Effects of double mutation at two distant IgE-binding sites in the three-dimensional structure of the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activity

Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.     

Reactivities of mutants of a major house dust mite allergen Der f 2 to mouse anti-Der f 2 monoclonal antibodies analyzed by immunoblotting

A total of sixteen recombinant variants of a major house dust mite allergen Der f 2, the wild-type Der f 2, six cysteine mutants, six proline mutants, and three lysine mutants, were expressed in Escherichia coli. The cells were solubilized and run on SDS-PAGE under reducing conditions. Epitopes for five mouse anti-Der f 2 monoclonal antibodies, 1B2, 7C10, 13A4, 15E11, and 18G8, to the recombinant Der f 2 variants were characterized by immunoblot analysis.     

Structure of the house dust mite allergen Der f 2: implications for function and molecular basis of IgE cross-reactivity

The X-ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 A resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti-parallel beta-sheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross-reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.     

Expression of house dust mite allergens Der f 1 and Der f 2 in leaves of Nicotiana benthamiana

In test systems for diagnostics of type I allergy, recombinant allergens in native conformation are used, because immunoglobulins E (IgE), responsible for the development of this type of allergy, recognize conformational epitopes of protein allergens. To obtain recombinant allergens of the Dermatophagoides farinae house dust mites (HDM), Der f 1 and Der f 2, two systems of heterological expression were used: Escherichia coli and Nicotiana benthamiana plants. IgE from sera of children allergic to HDM were shown to recognize the recombinant Der f 2 protein synthesized in both E. coli and N. benthamiana plants, as well as the recombinant Der f 1 protein produced in N. benthamiana plants, while mature form of Der f 1 produced in E. coli did not interact with IgE.     

Unlocking the allergenic structure of the major house dust mite allergen der f 2 by elimination of key intramolecular interactions

We report on the structural background of the remarkable reduction of allergenicity in engineering of the major house dust mite allergen Der f 2. Disruption of intramolecular disulfide bonds in Der f 2 caused extensive conformational change that was monitored by circular dichroism and gel-filtration analysis. The degree of conformational change correlated well with the degree of reductions in the capacity to bind IgE and to induce histamine release from basophils in mite-allergic patients. Loosening the rigid tertiary structure by elimination of key intramolecular interactions is an effective strategy to reduce the number of high affinity IgE epitopes of allergen vaccine.     

Sequence polymorphisms of Der f 1, Der p 1, Der f 2 and Der p 2 from Korean house dust mite isolates

Amino acid sequence variations have possible influences on the allergenicity of allergens and may be important factors in allergen standardization. This study was undertaken to investigate the sequence polymorphisms of group 1 and 2 allergens from Korean isolates of the house dust mites Dermatophagoides farinae and D. pteronyssinus. cDNA sequences encoding group 1 and 2 allergens were amplified by RT-PCR and compared the deduced amino acid sequences. Der f 1.0101, which appeared in 64.0 % of the 50 sequences analyzed, was found to be predominant. Among the Der p 1 sequences, Der p 1.0102 and 1.0105 were predominant (58 %). Among the Der f 2 sequences, Der f 2.0102 (40.7 %) and a new variant with Gly at position 42 (27.8 %) were predominant. The deduced amino acid sequences of 60 Der p 2 clones were examined, and 28 variants with 1-5 amino acid substitutions were found. Interestingly, all of the Der p 2 sequences had Thr instead of Lys at position 49. Two variants (Leu40, Thr49, and Asn114 (26.6 %); Val40, Thr49, and Asn114 (20.0 %)) were found to be the most predominant forms of Der p 2. Der p 1 has a high rate of sporadic substitutions and the group 2 allergens show a more regular pattern with orderly associations of amino acid substitutions. Der f 1 and Der p 2 from Korean mite isolates have unique amino acid sequence polymorphisms. These findings provide important data for house dust mite allergen standardization.     

Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae.

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.     

