title: Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy.
abstract: BACKGROUND
  Profilins are ubiquitous panallergens that have been extensively characterized; yet, their clinical relevance is still unclear.
  
  
  OBJECTIVE
  The aim of the present study was to produce recombinant apple profilin (rMal d 4) and to evaluate its allergenic activity and its potency for component-resolved allergy diagnosis.
  
  
  METHODS
  Complementary DNA-derived Mal d 4 was cloned, expressed in Escherichia coli and subsequently purified via poly (l-proline) sepharose. A total of 28 sera from apple-allergic patients were used for IgE-ELISA, immunoblot, RAST and basophil histamine release (BHR) test. In addition, skin prick tests (SPTs) were performed in five patients.
  
  
  RESULTS
  Four different complementary DNA coding for apple profilin, Mal d 4, each with an open reading frame of 393 nucleotides, were identified. One isoform Mal d 4.0101 was expressed in Escherichia coli and subsequently purified. Mass spectroscopy revealed the expected mass of 13.826 for rMal d 4.0101, and circular dichroism analysis data were typical for a folded protein and small-angle X-ray scattering measurement identified the protein as a monomer. All the serum samples displayed IgE binding to rMal d 4.0101 in IgE ELISA, immunoblot and RAST. In immunoblotting, IgE binding to natural Mal d 4 was partially/completely inhibited by preincubation with rMal d 4.0101, and RAST values to apple extract were significantly reduced upon serum pretreatment with rMal d 4.0101. SPTs and BHR assays using purified rMal d 4.0101 were positive. Purified rMal d 4.0101 was destroyed within seconds when subjected to pepsin digestion.
  
  
  CONCLUSIONS
  Apple profilin complementary DNAs were identified. The physicochemical and allergenic properties of purified recombinant Mal d 4.0101 were evaluated showing that the recombinant protein was equal to the natural protein as shown by inhibition assays. Thus, Mal d 4 represents another example suitable for component-resolved diagnosis of food allergy.
authors.affiliation: Department of Pathophysiology, Center of Physiology and Pathophysiology, Medical University Vienna, Vienna, Austria.
authors.forename: Y
authors.initials: Y
authors.lastname: Ma
authors.forename: L
authors.initials: L
authors.lastname: Zuidmeer
authors.forename: B
authors.initials: B
authors.lastname: Bohle
authors.forename: S T H
authors.initials: ST
authors.lastname: Bolhaar
authors.forename: G
authors.initials: G
authors.lastname: Gadermaier
authors.forename: E
authors.initials: E
authors.lastname: Gonzalez-Mancebo
authors.forename: M
authors.initials: M
authors.lastname: Fernandez-Rivas
authors.forename: A C
authors.initials: AC
authors.lastname: Knulst
authors.forename: M
authors.initials: M
authors.lastname: Himly
authors.forename: R
authors.initials: R
authors.lastname: Asero
authors.forename: C
authors.initials: C
authors.lastname: Ebner
authors.forename: R
authors.initials: R
authors.lastname: van Ree
authors.forename: F
authors.initials: F
authors.lastname: Ferreira
authors.forename: H
authors.initials: H
authors.lastname: Breiteneder
authors.forename: K
authors.initials: K
authors.lastname: Hoffmann-Sommergruber
mesh_terms: D052179:Antigens, Plant / Q000276:immunology / Q000302:isolation & purification*; D015549:Basophil Degranulation Test; D019149:Bioreactors; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D005512:Food Hypersensitivity / Q000175:diagnosis*; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000175:diagnosis*; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood; D027845:Malus; D051304:Profilins / Q000235:genetics; D011852:Radioallergosorbent Test; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012882:Skin Tests; D013057:Spectrum Analysis
chemical_list: D052179:Antigens, Plant; D018076:DNA, Complementary; C518790:Mal d 4 allergen, Malus domestica; D051304:Profilins; D011994:Recombinant Proteins; D007073:Immunoglobulin E