Characterization of enolase allergen from Rhodotorula mucilaginosa.

摘要

Rhodotorula mucilaginosa (also known as R. rubra) is among the most commonly found yeast strains in our environment. However, allergens from R. mucilaginosa have not yet been characterized at the molecular level. The purpose of this study was to characterize the enolase allergen from R. mucilaginosa and examine the allergenic/antigenic cross-reactivity among fungal enolases. The full-length cDNA encoding the R. mucilaginosa enolase was isolated through the reverse transcriptase-polymerase chain reaction in conjunction with the 5'-end and 3'-end rapid amplification cDNA end reactions. The corresponding natural enolase from R. mucilaginosa was identified using two-dimensional gel electrophoresis and N-terminal amino acid sequence analysis. The results showed that the enolase from R. mucilaginosa is a protein of 439 residues and is encoded by a cDNA of 1497 bp. It shares high sequence identity with enolase allergens from Candida albicans (85%), Saccharomyces cerevisiae (76%), Penicillium citrinum (76%), Aspergillus fumigatus (76%), Cladosporium herbarum (76.5%), and Alternaria alternata (74%). A 47-kD component in the R. mucilaginosa extracts was found to react with IgE or rabbit anti-enolase antiserum and has an N-terminal amino acid sequence identical to that deduced from the isolated enolase cDNA. Sera from three (21%) of 14 allergic patients sensitized to R. mucilaginosa showed IgE binding to this 47-kD R. mucilaginosa component and the His-tagged recombinant enolase. A rabbit antiserum against the P. citrinum enolase and a monoclonal antibody (MoAb; Afueno 8) against the A. fumigatus enolase reacted with all 5 fungal enolases tested. However, an MoAb (E2a) generated by using the Saccharomyces enolase as antigen could only recognize the immunizing enolase. In addition, heterogeneity in immunoblot profiles of IgE antibodies in serum samples from 9 allergic patients against 5 different fungal enolases tested was also observed. The presence of IgE cross-reactivity among enolase allergens from R. mucilaginosa, C. albicans and P. citrinum was detected by immunoblot inhibition. In conclusion, a new and cross-reactive enolase allergen from R. mucilaginosa (Rho m 1) was identified. Although enolases are highly conserved allergens among different fungal species, most of the allergic patients examined in this study differed in their IgE reactivity to the 5 different fungal enolases tested. The results obtained will be of value in understanding the role of enolase allergen in clinical mould allergy.