Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics.
Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.
Reuter
Andreas
A
Paul-Ehrlich-Institut, Division of Allergology, D-63225 Langen, Germany.
Fortunato
Donatella
D
Garoffo
Lorenza Perono
LP
Napolitano
Lorenzo
L
Scheurer
Stephan
S
Giuffrida
Maria G
MG
Vieths
Stefan
S
Conti
Amedeo
A
D000485:Allergens / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D015723:Gene Library; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011058:Pollen; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D027824:Rosaceae / Q000235:genetics / Q000276:immunology*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000485:Allergens; D019151:Peptide Library; D010940:Plant Proteins; D020033:Protein Isoforms; D007073:Immunoglobulin E