cDNA cloning, biological and immunological characterization of the alkaline serine protease major allergen from Penicillium chrysogenum

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.     

Molecular and structural analysis of immunoglobulin E-binding epitopes of Pen ch 13, an alkaline serine protease major allergen from Penicillium chrysogenum

BACKGROUND: Through proteomic and genomic approaches we have previously identified and characterized an alkaline serine protease that is a major allergen (88% frequency of IgE binding) of Penicillium chrysogenum (Pen ch 13). OBJECTIVE: The aim of the present study is to identify the linear IgE-binding epitopes of Pen ch 13. METHODS: IgE-binding regions were identified by dot-blot immunoassay using 11 phage-displayed peptide fragments spanning the whole molecule of Pen ch 13. The minimal epitope requirements for IgE binding were further defined with overlapping peptides synthesized on derivatized cellulose membranes using SPOTs technology. The critical residues on the immunodominant epitopes were mapped through site-directed mutagenesis. The locations of the IgE epitopes identified were correlated with a three-dimensional structure of Pen ch 13. RESULTS: IgE antibodies in 35 serum samples reacted with at least one of the 11 peptide fragments of Pen ch 13. Peptide f-2n (residues 31-61) showed a high-intensity and the highest frequency (77%) of IgE binding. The frequencies of IgE binding to peptide f-4 (residues 93-133), f-1 (residues 1-37) and f-7 (residues 168-206) were 51%, 34% and 31%, respectively. SPOTs assay narrowed down the region of IgE binding of f-2n to residues 48-55 (GHADFGGR). Three, two and one epitope(s) that are four to nine amino acids in length, within f-4, f-1 and f-7, respectively, were found. Site-directed mutagenesis of Pen ch 13 revealed that substitution of His49 and/or Phe52 on Pen ch 13 with methionine resulted in proteins with drastic loss of IgE binding in seven sera tested. Proteins with amino acid replacements at residues 15-18 (RISS), or at residues 112 (I) and 116 (D) have lower IgE-binding reactivity in one of the two patient's sera tested. Substituting residues 117 (W), 119 (V) and 120 (K) also block most of the IgE binding in one of the two patient's sera tested. In addition, replacing residues 203 (V) and 204 (D) along with a deletion at residue 206 (Y) diminished the IgE binding in two serum samples tested. A model was constructed based on the structure of P. cyclopium subtilisin protease that has >90% (256 out of 283 amino acids) sequence identity with Pen ch 13. The major epitope (GHADFGGR) on Pen ch 13 formed a loop-like structure and was located at the surface of the allergen. CONCLUSIONS: Several linear IgE-reactive epitopes and their critical core amino acid residues were identified for the Pen ch 13 allergen. The major linear IgE-binding epitope, 48GHADFGGR55, formed a loop-like structure at the surface of the allergen. Substitution of His49 and/or Phe52 with methionine significantly reduced IgE-binding to Pen ch 13. Mapping of these results on a 3D model of the allergen provides valuable information about the molecular basis of allergenicity for Pen ch 13 and for designing specific immunotherapeutics.     

Characterization of a monoclonal antibody (P40) against the 68 kD major allergen of Penicillium notatum

A monoclonal antibody (MoAb P40) against the 68 kD major allergen of Penicillium notatum (P. notatum) was obtained by immunizing the mouse with a crude extract of P. notatum. Analysed by two-dimensional gel electrophoresis and immunoblotting, P40 reacted with two different isoforms of the 68 kD component of P. notatum with pIs of 5.4 and 5.5. In addition to P. notatum, P40 showed positive ELISA activity to Aspergillus fumigatus (A. fumigatus) but not to components of six other fungi including Alternaria porri, Cladosporium cladosporoides, Aureobasidium pullulans, Fusarium solani, Rhizopus arrhizus and Candida albicans. Analysed by ELISA, MoAb P40 also showed positive activity to two (P. frequentans and P. roseopurpureum) of the 10 other Penicillium species and two (A. terreus and A. flavus) of the four other Aspergillus species tested. SDS-PAGE and immunoblotting studies demonstrated P40 positive reactivity to components with MW of about 67 kD in all these Penicillium and Aspergillus species with positive ELISA activity to P40. Furthermore, immunoblotting activity of MoAb P40 to the 67 kD component of A. niger was also observed. The epitope of the 68 kD allergen of P. notatum recognized by MoAb P40 was resistant to treatment of periodate oxidation with concentration of NaIO4 up to 20 mM. This MoAb may thus be useful in the characterization and purification of the 68 kD allergen from crude extracts, and in the molecular cloning of allergen genes.     

