused the common allergen proteins that can be identified in a variety of plants [ 29 – 31 ].…”

Human Monoclonal IgE Antibodies—a Major Milestone in Allergy

“… Creating hybrid molecules or chimeras, by grafting an area containing conformational or linear epitopes into a homologous nonallergenic molecule. Conformational IgE epitopes have been localized on surface patches of the allergen by comparing IgE binding to the chimeras [ 75 , 76 •]. Using NMR-based hydrogen/deuterium exchange to identify areas containing conformational epitopes.…”

Identification and biochemical characterization of Asp t 36, a new fungal allergen from Aspergillus terreus

“…Once identified, preparing recombinant allergens helps in determining the structure and identifying possible epitopes (11). Thereafter, further studies can be done by manipulating the epitopes to develop hypoallergenic vaccines for immunotherapy regimes (12). As human experimentation is ethically restrictive, mice have served as models for many allergic diseases.…”