IgE-binding proliferative responses and skin test reactivity to Cop c 1, the first recombinant allergen from the basidiomycete Coprinus comatus

Abstract

Background: Basidiomycetes spores are ubiquitously distributed, found throughout the year in outdoor and indoor air, and represent relevant sources of aeroallergens associated with allergy and asthma.

Objective: Cloning and characterization of Coprinus comatus (shaggy cap mushroom) allergens is essential to elucidate their molecular characteristics and to improve the diagnosis of allergy.

Methods: A complementary DNA (cDNA) library of C comatus displayed on phage surface was screened with sera of basidiomycete-sensitized individuals. Subcloning and high-level expression of one of the enriched cDNAs allowed the isolation of a [His](6)-tagged recombinant protein formally termed rCop c 1. The allergenic properties of rCop c 1 were investigated in vitro by ELISA, inhibition experiments, immunoblots, and proliferation assays and in vivo by skin tests.

Results: The rCop c 1-encoding cDNA spans 435 bp and contains an open reading frame of 246 bp, predicting a protein of 8.96 kd without significant sequence homology to known proteins. Immunoblots with [His](6)-rCop c 1 fusion protein show a background free IgE-binding band of the expected size that can be completely inhibited by crude C comatus extracts in ELISA. rCop c 1 induced specific proliferative responses in PBMCs of C comatus-sensitized individuals. The incidence of sensitization to rCop c 1 among 92 sera of basidiomycete-sensitized individuals tested in ELISA was 25%, indicating that Cop c 1 is an intermediate allergen. However, prick tests showed that less than 2 pmol of the rCop c 1 protein was able to induce strong specific skin reactions in sensitized individuals.

Conclusions: rCop c 1, the first cloned allergen from the genus Coprinus, fulfills all the criteria required to be classified as a clinically relevant allergen. The data demonstrate at the molecular level the presence of sensitizing molecules among Basidiomycetes, the most important source contributing to the total spore load in the outdoor air.