Cross-reactivity studies of a new group 2 allergen from the dust mite Glycyphagus domesticus , Gly d 2, and group 2 allergens from Dermatophagoides pteronyssinus, Lepidoglyphus destructor , and Tyrophagus putrescentiae with recombinant allergens

Gafvelin, Guro; Johansson, E.; Lundin, Anna; Smith, Anderson D.; Chapman, Martin D.; Benjamin, David C.; Derewenda, Urszula; Hage, Marianne van

doi:10.1067/mai.2001.112264

Cited by 52 publications

(32 citation statements)

References 42 publications

(39 reference statements)

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“…Their IgE epitopes are dependent on the integrity of the conformational structure rather than the contiguous sequence of amino acids [10,11,12,13,14,15,16]. Their amino acid sequences are homologous to other group 2 allergens from different mite species, esr16 found in moth trachea, and Niemann-Pick type C2 disease protein (NPC2/HE1) [17,18,19,20,21,22]. Recently, a structural relationship between the Der f 2 homologues and MD-2, which associates with Toll-like receptor 4, was indicated [23].…”

Section: Introductionmentioning

confidence: 99%

Dilution Method to Refold Bacterially Expressed Recombinant Der f 2 and Der p 2 to Exhibit the Secondary Structure and Histamine-Releasing Activity of Natural Allergens

Takai

Takaoka²,

Yasueda³

et al. 2005

Int Arch Allergy Immunol

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Background: Structurally refolded recombinant forms of major house dust mite group 2 allergens, Der f 2 and Der p 2, expressed in Escherichia coli, were prepared by solubilizing the insoluble products with urea and subsequently dialyzing against buffer. In this study, we determined conditions for refolding the urea-denatured recombinant Der f 2 and Der p 2 by one-step dilution as an alternative to dialysis, which requires several steps of handling and much time and cost. Methods: The insoluble bacterial product containing recombinant Der f 2 was solubilized with a buffer containing 8 M urea, and the solution was diluted to various urea concentrations. The refolding efficiency in each dilution was estimated from the height of the peak corresponding to the folded recombinant Der f 2 and that containing the aggregated form on anion exchange chromatography. The structure and allergenicity of the purified recombinant Der f 2 and Der p 2 refolded using the dilution method were analyzed based on circular dichroism and a basophil histamine-releasing assay, respectively. Results: Although the refolding efficiency decreased as the urea concentration in the dilution increased, experimental conditions whereby the protein and urea concentrations in the dilution were less than 0.5 mg/ml and 0.8 M, respectively, achieved maximum refolding efficiency. The recombinant allergens prepared by the dilution method exhibited the secondary structure and histamine-releasing activity of natural allergens purified from mite culture. Conclusions: The dilution method established in this study is more convenient in terms of handling, time, and cost than the dialysis method and will be useful for large-scale production and for the preparation of numbers of mutants to analyze IgE epitopes.

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Section: Introductionmentioning

confidence: 99%

Dilution Method to Refold Bacterially Expressed Recombinant Der f 2 and Der p 2 to Exhibit the Secondary Structure and Histamine-Releasing Activity of Natural Allergens

Takai

Takaoka²,

Yasueda³

et al. 2005

Int Arch Allergy Immunol

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“…Mites are a common cause of allergic disease world-wide (45,46), and group 2 allergens are known to be major allergens in several mite species including Dermatophagoides spp, L. destructor (47) and G. domesticus (37). Allergen-specific immunotherapy to mite allergy is routinely carried out using mite extracts, but there have been several recent attempts to develop improved and standardized allergen preparations based on recombinant allergens.…”

Section: Discussionmentioning

confidence: 99%

“…In this study we applied multigene recombination to three group 2 mite allergen genes, two isoforms of the major allergen from the dust mite Lepidoglyphus destructor, Lep d 2 (36) and a group 2 allergen from the related dust mite Glycyphagus domesticus, Gly d 2 (37 …”

