Background:Cladosporium is an important allergenic fungus worldwide. We report here a major allergen of C. cladosporioides.Methods: Major C. cladosporioides allergens were characterized by immunoblotting, N-terminal amino acid sequencing, protein purification and cDNA cloning. Results: Seventy-four sera (38%) from 197 bronchial asthmatic patients demonstrated IgE binding against C. cladosporioides extracts. Among these 74 sera, 41 (55%) and 38 (51%) showed IgE binding against a 36- and a 20-kDa protein of C. cladosporioides, respectively. Both IgE-reacting components reacted with FUM20, a monoclonal antibody against fungal serine proteases. N-terminal amino acid sequencing results suggest that they are vacuolar serine proteases, and the 20-kDa component is possibly a degraded product of the 36-kDa allergen. A corresponding 5′-truncated 1,425-bp cDNA fragment was isolated. The mature protein after N-terminal processing starts with an N-terminal serine that is the ninth residue encoded by the 5′-truncated cDNA. The protein sequence deduced shares 69–72% sequence identity with Penicillium vacuolar serine proteases and was designated as Cla c 9. The purified 36-kDa Cla c 9 allergen showed proteolytic activity with peptide Z-Ala-Ala-Leu-pNA as substrate. IgE cross-reactivity was detected between the purified Cla c 9 and serine protease allergens from Aspergillusfumigatus and Penicillium chrysogenum.Conclusion: We identified a vacuolar serine protease as a major allergen of C. cladosporioides (Cla c 9) and a major pan-allergen of prevalent airborne fungi. IgE cross-reactivity among these highly conserved serine protease pan-fungal allergens was also detectable.

Background: Alkaline/vacuolar serine proteases comprise a major group of pan-fungal allergens from several prevalent airborne fungal species. It is of importance to characterize antigenic determinant(s) recognized by monoclonal antibodies against these major allergens. Methods: The antigenic determinant of fungal serine proteases recognized by a monoclonal antibody, FUM20, was analyzed by dot immunoassay of synthetic peptides immobilized on cellulose membrane. Results obtained were confirmed by wild-type recombinant protease and its mutants. The epitopes were mapped to the structure of serine proteases by molecular modeling. Results: A linear epitope encompassing 9 amino acids from Pen ch 18 (⁶EKNAPWGLA¹⁴) binds FUM20. The corresponding peptide (⁵AKGAPWGLA¹³) from Rho m 2 also binds FUM20. Substitution of K6, P9 or W10 with alanine in this peptide resulted in drastic loss of FUM20 binding. Rho m 2 mutants with single K6A, P9A, P9G, W10A or W10F substitute showed negative immunoblot reactivity against FUM20. However, the Rho m 2 K6R mutant can bind FUM20. Three-dimensional structural models of the FUM20 antigenic determinants on serine proteases were constructed. The lysine residue critical for FUM20 interaction is on the surface of the proteases and solvent accessible. The critical core residue proline is located at the beginning of an α-helix. Conclusions: The lysine, proline and tryptophan residues located on the N-terminal region of fungal serine proteases are critical core amino acid residues recognized by FUM20, a monoclonal antibody against serine protease pan-fungal allergens. These findings advance our understanding of the antigenic structures responsible for the antigenicity of serine protease allergens.

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