Thioredoxin-linked mitigation of allergic responses to wheat

Abstract

Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert SOS to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat f lour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the ␣ and ␤ fractions were most potent, followed by the ␥ and types. Thioredoxin mitigated the allergenicity associated with the major protein fractions-i.e, the gliadins (including the ␣, ␤, and ␥ types) and the glutenins-but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADPthioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously-i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, moredigestible foods.

Next-generation protein-rich potato expressing the seed protein gene AmA1 is a result of proteome rebalancing in transgenic tuber

“…The potent food allergens are known to be very stable in in vitro pepsin digestion, whereas most dietary proteins are readily digestible (27). The pepsin digestibility assay is thus considered one of the major ways to identify food allergens (28)(29)(30). To determine the relative stability of AmA1 to the extremes of pH and pepsin protease encountered in the mammalian digestive tract, the protein extracts of transgenic tubers were subjected to pepsin digestibility.…”

A Pepsin-Resistant 20 kDa Protein Found in Red Kidney Bean (Phaseolus vulgarisL.) Identified as Basic Subunit of Legumin

“…3) The disulfide bonds in the bean proteins were examined by the fluorescent labeling method. 4) Bean flour (200 mg) was mixed with 2 ml of extraction buffer (30 mM Tris-HCl, pH 7.9, containing 1 mM PMSF and 0.02% NaN 3 ), and centrifuged at 10;000 Â g for 15 min. Forty ml of supernatant (100 mg protein) was incubated for 20 min at room temperature with the following reduction reagents or enzymes in a final volume of 100 ml: (i) 0.5 mM DTT, (ii) 10 mM mercaptoethanol, (iii) 0.5 mM DTT and 1.6 mg thioredoxin, (iv) 2.5 mM NADPH, 1.6 mg thioredoxin, and 1.4 mg thioredoxin reductase, and (v) 2.5 mM NADPH, 2 mM glutathion, and 1 mg glutathione reductase.…”

Protein digestibility and relevance to allergenicity.

“…Buchanan and colleagues (Buchanan et al 1997;del Val et al 1999) reported that when structure of the major allergens from these foods is disrupted by reduction of disulfide bonds, the allergens were strikingly sensitive to pepsin digestion and lost their ability to elicit allergic reactions in previously sensitized dogs. Standardization of the assay conditions (such as pepsin concentration, pH, and temperature) has been described in the U.S. Pharmacopia (1990) and is sometimes referred to as SGF.…”