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confidence: 99%

Hypoallergens for Allergen-specific Immunotherapy by Directed Molecular Evolution of Mite Group 2 Allergens

Gafvelin

Parmley

Neimert-Andersson

et al. 2007

Journal of Biological Chemistry

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Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wildtype allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.The large increase in allergic diseases observed during the past decades has urged the development of safe and efficient treatments of allergy. Many new concepts based on targeting the underlying immunological causes of IgE-mediated allergy have been suggested (1-5). However, the only treatment that causes a long-lasting relief of symptoms is allergen-specific immunotherapy (ASIT).3 Today ASIT is based on the repeated administration of allergen extracts prepared from the allergen source. Although successful clinical outcomes are well documented, several problems are associated with allergen extractbased ASIT, including the risk of inducing local and systemic side effects and induction of new sensitizations (6, 7).Large batches of well defined single allergen components can be produced using recombinant techniques, making it feasible to solve problems linked to allergen extract-based ASIT. The most severe side effects of ASIT are caused by the binding of injected allergen to allergen-specific IgE on high affinity Fc⑀RI receptor-bearing effector cells, leading to cross-linking of the Fc⑀RI receptors, degranulation, and release of anaphylactogenic mediators. Therefore, allergens with reduced IgE binding capacity have been proposed to improve the safety of ASIT (8...

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“…[31]. The crystal structure of Der p 2 has been used in standard homology modelling to predict the secondary and tertiary structure of Lep d 2.0101 [32]. According to this model, amino acids [25].…”

Section: Discussionmentioning

confidence: 99%

Lep d 2 polymorphisms in wild and cultured Lepidoglyphus destructor mites

Kaiser

Gafvelin

Johansson

et al. 2003

European Journal of Biochemistry

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We have previously cloned, expressed and characterized two variants of the major allergen Lep d 2 from cultured Lepidoglyphus destructor mites. These variants, Lep d 2.0101 and Lep d 2.0201, differ at 13 amino acid positions. In this study we investigated Lep d 2 sequence diversity between wild and cultured mites. PCR, Southern blot and DNA sequence analysis revealed the presence of two different Lep d 2 genes, one with and one without an intron. In addition, two new variants of Lep d 2, Lep d 2.0102 and Lep d 2.0202, were found at different frequencies in wild and cultured mites. When we expressed the Lep d 2 variants and compared their IgE binding properties by ELISA inhibition, we found that Lep d 2.0102 was a more potent inhibitor than Lep d 2.0101, and to a lesser extent Lep d 2.0202 was more potent than Lep d 2.0201. Long‐term cultures of peripheral blood mononuclear cells were used to assess the ability of the expressed Lep d 2 variants to induce cytokine release. Although cells from different individuals released different amounts of interferon‐γ and interleukin‐5, no consistent cytokine release pattern could be linked to any specific Lep d 2 variant. In conclusion, we show that both cultured and wild Lepidoglyphus destructor mites contain the same pattern of polymorphism. Furthermore, this Lep d 2 sequence diversity seems not to have any significant impact on the allergens IgE binding or its ability to induce T cell cytokine release.

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Cross-reactivity studies of a new group 2 allergen from the dust mite Glycyphagus domesticus , Gly d 2, and group 2 allergens from Dermatophagoides pteronyssinus, Lepidoglyphus destructor , and Tyrophagus putrescentiae with recombinant allergens

Cited by 52 publications

References 42 publications

Dilution Method to Refold Bacterially Expressed Recombinant Der f 2 and Der p 2 to Exhibit the Secondary Structure and Histamine-Releasing Activity of Natural Allergens

Dilution Method to Refold Bacterially Expressed Recombinant Der f 2 and Der p 2 to Exhibit the Secondary Structure and Histamine-Releasing Activity of Natural Allergens

Hypoallergens for Allergen-specific Immunotherapy by Directed Molecular Evolution of Mite Group 2 Allergens

Lep d 2 polymorphisms in wild and cultured Lepidoglyphus destructor mites

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