Sequence analysis of cDNA coding for a major house dust mite allergen, Der p 1. Homology with cysteine proteases.
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ABSTRACT:
SUBMITTER: Chua KY
PROVIDER: S-EPMC2188807 | biostudies-other | 1988 Jan
REPOSITORIES: biostudies-other
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Sequence analysis of cDNA coding for a major house dust mite allergen, Der p 1. Homology with cysteine proteases.
Chua K Y KY Stewart G A GA Thomas W R WR Simpson R J RJ Dilworth R J RJ Plozza T M TM Turner K J KJ
The Journal of experimental medicine 19880101 1
A cDNA clone coding for Der p 1, a major allergen from the house dust mite Dermatophagoides pteronyssinus, has been sequenced. It codes for a 222 residue mature protein with a derived molecular weight of 25,371 and contains 1 potential N-glycosylation site. In addition, the cDNA appears to code for a 13 residue proregion, and an incomplete signal peptide. The deduced sequence shows a high degree of homology with animal and plant cysteine proteases, particularly in the region of the contact resid ...[more]
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Project description:Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5 Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identified as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specific major epitope residues in an allergen and the sIgE level of the atopic population.
| S-EPMC6426935 | biostudies-literature
Project description:House dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P < 0.001). Atopic individuals who were only sensitized to HDM have a significantly higher number of IgE-binding residues than the individuals who were polysensitized to HDM and other crude allergens (P < 0.05). Individuals with allergic multimorbidity and moderate-to-severe allergic rhinitis also have a higher number of IgE-binding residues compared to those with single allergic disease and mild allergic rhinitis. The results prompt us to hypothesize that the individuals who have a higher number of IgE-binding residues may face a bigger challenge to be treated through immunotherapy due to the complexity in designing an effective hypoallergen with a high number of IgE-binding residues. We propose that the development of a refined molecular diagnostic assay, which includes alanine substitution of surface-exposed residues could be a more precise diagnostic strategy to identify all the IgE-binding residues of a major allergen for an atopic individual and the development could be another new dimension in allergy diagnosis and allergen immunotherapy treatment.
| S-EPMC7806929 | biostudies-literature
Project description:Previously, a ubiquinol‑cytochrome c reductase binding protein (UQCRB) homolog was identified in the house dust mite (HDM) species Dermatophagoides farinae (Der f) as a major allergen. In the present study, the immunodominant immunoglobulin E (IgE) epitope of the protein Der f 24 was investigated. Analysis of the homologous amino acid (aa) sequences in Der f and human UQCRB was performed. Four different recombinant Der f 24 and hybrid proteins formed by integrating Der f and human UQCRB sequences were expressed in Escherichia coli, purified using Ni‑NTA resins, and IgE‑binding activity was determined using IgE‑western blotting and enzyme‑linked immunosorbent assay (ELISA) experiments. IgE epitopes were further identified by IgE‑dot blotting and IgE‑ELISA with synthetic polypeptides and HDM‑allergic sera. Three‑dimensional (3D) structural modeling was used to analyze the position of the immunodominant IgE epitope. The amino acid sequence homology between Der f 24 and the human UQCRB protein was determined to be 39.34%. IgE‑ELISA and western blot analysis showed that all of the Der f‑human UQCRB hybrid proteins generated, except for the one lacking 59 residues of the N‑terminal region of Der f 24, were bound by allergic serum IgE. A synthetic polypeptide consisting of 32 residues of the N‑terminal reacted with IgEs from HDM‑allergic sera and could be used to generate high titer specific IgG or specific IgE antibodies in immunized mice. The 32‑aa N‑terminal region of Der f 24 was localized to a structural protrusion, which may facilitate specific IgE‑binding. These results indicate that the immunodominant IgE epitope of Der f 24 is located mainly in a 32‑residue region of the N‑terminus. These findings may inform the mechanisms of HDM allergy sensitization and allergy immunotherapy development.
