Crystal structure of a dimerized cockroach allergen Bla g 2 complexed with a monoclonal antibody

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-angstroms resolution. Bla g 2 binds to the antibody through four loops that include residues 60-70, 83-86, 98-100, and 129-132. Cation-pi 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 alpha-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 beta-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.     

Carbohydrates contribute to the interactions between cockroach allergen Bla g 2 and a monoclonal antibody

The crystal structure of a murine mAb, 4C3, that binds to the C-terminal lobe of the cockroach allergen Bla g 2 has been solved at 1.8 ? resolution. Binding of 4C3 involves different types of molecular interactions with its epitope compared with those with the mAb 7C11, which binds to the N-terminal lobe of Bla g 2. We found that the 4C3 surface epitope on Bla g 2 includes a carbohydrate moiety attached to Asn(268) and that a large number of Ag-Ab contacts are mediated by water molecules and ions, most likely zinc. Ab binding experiments conducted with an enzymatically deglycosylated Bla g 2 and a N268Q mutant showed that the carbohydrate contributes, without being essential, to the Bla g 2-4C3 mAb interaction. Inhibition of IgE Ab binding by the mAb 4C3 shows a correlation of the structurally defined epitope with reactivity with human IgE. Site-directed mutagenesis of the 4C3 mAb epitope confirmed that the amino acids Lys(251), Glu(233), and Ile(199) are important for the recognition of Bla g 2 by the 4C3 mAb. The results show the relevance of x-ray crystallographic studies of allergen-Ab complexes to identify conformational epitopes that define the antigenic surface of Bla g 2.     

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

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 approximately 48 mg x 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.     

Mechanisms of allergen-antibody interaction of cockroach allergen Bla g 2 with monoclonal antibodies that inhibit IgE antibody binding

Background

Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2.

Methodology/Principal Findings

Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab′ fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-π interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients.

Conclusions/Significance

Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates.     

Crystal structure of cockroach allergen Bla g 2, an unusual zinc binding aspartic protease with a novel mode of self-inhibition

The crystal structure of Bla g 2 was solved in order to investigate the structural basis for the allergenic properties of this unusual protein. This is the first structure of an aspartic protease in which conserved glycine residues, in two canonical DTG triads, are substituted by different amino acid residues. Another unprecedented feature revealed by the structure is the single phenylalanine residue insertion on the tip of the flap, with the side-chain occupying the S1 binding pocket. This and other important amino acid substitutions in the active site region of Bla g 2 modify the interactions in the vicinity of the catalytic aspartate residues, increasing the distance between them to approximately 4A and establishing unique direct contacts between the flap and the catalytic residues. We attribute the absence of substantial catalytic activity in Bla g 2 to these unusual features of the active site. Five disulfide bridges and a Zn-binding site confer stability to the protein, which may contribute to sensitization at lower levels of exposure than other allergens.     

Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2

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-π interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A/K251A/F162Y showed an ∼100-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.     

Sequence analysis of the first complete cDNA clone encoding an American cockroach Per a 1 allergen

C3, designated as Per a 1.0103 according to WHO/IUIS nomenclature, encoding a novel American cockroach allergen of 395 aa (44.6 kDa), is the first complete cDNA clone with a translatable immunoreactive protein among the reported group 1 cockroach allergens. Its deduced amino acid sequence possesses a signal sequence, phosphorylation sites, mitochondrial energy transfer protein signatures, and four repeats each sharing striking similarity with the corresponding repeats of the other cockroach allergens. The latter similarity suggests that the group 1 cockroach allergens might have evolved from a primordial mitochondrial sequence by exon duplication.     

变应原疫苗的标准化

全球范围生产和销售变应原疫苗,用来诊断和治疗IgE抗体介导的疾病.变应原疫苗是天然物质的混合物.每一种变应原提取物含有蛋白质、碳水化合物、酶、色素等物质,其中,具有变应原活性的物质只占一小部分.传统的变应原疫苗用投料比(重量/体积)标示疫苗,也有用凯氏定氮法测定蛋白氮标示疫苗,但是这两种标示疫苗的方法与疫苗的活性没有必然的关系.缺乏一致的方法来维持产品的一致性,疫苗批与批之间多样化是显然的.所以变应原疫苗的标准化是必须和必然的.变应原疫苗的标准化主要通过建立内部参考品,从变应原疫苗总蛋白组分、总蛋白含量、主要致敏蛋白的组成、主要致敏蛋白的含量、体内体外实验测定总活性这几个方面,每批产品与内部参考品相比较,达到产品批与批之间的一致性.在美国和欧洲的变应原标准化存在一定的差异,本文从美国和欧洲变应原疫苗的标准化历程介绍变应原疫苗标准化的方法和发展趋势.     

