The crystal structure of the common house mite (Dermatophagoides sp.) Der p 2 allergen was solved at 2.15Å 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 β-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.

BACKGROUND:Dust mites are important inducers of allergic disease. Group 2 allergens are recognized as major allergens in several mite species, including Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and Tyrophagus putrescentiae. No allergens have thus far been characterized on the molecular level from the dust mite Glycyphagus domesticus. OBJECTIVE:We sought to examine the cross-reactivity among group 2 allergens of G domesticus, L destructor, T putrescentiae, and D pteronyssinus. METHODS:A group 2 allergen from G domesticus, Gly d 2, was cloned and expressed as a recombinant protein. Cross-reactivity between Gly d 2 and 3 other group 2 allergens, Lep d 2, Tyr p 2, and Der p 2, was studied by using individual sera and a serum pool RAST-positive to G domesticus, L destructor, T putrescentiae, and D pteronyssinus. Recombinant allergens were used as inhibitors of IgE binding in immunoblotting experiments. Molecular modeling on the basis of the Der p 2 structure was carried out for Gly d 2, Lep d 2, and Tyr p 2. RESULTS:Two cDNAs encoding isoforms of Gly d 2 were isolated, but only the Gly d 2.02 isoform was used in this study. Sixteen of 17 subjects had IgE to Gly d 2. The protein sequence of Gly d 2 revealed 79% identity to Lep d 2 and 46% and 41% identity to Tyr p 2 and Der p 2, respectively. Extensive cross-reactivity was demonstrated among Gly d 2, Lep d 2, and Tyr p 2, but little cross-reactivity was found between these allergens and Der p 2. According to the tertiary structure of Der p 2 and 3-dimensional models of Gly d 2, Lep d 2, and Tyr p 2, differences reside mainly in surface-exposed residues. CONCLUSION:Gly d 2 showed high sequence homology to Lep d 2. Cross-reactivity was observed between Gly d 2, Lep d 2, and Tyr p 2, but only limited cross-reactivity was demonstrated between these 3 allergens and Der p 2.

Background: Mite group 2 allergens Der p 2, Der f 2, and Eur m 2 are 14-kDa proteins of unknown function that share 83% to 85% amino acid sequence identity. Isoforms of the allergens within each genus have been identified which differ by 3 or 4 amino acids, but little is known of the influence of group 2 polymorphisms on human IgE antibody binding. Objective: The purpose of this study was to investigate the importance of interspecies and isoform substitutions on murine mAb and IgE antibody binding and on the molecular structure of the group 2 allergens. Methods: Site-directed mutagenesis was used to incorporate the isoform amino acid substitutions onto the Der p 2.0101 sequence. Recombinant allergens were expressed and purified from Escherichia coli and used to evaluate antibody binding by enzyme-linked immunosorbent assay (ELISA). Molecular modeling of the tertiary structure was used to analyze structural differences between the various group 2 allergens. Results: The substitution of asparagine for aspartic acid at position 114 restored mAb binding of rDer p 2.0101; the other Der p 2 isoforms and the 3 rDer f 2 isoforms also reacted in the 2-site ELISA. The correlation of IgE binding to the Der p 2 isoforms was excellent and tended to be higher in the isoforms with the asparagine 114 substitution (r 2 = 0.87 vs r 2 = 0.95). rEur m 2.0101 bound to all mAb except 7A1; when compared with rDer p 2 for IgE binding, rEur m 2.0101 gave a correlation coefficient of r 2 = 0.68. Molecular modeling revealed that Eur m 2 and the storage mite homologs Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2. Eur m 2 has a conserved surface, whereas Lep d 2 and Tyr p 2 present most of the amino acid substitutions on this surface. Lep d 2 and Tyr p 2 did not react with mAb or with sera from patients with IgE to Dermatophagoides species. Conclusion: The isoform substitutions of rDer p 2 can be distinguished by mAb. The allergenic cross-reactivity between Der p 2, Der f 2, and Eur m 2 is a direct result of the conserved antigenic surface, whereas the lack of cross-reactivity with Lep d 2 and Tyr p 2 is a result of the multiple substitutions across this surface. (J Allergy Clin Immunol 2001;107:977-84.)