| S-EPMC6777665 | biostudies-literature
Project description:BackgroundDespite the high prevalence of allergic asthma, currently, avoidance of the responsible allergens, which is nearly impossible for allergens such as house dust mite (HDM), remains among the most effective treatment. Consequently, determination of the immunogenic epitopes of allergens will aid in developing a better understanding of the condition for diagnostic and therapeutic purposes. Current methods of epitope identification, however, only evaluate immunoglobulin E-epitope binding interactions, which is not directly related to epitope immunogenicity.ObjectiveTo determine and rank the immunogenicity of the epitopes of major HDM allergen, Der p 2.MethodsWe performed degranulation assays with RBL-SX38 cells primed using patient plasma and challenged with nanoallergens which multivalently displayed epitopes to study the relative immunogenicity of various epitopes of Der p 2. Nanoallergens were used to evaluate epitopes individually or in combination.ResultsWhen evaluated using 3 patient samples, 3 epitopes in 2 distal regions of Der p 2 were identified as highly immunogenic when presented in combination, whereas no individual epitope triggered relevant degranulation. One of the epitopes (69-DPNACHYMKCPLVKGQQY-86) was identified to be cooperatively immunogenic when combined with other epitopes.ConclusionOur study highlights the importance of conformational epitopes in HDM-related allergies. This study also provides further evidence of the versatility of nanoallergens and their value for functional characterization of allergy epitopes, by ranking the Der p 2 epitopes according to immunogenicity. We believe that nanoallergens, by aiding in identification and understanding of immunogenic epitopes, will provide a better understanding of the manifestation of the allergic condition and potentially aid in developing new treatments.
| S-EPMC9808607 | biostudies-literature
Project description:Environmental proteases have been widely associated to the pathogenesis of allergic disorders. Der p 1, a cysteine-protease from house dust mite (HDM) Dermatophagoides pteronyssinus, constitutes one of the most clinically relevant indoor aeroallergens worldwide. Der p 1 protease activity depends on the redox status of its catalytic cysteine residue, which has to be in the reduced state to be active. So far, it is unknown whether Der p 1-protease activity could be regulated by host redox microenvironment once it reaches the lung epithelial lining fluid in addition to endogenous mite components. In this sense, Glutathione-S-transferase pi (GSTpi), an enzyme traditionally linked to phase II detoxification, is highly expressed in human lung epithelial cells, which represent the first line of defence against aeroallergens. Moreover, GSTpi is a generalist catalyst of protein S-glutathionylation reactions, and some polymorphic variants of this enzyme has been associated to the development of allergic asthma. Here, we showed that human GSTpi increased the cysteine-protease activity of Der p 1, while GSTmu (the isoenzyme produced by the mite) did not alter it. GSTpi induces the reduction of Cys residues in Der p 1, probably by rearranging its disulphide bridges. Furthermore, GSTpi was detected in the apical medium collected from human bronchial epithelial cell cultures, and more interesting, it increased cysteine-protease activity of Der p 1. Our findings support the role of human GSTpi from airways in modulating of Der p 1 cysteine-protease activity, which may have important clinical implications for immune response to this aeroallergen in genetically susceptible individuals.
| S-EPMC6597738 | biostudies-literature
Project description:Der p 23, a new, major house dust mite (HDM) allergen that is recognized by >70% of HDM-allergic patients, has high allergenic activity and, therefore, must be considered an important component for HDM-specific immunotherapy. We constructed and characterized a hypoallergenic Der p 23 vaccine for HDM immunotherapy. Three nonallergenic peptides from the C-terminal IgE epitope-containing part of Der p 23 (P4, P5) and P6, a mutant peptide containing serines instead of cysteines, were identified. Peptides were fused to the hepatitis B virus-derived PreS domain as recombinant fusion proteins (i.e., PreS-2XP4P5 and PreS-4XP6) that were expressed in Escherichia coli and purified to homogeneity. Compared with Der p 23, PreS-2XP4P5 and PreS-4XP6 showed no relevant IgE reactivity and exhibited considerably reduced allergenic activity in basophil activation tests using blood from HDM-allergic patients. Upon immunization of rabbits, only PreS-2XP4P5 induced high levels of Der p 23-specific IgG Abs that inhibited binding of patients' IgE to Der p 23, comparable to IgG Abs induced with Der p 23, whereas Abs induced with PreS-4XP6 had only low blocking capacity. Additionally, IgG Abs induced with PreS-2XP4P5 inhibited Der p 23-induced basophil activation comparable to IgG Abs induced with Der p 23. Compared with Der p 23, PreS-2XP4P5 induced lower T cell proliferation but higher levels of the tolerogenic cytokine IL-10 and the Th1 cytokine IFN-? in PBMCs from HDM-allergic patients, indicating an immunomodulatory capacity of the fusion protein. Therefore, PreS-2XP4P5 represents a promising candidate for immunotherapy of HDM-allergic patients.
