Identification of Epicoccum purpurascens allergens by two-dimensional immunoblotting and mass spectrometry.
Sensitization to allergens of Epicoccum purpurascens is reported from many places world over. Previously, the IgE binding proteins of Epicoccum were studied by one-dimensional immunoblotting (1-DE). However, the allergens recognized by 1-DE may be a mixture of proteins appearing at the same molecular weight. In the present study, IgE-binding components of E. purpurascens were detected using two-dimensional immunoblotting and mass spectrometry. Approximately 250 protein spots were identified by Coomassie staining in the range of 14-110kDa and pI 3.5-9.5. Of these, 147 showed specific IgE reactivity with pooled patients' sera whereas 16 protein components reacted with more than 50% of the individual patients' sera. The frequency of IgE reactivity was highest (87.5%) for a 25-kDa protein (pI-7.85). Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of 16 protein components led to identification of 6 important allergens as phospho-glycerate kinase (PGK), RNA-dependent RNA polymerase, pyrroline-5-carboxylate dehydrogenase, 40S ribosomal protein and two proteins of unknown functions. The major allergens identified may be assessed for cross reactivity with other fungi. In conclusion, 2-DE followed by immunoblotting allowed characterization of E purpurascens allergens. Six new allergens have been identified using this technique and mass spectrometry. The availability of such relevant allergens would help in component-based diagnosis and therapy of fungal allergy.
Kukreja
Neetu
N
Institute of Genomics and Integrative Biology (CSIR), Delhi University Campus, Mall Road, Delhi-110007, India.
Singh
B P
BP
Arora
Naveen
N
Gaur
S N
SN
Sridhara
S
S
D000328:Adult; D000485:Allergens / Q000276:immunology / Q000302:isolation & purification*; D001203:Ascomycota / Q000276:immunology* / Q000302:isolation & purification; D005260:Female; D006801:Humans; D006967:Hypersensitivity / Q000097:blood / Q000276:immunology*; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood* / Q000276:immunology; D008297:Male; D012882:Skin Tests; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000485:Allergens; D007073:Immunoglobulin E