Epitope mapping of the Dermatophagoides pteronyssinus house dust mite major allergen Der p II using overlapping synthetic peptides

304

254

The examination of house dust mite extracts has indicated that over 30 different proteins can induce IgE antibody in patients allergic to the house dust mite. There are however dominant specificities especially the group 1 and 2 allergens which can account for much of the allergenicity of extracts. Of the 19 denominated allergens, the major IgE binding has been reported for the group 1, 2, 3, 9, 11, 14 and 15 allergens. The high-molecular-weight group 11, 14 and 15 allergens have only recently been described and although high IgE binding has been anticipated from immunoblotting, there is a need for considerable corroboration. Similarly, the study of the group 3 and 9 serine protease allergens has been incomplete. The group 4, 5, 7 and 8 allergens have shown intermediate IgE binding and the group 10 tropomyosins are of interest because of their potential cross-reactivity with allergen from disparate species. Although the progress with the production of recombinant group 1 allergens has been recent, many of the allergens can be produced as high IgE-binding polypeptides. The tertiary structure of the group 2 allergens has been determined from recombinant proteins and they are an excellent model for the investigation of modified allergens. An unexpected property of the group 1, 2 and 3 allergens has been the high degree of polymorphism found by cDNA analysis. It has however been possible to identify sequences to represent the variation in the natural allergens. The group 7 and 14 allergens show secondary modifications which vary in different extracts creating batch variation. While some estimate of the importance of allergens can be obtained from IgE binding, few analyses of T-cell responses have been made and these regulate both the development of, and the protection from sensitization.

Section: Recombinant Mite Allergensmentioning

confidence: 99%

Characterization and Immunobiology of House Dust Mite Allergens

Thomas¹,

Smith²,

Hales³

et al. 2002

304

254

“…Natural and recombinant Der f 2 have three disulfide bonds [8,9,10,11]. Their IgE epitopes are dependent on the integrity of the conformational structure rather than the contiguous sequence of amino acids [10,11,12,13,14,15,16]. Their amino acid sequences are homologous to other group 2 allergens from different mite species, esr16 found in moth trachea, and Niemann-Pick type C2 disease protein (NPC2/HE1) [17,18,19,20,21,22].…”

Section: Introductionmentioning

confidence: 99%

Dilution Method to Refold Bacterially Expressed Recombinant Der f 2 and Der p 2 to Exhibit the Secondary Structure and Histamine-Releasing Activity of Natural Allergens

Takai

Takaoka²,

Yasueda³

et al. 2005

Background: Structurally refolded recombinant forms of major house dust mite group 2 allergens, Der f 2 and Der p 2, expressed in Escherichia coli, were prepared by solubilizing the insoluble products with urea and subsequently dialyzing against buffer. In this study, we determined conditions for refolding the urea-denatured recombinant Der f 2 and Der p 2 by one-step dilution as an alternative to dialysis, which requires several steps of handling and much time and cost. Methods: The insoluble bacterial product containing recombinant Der f 2 was solubilized with a buffer containing 8 M urea, and the solution was diluted to various urea concentrations. The refolding efficiency in each dilution was estimated from the height of the peak corresponding to the folded recombinant Der f 2 and that containing the aggregated form on anion exchange chromatography. The structure and allergenicity of the purified recombinant Der f 2 and Der p 2 refolded using the dilution method were analyzed based on circular dichroism and a basophil histamine-releasing assay, respectively. Results: Although the refolding efficiency decreased as the urea concentration in the dilution increased, experimental conditions whereby the protein and urea concentrations in the dilution were less than 0.5 mg/ml and 0.8 M, respectively, achieved maximum refolding efficiency. The recombinant allergens prepared by the dilution method exhibited the secondary structure and histamine-releasing activity of natural allergens purified from mite culture. Conclusions: The dilution method established in this study is more convenient in terms of handling, time, and cost than the dialysis method and will be useful for large-scale production and for the preparation of numbers of mutants to analyze IgE epitopes.

“…For the allergens Der p 2 [11], Fel d 1 [12, 13], Amb a 3 [14], Hev b 1 [15], Chi t 1 [21]and Lol p 5 [16], epitope mapping using synthetic peptides has been previously reported. To determine the complete primary structure of the allergen and to obtain information on the IgE binding epitopes for T cell immunotherapy, epitope mapping is useful.…”

Section: Discussionmentioning

confidence: 99%

“…Epitope mapping with synthetic peptides has been demonstrated to be a suitable tool to locate some B cell epitopes on some allergens [11, 12, 13, 14, 15, 16]. In previous studies, the B cell epitopes on Cry j 1 were analyzed by the specific monoclonal antibodies (mAbs) to Cry j 1, and the presence of five identified epitopes was confirmed [17, 18].…”

Section: Introductionmentioning

confidence: 99%

Identification of a Sequential B-Cell Epitope on Major Allergen (Cry j 1) of Japanese Cedar <i>(Cryptomeria japonica)</i> Pollen in Mice

Tamura¹,

Sasaki

Inouye

et al. 2000

Background: Japanese cedar (Cryptomeria japonica; CJ) pollinosis is one of the most common allergic diseases in Japan. B cell epitopes on Cry j 1, a major allergen of CJ pollen, have been analyzed by the specific monoclonal antibodies to Cry j 1, and most of these epitopes may be conformational, but no previous report has addressed the analysis of sequential epitope mapping with synthetic peptides. The main purpose of the present study is to identify IgE and IgG B cell epitopes on Cry j 1 by using a synthetic peptide approach in mice. Methods: We synthesized 35 overlapping peptides that cover the entire length of Cry j 1 and examined whether mouse IgE and IgG antibodies produced by immunization with Cry j 1 reacted to the Cry j 1 peptides. Results and Conclusion: We found that mouse IgE and IgG antibodies reacted strongly to Cry j 1 peptide No. 15 (¹⁴¹GVEPVHPQDGDALTLRTATN¹⁶⁰), though those antibodies did not react with other peptides. IgE and IgG antibody binding to peptide No. 15 was completely inhibited by Cry j 1 and the peptide. To determine the minimum epitope in peptide No. 15, we conducted an ELISA inhibition test. IgE and IgG antibody binding to peptide No. 15 was inhibited by smaller peptides of this peptide. We found the core of the epitope to be ¹⁴⁵VHPQDGDA¹⁵².