A Recombinant Group 1 House Dust Mite Allergen, rDer f 1, with Biological Activities Similar to Those of the Native Allergen

303

254

The examination of house dust mite extracts has indicated that over 30 different proteins can induce IgE antibody in patients allergic to the house dust mite. There are however dominant specificities especially the group 1 and 2 allergens which can account for much of the allergenicity of extracts. Of the 19 denominated allergens, the major IgE binding has been reported for the group 1, 2, 3, 9, 11, 14 and 15 allergens. The high-molecular-weight group 11, 14 and 15 allergens have only recently been described and although high IgE binding has been anticipated from immunoblotting, there is a need for considerable corroboration. Similarly, the study of the group 3 and 9 serine protease allergens has been incomplete. The group 4, 5, 7 and 8 allergens have shown intermediate IgE binding and the group 10 tropomyosins are of interest because of their potential cross-reactivity with allergen from disparate species. Although the progress with the production of recombinant group 1 allergens has been recent, many of the allergens can be produced as high IgE-binding polypeptides. The tertiary structure of the group 2 allergens has been determined from recombinant proteins and they are an excellent model for the investigation of modified allergens. An unexpected property of the group 1, 2 and 3 allergens has been the high degree of polymorphism found by cDNA analysis. It has however been possible to identify sequences to represent the variation in the natural allergens. The group 7 and 14 allergens show secondary modifications which vary in different extracts creating batch variation. While some estimate of the importance of allergens can be obtained from IgE binding, few analyses of T-cell responses have been made and these regulate both the development of, and the protection from sensitization.

Section: Recombinant Mite Allergensmentioning

confidence: 99%

Characterization and Immunobiology of House Dust Mite Allergens

Thomas¹,

Smith²,

Hales³

et al. 2002

303

254

“…This prokaryotic expression system is still applied for the production of many different recombinant allergens [103, 104, 105, 106, 107, 108]. In more recent years, several allergens were produced in eukaryotic expression systems like the yeast Pichia pastoris [109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127], insect cells [128, 129, 130, 131, 132, 133, 134, 135, 136], and tobacco plants [137, 138]. These systems facilitate post-translational modifications that do not (easily) occur in E. coli , like disulfide-bridge formation, hydroxylation of prolines, glycosylation.…”

Section: Recombinant Allergens For Application In Diagnostics and Immmentioning

confidence: 99%

Carbohydrate Epitopes and Their Relevance for the Diagnosis and Treatment of Allergic Diseases

Ree¹

2002

173

119

Allergenicity of plant and invertebrate N-glycans has been shown to be caused by the presence of two typical nonmammalian substitutions: an α(1,3)-fucose linked to the proximal N-acetylglucosamine and a β(1,2)-xylose linked to the core mannose. IgE antibodies against these carbohydrate structures are induced upon exposure to pollen or after insect stings, and result in extensive cross-reactivity to plant and invertebrate foods. These cross-reactive IgE antibodies have been shown to possess variable degrees of biological activity, but have never been convincingly shown to induce clinical food allergy. The most likely explanation for this lack of clinical relevance has to be sought in a combination of epitope valency and antibody affinity. In diagnostic tests, these antibodies are at the basis of many false-positive test results for food allergy. Recombinant technologies offer the possibility to produce allergens that do not carry IgE-binding glycans. Whether their absence or presence is of importance for the application of recombinant allergens in immunotherapy is still largely unknown.

“…Recently, we and others reported on efficient systems that can be used to produce recombinant mature Der f 1 and Der p 1 with IgE-binding and proteolytic activities by acid treatment of the proforms expressed in yeast Pichia pastoris [30,31,32,33,34,35] or Escherichia coli [36]. Furthermore, we prepared recombinant Der p 1 and Der f 1 without yeast-derived hyperglycosylation [30,31,32] and demonstrated that they exhibited similar molecular sizes, secondary structures and allergenicities as their natural types [30].…”

Section: Introductionmentioning

confidence: 99%

Recombinant Der p 1 and Der f 1 with in vitro Enzymatic Activity to Cleave Human CD23, CD25 and α<sub>1</sub>-Antitrypsin, and in vivo IgE-Eliciting Activity in Mice

Takai¹,

Kato²,

Ota³

et al. 2005

Background: The major house dust mite group 1 allergens Der p 1 and Der f 1 are the most potent indoor allergens. Der p 1 cleaves human cell surface molecules, the low-affinity IgE receptor (CD23/FcΕRII), the α-subunit of the IL-2 receptor (CD25), and a protease inhibitor α₁-antitrypsin, and in vitro and in vivo studies suggested the importance of its proteolytic activity in the pathogenesis of allergy. Recently, we established an efficient system to prepare correctly folded active recombinant Der p 1 and Der f 1 (Der p 1-N52Q and Der f 1-N53Q) with similar molecular sizes, secondary structures and allergenicities as their natural types. To evaluate whether Der p 1-N52Q and Der f 1-N53Q are suitable for use in future in vitro and in vivo studies as alternatives to the natural types, we investigate their proteolytic activity to cleave the human proteins and IgE-eliciting activity in mice. Methods: Proteolytic activities of Der p 1-N52Q and Der f 1-N53Q against a short peptide substrate, a collagen substrate Azocoll, human CD23 and CD25 expressed on the cells and human α₁-antitrypsin were analyzed by kinetic assays for proteolysis of the fluorogenic or colorimetric substrates, flow cytometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Mice were intraperitoneally immunized with Der p 1-N52Q and Der f 1-N53Q adsorbed on Alum, and the serum IgE levels were measured by sandwich ELISA. Results: Der p 1-N52Q and Der f 1-N53Q showed proteolytic specificities against the short peptide substrate, Azocoll, human cell surface CD23 and CD25 and human α₁-antitrypsin, and elicited significant serum IgE levels in immunized mice. Conclusion: The recombinant forms, Der p 1-N52Q and Der f 1-N53Q, will be useful tools as alternatives to the natural Der p 1 and Der f 1 for various in vitro and in vivo analyses.