Determination of the N- and C-terminal sequences required to bind human IgE of the major house dust mite allergen Der f 2 and epitope mapping for monoclonal antibodies (original) (raw)
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Identification of immunodominant IgE epitopes of the major house dust mite allergen Der f 24
International Journal of Molecular Medicine, 2019
Previously, a ubiquinol-cytochrome c reductase binding protein (UQCRB) homolog was identified in the house dust mite (HdM) species Dermatophagoides farinae (der f) as a major allergen. In the present study, the immunodominant immunoglobulin E (IgE) epitope of the protein der f 24 was investigated. Analysis of the homologous amino acid (aa) sequences in der f and human UQcRB was performed. Four different recombinant der f 24 and hybrid proteins formed by integrating der f and human UQcRB sequences were expressed in Escherichia coli, purified using Ni-NTA resins, and IgE-binding activity was determined using IgE-western blotting and enzyme-linked immunosorbent assay (ELISA) experiments. IgE epitopes were further identified by IgE-dot blotting and IgE-ELISA with synthetic polypeptides and HdM-allergic sera. Three-dimensional (3d) structural modeling was used to analyze the position of the immunodominant IgE epitope. The amino acid sequence homology between der f 24 and the human UQcRB protein was determined to be 39.34%. IgE-ELISA and western blot analysis showed that all of the der f-human UQcRB hybrid proteins generated, except for the one lacking 59 residues of the N-terminal region of der f 24, were bound by allergic serum IgE. A synthetic polypeptide consisting of 32 residues of the N-terminal reacted with IgEs from HdM-allergic sera and could be used to generate high titer specific IgG or specific IgE antibodies in immunized mice. The 32-aa N-terminal region of der f 24 was localized to a structural protrusion, which may facilitate specific IgE-binding. These results indicate that the immunodominant IgE epitope of der f 24 is located mainly in a 32-residue region of the N-terminus. These findings may inform the mechanisms of HdM allergy sensitization and allergy immunotherapy development.
The Journal of Immunology, 2019
Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus. Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1–single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to ab...
Molecular determinants for antibody binding on group 1 house dust mite allergens
2012
Background: A unique, cross-reacting monoclonal antibody binds both Der f 1 and Der p 1. Results: A common epitope present on both Der f 1 and Der p 1 was identified and mutated. Conclusion: Mutagenesis and antibody binding analysis allowed identification of IgE antibody binding sites. Significance: The obtained data will lead to the production of hypoallergens with low IgE antibody binding capacity.
The molecular basis of antigenic cross-reactivity between the group 2 mite allergens
Journal of Allergy and Clinical Immunology, 2001
Background: Mite group 2 allergens Der p 2, Der f 2, and Eur m 2 are 14-kDa proteins of unknown function that share 83% to 85% amino acid sequence identity. Isoforms of the allergens within each genus have been identified which differ by 3 or 4 amino acids, but little is known of the influence of group 2 polymorphisms on human IgE antibody binding. Objective: The purpose of this study was to investigate the importance of interspecies and isoform substitutions on murine mAb and IgE antibody binding and on the molecular structure of the group 2 allergens. Methods: Site-directed mutagenesis was used to incorporate the isoform amino acid substitutions onto the Der p 2.0101 sequence. Recombinant allergens were expressed and purified from Escherichia coli and used to evaluate antibody binding by enzyme-linked immunosorbent assay (ELISA). Molecular modeling of the tertiary structure was used to analyze structural differences between the various group 2 allergens.