Background: The group 2 allergens Der p 2, Der f 2 and Eur m 2 are 14-kD proteins with >80% sequence identity. Isoforms within each genus have been identified which differ by 3–4 amino acids. The aim of this study was to investigate the importance of these substitutions to antibody binding. Methods: Recombinant allergens were expressed and purified from Escherichia coli. ELISA and skin testing were used to evaluate antibody binding. Molecular modeling of the tertiary structure was preformed to examine the location of substitutions. Results: The three Der f 2 isoforms and two of three of the Der p 2 isoforms reacted with all monoclonal antibodies (mAb). Der p 2.0101, the isoform with aspartate at position 114, bound all mAb except 1D8. Substitution of asparagine for aspartate restored binding of rDer p 2.0101 to mAb 1D8 and increased the correlation coefficient for IgE binding from 0.72 to 0.77. The three Der p 2 isoforms showed comparable skin test reactivity to nDer p 2 and commercial extract. rEur m 2.0101 bound to all mAb except 7A1 and when compared with rDer p 2 for IgE binding, r2 = of 0.58 (n = 72). Lep d 2 did not react with mAb or with Dermatophagoides spp. allergic sera. Modeling revealed that Eur m 2, Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2 and the substitutions are on the surface. Conclusions: mAb could distinguish isoform substitutions. IgE binding showed a good correlation among all isoforms, thus the recombinant allergens are useful for diagnosis.

The 29-kDa subtilase homologue, Tri r 2, derived from the dermatophyte fungus Trichophyton rubrum, exhibits unique immunologic characteristics in its ability to elicit immediate (IH) and delayed-type (DTH) hypersensitivity skin tests in different individuals. Thus, Tri r 2 provides a model for comparing the T cell repertoire in subjects with distinct immune responses to a single Ag, Recombinant Tri r 2 produced as a GST fusion protein in Escherichia coli stimulated strong in vitro lymphoproliferative responses in 10 IH and 10 DTH responders. Patterns of T cell epitope recognition were compared between skin test groups using 28 overlapping peptides (each in 12 replicate wells) derived from Tri r 2 to stimulate T lymphocyte proliferation in vitro. Peptide 5 (P5; aa 41-60) induced the strongest response in DTH subjects and showed the largest difference between DTH and IH responders in proliferation (mean standardized index, 2.22 and 0.82, respectively; p = 0.0047) and number of positive wells (81 vs 12). Responses to P5 were associated with diverse HLA haplotypes, These results showed that P5 contains an immunodominant epitope specifically associated with DTH and that this peptide is recognized in a permissive manner. Cross-validated linear discriminant analysis using T cell proliferative responses to two regions of Tri r 2 (aa 51-90 and 231-270) gave a 95% predictive accuracy for classification of subjects into IH or DTH groups. We conclude that different immune responses to Trichophyton are mediated by distinct T cell repertoires between individuals with IH and DTH reactions to Tri r 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.

The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs), The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway fur ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed.

The dermatophyte fungus Trichophyton exhibits unique immunologic properties by its ability to cause both immediate and delayed type hypersensitivity. An 83-kDa Trichophyton tonsurans allergen (Tri t 4) was previously shown to elicit distinct T lymphocyte cytokine profiles in vitro. The homologous protein, Tri r 4, was cloned from a Trichophyton rubrum cDNA library, and the recombinant protein was expressed in Pichia pastoris. This 726-amino acid protein contained an arrangement of catalytic triad residues characteristic of the prolyl oligopeptidase family of serine proteinases (Ser-Asp-His). In addition, a novel Trichophyton allergen, encoding 412 amino acids, was identified by its human IgE antibody-binding activity. Sequence similarity searches showed that this allergen, designated Tri r 2, contained all of the conserved residues characteristic of the class D subtilase subfamily (41-58% overall sequence identity). Forty-two percent of subjects with immediate hypersensitivity skin test reactions to a Trichophyton extract exhibited IgE antibody binding to a recombinant glutathione S-transferase fusion protein containing the carboxyl-terminal 289 amino acids of Tri r 2. Furthermore, this antigen was capable of inducing delayed type hypersensitivity skin test reactions. Our results define two distinct antigens derived from the dermatophyte Trichophyton that serve as targets for diverse immune responses in humans.

Sensitization to indoor allergens, especially those of the house dust mite, is strongly correlated with the development of asthma. We report the tertiary structure of the major house dust mite allergen, Der p 2, determined by NMR methods. The structure of Der p 2 is a beta-barrel and is composed of two three-stranded antiparallel beta-pleated sheets. This arrangement of beta-strands is similar to the immunoglobulin fold with respect to the orientation of the two sheets and the interactions of the strands. However, the three-dimensional structure of Der p 2 aligns equivalently with a number of proteins from different families within the immunoglobulin superfamily. The structural homology with the highest significance score from analysis by DALI is to Der f 2. Although Der p 2 and Der f 2 are 87% identical in amino acid sequence, they align in three dimensions rather poorly (4.85 A RMSD; Z-score, 8.58). This unexpected finding is likely due to the different solution conditions used during structure determination by NMR for both proteins. While the structural comparisons did not elucidate a clear homologue for the function of Der p 2 in mites, we report that Der p 2 is sequentially homologous to esr16. This is a protein from moths that is expressed coincident with molting. Thus, this homology has important ramifications for the study of mite allergy. The structure of Der p 2 provides a useful tool in the design of recombinant immunotherapeutics for the group 2 allergens.

