Effects of Ser47-Point Mutation on Conformation Structure and Allergenicity of the Allergen of Der p 2, a Major House Dust Mite Allergen

PURPOSE: Hypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined. METHODS: S47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined. RESULTS: S47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2. CONCLUSIONS: The mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.

Structures and Antigenic Epitopes of Dust Mite Allergens: Der p 2 and Der f 2: a Comparative Study / 现代医院

Objective To compare and analyze the primary and secondary structures and antigenic epitopes of the two allergens: Der p 2 and Der f 2. Methods The protein sequences of Der p 2 and Der f 2 were downloaded online. The primary and secondary structures of the dust mite allergens were compared and analyzed bioinformatically to determine the potential epitope and signal peptide sites. Results Both Der p 2 and Der f 2 contained 146 amino acids and 9 potential protein binding sites with a secondary structure that mainly contains [3 - sheets, and there might be signal peptides site at the 1st 17th segment of the N - terminus. B cell epitopes analysis revealed that both Der p 2 and Der f 2 have 9 potential linear B epitopes and 2 conformational B epitopes. NetMHCⅡserver prediction showed Der p 2 contains 6 high affinity sites, whereas Der f 2 0nly contains 5. Conclusion This study may lay the foundation for further research of the biochemical function of the 2 allergens and contribute to vaccine development for allergen - specific immunotherapy.

A specific immune therapeutic effect of Der p2 T cell epitope vaccine on asthma mice / 中国血吸虫病防治杂志

Objective To investigate the specific immune therapeutic effect of the T cell fusion peptide vaccine from group II allergens from Dermatophagoides pteronyssinus(Der p2). Methods Thirty mice were randomly divided into three groups, namely a negative control group(a PBS group),positive control group(an asthma group)and protein Der p2 T cell fusion epit?ope for specific immunotherapy(SIT)group(a Der p2 T group). The extract of house dust mites(HDM)was used to establish the asthmatic models in BALB/c mice,and the PBS group was always used with PBS buffer. Thirty minutes before spray inhala?tion from 25 to 27 days,the mice of the Der p2 T group were respectively injected subcutaneously with the therapeutic proteins for SIT,then the serum and bronchoalveolar lavage fluid(BALF)were collected. ELISA was used to assay the levels of IFN?γ, IL?4,and IL?13 in BALF,as well as serum levels of specific IgE and IgG2a. The lung tissue sections were stained with haematox?ylin and eosin(H&E)for pathological examinations. Results The ELISA detection revealed that the number of eosinophil in BALF of the asthmatic mice was(5.57 ± 0.64)× 105/ml,which was significantly higher than that in the PBS group[(0.50 ± 0.30)× 105/ml,P < 0.01],the number of eosinophil in the Der p2 T immunotherapy group decreased significantly[(3.45 ± 0.36)×105/ml,P<0.01]. The content of IFN?γin the PBS group,asthma group and Der p2 T group were(267.00 ± 21.98), (155.80 ± 20.53)pg/ml and(234.40 ± 24.46)pg/ml respectively. Compared with the asthma group,the mice with Der p2 T vac?cine specific immune treatment produced a high level of IFN?γ(P<0.01). The content of IL?4 in the PBS group,asthma group and Der p2 T group were(23.40 ± 5.96),(53.28 ± 8.26)pg/ml and(30.00 ± 5.50)pg/ml respectively. Compared with the asth?ma group,the content of IL?4 in the mice of the Der p2 T treatment group was significantly lower(P<0.01). Compared with the asthma group[(308.10 ± 28.32)pg/ml],the content of IL?13 in BALF of the mice in the Der p2 T treatment group was signifi?cantly decreased,which was[(174.50 ± 25.99)pg/ml,P<0.01]. The content of IL?13 in the PBS group was(95.99 ± 31.14) pg/ml. The lung tissue sections showed that the lung inflammation in the p2 T Der group was significantly less than that in the asthma group,and the inflammatory cell infiltration was significantly decreased,and airway epithelial construction remodeled. Conclusion The Der p2 T cell fusion epitope,which is as vaccines for specific immunotherapy with asthma models,can allevi?ate effectively allergic inflammation of airway and lung in the mice,and it may be used as a candidate vaccine for asthma.

Effect of Amino Acid Polymorphisms of House Dust Mite Der p 2 Variants on Allergic Sensitization

