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

Abstract

Summary 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.

Molecular basis of allergen cross-reactivity: Non-specific lipid transfer proteins from wheat flour and peach fruit as models

“…An in-house wheat extract of salt-soluble proteins, as well as rTri a 14, were obtained according to Palacin et al (2007Palacin et al ( , 2009, and rPru p 3 was produced and isolated following . Purified allergens were quantified by means of the commercial bicinchoninic acid test (Pierce, Cheshire, UK), and purity ascertained by a single band of the expected molecular size in SDS-PAGE analysis and a single and expected (Diaz-Perales et al, 2000Palacin et al, 2009) N-terminal amino acid sequence (10 residues).…”

“…Purified allergens were quantified by means of the commercial bicinchoninic acid test (Pierce, Cheshire, UK), and purity ascertained by a single band of the expected molecular size in SDS-PAGE analysis and a single and expected (Diaz-Perales et al, 2000Palacin et al, 2009) N-terminal amino acid sequence (10 residues).…”

“…However, an increasing body of experimental data point out to a complementary role as inhalant allergens, linked in some cases to plant food and pollen cross-reactions (Barber et al, 2008;Lauer et al, 2007;Zuidmeer and van Ree, 2007). In this context, wheat LTP Tri a 14 has been recently identified as a relevant allergen involved both in wheat food allergy and baker's asthma (Palacin et al, 2007(Palacin et al, , 2009Pastorello et al, 2007).…”

“…However, no proper panel of molecular tools (purified allergens) has been still developed for this allergy. A wheat LTP, namedTria 14, has been proposed as a potential useful diagnostic tool, based on its high sensitization rate (60% of sera with specific IgE to the allergen) and in vivo (SPT) positive response (62% of tested patients) (Palacin et al, 2007(Palacin et al, , 2009). Furthermore, a complex sensitization profile to Tri a 14 and peach Pru p 3 have been described in baker's asthma patients (Palacin et al, 2007(Palacin et al, , 2009, indicating that there are high or poor cross-reactivity between the two allergens (i.e.…”

A Baker with Asthma and Wheat Flour Food Allergy

“…A wide array of wheat flour proteins, both salt-soluble proteins and prolamins, and particularly members of the α-amylase inhibitor family and ω5-gliadin (in wheat-dependent exercise-induced anaphylaxis), have been linked to food wheat allergy (2)(3)(4). LTPs have (wheat flour Tri a 14) recently been identified as a relevant allergen associated with both baker's asthma and wheat IgE-mediated food allergy (3)(4)(5)(6). Several of the major salt-insoluble wheat flour proteins (prolamins) such as gliadin also appear to be implicated in baker's asthma and wheat mediated food allergy (7).…”