Molecular Cloning of a Major Cockroach (Blattella germanica) Allergen, Bla g 2

Arruda, L. Karla; Vailes, Lisa D.; Mann, Barbara J.; Shannon, John D.; Fox, Jay W.; Vedvick, Thomas S.; Hayden, Mary Lou; Chapman, Martin D.

doi:10.1074/jbc.270.33.19563

Cited by 166 publications

(151 citation statements)

References 43 publications

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“…Briefly, the DNA encoding for the mature form of Bla g 2 with a mutation (N93Q) in one of the three N-glycosylation motifs was inserted into the P. pastoris expression vector pGAPZ␣C (Invitrogen) for constitutive expression of the allergen. Recombinant Bla g 2 was expressed and purified from culture media by affinity chromatography over a 7C11 monoclonal antibody (mAb) 3 column as described (15,16). The purity of rBla g 2 was Ͼ95% by SDS-PAGE and mass spectroscopy.…”

Section: Methodsmentioning

confidence: 99%

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

Gustchina

Alexandratos

et al. 2008

Journal of Biological Chemistry

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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: Methodsmentioning

confidence: 99%

“…allergens from dust mite and cat (3)(4)(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.…”

mentioning

confidence: 99%

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

Gustchina

Alexandratos

et al. 2008

Journal of Biological Chemistry

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103

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“…The studies performed at The Johns Hopkins University were approved by the Human Investigation Committee, and blood was drawn after informed written consent was obtained from the patient. IgE antibody levels in sera ranged from 25 to 640 ng of total IgE/ml (average 1721 Ϯ 1722 ng/ml) (2), and 6 -164 ng of IgE against Bla g 2/ml (average 56 Ϯ 45 ng/ml), measured by mAb-based RIA (10). A pool was prepared by mixing sera from six cockroach allergic patients (total IgE, 42 ng/ml; range 25-219 ng/ml; Bla g 2-specific IgE of 33 ng/ml; range 45-164 ng/ml).…”

Section: Methodsmentioning

confidence: 99%

“…Natural or recombinant cockroach allergens have been used to evaluate their importance in allergic disease. Of all B. germanica allergens, patients show the highest prevalence of sensitization (ϳ55-70%) to Bla g 2, and this major allergen was therefore selected as the main focus of our studies (10,11). A Bla g 2 homolog from P. americana also showed a high prevalence of sensitization (81%) among patients with persistent asthma and rhinitis in Taiwan (7).…”

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Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2

Woodfolk¹,

Glesner²,

Wright³

et al. 2016

Journal of Biological Chemistry

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Bla g 2 is a major indoor cockroach allergen associated with the development of asthma. Antigenic determinants on Bla g 2 were analyzed by mutagenesis based on the structure of the allergen alone and in complex with monoclonal antibodies that interfere with IgE antibody binding. The structural analysis revealed mechanisms of allergen-antibody recognition through cation-interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A/K251A/F162Y showed an ϳ100-fold reduced capacity to bind IgE, while preserving the native molecular fold, as proven by x-ray crystallography. This mutant was still able to induce mast cell release. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and the CD4 ؉ T-cell phenotype in peripheral blood mononuclear cell cultures. Although T-cell activating capacity was similar for the KKF mutant and Bla g 2 based on CD25 expression, the KKF mutant was a weaker inducer of the Th2 cytokine IL-13. Furthermore, this mutant induced IL-10 from a non-T-cell source at higher levels that those induced by Bla g 2. Our findings demonstrate that a rational design of site-directed mutagenesis was effective in producing a mutant with only 3 amino acid substitutions that maintained the same fold as wild type Bla g 2. These residues, which were involved in IgE antibody binding, endowed Bla g 2 with a T-cell modulatory capacity. The antigenic analysis of Bla g 2 will be useful for the subsequent development of recombinant allergen vaccines.

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“…In the US, this problem is particularly important in inner city areas where infestation by the German cockroach (Blattella germanica) is common [2][3][4][5]. Several German cockroach allergens have been cloned including Bla g 1, Bla g 2, Bla g 4, Bla g 5 and Bla g 6 [6][7][8][9][10]. Bla g 1 is the only German cockroach allergen that shows antigenic cross-reactivity with an American cockroach (Periplaneta americana) allergen, Per a 1 [7,[11][12][13][14].…”

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IgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1

Pomés

Vailes

Helm

et al. 2002

European Journal of Biochemistry

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Sensitization to cockroach allergens is associated with the development of asthma. Bla g 1 is a German cockroach allergen that shows allergenic cross‐reactivity with American cockroach allergen, Per a 1, and has a molecular structure composed of multiple tandem amino‐acid repeats. Two consecutive repeats are not identical but form a duplex that constitutes a basic molecular unit of Bla g 1. By molecular mass, purified natural Bla g 1 would contain approximately two duplexes. We investigated the pattern of IgE antibody binding to this repeated structure, and whether one or two duplexes are sufficient for IgE binding. Recombinant (r)Bla g 1 duplexes were expressed in Escherichia coli and in Pichia pastoris, and analyzed for monoclonal antibody and IgE antibody binding by ELISA and/or immunoblotting. Optimal rBla g 1 expression was obtained using methanol‐inducible P. pastoris (> 95% pure protein, yield ≈ 48 mg·L−1), and rBla g 1 was produced as multiple molecular forms of molecular mass 43, 32, 21 and 6 kDa, that were the result of proteolytic cleavage. There was an excellent correlation between IgE antibody binding to natural and recombinant Bla g 1 (r = 0.91, n = 29, P < 0.001), and immunoblot analysis showed that a single Bla g 1 duplex was sufficient for IgE antibody binding. The rBla g 1 is suitable for structural studies and a candidate for clinical use in diagnosis of cockroach allergy and development of new forms of immunotherapy.

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Molecular Cloning of a Major Cockroach (Blattella germanica) Allergen, Bla g 2

Cited by 166 publications

References 43 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

Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2

IgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1

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