Research Articles| September 04 2009 Antigenic and Molecular Structure of the Mite Allergen Der p 2 Subject Area: Immunology and Allergy Alisa M. Smith; Alisa M. Smith aAsthma and Allergic Diseases Center and Departments of Search for other works by this author on: This Site PubMed Google Scholar Ernesto A. Taketomi; Ernesto A. Taketomi aAsthma and Allergic Diseases Center and Departments of Search for other works by this author on: This Site PubMed Google Scholar Geoffrey A. Mueller; Geoffrey A. Mueller cBiochemistry, University of Virginia School of Medicine, Charlottesville, Va., USA Search for other works by this author on: This Site PubMed Google Scholar Gordon S. Rule; Gordon S. Rule cBiochemistry, University of Virginia School of Medicine, Charlottesville, Va., USA Search for other works by this author on: This Site PubMed Google Scholar David C. Benjamin; David C. Benjamin bMicrobiology and Search for other works by this author on: This Site PubMed Google Scholar Martin D. Chapman Martin D. Chapman aAsthma and Allergic Diseases Center and Departments of Search for other works by this author on: This Site PubMed Google Scholar Int Arch Allergy Immunol (1997) 113 (1-3): 99–101. https://doi.org/10.1159/000237517 Article history Published Online: September 04 2009 Content Tools Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Alisa M. Smith, Ernesto A. Taketomi, Geoffrey A. Mueller, Gordon S. Rule, David C. Benjamin, Martin D. Chapman; Antigenic and Molecular Structure of the Mite Allergen Der p 2. Int Arch Allergy Immunol 31 December 1997; 113 (1-3): 99–101. https://doi.org/10.1159/000237517 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsInternational Archives of Allergy and Immunology Search Advanced Search Article PDF first page preview Close Modal Keywords: Dermatophagoides pteronyssinus, Antigenic determinant, IgE antibody, Mutagenesis, Antigenic variants, Tertiary structure 1997Copyright / Drug Dosage / DisclaimerCopyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. You do not currently have access to this content.

There exists a strong correlation between asthma and sensitization to indoor allergens. This study reports on the secondary structure of the major house dust mite allergen Der p 2, determined using heteronuclear NMR methods. The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter. The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST). The secondary structure was determined by examining chemical shifts, short and long range NOESYs, JHN-HA coupling constants, and amide exchange rates. From these data, it is clear that Der p 2 is composed of beta-sheets and random coil. Based on long range distance constraints, a number of beta-strands were aligned into two three-stranded, anti-parallel beta-sheets.

BACKGROUND AND OBJECTIVE:Chimeric mouse/human monoclonal IgG1 and IgG4 antibodies were developed against the house dust mite allergen Der p 2. These chimeric IgG antibodies, hIgG1-Dp2 A and hIgG4-Dp2 A, have the same binding characteristics as the previously reported chimeric hIgE-Dp2 A and are composed of the heavy chain variable domains and light chains of the original murine monoclonal antibody 2B12, whereas the heavy chain constant domains have been replaced by the human IgG1 or IgG4 heavy chain. The expression level of hIgG1-Dp2 A and hIgG4-Dp2 A was 1 and 3.5 microg/mL, respectively.METHODS AND RESULTS:Since all IgG in these culture supernatants is allergen-specific, they are useful reference reagents and enable the calculation of the amount of allergen specific IgG1 and IgG4 antibodies in absolute IgG amounts. The results obtained with two panels of sera from patients in immunotherapeutic treatment were evaluated and compared in Der p 2 IgE, IgG1 and IgG4 RAST and with reversed IgG4 RAST using labelled purified Der p 2. Close agreement between the results for the two IgG4 assays was found.CONCLUSION:With these chimeric reference reagents the quantities of isotype specific antiallergen antibodies can be calculated and compared.

