Molecular and immunological characterization and IgE epitope mapping of Pen n 18, a major allergen of Penicillium notatum.
The mould genus, Penicillium, is a significant source of environmental aero-allergens. A major allergen from Penicillium notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of Penicillium citrinum, followed by matrix-assisted laser-desorption ionization-time-of-flight MS analysis of the peptide digest. Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence. The cDNA encoded a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N- and C-terminal prosequences. The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi. The Pen n 18 coding sequence was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni(2+)-chelate affinity chromatography. On immunoblots, the purified recombinant protein specifically bound IgE from mould-allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P. citrinum, Pen c 18. When mapping of the allergenic epitopes was performed, at least nine different linear IgE-binding epitopes, located throughout the Pen n 18 protein, were identified. Of these, peptide C12, located in the N-terminal region of the molecule, was recognized by serum from 75% of the patients tested and therefore appears to be an immunodominant IgE-binding epitope.
Yu
Chia-Jung
CJ
Institute of Biochemistry & Molecular Biology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Taipei, Taiwan.
Chen
Yen-Ming
YM
Su
Song-Nan
SN
Forouhar
Farhad
F
Lee
Shu-Hua
SH
Chow
Lu-Ping
LP
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D018604:Epitope Mapping; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007073:Immunoglobulin E; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010408:Penicillium chrysogenum / Q000276:immunology*; D010449:Peptide Mapping
D000485:Allergens; D005656:Fungal Proteins; C455980:Pen n 18 protein, Penicillium chrysogenum; D007073:Immunoglobulin E