Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans
Lucrezia Morticelli1, Charlotte Rossdam2, Samanta Cajic3,4, Dietmar Böthig5, Mikhail Magdei1, Sugat R Tuladhar5, Björn Petersen6, Konrad Fischer7, Erdmann Rapp3,4, Sotirios Korossis8, Axel Haverich1,5, Angelika Schnieke7, Heiner Niemann9, Falk Buettner2, Andres Hilfiker1,5.
1Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Hannover, Germany; 2Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany; 3Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; 4glyXera GmbH, Magdeburg, Germany; 5Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany; 6Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee/Neustadt am Ruebenberge, Germany; 7Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany; 8Cardiopulmonary Regenerative Engineering (CARE) Group, Centre for Biological Engineering (CBE), Loughborough University, Loughborough, United Kingdom; 9Clinic for Gastroenterology, Hepatology & Endocrinology, Hannover Medical School, Hannover, Germany
Introduction: The use of pig-derived xenogeneic tissue for clinical application could eliminate the ever-growing scarcity of human donor organs. The glycans Galα1–3Gal (α-Gal epitope) and N-glycolylneuraminic acid (Neu5Gc) largely contribute to the immunogenicity of porcine tissues and ultimately cause rejection of porcine xenografts in human. Knockout (KO) pigs lacking the expression of N-acetyllactosaminide alpha-1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydrolase (CMAH), which are essential enzymes for the synthesis of α-Gal and Neu5Gc, respectively, have been produced for the production of safer xenografts.
Method: Here, we investigated the N-glycome and glycosphingolipid glycosylation of native and decellularized porcine pericardia from wild type (WT), GGTA1-KO and GGTA1/CMAH-KO pigs via multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF).
Results: We detected biantennary and core-fucosylated N-glycans with immunogenic α-Gal- and Neu5Gc-epitopes at their termini on pericardium of WT pigs. The knockout of GGTA1 and GGTA1/CMAH led to complete absence of α-Gal- and α-Gal-/Neu5Gc-epitopes, respectively. On the other hand, increased relative levels of N-glycans terminating either with single galactoses as well as their derivatives elongated by one or two Neu5Ac were discovered in both, GGTA- and GGTA1/CMAH-KO pigs. Furthermore, sialylated N-glycans as well as the ganglioside Neu5Gc-GM3, terminating with Neu5Gc were increased in GGTA1-KO pigs but could not be detected in GGTA1/CMAH-KO pigs. Of note, glycosphingolipids could be efficiently removed by the process of detergent-based tissue decellularization.
Conclusion: There results show that deletion of GGTA1 or GGTA1/CMAH leads to a more human-like glycosylation pattern, but quantities of other porcine glycans were altered which in turn might affect immunogenicity of the porcine material.
This work was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 213602938 – TRR 127 for A.H., H.N. and F.F.R.B. (project C7) and for B.P., A.S. and K.F. (project Z3) and for Forschungsgruppe FOR2953 (Projektnummer: 409784463432218849) for F.F.R.B. (project P9; BU 2920/4-1)). The authors thank Astrid Oberbeck for excellent technical support.
