Study of alpha-Gal glycosylation of cow's milk proteins and allergological implications

Background: Alpha gal syndrome (AGS) is a mammalian meat allergy associated with tick bites and specific IgE antibodies to the oligosaccharide galactose-?-1,3-galactose (?-gal). The ?-gal epitope is abundantly expressed on glycolipids and glycoproteins from non-primate mammals, but not in humans, where the gene encoding ?-1,3-galactosyltransferase is not functional. AGS shows several exclusive features that make it different from common food allergies: i) it is characterized by late on-set symptoms, appearing 3 to 6 hours after mammalian meat consumption; ii) IgE antibodies are against a carbohydrate moiety rather than a protein epitope; iii) patients can develop AGS in late adulthood. The symptoms ranged from gastrointestinal diseases, urticaria, angioedema, to anaphylaxis. Commins et al. (2016) demonstrated that not only red meat, but also bovine milk, might contain ?-Gal-epitopes. In more recent studies, in fact, the 10-20% of AGS patients even react to milk. Combining the recent findings of Perusko et al. (2020) about milk involvement in AGS and the considerations of Román-Carrasco et al. (2019), about the role of lipids in eliciting ?-Gal reaction, we investigated the milk fat globule protein (MFGP) fraction, to discover ?-Gal carrying proteins and to evaluate their immune-recognition by AGS patients. Method: The milk protein fractions including Milk Fat Globule Proteins (MFGP) were extracted from fresh cow's milk and separated by LDS-PAGE under reducing condition. The use of magnetic beads was useful to obtain a successful purification of the ?-gal glycosylated proteins. The anti-?-Gal monoclonal antibody and the serum of 10 patients with a documented ?-gal syndrome were tested by immunoblotting and the extracted proteins were identified by means of LC-HR MS/MS. Results: The immunoblotting showed that the MFGP fraction is ?-gal glycosylated. Both the anti-?-Gal monoclonal antibody and the IgE of patients with ?-Gal syndrome showed reactivity towards a high molecular weight protein (>200 kDa). Conclusion: With the first results obtained from LC-MS/MS identifications, it seems that the high molecular weight protein could be an aggregate of at least two different proteins recognized by the AGS patients' IgEs. These results suggest that it is important to investigate further the risk associated to milk consumption for patients suffering from ?-Gal syndrome.