1Present address: CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain.
Recombinant lipid transfer protein Tri a 14: a novel heat and proteolytic resistant tool for the diagnosis of baker's asthma
Article first published online: 26 MAY 2009
© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd
Clinical & Experimental Allergy
Volume 39, Issue 8, pages 1267–1276, August 2009
How to Cite
Palacin, A., Varela, J., Quirce, S., Del Pozo, V., Tordesillas, L., Barranco, P., Fernandez-Nieto, M., Sastre, J., Diaz-Perales, A. and Salcedo, G. (2009), Recombinant lipid transfer protein Tri a 14: a novel heat and proteolytic resistant tool for the diagnosis of baker's asthma. Clinical & Experimental Allergy, 39: 1267–1276. doi: 10.1111/j.1365-2222.2009.03280.x
- Issue published online: 9 JUL 2009
- Article first published online: 26 MAY 2009
- Submitted 5 December 2008; revised 30 March 2009; accepted 1 April 2009
- baker's asthma;
- lipid transfer protein;
- molecular diagnosis;
- proteolytic resistance;
- recombinant allergen;
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.