Effects of omalizumab, a humanized monoclonal anti-IgE antibody, on nasal reactivity to allergen and local IgE synthesis.

BACKGROUND: Treatment with omalizumab has been shown to reduce serum free IgE concentrations and to have beneficial effects on allergic airway disease. However, its effect on IgE synthesis is unknown. OBJECTIVE: To determine whether omalizumab therapy affects nasal reactivity to allergen and local IgE production. METHODS: Nineteen patients with perennial allergic rhinitis were treated with intravenous omalizumab every 2 weeks for 26 weeks in an open-label study. Serum free and total IgE concentrations were measured at baseline and every 2 weeks throughout the study. Nasal challenge to dust mite allergen was performed at baseline and after 12 and 24 weeks of treatment. Nasal lavage fluid obtained before and after each nasal challenge was evaluated for mite-specific antibodies, plaque-forming cells, and productive epsilon messenger RNA (mRNA). RESULTS: During treatment, serum free IgE concentrations were decreased by 97% to 99%, and the nasal response to allergen challenge was significantly reduced on days 80 and 164. The postchallenge increase in nasal lavage mite specific IgE was significantly reduced by treatment with omalizumab on day 168. IgE plaque-forming cells and productive epsilon mRNA were not significantly affected by omalizumab treatment. CONCLUSIONS: Omalizumab treatment markedly reduced serum free IgE and the clinical response to nasal allergen challenge. However, the absence of an effect on IgE-secreting B cells and epsilon mRNA in nasal lavage fluid suggests that omalizumab treatment for 6 months does not significantly modulate synthesis of nasal IgE.     

Distribution of IgE in a community population sample: correlations with age,sex, and allergen skin test reactivity

The distribution of total serum IgE determined by the paper radioimmunosorbent test (PRIST) is examined in a large random stratified community population. Prior to logarithmic conversion the distribution of this immunoglobulin is not normal, with almost 40% of values below 20 lU/ml. A normal distribution occurs following such conversion, with a geometric mean value of 32.1 lU/ml. Both age and sex, in addition to atopic status, relate to IgE level. In both sexes highest levels occur among 6- to 14-year-olds, and males have higher levels than females at any given age. Women over age 75 yr have the lowest levels (geometric mean 9.2 lU/ml). Subjects with positive skin test results have several times the concentration of IgE as their nonatopic counterparts.     

Association between IgE response against Bet v I, the major allergen of birch pollen, and HLA-DRB alleles.

The association of the human IgE response against bet v I, the major allergen of birch pollen, and the HLA-DR and DQ phenotype was studied. Birch pollen allergic patients showed a typical case history, positive skin-prick test, and positive RAST with birch pollen extracts. They were divided into two groups. Group I (n = 37) consisted of individuals generating IgE antibodies that selectively reacted with Bet v I. Their serum IgE did not react with minor allergens from birch pollen as tested by immunoblot analysis, nor did they show a response against allergens from a panel of grass and other tree pollen or perenial allergens from animals and fungi as determined by skin-prick test. Patients belonging to group II (n = 34) possessed IgE reacting with Bet v I plus one or more additional allergens. The control group consisted of 637 healthy blood donors. Comparison of the frequencies of RFLP-defined HLA-DR and DQ alleles in patients and the control group revealed that the distribution of DRB3 alleles in group I patients differed significantly from that in the control group: A higher frequency of the DRw52a/c alleles in comparison to the control group (P_corr < 0.02) was observed. In addition, alleles defined by nucleotide sequences coding for the amino acid sequence tyrosine-phenylalanine-histidine at positions 30–32 of the β chain of DR molecules were found with a higher frequency in patients group I(P_corr < 0.02), too. These alleles comprises DRw52a/c and some DRB1 alleles. In patients of group II, no significant differences in the distribution of class II alleles in comparison with the control group were observed. The fact that the association with DRB alleles is observed only in patients with a selective allergen-specific IgE response could mean that, among the genes that contribute to disease susceptibility, the class II genes play a more dominant role in these patients. In conclusion, certain allele encoded in DRB3 and DRB1 genes of the HLA compex might play a functional role in birch pollen allergy.     

