title: Molecular cloning and immunological characterisation of potential allergens from the mould Fusarium culmorum.
abstract: BACKGROUND
  High quality and stability are essential requirements of commercial allergen preparations. Recently we have demonstrated the very low stability of protein allergens in an extract of the ubiquitous mould Fusarium culmorum.
  
  
  OBJECTIVE
  The present study was performed to identify, isolate and characterise allergens of F. culmorum as a basis for a stable allergenic reference material. In addition, the significance of IgE binding to carbohydrate structures in the natural allergen source was investigated.
  
  
  METHODS
  Sera of 52 subjects with suspected mould allergy were used to determine the IgE binding capacity of a commercial F. culmorum extract and an in-house extract by immunoblotting and enzyme allergo sorbent test (EAST). Binding of IgE-antibodies to putative carbohydrate structures located on glycoproteins was verified by periodate treatment of blot strips prior to immunodetection. A complementary (c)DNA expression library of F. culmorum was prepared and screened for IgE-binding clones using sera from F. culmorum-sensitive individuals. Positive clones were isolated, and the open reading frames were subcloned into expression vectors to produce recombinant proteins in E. coli. The recombinant proteins were tested for their IgE reactivity by immunoblotting and EAST.
  
  
  RESULTS
  Using the in-house extract for EAST and immunoblot experiments 44% (23/52) of the sera were found to contain F. culmorum-specific IgE antibodies. Compared to the in-house extract, nearly all IgE-reactivties in the range of 15-30kD were lacking in the commercial preparation as examined by immunoblot analysis and only 10% (5/52) of the sera were found to contain F. culmorum-specific IgE by EAST. IgE binding to putative carbohydrate structures was observed in the high molecular weight range in approximately 50% (12/23) of the IgE-positive sera by both extracts. Three IgE binding clones were isolated from the cDNA-library. One clone (Fus c 1) is homologous to the highly conserved 60S acidic ribosomal protein P2 described as minor allergen in other moulds. The second (Fus c 2) shows high similarity (64%) to a respiratory allergen from the basidiomycete Coprinus comatus (Cop c 2). The third clone (Fus c 3) was not related to known proteins. With sera from 26 individuals sensitised to F. culmorum the IgE prevalence of recombinant proteins rFus c 1, rFus c 2 and rFus c 3 was found to be 35, 50, and 15%, respectively.
  
  
  CONCLUSIONS
  F. culmorum may represent an underestimated source of aeroallergens. In contrast to highly labile and poorly standardised F. culmorum extracts, the new recombinant allergens may serve as stable allergenic reference material. A combination of rFus c 1 and rFus c 2 is suitable to diagnose 81% of F. culmorum-sensitised subjects. IgE reactivity to putative carbohydrate structures is relatively frequent, and can not be detected by these allergens.
authors.affiliation: Department of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Stasse 51-59, D-63225, Langen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Hoff
authors.forename: B K
authors.initials: BK
authors.lastname: Ballmer-Weber
authors.forename: B
authors.initials: B
authors.lastname: Niggemann
authors.forename: A
authors.initials: A
authors.lastname: Cistero-Bahima
authors.forename: M
authors.initials: M
authors.lastname: San Miguel-Moncín
authors.forename: A
authors.initials: A
authors.lastname: Conti
authors.forename: D
authors.initials: D
authors.lastname: Haustein
authors.forename: S
authors.initials: S
authors.lastname: Vieths
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000595:Amino Acid Sequence; D000946:Antigens, Fungal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D002241:Carbohydrates / Q000276:immunology; D003001:Cloning, Molecular; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005670:Fusarium / Q000276:immunology*; D015723:Gene Library; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000276:immunology
chemical_list: D000485:Allergens; D000946:Antigens, Fungal; D002241:Carbohydrates; D000939:Epitopes; D006023:Glycoproteins; D010940:Plant Proteins; D007073:Immunoglobulin E