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Notes: Background The allergen Der p 7 from Dermatophagoides pteronyssinus has been defined by molecular cloning and shown to be an important specificity in 50% of miteallergic patients.Objective This study compares the cDNA sequence and serological reactivity of Der f 7 from D. farinae with Der p 7. Method cDNA encoding Der f 7 was amplified by polymerase chain reaction. sequenced and expressed as a fusion with glutathione-S-transferase for IgE and monoclonal antibody binding studies.Results Der f 7 cDNA encoded a 213 polypeptide containing a predicted 17 amino acid leader sequence, no cysteines and a single N-glycosylation site similar to Der p 7. The predicted 196 residue mature polypeptide had 86% identity lo Der p 7 and a calculated molecular weight of 22 34SDa. No homologues were found in searches of the data banks. The Der f 7 fusion protein showed a single band of 46 k Da by sodium dodccyl snlfute-polyaerylamide gel electrophoresis (SDS-PAGF) and reacted with IgE antibodies in 19/41 (46%) of sera from asthmatic children. The degree of binding was usually 30% of that to Der p 7 consistent with the exposure of the patients to D. Pteronyssinus. Monoclonal antibodies (WH9 and WH22) against Der p 7 reacted with Der f 7 but inhibition studies showed a 10-fold difference in reactivity.Conclusion Der f 7 has a predicted 213 residue polypeptide with 86% homology and serological cross reactivity to Der p 7.

Notes: 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.

Notes: Aspergillus species are common airborne fungi that have been identified as causative agents of extrinsic bronchial asthma. More than 10 allergens from A. fumigatus have been recently characterized by cDNA cloning.The objective of this study is to identify A. fumigatus allergens through immunoblot analysis using sera from asthmatic patients.IgE-binding components of A. fumigatus and IgE cross-reactivity among allergens of different prevalent airborne fungal species were analysed by immunoblot and immunoblot inhibition, respectively, using sera from asthmatic patients. The N-terminal amino acid sequences of major allergens identified were determined by Edman degradation.Among two batches (70 and 41 sera) of asthmatic sera tested, 19 (27%) and 14 (34%), respectively, have IgE immunoblot reactivity towards components of A. fumigatus. A 34-kDa protein that reacts with IgE antibodies in 15 (79%) and 11 (79%) of the 19 and 14 positive samples, respectively, may be considered a major allergen of A. fumigatus. The N-terminal amino acid sequences of the 34 kDa major allergen and the 30.5 and 30 kDa IgE-binding components of A. fumigatus showed sequence identity to that of the vacuolar serine proteinase from A. fumigatus. The results from immunoblot inhibition show IgE cross-reactivity among major allergens of A. fumigatus, P. notatum and P. oxalicum.Results obtained suggest that the 34 kDa major allergen of A. fumigatus may be a vacuolar serine proteinase. There is IgE cross-reactivity among serine proteinase allergens of A. fumigatus, P. notatum and P. oxalicum.

Notes: Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species.〈section xml:id="abs1-2"〉〈title type="main"〉ObjectiveThe object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens.〈section xml:id="abs1-3"〉〈title type="main"〉MethodsBALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis.〈section xml:id="abs1-4"〉〈title type="main"〉ResultsFour (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP.〈section xml:id="abs1-5"〉〈title type="main"〉ConclusionFive monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts.

Notes: The house dust mite allergen Der p 7, which was defined by cDNA cloning, has been shown to react with about 50% of allergic sera and corresponds to or is antigenically related to at least three different sized components in mite extracts. To characterize these entities, monoclonal antibodies (MoAbs) were generated by immunizing BALB/c mice affinity-purified Der p 7-GST (glutathione S-transferase) fusion protein. MoAbs WH9 and WH22 showed positive reactivity to recombinant Der p 7 negative reactivity to GST and the Der p 5-GST fusion protein in ELISA and immunoblotting. The specificity of both MoAbs was confirmed by inhibition of the ELISA activity by recombinant Der p 7 but not by the recombinant Der p 5. Immunoblot analysis demonstrated that both MoAbs showed reactivities to components with molecular weights (mol. wt.) of 31, 30 and 26kDa reactive to both MoAbs. At least six major forms with different pI or size were indicated by 2-D gel analysis. In addition to characterization of Der p 7, both MoAbs may also be considered for use in the standardization of Der p 7 in mite extracts.