Rationally designed hypoallergenic mutant variants of the house dust mite allergen Der p 21

BackgroundAllergic diseases figure among the most common immune-mediated diseases worldwide, affecting more than 25% of the world's population. Allergic reactions can be triggered by house dust mite (HDM) allergens, of which the so-called group 21 of allergens is considered as clinically relevant.MethodsHerein, we used a structural bioinformatics and immunoinformatics approach to design hypoallergenic mutant variants of the Der p 21 allergen of Dermatophagoides pteronyssinus, which were then recombinantly expressed in bacteria and tested for their IgE-reactivities. For this, we scanned the wild-type Der p 21 protein for all possible single amino acid substitutions in key IgE-binding regions that could render destabilization of the major epitope regions.ResultsFour main substitutions (D82P, K110G, E77G, and E87S) were selected to build mutant variants of the Der p 21 allergen, which were produced in their recombinant forms; two of these variants showed reduced reactivity with IgE. Molecular dynamic simulations and immune simulations demonstrated the overall effects of these mutations on the structural stability of the Der p 21 allergen and on the profile of immune response induced through immunotherapy.ConclusionsWhen produced in their recombinant forms, two of the Der p 21 mutant variants, namely proteins K110G and E87S, showed significantly reduced IgE reactivities against sera from HDM-allergic individuals (n = 20; p < 0.001).General significanceThis study successfully translated a rational in silico mutagenesis design into low IgE-binding mutant variants of the allergen rDer p 21. These novel hypoallergens are promising to compose next-generation allergen-immunotherapy formulations in near future.     

Crystal structure and some properties of a major house dust mite allergen, Derf 2

Pyroglyphid house dust mites are a major source of allergens in house dust. Mite allergens sensitize and induce asthma, rhinitis, and eczema in a large portion of patients with allergic diseases. Here, the crystal structure of a major mite allergen, Derf 2, derived from Dermatophagoides farinae was solved by single isomorphous replacement method with anomalous scattering (SIRAS) at 2.1A resolution. The present study also demonstrated that the conformation of the allergen was critical in the determination of Th1/Th2 shift based on physicochemical and immunological analyses. This indicates that rigidly folded and singly dispersed structure is essentially required for the generation of Th2 type cells by the allergen, while conformational variant protein leads to Th1 skewing, irrespective of the same amino acid sequence. This structure/function relationship may allow us to develop a novel strategy for hyposensitization therapy in patients with allergic diseases triggered by house dust mite allergens.     

Molecular cloning, expression, sequence analyses of dust mite allergen Der f 6 and its IgE-binding reactivity with mite allergic asthma patients in southeast China

We report the cloning and molecular characterization of a full-length cDNA encoding house dust mite allergen, Der f 6 from D. farinae isolated in China. The full-length Der f 6 cDNA was obtained with 840 nucleotides long. Nucleotide sequencing analyses showed a total of 36 mutations in five Der f 6 cDNA clones, corresponding to 23 incompatible amino acid residues. Recombinant Der f 6 (rDer f 6) protein was successfully expressed in and purified from E. coli BL21. Among 20 asthmatic patients, 45% was positive to rDer f 6 by ELISA. Bioinformatics analyses revealed that the mature Der f 6 was a hydrophobic and extracellular protein with chymotrypsin-like serine protease activity, its secondary structure was composed of alpha helix (7.69%), extended strand (34.62%), random coils (57.69%), and the similarity of Der f 6 to Blo t 6, Sui m 6, Der f 3 and Der f 9 was 64, 65, 35, and 38%, respectively.     

Hydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10-100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2-3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy.     

IgE and IgG binding of peptides expressed from fragments of cDNA encoding the major house dust mite allergen Der p I

Large peptides expressed from cDNA fragments of a clone encoding the mite allergen Der p I were able to bind IgE and IgG in sera from allergic individuals. The binding was found for peptides from sequences throughout the molecule, with at least five regions, comprising residues 1-56, 53-99, 98-140, 166-194, and 188-222. The only limitation was that more than 30 amino acid residues were required for consistent binding. Each of seven sera examined showed a different profile of antibody binding to the peptides. For the most part the pattern of IgE and IgG binding to the peptides for each serum was similar, demonstrating a concordant repertoire. In 5/7 sera, however, IgG bound to some peptides which had little or no IgE binding activity, thus showing more diverse specificities. It is suggested that some divergence of repertoire can develop during the maturation of the B cell response.     