| S-EPMC4582415 | biostudies-literature
Project description:BackgroundSeveral studies indicate that Der p 7 is an important and clinically relevant allergen of Dermatophagoides pteronyssinus which should be included in vaccines for treatment of house dust mite (HDM) allergy. Aim of this study was to characterize the IgE epitopes of Der p 7.MethodsRecombinant Der p 7 was expressed and purified, analyzed for fold by circular dichroism and tested for its allergenic activity by basophil activation. Seven overlapping, surface-exposed peptides (P1-P7) with a length of 27 to 37 amino acids, which spanned the Der p 7 sequence, were synthesized and tested for IgE reactivity and allergenic activity by basophil activation assay. Carrier-bound peptides were studied for their ability to induce allergen-specific IgG antibodies in rabbits. Peptide-specific antibodies were used to inhibit allergic patients` IgE binding to Der p 7 by ELISA for mapping of IgE epitopes.ResultsrDer p 7 showed high allergenic activity comparable with Der p 5, Der p 21, and Der p 23. None of the seven tested peptides showed any IgE reactivity or allergenic activity when tested with HDM- allergic patients indicating lack of sequential IgE epitopes on Der p 7. IgE inhibition experiments using anti-peptide specific IgGs and molecular modeling enabled us to identify discontinuous, conformational IgE epitopes of Der p 7.Conclusion and clinical relevanceIgE epitopes of Der p 7 belong to the conformational and discontinuous type whereas sequential Der p 7 peptides lack IgE reactivity. It should thus be possible to construct hypoallergenic vaccines for Der p 7 based on carrier-bound allergen peptides.
| S-EPMC8241568 | biostudies-literature
Project description:BackgroundThe house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated.ObjectiveTo perform a detailed characterization of Der p 18 on a molecular, structural and immunological level.MethodsDer p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments.ResultsRecombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients.ConclusionDer p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.
| S-EPMC4993390 | biostudies-literature
Project description:House dust mites (HDMs) belong to the most potent indoor allergen sources worldwide and are associated with allergic manifestations in the respiratory tract and the skin. Here we studied the importance of the high-molecular-weight group 11 allergen from Dermatophagoides pteronyssinus (Der p 11) in HDM allergy. Sequence analysis showed that Der p 11 has high homology to paramyosins from mites, ticks, and other invertebrates. A synthetic gene coding for Der p 11 was expressed in Escherichia coli and rDer p 11 purified to homogeneity as folded, alpha-helical protein as determined by circular dichroism spectroscopy. Using antibodies raised against rDer p 11 and immunogold electron microscopy, the allergen was localized in the muscle beneath the skin of mite bodies but not in feces. IgE reactivity of rDer p 11 was tested with sera from HDM-allergic patients from Europe and Africa in radioallergosorbent test-based dot-blot assays. Interestingly, we found that Der p 11 is a major allergen for patients suffering from atopic dermatitis (AD), whereas it is only a minor allergen for patients suffering from respiratory forms of HDM allergy. Thus, rDer p 11 might be a useful serological marker allergen for the identification of a subgroup of HDM-allergic patients suffering from HDM-associated AD.
| S-EPMC4636057 | biostudies-literature
Project description: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.
| S-EPMC6267192 | biostudies-literature