IgE binding structures of the major house dust mite allergen Der p I.

The group I allergens Der p I and Der f I are potent allergens of mites from the genus Dermatophagoides. IgE radioimmune dot blots and immunoabsorption with recombinant peptides have been used to define areas of antigenicity. Four linear binding regions comprising residues 15-33, 60-80, 81-94 and 101-111 were found in the N terminal domain and one, 155-187, in the C-terminal domain, but direct evidence for their discontinuous nature is shown. Firstly, the binding activity of residues 60-80 required either C- or N-terminal flanking sequences to express reactivity and secondly a discontinuous determinant was directly demonstrated by the two non-overlapping peptides 53-99 and 101-154 which significantly cross absorbed specificities to one another. This also indicates considerable homogeneity in the antibodies recognising these peptides. The IgE binding peptides could be located to equivalent residues on the X-ray crystallographic structure of the homologous proteins actinidin and papain. The residues 81-94 and 101-111 which gave strong reactivity were located on a flexible loop connecting the domains and represent areas in which synthetic peptides could be expected to retain activity.     

The proteolytic activity of the major dust mite allergen Der p 1 enhances the IgE antibody response to a bystander antigen

BACKGROUND: We have recently demonstrated that immunization of mice with proteolytically active Der p 1, the major dust mite allergen, results in a significant enhancement in total and Der p 1-specific IgE synthesis compared to mice immunized with Der p 1 that has been irreversibly blocked with the cysteine protease inhibitors E-64 and iodoacetamide. Thus, the demonstration that the proteolytic activity of Der p 1 enhances total IgE production, apart from increasing Der p 1-specific IgE, suggests that this allergen may have an IgE-specific adjuvant effect. OBJECTIVE: To determine if the proteolytic activity of Der p 1 has an IgE-specific adjuvant effect. METHODS: We have examined this concept in experiments whereby ovalbumin, used as a bystander antigen, was injected alone or coinjected with either proteolytically active or inactive Der p 1 into groups of mice and IgE and IgG antibody responses were measured. RESULTS: Here we demonstrate for the first time that the proteolytic activity of Der p 1, when given at 10-fold higher concentration, enhances the IgE antibody response to ovalbumin. CONCLUSIONS: These findings show that the proteolytic activity of Der p 1 leads to the augmentation of IgE antibody responses to itself and to other allergens present in the microenvironment.     

Structure of the major cat allergen Fel d I in different allergen sources: an immunoblotting analysis with monoclonal antibodies against denatured Fel d I and human IgE.

In this paper we show the reactivity of monoclonal antibodies (mAbs) and human IgE with Fel d I from different allergen sources in reduced SDS-PAGE immunoblots. By SDS-PAGE analysis of affinity-purified 125I-Fel d I, a 14- to 20-kD band was found, which dissociated under reducing conditions into a 4- to 5-kD chain (chain 1) and a 11- to 15-kD chain (chain 2). In initial immunoblotting experiments with mAbs against Fel d I however, only chain 1 was detected, while the mAbs lost activity upon reduction of Fel d I. Therefore mAbs were raised against reduced and alkylated Fel d I. Two of the four mAbs to 'denatured' Fel d I that were obtained did react with chain 2 on an immunoblot under reducing conditions; the other two reacted with chain 1. The mAbs did not react with native Fel d I. With these mAbs and human IgE, differences between allergen source materials in blot patterns of Fel d I were detected. A variable molecular weight for the protein stained with mAb antichain 2 was found, and occasionally the presence of a 12-kD band stained with mAb antichain 1. Human IgE strongly bound to chain 1 of Fel d I, while only 2 out of 6 sera gave a strong reaction with chain 2. The additional 12-kD band was also recognized by human IgE. In a competitive radioimmunoassay with mAb antichain 1, differences in levels of 'denatured' Fel d I between commercial extracts were quantitated. In vitro 'denatured' Fel d I was generated under high pH conditions. The reactivity of human IgE with this 'denatured' Fel d I was demonstrated in indirect RAST experiments with mAb antichain 1. We conclude that mAb antichain 1 recognizes a form of Fel d I that is not detected by mAb antinative Fel d I, but does react with human IgE.     

