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

J Biol Chem. 2020 Feb 28;295(9):2736-2748. doi: 10.1074/jbc.RA119.011659. Epub 2019 Dec 27.

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.

Keywords: Rhi o 2; allergen; allergy; cyclophilin; epitope mapping; fungi; immunoglobulin E (IgE).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allergens / genetics
  • Allergens / immunology*
  • Amino Acid Sequence
  • Conserved Sequence
  • Cross Reactions / immunology*
  • Cyclophilins / genetics
  • Cyclophilins / immunology*
  • Cyclophilins / isolation & purification
  • DNA, Complementary
  • Epitopes / analysis*
  • Fungal Proteins / immunology
  • Humans
  • Hypersensitivity / diagnosis
  • Immunoglobulin E / immunology*
  • Peptide Fragments / immunology
  • Rhizopus / immunology*

Substances

  • Allergens
  • DNA, Complementary
  • Epitopes
  • Fungal Proteins
  • Peptide Fragments
  • Immunoglobulin E
  • Cyclophilins

Associated data

  • PDB/1IST