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

Chunsheng Jin,Michael E Breimer,Jining Liu,Cesare Galli,Reeja Maria Cherian,Niclas G Karlsson,Jan Holgersson,Heribert Playà-Albinyana

doi:10.1007/s10719-020-09931-1

Chunsheng Jin, Michael E Breimer + Show 6 more

Open Access

https://doi.org/10.1007/s10719-020-09931-1

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Abstract

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.

Highlights

More than 250,000 heart valves are replaced worldwide each year due to valvular heart disease [1]
The Pro Q Emerald staining of the porcine pulmonary and aortic valve tissues appeared similar even though the pulmonary valve lysate stained stronger than the aortic valve lysate (Fig. 1b)
Most of the work done to characterize the repertoire of xenogeneic carbohydrate determinants such as α-Gal, Neu5Gc and others, on animal donor tissue for bioprosthetic heart valves (BHV) has been performed using immunohistochemistry [8, 13, 18, 19]

Summary

Introduction

More than 250,000 heart valves are replaced worldwide each year due to valvular heart disease [1]. BHV are manufactured from porcine valvular tissue or porcine, bovine or equine pericardial tissue, which has been treated in a process that includes glutaraldehyde treatment [1,2,3]. The α-Gal determinant has been demonstrated on fibrocytes interspersed in the connective tissue of fixed and native porcine valves [13, 14] and patients receiving porcine BHV exhibited a rise in cytotoxic anti-Gal IgM antibodies [13]. Western blotting and liquid chromatography – tandem mass spectrometry was used to investigate the Nand O-glycomes in lysates of native porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia to determine the representation of protein-linked glycans focusing on defining the core chains carrying α-Gal and Neu5Gc xenogeneic determinants, as well as potentially new xenogeneic carbohydrate determinants

Experimental Procedures

Results

Discussion

Compliance with ethical standards