Identification of the major allergenic components in Blomia tropicalis and the relevance of the specific IgE in asthmatic patients.

BACKGROUND: Blomia tropicalis has been reported to be a clinically important allergen in house dust. High prevalence of sensitization to B. tropicalis has been noted in asthmatic patients in Taiwan; however, the allergenic components and its impact on asthmatic patients remain to be clarified. OBJECTIVE: To analyze the prevalence of IgE against B. tropicalis and each allergenic component in asthmatic patients. METHODS: A series of recombinant allergenic components were used for skin tests. The B. tropicalis specific IgE in the serum were measured using the Pharmacia CAP System and immunoblot analysis. RESULTS: A total of 131 patients were included in this study: 44% of these 131 patients were allergic to B. tropicalis, 43% of the 80 B. tropicalis-sensitive patients were allergic to Blo t 5, and 75% of the 65 Blo t 5-sensitive patients were allergic to Blo t 5 fragment 3 (Blo t 5 70-117). The sera IgE binding activity to B. tropicalis was repeatedly tested after Dermatophagoides pteronyssinus absorption, and results showed that most patients were concurrently sensitized to D. pteronyssinus and B. tropicalis. In addition, in 2 (18%) of 11 patients, the B. tropicalis sensitization was caused by the cross-reactivity of D. pteronyssinus. CONCLUSION: A high prevalence of B. tropicalis sensitization was detected in our asthmatic patients, and most of them were concurrently sensitized to D. pteronyssinus and B. tropicalis. The major allergenic component and its IgE binding fragments in Blo t 5 have been identified. These allergenic components can be used for the allergenic determination in B. tropicalis and for further immunotherapy.     

Characterization of exposure to low levels of viable Penicillium chrysogenum conidia and allergic sensitization induced by a protease allergen extract from viable P. Chrysogenum conidia in mice

BACKGROUND: Previous evidence by our laboratory has shown that mice inoculated with viable Penicillium Chrysogenum conidia or spores at levels comparable to those found in contaminated buildings induced spore antigen-specific allergic responses. We proposed that mice exposed to low levels of viable P. Chrysogenum conidia would not develop allergic symptoms. We also hypothesized that the symptoms induced by high numbers of conidia were the result of sensitization to allergens released by the conidia. METHODS: C57BL/6 and BALB/c mice were exposed to 1 x 10(2) viable P. Chrysogenum conidia by intranasal instillation weekly for a period of 11 weeks. C57BL/6 mice were also sensitized to a viable P. Chrysogenum conidia protease extract by intraperitoneal injections for a period of 6 weeks followed by intranasal challenge with protease extract, viable, or nonviable P. Chrysogenum conidia for 2 weeks. RESULTS: C57BL/6 mice inoculated with low numbers of conidia developed no significant lung inflammation or increased serum immunoglobulins. Mice sensitized to the protease extract and challenged with both protease extract and viable conidia produced significant increases in serum IgE and IgG1. Mice sensitized to and challenged with the protease extract developed significant eosinophilia and mucus hyperproduction as determined by bronchoalveolar lavage and histopathological examination of lung tissue. CONCLUSIONS: Mice did not develop allergic symptoms in response to challenge with low levels of P. Chrysogenum conidia. Protease allergens from viable conidia induced specific allergic responses in mice, indicating the importance of P. Chrysogenum conidia in allergic sensitization to the organism.     

