Supplementary information files for "Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans"

Supplementary files for article "Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans"<b>Background</b>: Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes <i>GGTA1</i> and <i>CMAH</i>, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection.<b>Methods</b>: The <i>N</i>-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), <i>GGTA1</i>-KO and <i>GGTA1</i>/<i>CMAH-</i>KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection.<b>Results</b>: We identified biantennary and core-fucosylated <i>N</i>-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in <i>GGTA1</i> and <i>GGTA1</i>/<i>CMAH</i>-KO pigs, respectively. Levels of <i>N</i>-glycans terminating with galactose bound in β(1-4)-linkage to <i>N</i>-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. <i>N</i>-glycans capped with Neu5Gc were increased in <i>GGTA1</i>-KO pigs compared to WT, but were not detected in <i>GGTA1</i>/<i>CMAH</i>-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and <i>GGTA1</i>-KO but not in <i>GGTA1</i>/<i>CMAH</i>-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans.<b>Conclusion</b>: Genetic deletion of <i>GGTA1</i> or <i>GGTA1</i>/<i>CMAH</i> removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.<br>