Molecular and immunological characterization of cytochrome c: a potential cross-reactive allergen in fungi and grasses

Background: Recombinant allergens are required for component-resolved diagnosis/therapy of allergic disorders. The study was aimed to express and characterize an allergenic protein from Curvularia lunata and study its cross-reactivity. Methods: A clone encoding a 12-kDa protein screened from the cDNA library of C. lunata was sequenced and expressed in pET22b+ vector. The purified protein was characterized by biophysical and immunological methods. Results: The sequence of gene encoding a 12-kDa protein showed homology to cytochrome c. It was expressed in Escherichia coli yielding 0.5 mg protein/l culture and designated as Cur l 3. The absorption and circular dichroism spectrum of Cur l 3 were similar to horse cytochrome c and the protein reacted with cytochrome c antibody. ELISA with different fungal-positive patients' sera showed >= 3 times specific IgE to Cur l 3 compared with healthy controls. Mice anti-Cur l 3 reacted with tropical and temperate grass extracts. Protein also reacted with grass-positive patients' sera. In vitro stimulation of peripheral blood mononuclear cells from C. lunata, fungi or grass-positive patients with it released significant levels of Th2 cytokines. In vivo testing of this protein in allergic patients showed marked positive skin reactivity in 60% fungal and 43% grass-positive cases. Cross inhibition assays (EC50) demonstrated allergenic cross-reactivity of Cur l 3 with fungi and grasses. Conclusions: Cytochrome c, a major allergen from C. lunata was cloned, sequenced and expressed. It was identified as a cross-reactive allergen among fungi and grasses and has potential for clinical application.