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 > or = 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 (EC(50)) 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.
Sharma
V
V
Institute of Genomics and Integrative Biology, Delhi, India.
Singh
B P
BP
Gaur
S N
SN
Arora
N
N
D000485:Allergens / Q000235:genetics / Q000276:immunology*; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D001249:Asthma / Q000175:diagnosis / Q000276:immunology; D003001:Cloning, Molecular; D003429:Cross Reactions; D045304:Cytochromes c / Q000235:genetics / Q000276:immunology*; D016207:Cytokines / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology; D006109:Poaceae / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012221:Rhinitis, Allergic, Perennial / Q000175:diagnosis / Q000276:immunology; D012882:Skin Tests
D000485:Allergens; D016207:Cytokines; D005656:Fungal Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D045304:Cytochromes c