Background:Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. Methods: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species – P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). Results: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116–125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12–73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. Conclusions: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.

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House dust mites have been reported as one of the most important allergens in Taiwan especially in asthmatic patients. This study was conducted to determine the allergenicity of Blomia tropicalis and sensitization of asthmatic patients in Taiwan. Serial dust samples were collected every month between July 1993 and June 1994 from 13 houses of mite-allergic patients. About 1 m² surface area of a quilt was vacuumed. The floating method was used to collect mites, then identification and counting were performed. Results showed that Dermatophagoides pteronyssinus and B. tropicalis were the two most common species of mites found in allergenic patients’ houses in Taipei. D. pteronyssinus accounted for 52.1% of the total number of mites and was found in every house. B. tropicalis, although not present in every sample, accounted for 44.3% of the total number of mites. The skin test positive reaction to B. tropicalis, D. pteronyssinus and Dermatophagoides farinae were 73.3, 88.3 and 85.0% as determined from 60 allergic patients who attended our allergy clinics. The extract prepared from B. tropicalis was used to determine the allergenicity and contained at least 30 protein components when silver stained. The most frequently detected allergens were proteins with molecular weights of 14.3, 106.5, 94.0, 72.0, 91.9, 63.7, 100.3, 43.6, 27.3, 62.0, 34.7, 18.3, 41.1 and 21.9 kD. The frequency of IgE binding of patient sera to those proteins were 87.0, 65.2, 56.5, 43.4, 39.1, 39.1, 34.8, 30.4, 30.4, 17.4, 17.4, 17.4, 13.0 and 8.7%. The results from immunoblot inhibition showed that there was IgE cross-reactivity among the B. tropicalis and D. pteronyssinus. However, there were two major allergenic components of B. tropicalis not inhibited by D. pteronyssinus with molecular weights of about 14.3 and 27.3 kD. The use of B. tropicalis extract for diagnostic purposes to identify patients with specific sensitivity should be considered in Taiwan.

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