Burlak Christopher, Bern Marshall, Brito Alejandro E, Isailovic Dragan, Wang Zheng-Yu, Estrada Jose L, Li Ping, Tector A Joseph
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Xenotransplantation. 2013 Sep-Oct;20(5):277-91. doi: 10.1111/xen.12047. Epub 2013 Sep 5.
The temporary or long-term xenotransplantation of pig organs into people would save thousands of lives each year if not for the robust human antibody response to pig carbohydrates. Genetically engineered pigs deficient in galactose α1,3 galactose (gene modified: GGTA1) and N-glycolylneuraminic acid (gene modified: CMAH) have significantly improved cell survival when challenged by human antibody and complement in vitro. There remains, however, a significant portion of human antibody binding.
To uncover additional xenoantigens, we compared the asparagine-linked (N-linked) glycome from serum proteins of humans, domestic pigs, GGTA1 knockout pigs, and GGTA1/CMAH knockout pigs using mass spectrometry. Carbohydrate structures were determined with assistance from GlycoWorkbench, Cartoonist, and SimGlycan software by comparison to existing database entries and collision-induced dissociation fragmentation data.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of reduced and solid-phase permethylated glycans resulted in the detection of high-mannose, hybrid, and complex type N-linked glycans in the 1000-4500 m/z ion range. GGTA1/CMAH knockout pig samples had increased relative amounts of high-mannose, incomplete, and xylosylated N-linked glycans. All pig samples had significantly higher amounts of core and possibly antennae fucosylation.
We provide for the first time a comparison of the serum protein glycomes of the human, domestic pig, and genetically modified pigs important to xenotransplantation.
如果不是因为人体对猪碳水化合物产生强烈的抗体反应，将猪器官临时或长期异种移植到人体中每年可挽救数千人的生命。缺乏α1,3半乳糖（基因修饰：GGTA1）和N-羟乙酰神经氨酸（基因修饰：CMAH）的基因工程猪在体外受到人抗体和补体攻击时，细胞存活率有显著提高。然而，仍有相当一部分人抗体会与之结合。
为了发现其他异种抗原，我们使用质谱法比较了人类、家猪、GGTA1基因敲除猪和GGTA1/CMAH基因敲除猪血清蛋白中的天冬酰胺连接（N-连接）聚糖组。通过与现有数据库条目和碰撞诱导解离碎片数据进行比较，借助GlycoWorkbench、Cartoonist和SimGlycan软件确定碳水化合物结构。
对还原和固相全甲基化聚糖进行基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）分析，在1000-4500 m/z离子范围内检测到高甘露糖型、杂合型和复合型N-连接聚糖。GGTA1/CMAH基因敲除猪样本中高甘露糖型、不完全型和木糖基化N-连接聚糖的相对含量增加。所有猪样本中核心岩藻糖基化以及可能的触角岩藻糖基化含量均显著更高。
我们首次比较了对异种移植重要的人类、家猪和转基因猪的血清蛋白聚糖组。
