Recombinant lipid transfer protein Tri a 14: A novel heat and proteolytic resistant tool for the diagnosis of baker's asthma

A. Palacin, J. Varela, S. Quirce, V. Del Pozo, L. Tordesillas, P. Barranco, M. Fernandez-Nieto, J. Sastre, A. Diaz-Perales, G. Salcedo

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

Background Baker's asthma is an important occupational allergic disease. Wheat lipid transfer protein (LTP) Tri a 14 is a major allergen associated with wheat allergy. No panel of wheat recombinant allergens for component-resolved diagnosis of baker's asthma is currently available. Objective To evaluate the potential role of recombinant Tri a 14 as a novel tool for the diagnosis of baker's asthma, and to test the heat and proteolytic resistance of the wheat LTP allergen. Methods A cDNA encoding Tri a 14 was isolated and sequenced, the recombinant allergen produced in Pichia pastoris and purified by chromatographic methods. Physicochemical and immunological comparison of the natural and recombinant forms of Tri a 14 was carried out by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry, circular dichroism (CD) analysis, IgE immunodetection, and specific IgE determination and ELISA-inhibition assays using a pool or individual sera from 26 patients with baker's asthma. Thermal denaturation and simulated gastrointestinal digestion of both Tri a 14 forms were checked by spectroscopic and electrophoretic methods, respectively, and biological activity by basophil activation test (BAT). Results Natural and recombinant Tri a 14 were similarly folded, as indicated by their nearly identical CD spectra and heat denaturation profiles. A high interclass correlation coefficient (0.882) was found between specific IgE levels to both Tri a 14 proteins in individual sera from baker's asthma patients, but a slightly lower IgE-binding potency of rTri a 14 was detected by ELISA-inhibition assays. Natural and recombinant Tri a 14 elicited positive BAT in two and one out of three patients, respectively. Heat denaturation profiles and simulated gastrointestinal digestion assays indicated that Tri a 14 displayed a high heat and digestive proteolytic resistance, comparable to those of peach Pru p 3, the model food allergen of the LTP family. Conclusions Recombinant Tri a 14 is a potential tool for baker's asthma diagnosis, based on its physicochemical and immunological similarity with its natural counterpart. Wheat Tri a 14 shows a high thermal stability and resistance to gastrointestinal digestion.

Original languageEnglish
Pages (from-to)1267-1276
Number of pages10
JournalClinical and Experimental Allergy
Volume39
Issue number8
DOIs
StatePublished - Aug 2009
Externally publishedYes

Keywords

  • Baker's asthma
  • Lipid transfer protein
  • Molecular diagnosis
  • Proteolytic resistance
  • Recombinant allergen
  • Wheat

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