德国小蠊变应原Bla g 8的克隆表达及进化分析

通过5’-RACE获得德国小蠊变应原Bla g 8基因的全长cDNA序列,进行生物信息学分析,构建原核表达载体,诱导重组蛋白表达,建立系统进化树,为进一步研究奠定基础。通过5’-RACE技术,PCR扩增获取编码德国小蠊变应原Bla g 8蛋白的全长cDNA序列;采用生物信息学方法分析预测Bla g 8蛋白的信号肽、疏水性、跨膜区、二级结构、三级结构;建立系统进化树;构建原核表达载体pET32a-B8,IPTG诱导重组蛋白表达,并用His-tag抗体Western blotting验证。结果显示,获得编码德国小蠊变应原Bla g 8的全长cDNA序列,其完整阅读框含618个碱基,编码205个氨基酸。序列分析显示该蛋白,肌球蛋白轻链,具有EF手蛋白保守功能域。IPTG诱导获得重组蛋白。获得德国小蠊Bla g 8的完整cDNA序列,成功构建重组原核表达质粒pET32a-B8,并表达出融合蛋白。     

Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients' IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients' IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen's three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.     

Reactivity of German Cockroach Allergen, Bla g 2, Peptide Fragments to IgE Antibodies in Patients' Sera

Bla g 2 is a cockroach allergen of great importance. This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique. Approximately 50% of tested sera showed IgE reactivity to Pichia-expressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2). Only 5.3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients'' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.     

Inhibition of adjuvant arthritis by a DNA vaccine encoding human heat shock protein 60

Adjuvant arthritis (AA) is an autoimmune disease inducible in rats involving T cell reactivity to the mycobacterial 65-kDa heat shock protein (HSP65). HSP65-specific T cells cross-reactive with the mammalian 60-kDa heat shock protein (HSP60) are thought to participate in the modulation of AA. In this work we studied the effects on AA of DNA vaccination using constructs coding for HSP65 (pHSP65) or human HSP60 (pHSP60). We found that both constructs could inhibit AA, but that pHSP60 was more effective than pHSP65. The immune effects associated with specific DNA-induced suppression of AA were complex and included enhanced T cell proliferation to a variety of disease-associated Ags. Effective vaccination with HSP60 or HSP65 DNA led paradoxically to up-regulation of IFN-gamma secretion to HSP60 and, concomitantly, to down-regulation of IFN-gamma secretion to the P180-188 epitope of HSP65. There were also variable changes in the profiles of IL-10 secretion to different Ags. However, vaccination with pHSP60 or pHSP65 enhanced the production of TGFbeta1 to both HSP60 and HSP65 epitopes. Our results support a regulatory role for HSP60 autoreactivity in AA and demonstrate that this control mechanism can be activated by DNA vaccination with both HSP60 or HSP65.     

Therapeutic DNA vaccine encoding peptide P10 against experimental paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.     

Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen

Inhalation of allergens produced by the American cockroach (Periplaneta americana) induces IgE Ab production and the development of asthma in genetically predisposed individuals. The cloning and expression in Escherichia coli of P. americana tropomyosin allergen have been achieved. The protein shares high homology with other arthropod tropomyosins (80% identity) but less homology with vertebrate ones (50% identity). The recombinant allergen was produced in E. coli as a nonfusion protein with a yield of 9 mg/l of bacterial culture. Both natural and recombinant tropomyosins were purified by isoelectric precipitation. P. americana allergen 1 (Per a 1) and Per a 7 (tropomyosin) are to date the only cross-reacting allergens found in cockroaches. ELISA and Western blot inhibition experiments, using natural and recombinant purified tropomyosins from shrimp and cockroach, showed that tropomyosin induced cross-reactivity of IgE from patients allergic to these allergens, suggesting that this molecule could be a common allergen among invertebrates.     

Immune response induced by candidate Sarcoptes scabiei var. cuniculi DNA vaccine encoding paramyosin in mice

Sarcoptes scabiei is the causal agent of the highly contagious disease sarcoptic mange (scabies) that affects animals and humans worldwide. An increasing number of cases of treatment failure is being reported because of drug resistance. The development of a specific vaccine would be a sustainable option for control of this disease. In this study, we cloned and expressed a S. scabiei gene encoding paramyosin (PAR) and investigated the immune response elicited by DNA encoding PAR in mice. The ability of the DNA vaccine to express antigen in COS-7 cells was confirmed by RT-PCR and IFA. The immune response induced by DNA vaccine was investigated by ELISA, splenocyte proliferation assay, and cytokine production assay. Compared to the pVAX1 control group, the PAR DNA vaccination group showed the higher levels of IgG, IgG1, IgG2a, IgE, IgM, stronger lymphocyte proliferation in mouse spleen, and larger production of IL-2, IL-4, IL-5, and IFN-γ in the supernatant of cultures from splenocytes. These results indicated that the PAR DNA vaccine induced a mixed Th1/Th2 response in mice. In conclusion, our results revealed that the S. scabiei PAR DNA vaccine induced both a humoral and cellular immune response, which would provide basic data for the further study to develop an effective vaccine against sarcoptic mange.     

美洲大蠊变应原Cr PI的表达、纯化与免疫学特性鉴定

以阳性噬菌体克隆为模板,通过PCR扩增出目的基因片段并克隆入T载体,经测序证实为美洲大 蠊Periplaneta americana变应原Cr PI后,将该基因亚克隆入表达载体pGEX-5X-1。美洲 大蠊变应原Cr PI在大肠杆菌中得到高效表达,但主要以包涵体形式存在于沉淀中。目的蛋白溶 于6 mol/L盐酸胍并经稀释复性后,经Glutathione Sepharose^TM4B亲和层析,纯度达 90%以上。以蟑螂过敏病人血清进行免疫印迹检测,结果显示重组变应原具有良好的IgE结合活 性。