A Vacuolar Serine Protease (Rho m 2) Is a Major Allergen of <i>Rhodotorula mucilaginosa </i>and Belongs to a Class of Highly Conserved Pan-Fungal Allergens

Abstract

Background:Rhodotorula mucilaginosa is one of the most frequently encountered species of yeasts in our environment. We reported here a major allergen of R. mucilaginosa. Methods: A major R. mucilaginosa allergen (Rho m 2) was characterized by two-dimensional (2D) immunoblotting, protein sequencing, cDNA cloning and IgE cross-reactivity with fungal serine proteases. Results: Fourty-four sera (28%) from 157 bronchial asthmatic patients showed IgE-immunoblot reactivity against R. mucilaginosa extract. Among these 44 sera, 25 (57%) demonstrated IgE binding against a 31-kDa protein of R. mucilaginosa. Protein sequencing results suggest that it is a vacuolar serine protease. The corresponding cDNA clone encoding a mature protein of 312 residues was isolated. It shares 67–68% sequence identity with vacuolar serine protease allergens from three different Penicillium species (Pen ch 18, Pen o 18 and Pen c 18) and designated as Rho m 2 by the Allergen Nomenclature Committee. The native and recombinant Rho m 2 react with IgE antibodies and monoclonal antibody (MoAb) FUM20 against fungal serine proteases. IgE cross-reactivity between nRho m 2 and nPen ch 18 was observed. It was also detectable between rRho m 2 and rPen o 18. Conclusion: Our results suggest that R. mucilaginosa may also be a significant causative agent of human respiratory allergic disorders. We identified a vacuolar serine protease as a major allergen of R. mucilaginosa (Rho m 2) and a pan allergen of prevalent airborne fungal species. We detected IgE cross-reactivity among these highly conserved serine protease pan-fungal allergens.

Lys, Pro and Trp Are Critical Core Amino Acid Residues Recognized by FUM20, a Monoclonal Antibody against Serine Protease Pan-Fungal Allergens

“…A plasmid carrying the coding sequence of Rho m 2 is available in the laboratory [9] and used as template for polymerase chain reactions (PCR). Rho m 2 mutants carrying a single amino acid substitute at K6, P9 or W10 were generated with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA) according to the manufacturer’s instructions.…”

“…Recombinant proteins were expressed with an N-terminal His 6 -tag and purified in denatured form with Ni-NTA resin as described [9]. Affinity-purified proteins were further separated on denaturing polyacrylamide gel and transferred electrophoretically onto polyvinylidene difluoride membranes (0.45 µm; Millipore, Bedford, Mass., USA).…”

“…We have recently identified alkaline and/or vacuolar serine proteases as a group of highly conserved pan-fungal allergens of prevalent airborne Penicillium and Aspergillus species [6,7,8] as well as the frequently encountered yeast Rhodotorula mucilaginosa [9]. Our immunoblotting data showed that IgE antibodies against components of these fungal species could be detected in 15–30% of serum samples from asthmatic patients [6,7,8,9].…”

“…Our immunoblotting data showed that IgE antibodies against components of these fungal species could be detected in 15–30% of serum samples from asthmatic patients [6,7,8,9]. Most of the positive serum samples showed IgE binding to the 32-/ 34-kDa serine protease(s) with a >60% frequency in different fungal species tested [6,7,8,9]. …”

“…It was found that mAb FUM20 reacted with the vacuolar serine proteases from R. mucilaginosa (Rho m 2), P. chrysogenum (Pen ch 18) , P. citrinum (Pen c 18) , P. oxalicum (Pen o 18) , A. fumigatus (Asp f 18), and the alkaline serine proteases from A. fumigatus (Asp f 13) and A. flavus (Asp fl 13) [9, 10]. However, it does not react with the alkaline serine proteases from P. chrysogenum (Pen ch 13) and P. citrinum (Pen c 13) [10] (table 1).…”