Allergenicity and antigenicity of wild-type and mutant, monomeric, and dimeric carrot major allergen Dau c 1: Destruction of conformation, not oligomerization, is the roadmap to save allergen vaccines

Reese, Gerald; Ballmer‐Weber, Barbara; Wangorsch, Andrea; Randow, Stefanie; Vieths, Stefan

doi:10.1016/j.jaci.2006.11.699

Cited by 41 publications

(37 citation statements)

References 26 publications

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“…Recently, the crystal structure of a complex of ␤-lactoglobulin and Fab from IgE antibodies of a combinatorial scFv phage display library showed that the allergen is a dimer, and that the Fab fragments are located in positions such that the dimeric ␤-lactoglobulin would be able to cross-link two IgE antibodies (11). Our results suggest that allergen dimerization plays a role in allergenicity to Bla g 2, by increasing IgE antibody cross-linking, in agreement with a study with engineered dimers of carrot allergen Dau c 1 (28). Oligomerization of allergens in their natural form is very common (e.g.…”

Section: Discussionsupporting

confidence: 89%

Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody

Gustchina

Alexandratos

et al. 2008

Journal of Biological Chemistry

103

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The crystal structure of a 1:1 complex between the German cockroach allergen Bla g 2 and the Fab fragment of a monoclonal antibody 7C11 was solved at 2.8-Å resolution. Bla g 2 binds to the antibody through four loops that include residues 60 -70, 83-86, 98 -100, and 129 -132. Cation-interactions exist between Lys-65, Arg-83, and Lys-132 in Bla g 2 and several tyrosines in 7C11. In the complex with Fab, Bla g 2 forms a dimer, which is stabilized by a quasi-four-helix bundle comprised of an ␣-helix and a helical turn from each allergen monomer, exhibiting a novel dimerization mode for an aspartic protease. A disulfide bridge between C51a and C113, unique to the aspartic protease family, connects the two helical elements within each Bla g 2 monomer, thus facilitating formation of the bundle. Mutation of these cysteines, as well as the residues Asn-52, Gln-110, and Ile-114, involved in hydrophobic interactions within the bundle, resulted in a protein that did not dimerize. The mutant proteins induced less ␤-hexosaminidase release from mast cells than the wild-type Bla g 2, suggesting a functional role of dimerization in allergenicity. Because 7C11 shares a binding epitope with IgE, the information gained by analysis of the crystal structure of its complex provided guidance for site-directed mutagenesis of the allergen epitope. We have now identified key residues involved in IgE antibody binding; this information will be useful for the design of vaccines for immunotherapy.Cockroach allergy is associated with the development of asthma and is a risk factor for emergency room admission of asthmatic patients, especially among inner city children living in low-income houses infested with cockroaches (1, 2). Cockroaches release allergens to the environment, which are carried by particles (5-40 m of diameter) that reach the lung by inhalation. Bla g 2 is one of the most important cockroach allergens, eliciting production of specific IgE in ϳ70% of cockroach-allergic patients at exposure levels that are 10 -100-fold lower than those from other common indoor allergens from dust mite and cat (3-5). Exposure to Bla g 2 results in cross-linking of IgE bound to the surface of mast cells or basophils from sensitized patients (i.e. in immunological terms, cross-linking refers to the non-covalent linkages between the allergen and two IgE molecules at the surface of mast cells or basophils), and induces release of potent mediators (histamine, leukotrienes, prostaglandins, etc.) of allergic reactions.We recently solved the crystal structure of Bla g 2, confirming that the overall fold of this allergen corresponds to that of pepsin-like aspartic proteases, and revealing structural elements that explain why it is enzymatically inactive (6, 7). Bla g 2 contains important amino acid substitutions in the area corresponding to the catalytic site. These modifications impair enzymatic function, and the allergen did not show proteolytic activity in standard in vitro assays using casein and hemoglobin as substrates (7). Bla g 2 belongs to a gro...

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Section: Discussionsupporting

confidence: 89%

Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody

Gustchina

Alexandratos

et al. 2008

Journal of Biological Chemistry

103

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“…This effect would not necessarily always occur in artificially oligomerized recombinant allergens. Oligomers engineered artificially from a wild type that is not an oligomer may have reduced allergenicity if misfolding affects the conformation of IgE epitopes or if some epitopes are hidden in the oligomeric interface [61,62]. …”

Section: Relevance Of Oligomerization For Allergic Diseasementioning

confidence: 99%

Relevant B Cell Epitopes in Allergic Disease

Pomés¹

2009

Int Arch Allergy Immunol

131

106

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The 3-dimensional structure of an allergen defines the accessible parts on the surface of the molecule or epitopes that interact with antibodies. Mapping the antigenic determinants for IgE antibody binding has been pursued through strategies based on the use of overlapping synthetic peptides, recombinant allergenic fragments or unfolded allergens. These approaches led to the identification of mostly linear epitopes and are useful for food allergens that undergo digestion or food processing. For inhaled allergens, conformational epitopes appear to be the primary targets of IgE responses. Knowledge of the molecular structure of allergens alone and in complex with antibodies that interfere with IgE antibody binding is important to understand the immune recognition of B cell-antigenic determinants on allergens and the design of recombinant allergens for immunotherapy. Starting with the molecular cloning and expression of allergens, and with the advent of X-ray crystallography and nuclear magnetic resonance techniques, we have been able to visualize conformational epitopes on allergens.

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“…Several strategies have been proposed to reduce allergenicity, including extended deletions of B cell epitopes identified by peptide scanning of the linear sequence [31], multiple mutations predicted to alter the architecture of the protein [29,32,33,34] and oligomerization [35,36,37,38,39]. Along this line, Vieths’ group, using as a model the Daucus carota major allergen Dau c 1, confirmed that disruption of protein conformation is the roadmap to safe allergen vaccines [40]. Nonetheless, allergen variants may turn out not to be ideal for immunotherapy when the extended deletions, required to reduce their allergenicity, destroy crucial T cell epitopes [41,42].…”

Section: Introductionmentioning

confidence: 99%

In Search of a Vaccine for Mouse Allergy: Significant Reduction of Mus m 1 Allergenicity by Structure-Guided Single-Point Mutations

Ferrari

Breda

Longhi

et al. 2011

Int Arch Allergy Immunol

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Background: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. Methods: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. Results: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. Conclusions: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.

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Allergenicity and antigenicity of wild-type and mutant, monomeric, and dimeric carrot major allergen Dau c 1: Destruction of conformation, not oligomerization, is the roadmap to save allergen vaccines

Cited by 41 publications

References 26 publications

Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody

Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody

Relevant B Cell Epitopes in Allergic Disease

In Search of a Vaccine for Mouse Allergy: Significant Reduction of Mus m 1 Allergenicity by Structure-Guided Single-Point Mutations

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