Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties

Abstract

Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten.

Figure 1. Structural changes in gluten proteins by transamidation of wheat flour and gluten under non-reducing conditions.

(a) Reduced and alkylated glutenin subunit electrophoretic patterns of wheat flour and gluten, original and derivatised with mTG alone and with K-C₂H₅ and n-butylamine as amine nucleophiles under non-reducing conditions. (b) Gliadin electrophoretic patterns of wheat flour and gluten, original and derivatised with mTG alone and with K-C₂H₅ and n-butylamine as amine nucleophiles under non-reducing conditions. Lane 1, F; Lane 2, FB 50X; Lane 3, FB 5X; Lane 4, FK-C₂H₅ 5X; Lane 5, FmTG; Lane 6, G; Lane 7, GB 50X; Lane 8, GB 5X; Lane 9, GK-C₂H₅ 5X; Lane 10, GmTG. Reversed-phase HPLC results for gliadins (c,d) and glutenins (e,f) extracts of wheat flour and gluten, original and derivatised with mTG alone and with K-C₂H₅ and n-butylamine as amine nucleophiles under non-reducing conditions. Wheat flour chromatograms are represented for a maximum absorbance of 1.0 and gluten chromatograms are represented for a maximum absorbance of 2.5. ω5, ω1,2, α/β and γ represent the different identified gliadin proteins, and HMW and LMW represent the different glutenin subunits. Absorbance was registered at 210 nm. For sample nomenclature please consult Fig. 6. (g) Reactions catalysed by microbial transglutaminase and the end-products.

Figure 2. Cross-linking of gluten decreases toxic epitopes amount and affect negatively the rheological properties of gluten.

Amino acid composition determined after enzymatic hydrolysis of wheat flour (a–c) and gluten (d–f), original and derivatised with mTG alone and with K-C₂H₅ and n-butylamine as amine nucleophiles under non-reducing conditions. For (a–f) error bars represent the standard deviation *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 (n = 3). (g) R5 reactive epitopes’ content (mg of gliadin per kg of product) of wheat flour and gluten, original and derivatised with mTG alone and with K-C₂H₅ and n-butylamine as amine nucleophiles under non-reducing conditions after peptic-tryptic digestion. n = 2 different experiments (each with four replicates) and error bars represent the s.d. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Micro-extension tests with dough prepared from wheat flour, original and derivatised with mTG alone and with K-C₂H₅ and n-butylamine as amine nucleophiles under non-reducing conditions. (h): F; FK-C₂H₅; FB 5X; FB 50X; FmTG; F + mTG. Or, under reducing conditions. (i): FR; FRB 50X; FRmTG. (j) Rheological properties of dough prepared from wheat flour, original and derivatised with mTG alone and with K-C₂H₅ and n-butylamine as amine nucleophiles under non-reducing and reducing conditions. ^#For sample nomenclature please consult Fig. 6. The columns values with the same letter are not statistically significant, p < 0.05 (n = 2).

Figure 3. Reducing conditions favour the transamidation of gluten proteins and decreases further the amount of toxic epitopes.

(a) GC-MS chromatogram for the amino acid analysis of FR, FRB 50X and FRmTG. Indicated are the peaks of glutamic acid (Glu), phenylalanine (Phe), glutamine (Gln), γ-glutamyl-n-butylamine (γ-Glu-n-butylamine) and lysine (Lys). EI mass spectra of γ-Glu-n-butylamine residue is shown. (b) γ-Glu-n-butylamine residue formation under non-reducing and reducing conditions. For b, experiments were run in triplicate, and error bars represent the s.d. Amino acid composition determined after enzymatic hydrolysis of wheat flour (c–e) and gluten (f–h), original and derivatised with mTG alone and with n-butylamine as amine nucleophile under reducing conditions. For (c–h), error bars represent the standard deviation *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 (n = 3). (i) R5 reactive epitopes’ content (mg of gliadin per kg of product) of wheat flour and gluten, original and derivatised with mTG alone and with n-butylamine as amine nucleophile under reducing conditions after peptic-tryptic digestion. n = 2 different experiments (each with four replicates) and error bars represent the s.d. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. For sample nomenclature please consult Fig. 6.

Figure 4. Structural changes in gluten proteins by transamidation of wheat flour and gluten under reducing conditions.

(a) Reduced and alkylated glutenin subunit electrophoretic patterns of wheat flour and gluten (as denoted), original and derivatised with mTG alone and with n-butylamine as amine nucleophile with increasing concentrations under reducing conditions. (b) Gliadin electrophoretic patterns of wheat flour and gluten (as denoted), original and derivatised with mTG alone and with n-butylamine as amine nucleophile with increasing concentrations under reducing conditions. Reversed-phase HPLC results for gliadins (c,d) and glutenins (e,f) extracts of wheat flour and gluten, original and derivatised with mTG alone and with n-butylamine as amine nucleophile under reducing conditions. Wheat flour chromatograms are represented for a maximum absorbance of 1.0 and gluten chromatograms are represented for a maximum absorbance of 2.5. ω5, ω1, 2, α/β and γ represent the different identified gliadin proteins, and HMW and LMW represent the different glutenin subunits. Absorbance was registered at 210 nm. For sample nomenclature please consult Fig. 6.

Figure 5. Proteomics and cytotoxicity analysis of n-butylamine bioconjugated gluten.

Two-dimensional electrophoresis pattern (IEF × SDS-PAGE) of the gluten, original (a) and derivatised with n-butylamine (b) as amine nucleophile under reducing conditions. The arrowheads point to the excised and identified protein spots (please see Table 1, for spot identification). The dashed rectangle indicates protein aggregates. Tandem mass (MS/MS) spectra of (c) α-/β-gliadin derived tryptic digest peptide [precursor ion at m/z (+2) = 1396.2] from spot 7/8, excised from 2-DE gel of GRB 50X; (d) HMW-GS derived tryptic digest peptide [precursor ion at m/z (+2) = 1024.5] from spot 1, excised from 2-DE gel of GRB 50X. * indicates the modified glutamine residue. Alamar Blue (AB) assay for cell viability quantification using Caco-2 cells, after 24 h (e) or 48 h (f) treatment with different concentrations of peptic-tryptic digests of wheat flour and gluten, original and derivatised with n-butylamine, as amine nucleophile, under reducing conditions. For sample nomenclature please consult Fig. 6. A total of n = 3 different experiments (each with four replicates), from different cell passages. Error bars represent the standard deviation Empty bars () denote 0.5 mg/mL; and filled bars () denote 1.0 mg/mL.

Figure 6. Reaction conditions of transamidation of wheat flour and gluten.

The nomenclature and reaction conditions of the derivatised and control products. “F” denotes wheat flour and “G” denotes gluten. “R” denotes reducing conditions, “mTG” microbial transglutaminase, “B” n-butylamine and “K-C₂H₅” L-lysine ethyl ester. “1X”, “5X” and “50X” denote the different concentrations of amine nucleophile used.