The Fv fragment of the monoclonal antibody D1.3 was expressed in bacteria. Standard triple resonance techniques were used to obtain the NMR resonance assignments for 211 out of 215 backbone 15N/NH atoms for D1.3 Fv. Using these assignments, hydrogen exchange rates are measured for 82 amide hydrogen atoms in D1.3 Fv free and bound to hen egg-white lysozyme. Upon binding to antigen, exchange rates are decreased for residues throughout the Fv. Many of these residues are located remote from the site of interaction with the antigen. These changes are larger than previously observed for the antigen portion of the complex. Evidently, the β-sheet structure of the Fv propagates the effects of binding more efficiently than the antigen. These effects are compared between the three different polypeptide chains that make up the complex. These data suggest that reduced dynamics are a general feature of antibody binding to antigen.

Allergic reactions to cockroach (CR) were first described over 30 years ago by Bernton and Brown, amongst patients presenting to their allergy clinic in New York (1). These observations were followed up by several groups, notably by Dr. Kang and colleagues in Chicago in the 1970’s, and it is now well established that CR cause IgE antibody responses; that these responses are often associated with asthma; and that the prevalence of CR asthma in some populations in the U.S. is comparable to that of other common aeroallergens, such as dust mite, cat, and pollens (2, 3). Atopic individuals who live in CR infested housing become sensitized by inhalation of potent CR allergens and produce vigorous IgE antibody (ab) responses. There is a strong association between the development of IgE ab to CR and asthma. In some towns and cities in the U.S., up to 60% of house dust allergic, asthmatic patients become sensitized to CR (2–6). Allergic reactions to CR are not confined to urban or inner city populations, but occur wherever housing conditions sustain extensive cockroach infestation. For example, in Louisville, Kentucky, no differences were found between the prevalence of CR allergy in patients living in inner city or rural areas, with ∼40% prevalence of positive skin test reactivity in both groups being reported in a 10 year retrospective study (7). IgE ab to CR is an important risk factor for Emergency Room admission with asthma and is most commonly found among lower socio economic groups living in sub-standard housing, which in the U.S. often comprise a high proportion of minority groups (African Americans and Hispanics (8–9).

The binding of anti-lysozyme monoclonal antibodies, D44.1 or D1.3, to their antigen reduces the rate of exchange for many amide hydrogens in lysozyme. The D44.1 antibody contacts a similar region of lysozyme to the HyHEL-5 antibody, while the D1.3 antibody binds to the side of lysozyme which is opposite to the HyHEL-5 and D44.1 epitopes. We compare the effects of binding these antibodies on amide hydrogen exchange rates in lysozyme. These comparisons suggest that there are regions of lysozyme that fluctuate in a coordinated manner such that the effects of binding can be propagated to regions that are distant from the epitope. The activation enthalpies for hydrogen exchange for 36 of the 126 amide hydrogens in lysozyme and for 25 of 126 lysozyme amide hydrogens in the lysozyme-D1.3 complex are also reported. These data suggest that the reduction in amide hydrogen exchange rates upon antibody binding reflect changes in the dynamics of the antigen. These changes contribute to a reduction in the specific heat capacity upon binding.

An allergen cloned from a Blattella germanica (German cockroach) cDNA library, encoded a 182-amino acid protein of 20,904 Da. This protein, designated B. germanica allergen 4 (Bla g 4), was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by affinity chromatography and high-performance liquid chromatography. The prevalence of serum IgE antibody to recombinant Bla g 4 in 73 cockroach allergic patients with asthma ranged from 40% (antigen binding radioimmunoassay) to 60% (plaque immunoassay). Cockroach allergic patients gave positive intradermal skin tests to recombinant Bla g 4 at concentrations of 10(-3)-10(-5) mu g/ml, whereas non-allergic controls, or cockroach allergic patients with no detectable serum IgE antibody to Bla g 4, gave negative skin tests to 1 mu g/ml. Polymerase chain reaction and Southern analysis identified a 523-base pair DNA encoding Bla g 4 in both B. germanica and Periplaneta americana (American cockroach). However, Northern analysis showed that mRNA encoding Bla g 4 was transcribed in B. germanica but not in P. americana, suggesting that allergen expression was species specific. Sequence similarity searches showed that Bla g 4 was a ligand binding protein or calycin and unexpectedly revealed that this family contained several important allergens: beta-lactoglobulin, from cow milk, and rat and mouse urinary proteins. Although the overall sequence homology between these proteins was low (similar to 20%), macromolecular modeling techniques were used to generate two models of the tertiary structure of Bla g 4, based on comparisons with the x-ray crystal coordinates of bilin binding protein and rodent urinary proteins. The results show that members of the calycin protein family can cause IgE antibody responses by inhalation or ingestion and are associated with asthma and food hypersensitivity.