Background: Several proteins from Curvularia lunata have been identified as important fungal allergens. It will be worthwhile to study the functional aspects of these allergens. The present study aimed at purifying a major allergen and determining its biological function.
Methods: Concanavalin A and Superdex 75 were used to purify Cur l 1 major allergen from C. lunata. Cur l 1 activity was determined qualitatively and quantitatively. Serine protease inhibitors and specific substrate was used to determine the biological function of the protein.
Results: Concanavalin A-bound fraction showed five allergenic proteins, which on Superdex G-75 purification gave a homogenous Cur l 1 protein. Cur l 1 showed IgE reactivity with 80% of the C. lunata hypersensitive patient's sera indicating it to be a major allergen. It showed protease activity on different substrates. Cur l 1's amino terminal sequence, GLTQKSAPWGLGADTIVAVELDSY, showed homology with the alkaline serine protease precursor. Phenylmethylsulfonylfluoride, pefabloc, aprotinin and leupeptin inhibited 70-80% enzymatic activity of Cur l 1 and no inhibition was observed with ethylenediaminetetraacetic acid (EDTA). A dose-dependent hydrolysis of Nalpha-benzoyl-l-arginine ethyl ester-hydrochloride, a specific serine protease substrate was obtained with Cur l 1.
Conclusion: A major glycoprotein allergen Cur l 1 was purified to homogeneity from C. lunata. Amino terminal sequence and biochemical assays identified it as a serine protease.