The T cell reactivity against the major merozoite protein of Plasmodium falciparum.

We have undertaken a systematic search for T cell epitopes within the sequence of the major merozoite surface antigen (GP190) of Plasmodium falciparum. Recombinant polypeptides expressed in E. coli were used to evaluate the reactivity of peripheral blood mononuclear cells (PBMC) from both inhabitants of a rural community of West Africa exposed to P. falciparum transmission and from German patients with diagnosis of acute malaria. Although the proliferative response of the PBMC was in most cases very low, several T cell clones could be established. Deletion analysis of each gp190-derived polypeptide allowed the identification of six different T cell epitopes. Epitopes could be mapped within the dimorphic region of gp190, which also contains the sequences most frequently recognized by sera from adult individuals living in endemic areas.     

In vitro production of IgE by human peripheral blood mononuclear cells. IV. Modulation by allergen of the spontaneous IgE antibody biosynthesis.

Peripheral blood lymphocytes (PBL) from a proportion of grass-sensitive patients, studied during or immediately after the grass pollination period, showed spontaneous production in vitro of grass-specific IgE antibody, whereas PBL from atopic patients sensitive to allergens other than grass pollens or non-atopic individuals did not. Pre-incubation of IgE antibody producing PBL from grass-sensitive patients with minute amounts of a mixed grass pollen (MGP) extract or Rye grass antigen Group I (Rye I) usually resulted in a reduction of the spontaneous production in vitro of IgE protein and in a marked inhibition of the spontaneous production in vitro of grass-specific IgE antibody. This antigen-specific inhibition was not mediated by T lymphocytes, but it was apparently due to a signal directly delivered by antigen to the spontaneously IgE antibody producing cells. The results support the concept that the activity of cells responsible for the persistent IgE antibody formation in vitro in atopic patients can be modulated by antigen.     

Characterization of IgE epitopes of Cuc m 2, the major melon allergen,and their role in cross‐reactivity with pollen profilins

Background Plant profilins are described as minor allergens, although with some exceptions in foods such as melon, watermelon or orange. In fact, they could be responsible for many cross‐reactions among distantly related species. This is likely to be a consequence of the presence of common epitopes. Objective To characterize the B epitopes of Cuc m 2, a model of plant food profilin, using phage display techniques and to compare with other profilins, such as those of timothy grass and birch pollen, and human I profilin, to understand the mechanism of cross‐reaction among members of this family. Methods IgE of melon‐allergic patients was used to select clones from a phage display 12 mer peptide library. After two rounds of screening, Cuc m 2‐specific clones were eluted and the DNA insertion sequenced. The residues of each clone were mapped on the Cuc m 2 surface to define a mimotope, which was also localized on the three‐dimensional surfaces of other profilins. Results Seventeen melon‐allergic patients were selected. Sera from each of them recognized the melon profilin, Cuc m 2, but the majority also recognized Phl p 12 or Bet v 2, timothy grass‐, and birch‐pollen profilins, respectively. A Cuc m 2 mimotope was defined and mapped onto its surface giving the following sequence: S₂W₃A₅Y₆D₉H₁₀T₁₁₁P₁₁₂G₁₁₃Q₁₁₄N₁₁₆M₁₁₇R₁₂₁L₁₂₂. The homologous residues in Phl p 12 and Bet v 2 had almost identical sequences. By contrast, the homologous sequence in human profilin showed many differences. Conclusions The identified mimotope could be involved in cross‐reactions among food and pollen profilins. Many of these cross‐reactions observed in the clinical realm could be explained by the presence of a common epitope found in food and pollen allergens. A new strategy of immunotherapy based on this IgE region could be used in alternative immunotherapy strategies. Cite this as: L. Tordesillas, L. F. Pacios, A. Palacín, J. Cuesta‐Herranz, M. Madero and A. Díaz‐Perales, Clinical & Experimental Allergy, 2010 (40) 174–181.