Notes: To characterize the 68 kDa allergen of Penicillium notatum (also known as P. chrysogenum), a molecular antibody (MoAb) (P40) was previously generated. For cDNA cloning, three more MoAbs (3F, 5A3, 5G2) were generated in the present study. A mixture of all the four MoAbs was used in cloning of the gene coding for the 68 kDa allergen from a λgt11 cDNA library of P. chrysogenum. A cDNA clone (A6) with DNA insert of about 0.5 kb which encodes for the 3′-terminal nucleotide sequence of the 68 kDa allergen was obtained. The cloned sequence contained two putative N-glycosylation sites. The reduction in molecular weight from 68 to 62 kDa in immunoblotting after treatment of the crude extract of P. notatum with N-glycosidase F indicates that the 68 kDa allergen is a glycoprotein. Nucleotide sequence determination showed that 188 (54%) of the 348 nucleotides of the cDNA sequence obtained were identical to the same region of the nucleotide sequence of the beta-N-acetylglucosaminidase gene of Candida albicans. Although the cDNA clone obtained did not encode the full-length gene of the 68 kDa allergen, polypeptide expressed from the A6 cDNA showed positive immunological reactivities to all four MoAbs used in the cloning experiment and to IgE antibodies in sera of asthmatic patients. There was a loss of immunoblotting activity to the 68 kDa component after absorption of MoAb P40-containing culture supernatant with filters blotted on plaque lawns of cDNA clone A6. Moreover, the immunoblotting activity remained when the MoAbs affinity-purified with filters containing polypeptides encoded by the cDNA insert of clone A6 were used. These two observations indicate that clone A6, which encodes 117 amino acid residues of a putative 560-residue polypeptide, is a cDNA clone of the 68 kDa component of P. notatum. In conclusion, results obtained from cloning and characterization of a partial cDNA clone described in this study suggest that the 68 kDa allergen of P. notatum is a beta-N-acetylglucosaminidase.

Notes: Background Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. However, detailed studies on allergens of this ubiquitous Penicillium species are still lacking.Objective For the characterization of allergens of this prevalent Penicillium species, molecular cloning and expression of the allergen genes of P. citrinum were performed in the present study.Methods Molecular cloning of the allergen genes was performed by using a λUni-Zap XR cDNA library of P. citrinum and serum from an asthmatic patient. The cloned cDNA was excised from the phage vector as a recombinant pBluescript phagemid and sequenced. The cDNA of the IgE-binding clone was expressed as fusion protein with the gultathione-S-transferase. The corresponding natural allergen was analysed by absorption immunoblotting using monoclonal antibody and serum from asthmatic patient. The frequency of IgE-binding to the allergen cloned was analysed by dot immunoassay using recombinant allergen and by immunoblotting using the whole extract of P. citrinum.Results In the screening of cDNA library of P. citrinum using serum from an asthmatic patient, IgE-binding cDNA clones designated SC4 and XL were obtained. The 5′-truncated, 0.7-kb and 1.7-kb cDNA inserts of clones SC4 and XL contained open reading frames of 163 and 503 amino acids, respectively. On alignment, the deduced amino acid sequences showed that 97 (60%) of the 163 amino acids and 376 (75%) of the 503 amino acids were identical to the corresponding amino acid sequence of the human heat shock protein in the hsp70 family. Both recombinant SC4 and XL showed positive SDS-PAGE-immunoblot reactivity to a monoclonal antibody MA3-006 against the human hsp 70 protein. For characterization of the corresponding natural allergen, immunoblotting reactivities of MA3–006 and IgE antibodies to the 70kDa component of P. citrinum have been shown to be disappeared after absorption of these antibodies with the recombinant SC4 protein. Sera from 14 (41%) of 34 Penicillium-allergic patients showed IgE-binding to the recombinant XL protein and the 70kDa component in the extract of P. citrinum.Conclusion Results obtained suggest that hsp 70 is an allergen of P. citrinum and that clones SC4 and XL contain partial cDNAs of this allergen gene.

