Peptidomics of an in vitro digested α-Gal carrying protein revealed IgE-reactive peptides

The mammalian carbohydrate galactose-α1,3-galactose (α-Gal) causes a novel form of food allergy, red meat allergy, where patients experience severe allergic reactions several hours after red meat consumption. Here we explored gastric digestion of α-Gal glycoproteins using an in vitro model. Bovine thyroglobulin (BTG), a typical α-Gal carrying glycoprotein, was digested with pepsin. The resulting peptides were characterised by SDS PAGE, immunoblot and ImmunoCAP using sera from 20 red meat allergic patients. During pepsinolysis of BTG, a wide range of peptide bands was observed of which 14 to 17 kDa peptides remained stable throughout the gastric phase. The presence of the α-Gal epitope on the obtained peptides was demonstrated by an anti-α-Gal antibody and IgE from red meat allergic patients. The α-Gal digests were able to inhibit up to 86% of IgE reactivity to BTG. Importantly, basophil activation test demonstrated that the allergenic activity of BTG was retained after digestion in all four tested patients. Mass spectrometry-based peptidomics revealed that these peptides represent mostly internal and C-terminal parts of the protein, where the most potent IgE-binding α-Gal residues were identified at Asn₁₇₅₆, Asn₁₈₅₀ and Asn₂₂₃₁. Thus allergenic α-Gal epitopes are stable to pepsinolysis, reinforcing their role as clinically relevant food allergens.