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"Background: 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 GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection.Methods: The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection.Results: We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in β(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans.Conclusion: Genetic deletion of GGTA1 or GGTA1/CMAH 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.论文《猪GGTA1或CMAH/GGTA1基因敲除与新聚糖（neo-glycans）的出现相关》的补充材料： 背景：猪源组织有望解决移植领域人类供体器官短缺的问题。然而，由GGTA1和CMAH基因编码的酶合成的末端含α-半乳糖（α-Gal）与N-羟乙酰神经氨酸（Neu5Gc）的聚糖，已被证实是猪组织免疫原性的主要诱因，最终会引发异种移植排斥反应。 方法：本研究采用多重毛细管凝胶电泳联合激光诱导荧光检测技术，对野生型（WT）、GGTA1基因敲除型（GGTA1-KO）以及GGTA1/CMAH双基因敲除型猪的天然及脱细胞猪心包组织的N-糖组（N-glycome）和糖鞘脂组（glycosphingolipidome）进行了分析。 结果：本研究在野生型猪心包组织中鉴定出两类带有免疫原性表位的N-聚糖：其一为以α-Gal为末端的双天线型及核心岩藻糖基化N-聚糖，此类聚糖在GGTA1-KO猪中完全缺失；其二为同时带有α-Gal与Neu5Gc表位的此类聚糖，此类聚糖在GGTA1/CMAH-KO猪中未被检出。在两个基因敲除组中，以β(1-4)糖苷键连接于N-乙酰葡糖胺的半乳糖为末端的N-聚糖，及其被N-乙酰神经氨酸（Neu5Ac）延伸的衍生物的表达水平均显著升高。与野生型猪相比，GGTA1-KO猪中以Neu5Gc封端的N-聚糖水平有所上升，但此类聚糖在GGTA1/CMAH-KO猪中未被检测到。类似地，神经节苷脂Neu5Gc-GM3仅在野生型和GGTA1-KO猪中被检出，在GGTA1/CMAH-KO猪中未见存在。本研究采用的基于去污剂的脱细胞工艺可高效去除糖鞘脂聚糖。 结论：GGTA1单基因或GGTA1/CMAH双基因的敲除可去除特定的免疫原性表位，使猪组织的糖基化模式更趋近于人类，但同时也会改变其他具有潜在免疫原性的猪源聚糖的分布与表达水平。