Recombinant lipid transfer protein Tri a 14: a novel heat and proteolytic resistant tool for the diagnosis of baker's asthma.
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.
Palacin
A
A
Unidad de Bioquimica, Departamento de Biotecnología, E.T.S. Ingenieros Agronomos, Madrid, Spain.
Varela
J
J
Quirce
S
S
del Pozo
V
V
Tordesillas
L
L
Barranco
P
P
Fernandez-Nieto
M
M
Sastre
J
J
Diaz-Perales
A
A
Salcedo
G
G
D000328:Adult; D000485:Allergens / Q000097:blood / Q000737:chemistry / Q000276:immunology; D001249:Asthma / Q000175:diagnosis* / Q000276:immunology; D002352:Carrier Proteins / Q000097:blood / Q000737:chemistry* / Q000276:immunology; D002942:Circular Dichroism; D005260:Female; D005433:Flour; D005512:Food Hypersensitivity / Q000175:diagnosis* / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D019570:Inhalation Exposure; D008297:Male; D008875:Middle Aged; D009784:Occupational Diseases / Q000175:diagnosis* / Q000276:immunology; D011489:Protein Denaturation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000276:immunology; D014908:Triticum / Q000737:chemistry / Q000276:immunology*
D000485:Allergens; D002352:Carrier Proteins; D011994:Recombinant Proteins; C030363:lipid transfer protein; D007073:Immunoglobulin E