Identification by mass spectrometry and immunoblotting of xenogeneic antigens in the N - and O -glycomes of porcine, bovine and equine heart tissues

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N -glycans were released by PNGase F digestion and O -glycans by β-elimination. Released oligosaccharides were analyzed by liquid chromatography – tandem mass spectrometry. In total, 102 N -glycans and 40 O -glycans were identified in animal heart tissue lysates. The N - and O -glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N - and O -linked glycans, N,N´ -diacetyllactosamine (LacdiNAc) on N -glycans only and sulfated O -glycans. The relative amounts of α-Gal-containing N -glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O -glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N -glycans. The identified N- and O- linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.