2007
directed mutagenesis 5.6 Native Der p 2 and two other allergens of the ML domain family bind to cholesterol 5.7 Docking of cholesterol on Der p 2 viii 5.8 Discussion Chapter 6: IgE epitope mapping of Der p 2 6.1 Introduction 6.2 Design and production of Der p 2 alanine mutants with single amino acid substitution. 6.3 Reaction profile of dust mite allergic patients from Singapore and Italy 6.4 IgE epitope mapping of Der p 2 in the Singaporean and Italian populations based on sensitization profiles. 6.5 Evaluation of the changes in secondary structures of mutant E102A and unfolded Der p 2 6.6 Discussion Chapter 7: Evaluation of hypoallergen vaccine candidates for Der p 2 7.1 Introduction 7.2 Specific IgE binding to site directed mutants of Der p 2 in five allergic individuals 7.3 Skin Prick Test 7.4 Mouse IgG antibodies raised against mutant E102A and unfolded Der p 2 are able to block allergic individuals' IgE binding to WT Der p 2 7.5 T cell reactivity and cytokine profile 7.6 Discussion ix Chapter 8: Conclusion and future direction 8.1 Conclusion 8.2 Future directions References Appendix I
Molecular Immunology, 1999
Allergen-speci®c immunotherapy, in which repeated injections of allergens over prolonged periods are used to induce tolerance, has proven an eective treatment of allergy. A major side eect of allergen-speci®c immunotherapy is anaphylactic reaction. House dust mite allergens are major causative factors associated with various allergic diseases. Der f 2 is the major house dust mite allergen composed of 129 amino acid residues. Analysis using deletion mutants of Der f 2 suggested that T-cell epitopes of Der f 2 were multiple in mite-allergic patients. We found that some IgE epitopes were renatured by dialysis of a mixture of two denatured C-and N-terminal deletion mutants, 1±112 and 85±129 in 13 patients out of 14. On the other hand, IgE binding activity was negative in the separately dialyzed fragments and their mixture in each patient tested. Furthermore, we demonstrated that neither of the two separately prepared polypeptides induced in vivo skin prick test reactivity. These ®ndings are important for improvement of T-cell targeting allergen-speci®c immunotherapy and development of monovalent IgE haptens. The use of combinations of overlapping non-anaphylactic fragments of allergen covering all of the T-cell epitopes achieves the removal of IgE reactivity, the cause of harmful anaphylactic reactions, without aecting the T-cell reactivity essential for immunotherapy, oering potentially safer and more eective treatment for allergic disease.
FEBS Letters, 2005
The X-ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 Å resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti-parallel bsheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross-reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.
Characterization of Der f 22 - a paralogue of the major allergen Der f 2
Scientific reports, 2018
We previously identified an expressed sequence tag clone, Der f 22, showing 41% amino acid identity to published Der f 2, and show that both genes are possible paralogues. The objective of this study was to characterize the genomic, proteomic and immunological functions Der f 22 and Der f 2. The full-length sequence of Der f 2 and Der f 22 coded for mature proteins of 129 and 135 amino acids respectively, both containing 6 cysteine residues. Phylogenetic analysis of known group 2 allergens and their homologues from our expressed sequence tag library showed that Der f 22 is a paralogue of Der f 2. Both Der f 2 and Der f 22 were single gene products with one intron. Both allergens showed specific IgE-binding to over 40% of the atopic patients, with limited of cross-reactivity. Both allergens were detected at the gut region of D. farinae by immunostaining. Der f 22 is an important allergen with significant IgE reactivity among the atopic population, and should be considered in the diag...
International Archives of Allergy and Immunology
Background: The aim of this work was to understand the molecular features that trigger the cross-reactivity observed between Der p 5 from Dermatophagoides pteronyssinus, Blo t 5 from Blomia tropicalis, and Der f 5 from D. farinae. Methods: We collected serum from 60 house dust mite (HDM)allergic patients residing in the Dellys area of Boumerdès province in northern Algeria. The presence of specific IgE to Der p 5, Der f 5, and Blo t 5 was analyzed. We performed in silico analysis of the structure of the different allergens in order to identify epitopes that can elicit the cross-reactivity of the sera. Synthetic peptides corresponding to the linear epitope sequence of Der p 5, Der f 5, and Blo t 5 were used to evaluate its implication in the cross-reactivity between the allergens. We also modified the sequence of the conformational epitope of Der p 5 by site-directed mutagenesis to mimic Blo t 5. Results: Several sera of patients allergic to HDM contained specific IgE antibodies to Der p 5 and Blo t 5. We demonstrated that the linear epitope of Der p 5 and Blo t 5 is not involved in the cross-reactivity of the sera. Furthermore, mutations introduced in the sequence of Der p 5 to mimic Blo t 5 could not modulate the cross-reactivity between them. Conclusions: The major linear IgE epitopes of Der p 5 and Blo t 5 are involved in species-specific recognition. Our results may be useful for the development of a hypoallergenic vaccine against HDM group 5 allergens.