PURPOSE: The sequence variations of the Der p 2 allergen of Dermatophagoides pteronyssinus diverge along 2 pathways with particular amino acid substitutions at positions 40,47,111, and 114. The environmental prevalence and IgE binding to Der p 2 variants differ among regions. To compare IgE binding to Der p 2 variants between sera from Bangkok, Thailand and Perth, Western Australia with different variants and to determine the variant-specificity of antibodies induced by vaccination with recombinant variants. METHODS: The structures of recombinant variants produced in yeast were compared by circular dichroism and 1-anilinonaphthalene 8-sulfonic acid staining of their lipid-binding cavity. Sera from subjects in Bangkok and Perth where different variants are found were compared by the affinity (IC50) of IgE cross-reactivity to different variants and by direct IgE binding. Mice were immunized with the variants Der p 2.0101 and Der p 2.0110, and their IgG binding to Der p 2.0103, 2.0104, and 2.0109 was measured. RESULTS: The secondary structures of the recombinant variants resembled the natural allergen but with differences in ANS binding. The IC50 of Der p 2.0101 required 7-fold higher concentrations to inhibit IgE binding to the high-IgE-binding Der p 2.0104 than for homologous inhibition in sera from Bangkok where it is absent, while in sera from Perth that have both variants the IC50 was the same and low. Reciprocal results were obtained for Der p 2.0110 not found in Perth. Direct binding revealed that Der p 2.0104 was best for detecting IgE in both regions, followed by Der p 2.0101 with binding to other variants showing larger differences. Mouse anti-Der p 2.0101 antibodies had a high affinity of cross-reactivity but bound poorly to other variants. CONCLUSIONS: The affinity of IgE antibody cross-reactivity, the direct IgE binding, and the specificities of antibodies induced by vaccination show that measures of allergic sensitization and therapeutic strategies could be optimized with knowledge of Der p 2 variants.

Association of Single Nucleotide Polymorphisms in the MD-2 Gene Promoter Region With Der p 2 Allergy

PURPOSE: Sensitization to house dust mite (Dermatophagoides pteronyssinus) is a considerable risk factor for the progression of allergic disease. The group 2 allergen from Dermatophagoides pteronyssinus, Der p 2, is considered a major one in patients with specific immunoglobulin E (IgE) to Der p 2. Der p 2 has structural homology with myeloid differentiation 2 (MD-2), which is involved in the lipopolysaccharide-binding component of the Toll-like receptor 4 signaling pathway and the development of inflammation. The aim of this study was to examine the genetic association of single nucleotide polymorphisms (SNPs) in the promoter region of MD-2 with Der p 2-sensitive allergy. METHODS: We investigated associations between cohort's characteristics, including 280 allergic and 80 healthy subjects by examining total IgE, eosinophils, D. pteronyssinus-specific IgE, Der p 2-specific IgE, the number of IgE-producing B cells induced by Der p 2, and the odds ratio of allergic symptoms. RESULTS: Based on the 1,000 genome project data, the minor allele frequencies of the rs1809441 and rs1809442 are 0.467 and 0.474, respectively. However, the correlation of linkage disequilibrium (LD) between these 2 SNPs is D'=1, the genotype frequencies of the 2 MD-2 (LY96) SNPs (rs1809441 and rs1809442) that are located nearby were significantly different between allergic and health subjects: the TT genotype of rs1809441 and the GG genotype of rs1809442 were more frequent in allergic subjects than in healthy subjects (16.1% vs 2.5% in both genotypes). The allergic patients with these genotypes exhibited significantly higher levels of D. pteronyssinus-specific IgE and Der p 2-specific Ig E, and a larger number of Der p 2-activated B cells. In addition, these 2 SNPs in the MD-2 promoter region were significantly associated with the prevalence of nasal, skin, and asthmatic allergic symptoms. CONCLUSIONS: Our results indicated that 2 SNPs in the MD-2 promoter region were significantly associated with Der p 2-specific Ig E, and thereby suggest that these SNPs may play a major role in susceptibility to Der p 2-triggered immune responses in a Taiwanese population.

Enhanced Allergic Inflammation of Der p 2 Affected by Polymorphisms of MD-2 Promoter

PURPOSE: Myeloid differentiation-2 (MD-2) has been associated with endotoxin and inflammatory disorders because it can recognize lipopolysaccharide (LPS) binding and attenuate Toll-like receptor 4 (TLR4)-mediated signaling. However, its role in allergic inflammation has yet to be clarified. We examined whether single nucleotide polymorphisms (SNPs) in MD-2 promoter can affect MD-2 expression and aimed to clarify the relationship between Der p 2 allergy and SNPs of MD-2 promoter. METHODS: The function of SNPs of MD-2 promoter and the effects of cytokines and immunoglobulin on the secretion and mRNA expression were investigated in 73 allergic subjects with different MD-2 gene promoter variants. Peripheral blood mononuclear cells were cultured with or without LPS in the presence of Dermatophagoides pteronyssinus group 2 allergen (Der p 2), followed by mRNA extraction and cytokine expression analysis. The culture supernatants were collected for cytokine measurement. RESULTS: Patients with the MD-2 promoter SNPs (rs1809441/rs1809442) had increased mRNA expressions of MD-2, epsilon heavy chain of IgE (Cepsilon), and interleukin (IL)-8; however, only MD-2 and IL-8 were further up-regulated after Der p 2 stimulation. Patients with SNPs of MD-2 promoter tended to have high levels of IL-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-alpha after Der p 2 and LPS stimulation. Increased secretions of IL-6, IL-8, and IL-10 were found to be up-regulated by Der p 2 stimulation, and an increased secretion of IFN-gamma and decreased secretion of IL-4 were noted after LPS stimulation. CONCLUSIONS: The high levels of proinflammatory cytokines secreted by Der p 2 were predetermined by MD-2 promoter SNPs (rs1809441/rs1809442). Through cytokine secretion by Der p 2 and LPS, these SNPs may serve as an indicator of the pathological phenotype of Der p 2-induced allergic inflammation.

