Molecular and Immunological Characterization of a Wheat Serine Proteinase Inhibitor as a Novel Allergen in Baker's Asthma

Abstract

IgE-mediated sensitization to wheat flour belongs to the most frequent causes of occupational asthma. A cDNA library from wheat seeds was constructed and screened with serum IgE from baker's asthma patients. One IgE-reactive phage clone contained a full-length cDNA coding for an allergen with a molecular mass of 9.9 kDa and an isoelectric point of 6. According to sequence analysis it represents a member of the potato inhibitor I family, a group of serine proteinase inhibitors, and thus is the first allergen belonging to the group 6 pathogenesis-related proteins. The recombinant wheat seed proteinase inhibitor was expressed in Escherichia coli and purified to homogeneity. According to circular dichroism analysis, it represented a soluble and folded protein with high thermal stability containing mainly β-sheets, random coils, and an α-helical element. The recombinant allergen showed allergenic activity in basophil histamine release assays and reacted specifically with IgE from 3 of 22 baker's asthma patients, but not with IgE from grass pollen allergic patients or patients suffering from food allergy to wheat. Allergen-specific Abs were raised to localize the allergen by immunogold electron microscopy in the starchy endosperm and the aleuron layer. The allergen is mainly expressed in mature wheat seeds and, despite an ∼50% sequence identity, showed no relevant cross-reactivity with allergens from other plant-derived food sources such as maize, rice, beans, or potatoes. Recombinant wheat serine proteinase inhibitor, when used in combination with other specific allergens, may be useful for the diagnosis and therapy of IgE-mediated baker's asthma.

Molecular and Immunological Characterization of Tri a 36, a Low Molecular Weight Glutenin, as a Novel Major Wheat Food Allergen

“…Immature seeds were harvested 7,10,15,20,25,30, and 35 d after onset of pollination directly into liquid nitrogen and stored at 280˚C until use (20). Rye, barley, oat, spelt, and rice grains produced by Alnatura (Bickenbach, Germany) were bought at a local drugstore.…”

“…The structure of wheat allergens can be either identified by proteomic approaches (19) or by molecular cloning technologies (20). Diagnostic tests based on recombinant wheat allergens may be useful to discriminate between different forms of wheat hypersensitivity (21).…”

A Review: Biological and Technological Functions of Barley Seed Pathogenesis-Related Proteins (PRs)

“…A protease inhibitor from wheat, with high thermal stability, homologous with family potato inhibitor family I, was recently identified as a novel allergen in baker's asthma 63 .…”

Advances in allergen-microarray technology for diagnosis and monitoring of allergy: The MeDALL allergen-chip

“…Wheat allergens-A wheat cDNA library was screened with sera from wheat food allergic patients as described [28]. Recombinant Tri a 36 and Tri a 36 fragment (amino acids 191-369) [29], clones 37 (thioredoxin h), 38 (glutathione transferase), 112 (1-Cysperoxiredoxin), 123 (profilin) and 126 (dehydrin) as well as clone 10 (serine proteinase inhibitor) were cloned, expressed and characterized as described [28,30].…”

“…Recombinant Tri a 36 and Tri a 36 fragment (amino acids 191-369) [29], clones 37 (thioredoxin h), 38 (glutathione transferase), 112 (1-Cysperoxiredoxin), 123 (profilin) and 126 (dehydrin) as well as clone 10 (serine proteinase inhibitor) were cloned, expressed and characterized as described [28,30]. cDNA-sequences of IgE-reactive clones 79, 85 and 110 that showed partial sequence identities to gamma gliadins, respectively, cDNAs coding for Tri a 37, LTP, beta amylases and avenin-like protein were codon optimized for E. coli expression and cloned with an additional sequence coding for a hexa-histidine tag into the expression vector pET17b (Novagen).…”