Notes: Background The 33 kD component has been identified as a major allergen ofPenicillium citrinum, the most prevalent Penicillium species in the Taipei area of Taiwan.Objective This study analyses the isoforms, antigenic cross-reactivity and the N-terminal amino acid sequence of the 33 kD allergen of P. citrinum.Methods The composition of isoforms and antigenic cross-reactivity was analysed by SDS-PAGE and 2D-immunobIotting using MoAbs generated. The N-terminal sequence was analysed by using an automatic gas/liquid phase sequencer.Results Two MoAbs (55A and 34H) against the 33 kD allergen were generated in the present study. In addition to the 33 kD component. MoAb 34H also showed immunoblot reactivity to other components in the crude extract of P. citrinum. Analysed by 2D-immunoblotting. at least six different isoforms of the 33 kD component with pl values ranging from 6.75 to greater than 7.0 were shown to be reactive to both MoAbs and IgE antibodies in serum of an asthmatic patient. Different immunoblot patterns were observed when both MoAbs were reacted with four different strains of P. citrinum used in the present study. Among another six different Penicillium and four different Aspergillus species tested, only an immunoblot reactivity of MoAb 55 A to the 33 kD component of P. brevicompactum was observed. In 2D-immunoblotting. components of P. brevicompactum with an MW of about 33 kD and pi values similar to those of the 33 kD component of P. citrinum reacted with MoAb 55A and IgE antibodies in serum of the asthmatic patient. The N-terminal amino acid sequence of the 33 kD component of P. citrinum was determined to be ANVVQSNVP which was identical to the first 9 N-terminal amino acids of a heat-labile alkaline serine proteinase from P. citrinum.Conclusion Results obtained in the present study suggest that the 33 kD major allergen of P. citrinum may be an alkaline serine proteinase.

Notes: Background Penicillium species have been considered as important causative agents of extrinsic bronchial asthma. However, little is known about the allergens of these ubiquitous airborne fungal species.Objective This study cotnpares the allergenic profiles and allergenic crossreactivity among allergens of three prevaletit airborne Penicillium species.Methods IgE-binding Penicillium components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-immunoblotting using sera from 67 asthmatic patients. The presence of allergenic crossreactivity was analysed by immunoblot inhibition.Results Among the 67 serum samples tested, 15, 14 and 11 samples showed IgE reactivity to components of P. citrinun, P. notatum and P. brevicompactum, respectively. All 15 P. citrinum-positive serum samples showed IgE-binding to a 33 kDa extract component of this species. Thirteen (93%) of the 14 P. notatum-positive serum samples and 10 (91%) of the 11 P. brevicompactum-positive sera also showed IgE reactivity to components with a molecular weight of about 33 kDa in individual Penicillium species. All of the 10 P. brevicompactum 33 kDa component-positive serum samples showed IgE reactivity to the 33 kDa components of the other two Penicillium species tested. Dose-dependent inhibition of IgE-binding to these major allergens was observed when the positive serum sample was absorbed with different amounts of individual allergenic extract as well as with different amounts of extracts prepared from the other two Penicillium species.Conclusion Although different allergenie profiles were observed in the three different Penicillium species tested, results showed that there was an IgE crossreactivity among the 33 kDa group major allergens of P. citrinum, P. notatum and P. brevicompactum.

Notes: 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.