Der p2 Internalization by Epithelium Synergistically Augments Toll-like Receptor-Mediated Proinflammatory Signaling

PURPOSE: House-dust-mite (HDM) major allergen Der p2 shares homology and function with Toll-like receptor (TLR) signaling protein myeloid differentiation-2 (MD2) and may lead to airway inflammation. Should Der p2 be internalized by human airway epithelium, it has the theoretical propensity to potentiate epithelium activation. This study aimed to demonstrate the internalization of Der p2 by airway epithelium and to investigate the effects of Der p2 on MD2 expression and epithelium activation. METHODS: Internalization of recombinant, enhanced green fluorescent protein-labelled Der p2 (rDer p2-EGFP) into human airway epithelium (BEAS-2B) was tracked by laser confocal microscopy and confirmed by immunoblotting. Reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemical staining were used to determine the effect of Der p2 on MD2 expression in vitro and ex vivo. Expression of messenger RNA (mRNA) encoding receptors/cytokines was measured by RT-PCR. Secretion of interleukin-6/interleukin-8 (IL-6/IL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Internalization of Der p2 by BEAS-2B was confirmed by confocal microscopy and immunoblotting using rDer p2-EGFP and rDer p2, respectively. Expression of MD2 protein was increased in BEAS-2B and human nasal polyp airway epithelium cultured with rDer p2. Recombinant Der p2-cultured BEAS-2B caused little spontaneous IL-6/IL-8 secretion but significantly augmented by TLR ligand LPS. IL-6 secretion was up-regulated after MD2 transfection. Internalization of Der p2 was reduced by TLR2 RNA knockdown. Dexamethasone, calcitriol, anti-MD2/anti-TLR2 antibodies, and signalling inhibitors significantly reduced LPS+Der p2-induced IL-6/IL-8 secretion. CONCLUSIONS: Human airway epithelium may internalize Der p2, which potentiates the response to environmental proinflammatory stimuli through MD2 and TLRs. This study highlights a novel mechanism and alleviates IL-6/IL-8 secretion in mite-induced airway inflammation.

Detection of Serum IgE Specific to Mite Allergens by Immuno-PCR

BACKGROUND: Although a skin test is the primary option for detecting allergen-specific IgE in clinics, the serum IgE immunoassay is also important because it allows for the diagnosis of allergy without any accompanying adverse effect on the patient. However, the low detection limit of IgE levels by immunoassay may restrict the use of the method in some occasions, and improving its sensitivity would thus have a significant implication in allergy-immunology clinics. METHODS: In this study, we attempted to detect specific serum IgE by using immuno-polymerase chain reaction (IPCR) which combines the antigen-antibody specificity of enzyme-linked immunosorbent assays (ELISAs) with the amplification power of PCR. RESULTS: Our results demonstrated that Blo t5-specific serum IgE can be detected by IPCR with a 100-fold higher sensitivity than ELISA, and cross-reactivity of serum IgE to other mite allergens is able to be analyzed by using only 0.3 microliter of serum sample. Use of real-time IPCR seemed to permit more convenient determination of specific serum IgE as well. CONCLUSION: We believe that IPCR can serve as a valuable tool in determining specific serum IgE, especially when the amount of serum sample is limited.

The effect of Der p 2 antigen on the production of interferon gamma by CD3+T, CD56+NK and CD3+CD56+TNK cells from patients with mild persistent allergic asthma / 천식및알레르기

BACKGROUND: Asthma is a chronic inflammatory disease of the bronchial mucosa and is associated with excess production of Th2 cytokines (IL-4, IL-5) relative to Th1 cytokine (IFN-V). The NK cell and TNK cell are supposed to be involved in the pathogenesis of allergic inflammation by cytokine regulation. OBJECTIVES: The aim of this study was to investigate the effect of allergen (Der p 2) on the production of IFN-V by CD3+T cell, CD56+NK cell and CD3+CD56+TNK cells in patients with mild persistent asthma. METHOD: Peripheral blood mononuclear cells (PBMCs) from patients with mild persistent asthma (n=12) who were sensitive to dust mite, were cultured with or without Der p 2 for 3 days, and phorbol ester plus calcium ionophore and intracellular protein transport inhibitor were added 4 hours before staining. A three-color flow cytometric analysis was done to detect intracytoplasmic IFN-V, surface DC3 and CD56 antigen simultaneously. RESULTS: When PBMCs were cultured only in media, there were no significant differences in the percentage of IFN-V positive CD3+T cell, CD56+NK cell and CD3+CD56+TNK cells between asthmatic patients and normal subjects. However, there were significant decreases in the percent change of IFN-V positive CD3+T cell, CD56+NK cell and CD3+CD56+TNK cell in asthmatic patients comparde to normal subjects after stimulation of PBMCs with Der p 2. CONCLUSION: This study suggests that NK cell and TNK cell may participate in allergic reaction by IFN-V regulation.