title: cDNA cloning and immunologic characterization of Pen o 18, the vacuolar serine protease major allergen of Penicillium oxalicum.
abstract: Penicillium species are prevalent indoor airborne fungi that have been identified as causative agents of human extrinsic bronchial asthma. In the preparation 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 protease. The purpose of the present study was to define this major P. oxalicum allergen (Pen o 18) through cDNA cloning and immunologic characterization. The cDNA of Pen o 18 was isolated through a combination of reverse transcriptase-polymerase chain reaction and 5'- and 3'-rapid amplification cDNA ends reactions. The primers used in these reactions were constructed according to the internal amino acid sequences of Pen o 18 and the conserved amino acid sequences of fungal serine proteases. Our results showed that a 1897-bp cDNA with an open reading frame of 503 residues was isolated for the proenzyme of Pen o 18. The encoded protein has a 16-residue signal peptide and a 119-residue prosequence. On maturation, the protein has an N-terminal glutamate that is the 136th residue encoded by the cDNA. Apparently the precursor also undergoes C-terminal processing with the cleavage of about 47 amino acids. 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. Recombinant proteins (rPen o 18 and rPen c 18) with the putative mature N-termini and a his-tag were obtained by expressing the corresponding cDNAs in Escherichia coli. Serum samples from 7 asthmatic patients with immunoglobulin E reactivity to the 34-kd component of P. oxalicum also react to his-tagged recombinant Pen o 18. The presence of immunoglobulin E cross-reactivity between rPen o 18 and rPen c 18 was detected by immunoblot inhibition. Two monoclonal antibodies (PCM39 and FUM20) against fungal serine proteases react with rPen o 18, rPen c 18, and the 35/34-kd components in the corresponding crude fungal extracts. These components also react with immunoglobulin E antibodies in serum samples from asthmatic patients. In conclusion, results obtained confirm that the 34-kd major allergen of P. oxalicum is a vacuolar serine 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. Constructs beginning with mature N-terminal can be expressed in E. coli to produce recombinant polypeptides that are reactive to monoclonal antibodies or immunoglobulin E antibodies in serum samples from asthmatic patients. Results obtained may provide useful information and materials for preparation of standardized diagnostic reagents in clinical mold allergy.
authors.affiliation: Department of Medical Research, Veterans General Hospital-Taipei, Taiwan.
authors.forename: H D
authors.initials: HD
authors.lastname: Shen
authors.forename: C W
authors.initials: CW
authors.lastname: Wang
authors.forename: W L
authors.initials: WL
authors.lastname: Lin
authors.forename: H Y
authors.initials: HY
authors.lastname: Lai
authors.forename: M F
authors.initials: MF
authors.lastname: Tam
authors.forename: H
authors.initials: H
authors.lastname: Chou
authors.forename: S R
authors.initials: SR
authors.lastname: Wang
authors.forename: S H
authors.initials: SH
authors.lastname: Han
mesh_terms: D000328:Adult; D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000595:Amino Acid Sequence; D001249:Asthma / Q000097:blood; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood; D008969:Molecular Sequence Data; D010407:Penicillium / Q000276:immunology* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D014617:Vacuoles / Q000201:enzymology
chemical_list: D000485:Allergens; D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C421423:Pen o 18 protein, Penicillium oxalicum; D012697:Serine Endopeptidases