Pen c 1, a novel enzymic allergen protein from Penicillium citrinum

Abstract

A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum. The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.

Mold Allergen, Pen c 13, Induces IL-8 Expression in Human Airway Epithelial Cells by Activating Protease-Activated Receptor 1 and 2

“…We have previously cloned and purified an immunodominant allergen, Pen c 13, from Penicillium citrinum and, using a traditional caseinolytic analysis, characterized it as a serine protease and shown that it contains the conserved catalytic triad motif of serine proteases (27). In the present study, we showed that Pen c 13 induces proinflammatory cytokine release by human airway epithelial cells (HAECs) and activates PARs through the Ca 2ϩ -signaling pathway.…”

“…Pen c 13 was purified to homogeneity from P. citrinum culture medium as described previously (27). Briefly, Pen c 13 was isolated by a combination of anion-exchange (HiTrap DEAE-Sepharose Fast Flow; 0.7 ϫ 2.5 cm) and cation-exchange (HiTrap CM-Sepharose Fast Flow; 0.7 ϫ 2.5 cm) chromatography.…”

“…It was shown to be pure by sequencing of the 15 N-terminal amino acids, SDS gel electrophoresis, and liquid chromatography/tandem mass spectrometry (MS) analysis of the tryptic peptides. Its serine protease activity was measured using the azocasein assay as in our previous study (27).…”

Subcellular and Functional Proteomic Analysis of the Cellular Responses Induced by Helicobacter pylori

“…Cultures of transformed cells were grown to an A 600 of 0.6, and then expression was induced by addition of 1 mM isopropyl ␤-D-thiogalactoside for 3 h. The recombinant His 6 -ureaseA fusion protein was purified using a Ni 2ϩ chelate affinity column (19). The purified protein showed a single band on SDS-PAGE and was identified by mass spectrometry.…”

Allergy and Dermatophytes

“…Subtilisin Carlsberg (alcalase) produced by Bacillus licheniformis is frequently used in detergent formulations, and allergic respiratory reactions to alcalase have been reported among exposed factory workers (12,88). More recently, a novel subtilisin homologue with IgE antibody binding properties derived from the fungus Penicillium citrinum was identified (110). This allergen, Pen c 1, has a high degree of amino acid sequence identity to Tri r 2 (J. Woodfolk, unpublished data).…”