Department of Surgery, University of Minnesota, Minneapolis, MN, USA.
Department of Medical Microbiology and Immunology, University of Toledo Medical Center, Toledo, OH, USA.
Xenotransplantation. 2020 Nov;27(6):e12629. doi: 10.1111/xen.12629. Epub 2020 Jul 22.
Progress has been made in overcoming antibody-mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α-fucose as a glycan that was expressed in high levels on cells of GGTA1/CMAH KO pigs. To validate the α-fucose phenotype observed previously we compared lectin affinity toward human and pig serum glycoproteins by dot blot analysis and confocal microscopy. Human anti-fucose antibody isolated by affinity chromatography was tested for specificity to L-fucose by custom macroarray. The affinity and cytotoxicity of the isolated human anti-fucose antibody toward human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-fucose and may represent focal sites for anti-α-fucose antibody binding. The Isolated human anti-fucose IgA, IgG and IgM bound to GGTA1/CMAH KO pig PBMC and were cytotoxic. Interestingly, the isolated human IgG cross reacted with the methyl pentose, L-rhamnose. Human anti-fucose antibody bound and was cytotoxic to GGTA1/CMAH KO pig peripheral blood monocytes. We have shown that α-fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation.
通过删除产生独特碳水化合物表位的猪基因，在克服猪异种移植物的抗体介导排斥方面取得了进展。缺乏半乳糖 α-1,3-半乳糖（基因修饰：GGTA1）和 Neu5Gc（基因修饰：CMAH）的猪与人抗体结合的水平降低。以前我们发现 α-岩藻糖是 GGTA1/CMAH KO 猪细胞中高表达的聚糖。为了验证以前观察到的 α-岩藻糖表型，我们通过斑点印迹分析和共聚焦显微镜比较了凝集素对人血清和猪血清糖蛋白的亲和力。通过亲和层析分离的人抗岩藻糖抗体通过定制的宏阵列测试其对 L-岩藻糖的特异性。通过流式细胞术测定分离的人抗岩藻糖抗体对人 PBMC 和 GGTA1/CMAH KO 猪 PBMC 的亲和力和细胞毒性。斑点印迹和共聚焦分析支持我们之前的发现，即 α-岩藻糖在猪中的表达水平高于人类。猪肾肾小球和肾小管含有丰富的α-岩藻糖，可能代表抗α-岩藻糖抗体结合的焦点部位。分离的人抗岩藻糖 IgA、IgG 和 IgM 与人 PBMC 和 GGTA1/CMAH KO 猪结合并具有细胞毒性。有趣的是，分离的人 IgG 与甲基戊糖 L-鼠李糖发生交叉反应。人抗岩藻糖抗体与人 PBMC 和 GGTA1/CMAH KO 猪结合并具有细胞毒性。我们已经表明，α-岩藻糖是基因修饰猪的异种移植重要器官中细胞毒性人抗体的丰富靶标。
