title: Cloning and molecular and immunological characterisation of two new food allergens, Cap a 2 and Lyc e 1, profilins from bell pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum).
abstract: BACKGROUND
  Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food.
  
  
  OBJECTIVE
  The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins.
  
  
  METHODS
  cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergic patients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA.
  
  
  RESULTS
  The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomato profilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergic patients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin.
  
  
  CONCLUSIONS
  Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food.
authors.affiliation: Department of Chemistry and Biochemistry, University of Salzburg, Salzburg, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Willerroider
authors.forename: H
authors.initials: H
authors.lastname: Fuchs
authors.forename: B K
authors.initials: BK
authors.lastname: Ballmer-Weber
authors.forename: M
authors.initials: M
authors.lastname: Focke
authors.forename: M
authors.initials: M
authors.lastname: Susani
authors.forename: J
authors.initials: J
authors.lastname: Thalhamer
authors.forename: F
authors.initials: F
authors.lastname: Ferreira
authors.forename: B
authors.initials: B
authors.lastname: Wüthrich
authors.forename: O
authors.initials: O
authors.lastname: Scheiner
authors.forename: H
authors.initials: H
authors.lastname: Breiteneder
authors.forename: K
authors.initials: K
authors.lastname: Hoffmann-Sommergruber
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002212:Capsicum / Q000737:chemistry / Q000276:immunology*; D002850:Chromatography, Gel; D003285:Contractile Proteins; D004926:Escherichia coli / Q000235:genetics; D005512:Food Hypersensitivity / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D018551:Solanum lycopersicum / Q000737:chemistry / Q000276:immunology*; D008297:Male; D008840:Microfilament Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D008875:Middle Aged; D008969:Molecular Sequence Data; D051304:Profilins; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: D000485:Allergens; D003285:Contractile Proteins; D008840:Microfilament Proteins; D051304:Profilins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D012313:RNA