The Fate of IgE Epitopes and Coeliac Toxic Motifs during Simulated Gastrointestinal Digestion of Pizza Base

Abstract

Understanding how food processing may modify allergen bioaccessibility and the evolution of immunologically active peptides in the gastrointestinal tract is essential if knowledge-based approaches to reducing the allergenicity of food are to be realised. A soy-enriched wheat-based pizza base was subjected to in vitro oral-gastro-duodenal digestion and resulting digests analysed using a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The digestion profile of pizza base resembled that of bread crust where higher temperatures during baking reduced protein solubility but still resulted in the generation of a complex mixture of peptides. MS profiling showed numerous peptides carrying IgE epitopes, and coeliac toxic motifs were in excess of 20-30 residues long and were only released after either 120 min of gastric digestion or a combination of gastric and duodenal digestion. In silico prediction tools showed an overestimated number of cleavage sites identified experimentally, with low levels of atypical peptic and chymotryptic cleavage sites identified particularly at glutamine residues. These data suggest that such alternative pepsin cleavage sites may play a role in digestion of glutamine-rich cereal foods. They also contribute to efforts to provide benchmarks for mapping in vitro digestion products of novel proteins which form part of the allergenicity risk assessment.

Keywords: IgE epitope; allergen; allergenicity risk assessment; coeliac toxic motif; gluten; in vitro digestion; mass spectrometry; soy; wheat.

Figure 1

Wheat (black) and soy (red) allergens present in the WHO/IUIS allergen nomenclature database grouped according to Pfam clan [30]. Clans in the “other” category include CI-2, Glyco_hydro_tim, TIM_barrel, GADPH_aa-bio_dh, NADP_Rossmann, Thioredoxin, Zn_Beta_Ribbon and Bet_v_1_like.

Figure 2

SDS-PAGE profiles of the soluble fraction of in vitro oral, gastric and duodenal digests of pizza base. (A) Oral and gastric digestion stopped at the following digestion time points: ‘GU’- no pepsin 0.3-min control, ‘G0’—0.3 min, ‘G60’—60 min, ‘G120’—120 min. (B) G0 digests subjected to duodenal digestion stopped at duodenal digestion time points as follows: ‘D0’—0.3 min, ‘D60’—60 min, ‘D120’—120 min. (C) G60 digests subjected to duodenal digestion; timepoints are as in (B). Symbols indicate bands corresponding to enzymes included in the in vitro simulated digestion as follows: salivary α-amylase (★); pepsin (▲); pancreatic α-amylase (✦); chymotrypsin (■); trypsin (●); lysozyme (♦). Timepoints suffixed with * indicate duodenal digestion following 0.3 minutes of simulated gastric digestion.

Figure 3

Peptide profiles of pizza base digests defined by LC-MS/MS analysis. Number of identified wheat (A) and soy peptides (C). Peptide length distributions for wheat (B) and soy (D) peptides. Digestion time points are for gastric (G) and duodenal (D) digests after 0.3, 60 and 120 min.

Figure 4

Identification of wheat and soy proteins by mass spectrometry analysis of the soluble fraction of in vitro gastric (G) and duodenal (D) digestion samples. Filled bars—wheat; dotted bars—soy. (A) Total number of proteins identified by PEAKS with at least one unique peptide and FDR ≤ 1%. (B) Allergen isoforms were identified using the Aller-wheat and Aller-soy sequence databases. Digestion time points are as described in Figure 2C.

Figure 5

Bioaccessible wheat-derived peptides carrying IgE epitopes and coeliac toxic motifs in pizza base digests. (A) The number of non-unique IgE epitopes and coeliac toxic motifs found within identified peptides attributed to wheat throughout simulated digestion. (B,C) Length distribution of wheat peptides found containing non-unique IgE epitopes (B) and coeliac toxic motifs (C) throughout simulated digestion. Digestion time points are as described in Figure 2.

Figure 6

Peptide mapping of the LMW-GS allergen B2Y2Q7 throughout simulated digestion. Identified peptides were mapped onto the parent protein B2Y2Q7 at timepoints G60 (A), D0 (B) and D120 (C). The sequences bounded by a black box indicate the signal sequence, and those bounded in red and green represent IgE epitopes and coeliac toxic motifs, respectively. The domains with consensus repetitive peptides are shown in blue boxes. Endoprotease cleavage sites are denoted by red dots. Only the G60 digest is shown since it was similar to the G120 digest (Supplementary Figure S3A). Colour shading represents relative abundance of peptides calculated using spectral counting with the gradient from blue to red indicating increasing abundance.

Figure 7

Semi-quantitative analysis of peptide coverage of LMW GS B2Y2Q7 following simulated gastric (A) and gastro-duodenal digestion (B). Spectral counts [42] were plotted as a function of amino acid residue as follows: panel (A)—spectral counts of G60 labelled in black and G120 in red; panel (B)—D0 labelled in black, D60 in red and D120 in green.