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

Abstract

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.

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer

“…Of importance, the major peptides carrying the α-Gal epitope were detected at molecular weights between 25 and 55 kDa, although faintly stained, implicating a prolonged survival of proteins during gastric digestion and a prolonged availability for uptake by intestinal cells. Moreover, we have previously shown that α-Gal containing peptides resulting from pepsinolysis are allergenic, reinforcing their role as clinically relevant food allergens [18]. These findings are in accordance with a previous study that demonstrated that glycosylation protects the egg allergen ovomucoid from pepsinolysis [19].…”

Toward acellular xenogeneic heart valve prostheses: Histological and biomechanical characterization of decellularized and enzymatically deglycosylated porcine pulmonary heart valve matrices

“…Endoglycosidase H cleaves the linkage between the two N-acetylglucosamine residues in the diacetylchitobiose unit of high mannose and some hybrid types, but not of complex types of N-glycans, while PNGaseF cleaves N-glycans between the innermost GlcNAc and asparagine of all N-glycans [55][56][57]. The α-Gal epitope has been reported as being part of both complex and/or hybrid N-glycans 58. Hence, Endoglycosidase H has a limited specificityand is less suitable for an overall deglycosylation of N-glycans and, thus, for the α-Gal removal, in comparison with PNGaseF.…”

Immunoprofile of α‐Gal‐ and B‐antigen‐specific responses differentiates red meat‐allergic patients from healthy individuals

“…Allergen‐specific basophil degranulation was analysed by monitoring the basophil activation markers CD203c and CD63, as previously described . Heparinized venous blood samples from five red meat‐allergic patients, one atopic birch‐allergic patient, and one nonallergic control were analysed with HSA‐α‐Gal, HSA‐B‐antigen (Dextra Lab) and HSA.…”