title: Analysis of the structure and allergenicity of recombinant pro- and mature Der p 1 and Der f 1: major conformational IgE epitopes blocked by prodomains.
abstract: BACKGROUND
  The major house dust mite group 1 allergens Der p 1 and Der f 1, which belong to the papain-like cysteine protease family, are the most potent of indoor allergens. However, little information is available on the location of IgE epitopes.
  
  
  OBJECTIVE
  We investigated the allergenicities of recombinant proforms and mature forms of Der p 1 and Der f 1 to compare them with natural Der p 1 and Der f 1 and to obtain information on the conformational IgE-binding epitopes.
  
  
  METHODS
  Secreted pro-Der p 1 and pro-Der f 1 and their mutants without hyperglycosylation expressed in yeast were converted to mature forms. We purified the proforms and mature forms and analyzed their apparent molecular sizes and secondary structures by means of gel-filtration and circular dichroism analysis and their allergenicities by means of assays for IgE binding, IgE-binding inhibition, and basophil histamine release. The tertiary structure of pro-Der f 1 was predicted by molecular modeling.
  
  
  RESULTS
  The recombinant mature forms exhibited similar molecular sizes, secondary structures, and allergenicities as their natural types. On the other hand, their proforms exhibited different secondary structures and less allergenicities than the mature forms in all sera and volunteers tested. Molecular modeling revealed that the prosegment is anchored at the prosegment-binding loop and the substrate-binding cleft on the surface of the mature portion.
  
  
  CONCLUSIONS
  Our studies indicate that the prodomains of Der p 1 and Der f 1 reduce allergenicity and that the major conformational IgE epitopes commonly found in a broad population of patients exist within the 2 regions blocked by the prosegments. Recombinant Der p 1 and Der f 1 and the findings in the present study will be the basis for allergen standardization and the design of safer and more effective allergen vaccines.
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan. t-takai@med.juntendo.ac.jp
authors.forename: Toshiro
authors.initials: T
authors.lastname: Takai
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kato
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yasueda
authors.forename: Ko
authors.initials: K
authors.lastname: Okumura
authors.forename: Hideoki
authors.initials: H
authors.lastname: Ogawa
mesh_terms: D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry* / Q000276:immunology*; D002850:Chromatography, Gel; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D039741:Antigens, Dermatophagoides; D000939:Epitopes; D011994:Recombinant Proteins; D007073:Immunoglobulin E