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.
BACKGROUND:Baker's asthma is an important occupational allergic disease. Wheat lipid transfer protein (LTP) Tri a 14 is a major allergen associated with wheatallergy. 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 asthmapatients, 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. WheatTri a 14 shows a high thermal stability and resistance to gastrointestinal digestion.
Authors: H W Chu; C M Lloyd; W Karmaus; P Maestrelli; P Mason; G Salcedo; J Thaikoottathil; A J Wardlaw Journal: Clin Exp Allergy Date: 2010-11 Impact factor: 5.018
Authors: Susan M Tarlo; Jean-Luc Malo; Frédéric de Blay; Nicole Le Moual; Paul Henneberger; Dick Heederik; Monika Raulf; Christopher Carlsten; André Cartier Journal: Ann Am Thorac Soc Date: 2017-09
Authors: Arantxa Palacín; Cristina Gómez-Casado; Luis A Rivas; Jacobo Aguirre; Leticia Tordesillas; Joan Bartra; Carlos Blanco; Teresa Carrillo; Javier Cuesta-Herranz; Consolación de Frutos; Genoveva García Alvarez-Eire; Francisco J Fernández; Pedro Gamboa; Rosa Muñoz; Rosa Sánchez-Monge; Sofía Sirvent; María J Torres; Susana Varela-Losada; Rosalía Rodríguez; Victor Parro; Miguel Blanca; Gabriel Salcedo; Araceli Díaz-Perales Journal: PLoS One Date: 2012-12-14 Impact factor: 3.240
Authors: C Gómez-Casado; M Garrido-Arandia; P Gamboa; N Blanca-López; G Canto; J Varela; J Cuesta-Herranz; L F Pacios; A Díaz-Perales; L Tordesillas Journal: Clin Dev Immunol Date: 2013-11-14