A mouse model for in vivo tracking of the major dust mite allergen Der p 2 after inhalation

Inhaled environmental antigens, i.e. allergens, cause allergic symptoms in millions of patients worldwide. As little is known about the fate of an allergen upon inhalation, we addressed this issue for a major dust mite allergen, Der p 2. First, a model for Der p 2-sensitization was established in C57BL/6 J mice, in which sensitized mice mounted a Der p 2-specific IgE-response with eosinophilic lung inflammation after allergen challenge in the airways. In this model, we applied recombinant Der p 2 carrying a novel C-terminal tetrapeptide Sel-tag enabling labelling with the gamma-emitting radionuclide 75Se at a single selenocysteine residue ([75Se]Der p 2). In vivo tracking of intratracheally administered [75Se]Der p 2 using whole-body autoradiography revealed that [75Se]Der p 2-derived radioactivity persisted in the lungs of sensitized mice as long as 48 h. Radioactivity was also detected in kidneys, liver and in enlarged lung-associated lymph nodes. Interestingly, a larger proportion of radioactivity was found in the lungs of sensitized compared with nonsensitized mice 24 h after intratracheal instillation of [75Se]Der p 2. A radioactive protein corresponding to intact Der p 2 could only be detected in the lungs, whereas [75Se]Der p 2-derived radioactivity was recovered in known selenoproteins both in lung and other organs. Hence, using the recently developed Sel-tag method in a mouse model for Der p 2-sensitization, we could track the fate of an inhaled allergen in vivo. Based upon our findings, we conclude that the inflammatory state of the lung influences the rate of metabolism and clearance of Der p 2. Thus, an allergic response to the inhaled allergen may lead to prolonged retention of Der p 2 in the lung.     

Cloning and expression of cDNA encoding the complete prepro-form of an isoform of Der f 1, the major group 1 allergen from house dust mite Dermatophagoides farinae

cDNA clones encoding a major house dust mite allergen, Der f 1, were isolated from a Dermatophagoides farinae cDNA library by plaque immunoscreening using rabbit anti-Der f 1 serum. The sequences cover the complete open reading frame encoding the prepro-form. The sequence is different from previously reported cDNA of Der f 1 in six bases and the encoded amino acid sequence is different in two residues. Pro-forms of Der f 1 and its mutant, in which the N-glycosylation motif was disrupted, expressed in Pichia pastoris were converted to the mature forms by an in vitro activation process and they showed significant IgE-binding. The biologically active rDer f 1 molecules would be useful for diagnostic testing and allergen-specific immunotherapy. In contrast, Der f 1 directly expressed in Escherichia coli without the prosequence had very low IgE binding. The hypoallergenic Der f 1 polypeptide could be useful for safer and more effective immunotherapy.     

The crystal structure of a major dust mite allergen Der p 2, and its biological implications

The crystal structure of the common house mite (Dermatophagoides sp.) Der p 2 allergen was solved at 2.15 A resolution using the MAD phasing technique, and refined to an R-factor of 0.209. The refined atomic model, which reveals an immunoglobulin-like tertiary fold, differs in important ways from the previously described NMR structure, because the two beta-sheets are significantly further apart and create an internal cavity, which is occupied by a hydrophobic ligand. This interaction is structurally reminiscent of the binding of a prenyl group by a regulatory protein, the Rho guanine nucleotide exchange inhibitor. The crystal structure suggests that binding of non-polar molecules may be essential to the physiological function of the Der p 2 protein.     

Monoclonal antibodies to recombinant Der p 2, a major house dust mite allergen: specificity, epitope analysis and development of two-site capture ELISA.

House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophagoides pteronyssinus, were produced. Four monoclonal antibodies produced were species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides. A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p 2 in house dust. The sensitivity limit was 4 ng/ml with rDer p 2 and 8 micrograms/ml with the D. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p 2 could be useful for the environmental studies and for the standardization of mite allergen extracts.     

Relationship between the magnitude of IgE production in mice and conformational stability of the house dust mite allergen,Der p 2

BackgroundProtein antigens are degraded by endosomal protease in antigen presentation cell. T cells recognize peptides derived from antigen proteins bound to class II major histocompatibility complex molecules. We previously reported that an increase in the conformational stability of an antigen depressed its immunogenicity. However, there is little information on antigens with differences in molecular properties such as net charges and molecular weight.MethodsDenaturation experiments against guanidine hydrochloride. The serum IgE levels to protein antigens at 35 days after the first immunization analyzed using ELISA.ResultsThe Der p 2 mutations in which Ile13 is mutated to Ala (I13A) and Ala122 is mutated to Ile (A122I) were shown to have lower and higher conformational stability than the wild-type, respectively, by denaturation experiments. The amount of IgE production by the less stable I13A mutant was higher and that of the stable A122I mutant was lower than that of the wild-type.ConclusionOur results suggest that the increased conformational stability of Der p 2 depressed the IgE production in mice.General significanceThese findings should provide a milestone for the engineering of allergen vaccines.     

The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen

Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 A. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.     

A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen

Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa. Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, approximately 25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.