cDNA cloning and immunologic characterization of Pen o 18, the vacuolar serine protease major allergen of Penicillium oxalicum

Penicillium species are prevalent indoor airborne fungi that have been iden tified as causative agents of human extrinsic bronchial asthma. In the prep aration of standardized diagnostic reagents, it is imperative to define the allergens of these ubiquitous fungi. Results from our previous study on P. oxalicum suggest that the 34-kd major immunoglobulin E-reacting component of this prevalent Penicillium species is probably a vacuolar serine proteas e. The purpose of the present study was to define this major P. oxalicum al lergen (Pen o 18) through cDNA cloning and immunologic characterization. Th e cDNA of Pen a 18 was isolated through a combination of reverse transcript ase-polymerase chain reaction and 5'- and 3'-rapid amplification cDNA ends reactions. The primers used in these reactions were constructed according t o the internal amino acid sequences of Pen o 18 and the conserved amino aci d sequences of fungal serine proteases. Our results showed that a 1897-bp c DNA with an open reading frame of 503 residues was isolated for the proenzy me of Pen o 18. The encoded protein has a 16-residue signal peptide and a 1 19-residue prosequence. On maturation, the protein has an N-terminal glutam ate that is the 136th residue encoded by the cDNA. Apparently the precursor also undergoes C-terminal processing with the cleavage of about 47 amino a cids. The cDNA for Pen c 18 (the vacuolar serine protease allergen from P. citrinum) was also isolated for comparison. Contrary to a previous report, the C-terminal region of Pen c 18 is similar to that of Pen o 18. Recombina nt proteins (rPen o 18 and rPen c 18) with the putative mature N-termini an d a his-tag were obtained by expressing the corresponding cDNAs in Escheric hia call. Serum samples from 7 asthmatic patients with immunoglobulin E rea ctivity to the 34-kd component of P. oxalicum also react to his-tagged reco mbinant Pen o 18. The presence of immunoglobulin E cross-reactivity between rPen o 18 and rPen c 18 was detected by immunoblot inhibition. Two monoclo nal antibodies (PCM39 and FUM20) against fungal serine proteases react with rPen a 18, rPen c 18, and the 35/34-kd components in the corresponding cru de fungal extracts. These components also react with immunoglobulin E antib odies in serum samples from asthmatic patients. In conclusion, results obta ined confirm that the 34-kd major allergen of P. oxalicum is a vacuolar ser ine protease. The cDNAs of Pen o 18 and Pen c 18 encode precursor molecules that appear to undergo both N-terminal and C-terminal processing. Construc ts beginning with mature N-terminal can be expressed in E. coli to produce recombinant polypeptides that are reactive to monoclonal antibodies or immu noglobulin E antibodies in serum samples from asthmatic patients. Results o btained may provide useful information and materials for preparation of sta ndardized diagnostic reagents in clinical mold allergy.