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

Abstract

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...

Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2

“…Our approach, which was based on a combination of x-ray crystallography and site-directed mutagenesis, showed the conformational nature of IgE epitopes (13)(14)(15)(16). The strategies for mutagenesis in this study were based on experimentally determined structures of Bla g 2 alone or in complex with antibodies (13)(14)(15). Therefore, the three-dimensional structure of the protein, essential for induction of immune responses by inhaled allergens, was an important consideration in our study.…”

“…There are no computational tools to accurately predict conformational epitopes, and several prediction methods available identify only linear B-cell epitopes (41)(42)(43). Our approach, which was based on a combination of x-ray crystallography and site-directed mutagenesis, showed the conformational nature of IgE epitopes (13)(14)(15)(16). The strategies for mutagenesis in this study were based on experimentally determined structures of Bla g 2 alone or in complex with antibodies (13)(14)(15).…”

“…The primary objective was to mutate residues involved in different IgE antibody-binding sites that had been either previously identified by mutagenesis of two Bla g 2 epitopes defined by x-ray crystallography or newly identified in this study (13)(14)(15)(16). Our findings show mechanisms of allergen-antibody interaction and reveal that Bla g 2 mutants displaying the same overall fold as the wild type allergen display reduced capacity to bind IgE but retain the ability to activate T-cells and modulate their response.…”

Bla g 1 Allergen Levels in Zagreb Area Household Dust

“…Sensitisation and exposure data suggest that Bla g 2 induces strong IgE response at exposure levels that are often 10 to 100 times lower than levels of other common indoor allergens (9,13,42). However, group 1 allergens (Bla g 1 and Per a 1 produced by B. germanica and P. americana, respectively) share homology in their structure and signifi cant crossreactivity (7,8,11,12).…”

“…Cockroach infestation leads to accumulation of allergens in reservoir household dust (particles >10 μm in diameter) (7,12,13). Cockroach allergens have been located all over the insect, especially in the saliva, gut, faeces, cast skins, and all over their dead bodies (8,12).…”