Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species

Background:Penicillium citrinum and Aspergillus fumigatus are prevalent indoor airborne fungal species that have been implicated in human respiratory allergic disorders. It is important to understand the allergenic profile of these fungal species. The purpose of the present study is to characterize a newly identified enolase allergen from P. citrinum and A. fumigatus. Methods: Fungal proteins were separated by two-dimensional (2D) gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Protein spots that reacted with IgE antibodies in serum samples from asthmatic patients were identified and the N-terminal amino acid sequences were determined by Edman degradation. The peptide sequences obtained were utilized in cloning the cDNA of the allergen genes by reverse transcriptase-polymerase chain reaction and the 5′- and 3′-rapid amplification cDNA end reactions. Results: Our results from 2D immunoblotting identified a 47-kD IgE-reactive component in the extracts of P. citrinum and A. fumigatus. The N-terminal amino acid sequences of the 47-kD proteins are homologous to those of fungal enolases. The corresponding enolase cDNA from P. citrinum contains 1,552 bp and encodes a protein of 438 residues. In A. fumigatus, the isolated enolase cDNA has 1,649 bp and contains a 438-amino acid open reading frame. The deduced amino acid sequences of these two enolases have 94% identity. These enolases from P. citrinum and A. fumigatus were expressed in Escherichia coli as a His-tagged protein and designated as rPen c 22 and rAsp f 22, respectively. Sera from 7 (30%) of the 23 Penicillium-sensitized asthmatic patients showed IgE binding to the 47-kD P. citrinum component (Pen c 22) and rPen c 22. In addition, six of seven Pen c 22-positive serum samples have IgE immunoblot reactivity to the 47-kD A. fumigatus component (Asp f 22) and rAsp f 22. A polyclonal rabbit antiserum generated against the N-terminal peptide of Pen c 22 can react with Pen c 22, rPen c 22, Asp f 22 and rAsp f 22. In addition, the presence of IgE cross-reactivity between rPen c 22 and rAsp f 22 and between enolases from A. fumigatus and Alternaria alternata was also detected by immunoblot inhibition. Conclusions: These results demonstrated that a novel enolase allergen from P. citrinum (Pen c 22) and A. fumigatus (Asp f 22) was identified. In addition, IgE cross-reactivity between enolase allergens from A. fumigatus and P. citrinum and between enolases from A. fumigatus and A. alternata was also detected. Results obtained provide more information on fungal enolase allergens.

“…3). This protein has been identified as the vacuolar serine protease allergen of this Penicillium species by using serine protease-specific monoclonal antibodies [37](data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…The 34-kD and the 18-kD IgE-reacting components in figure 2 reacted with monoclonal antibodies FUM20 [37]and PCE2-11 [19](data not shown), respectively. They have been identified as the 34-kD vacuolar serine protease (Asp f 18) [16, 17]and the 18-kD peroxisomal membrane protein (Asp f 3) [20]allergens of A. fumigatus .…”

Section: Discussionmentioning

confidence: 99%

cDNA Cloning and Immunological Characterization of a Newly Identified Enolase Allergen from Penicillium citrinum and Aspergillus fumigatus

Lai¹,

Tam

Tang

et al. 2002

“…The pellet was dissolved in distilled water and dialyzed against 50 m M Tris-HCl, pH 8.5, before loading onto a DEAE SepraSorb cellulose ion exchange column (DE SepraSorb, Sepragen, Hayward, Calif., USA) that has been pre-equilibrated in the same buffer. Fractions with positive immunoblot reactivity to monoclonal antibody (MoAb) PCM10 against the alkaline serine protease allergens from Penicillium and Aspergillus species were collected and pooled [23]. The pooled fractions were then dialyzed against 10 m M sodium phosphate (pH 6.6) and further purified on a CM Sephadex C50 (Pharmacia Biotech, Uppsala, Sweden) cation exchanger that has been equilibrated with 10 m M sodium phosphate (pH 6.6).…”

Section: Methodsmentioning

confidence: 99%

Alkaline Serine Proteinase from Aspergillus fumigatus Has Synergistic Effects on Asp-f-2-Induced Immune Response in Mice

Kurup

Xia

Shen

et al. 2002

Background: Exposure to Aspergillus fumigatus allergens results in the sensitization and the development of allergic bronchopulmonary aspergillosis in susceptible individuals. Aspergillus antigen consists of a number of chemically diverse components and their cumulative or synergistic effect may result in disease. When mice were challenged with individual recombinant allergens, there was only reduced inflammation and immunological responses compared to the whole antigen. Various enzymes identified from A. fumigatus have been thought to cause airway damage. In the present study, we evaluated the effect of exposure to Asp f 13, an alkaline serine proteinase, and Asp f 2 in mice. Methods: BALB/c mice were challenged intranasally with Asp f 2 and Asp f 13 alone and in combination. The antibody response, pulmonary inflammation, and airway hyperreactivity were studied. Results: Results demonstrated no major difference in antibody response and airway responses among the different groups. The inflammatory responses in the lungs, however, showed marked differences in the various groups. Conclusion: In spite of the similar immunological responses in the different groups of mice studied, the results demonstrate enhanced inflammation in the lungs of mice exposed to a combination of both allergens. Allergens with proteinase activity have been found to be involved in airway inflammation and remodeling, which may also apply for Aspergillus-induced allergy.

“…The column was then eluted with a step gradient of 0.1, 0.2 and 0.3 M NaCl. The collected fractions were pooled according to their immunoblot reactivity to MoAb PCM10 against the alkaline serine protease allergens from Penicillium species [15]. The pooled fractions were then dialyzed against 10 m M sodium phosphate (pH 6.8) and further purified on a CM Sephadex C50 (Pharmacia Biotech, Uppsala, Sweden) cation exchanger that has been equilibrated with 10 m M sodium phosphate (pH 6.8).…”

Section: Methodsmentioning

confidence: 99%

“…They have been designated as the group 13 (alkaline serine protease) and the group 18 (vacuolar serine protease) allergens, respectively, for both fungal genera by the Allergen Nomenclature Subcommittee [14]. Monoclonal antibodies (MoAbs) against different epitopes on these fungal allergens have also been generated [9, 15]. Results from molecular characterization confirmed that the group 18 allergens were vacuolar serine proteases [16].…”

Section: Introductionmentioning

confidence: 99%

cDNA Cloning, Biological and Immunological Characterization of the Alkaline Serine Protease Major Allergen from Penicillium chrysogenum

Chou

Lai

Tam

et al. 2002

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.