Proteomic Profiling of Celiac-Toxic Motifs and Allergens in Cereals Containing Gluten

Abstract

Cereal-based foods can cause immune-mediated adverse reactions, including celiac disease and IgE-mediated allergies, but the potency of different cereal species to cause such reactions appears to vary, with oats being less celiac-toxic and allergenic than wheat. In order to define differences in the immunological potential of wheat, barley, rye, and oats, proteomic profiling of proteins carrying celiac-toxic motifs and allergens has been undertaken. Total protein extracts were subjected to chymotryptic digestion and analyzed using data-independent ion mobility mass spectrometry and a pipeline employing a curated gluten protein sequence database. Depending on the cereal species, 376-2769 proteins were identified, the majority being grain storage proteins. Relative quantitation of proteins containing celiac-toxic motifs showed that they were most abundant and diverse in wheat, with only a limited number, at much lower abundance, identified in oats. Allergens belonging to the seed storage prolamins were the most abundant, while allergens belonging to the α-amylase/trypsin inhibitor family associated with respiratory allergy were of only moderate abundance in comparison. Wheat allergen homologues were identified in other cereal species but at a very low level in oats. These data suggest that the relative risk of oats in the context of both celiac disease and IgE-mediated allergy is low.

Keywords: IgE epitope; Proteomics; barley; celiac-toxic motif; oats; rye; wheat.

Figure 1

Principal component analysis (PCA) of proteome profiles of wheat, barley, rye, and oats. PCA scores plot of UniProt protein accessions (lead accession from protein grouping) relative normalized abundances identified in wheat (T. aestivum; light blue circles), barley (H. vulgare; red circles), rye (S. cereale; dark blue circles), and oat (A. sativa; green circles) when searching against Viridiplantae (A; PC1 vs PC2, B: PC2 vs PC3) and tagging accessions identified in GluPro v 6.1 C; PC1 vs PC2, D; PC2 vs PC3). 3D loading plots are available in Figure S5 and Supporting Information Data File 3 -– Sheet 1.

Figure 2

Phylogenetic analysis and relative quantification of identified gluten proteins. Protein accessions tagged as gluten proteins in the GluPro curated gluten protein sequence databases were mapped either into the relevant GluPro phylogenetic tree (A, C, E, G) or Quant curves (B, D, F, H), where GluPro identifications were colored in pink and the total species specific identifications shown in green. (A, B) wheat—GluPro v 1.2; (C, D) barley—GluPro v 3; (E, F) rye—GluPro v 4; and (G, H) oats—GluPro v 5 (G). Protein accessions used to generate the figures were the lead accessions from protein grouping.

Figure 3

Distribution of celiac-toxic motifs in cereal seed proteomes. (A) Heat map of celiac-toxic motif-containing peptides retrieved from AllergenOnline and identified from discovery mass spectrometry (auto-scaled to features). Columns are specified by the classes, which were as follows: wheat—yellow; barley—red; rye—dark blue; oatsgreen. Relative normalized abundance of consensus celiac-toxic epitopes for DQ2.5 (B) and DQ2.2, DQ8 and DQ8.5 (C) α—α-gliadin; γ—γ-gliadin; ω—ω-gliadin; G-LLMW-GS; Horhordein, Sec—secalin; Ave—avenin; G-HHMW-GS. Cereal species are denoted by colored bars as in panel A. It was also clear that some peptides containing CTMs were present in combinations of wheat, barley, and rye, with varying abundances, but none were common to all three cereal species.

Figure 4

Identification and relative abundance of allergens associated with IgE-mediated allergies using the lead accession from the protein grouping. Yellow bars indicate the WHO allergen isoform, black bars are homologues identified through full sequence BLAST, green bars are those identified from a sliding 80mer window and FASTA, and pink bars are the accessions that were common between the two methods. Allergen sequences were retrieved from the WHO/IUIS allergen nomenclature database and homologues in other cereal species identified from discovery mass spectrometry. Allergens were grouped as being either gluten proteins (A), trypsin/α-amylase inhibitors (ATI) (B), or other (C). Homologues are identified as follows: wheat—Hom-W; barley—hom-B; rye—hom-R; oats—hom-O.