N-linked glycan profiling of GGTA1/CMAH knockout pigs identifies new potential carbohydrate xenoantigens.
N-linked glycan profiling of GGTA1/CMAH knockout pigs identifies new potential carbohydrate xenoantigens.
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DOI：
10.1111/xen.12047
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发表时间：
2013-09
影响因子：
3.9
通讯作者：
Tector AJ
中科院分区：
文献类型：
--
作者：
Burlak C;Bern M;Brito AE;Isailovic D;Wang ZY;Estrada JL;Li P;Tector AJ
如果不是人类对猪的碳水化合物产生了强烈的抗体反应，将猪器官暂时或长期异种移植到人类身上每年将挽救数千人的生命。当体外受到人抗体和补体攻击时，半乳糖α 1，3半乳糖（基因修饰：GGTA 1）和N-羟乙酰神经氨酸（基因修饰：CMAH）缺陷的基因工程猪显著提高了细胞存活率。然而，仍然存在相当大部分的人抗体结合。为了揭示另外的异种抗原，我们使用质谱法比较了来自人、家猪、GGTA 1敲除猪和GGTA 1/CMAH敲除猪的血清蛋白的天冬酰胺连接（N-连接）糖组。通过与现有数据库条目和碰撞诱导解离碎片化数据进行比较，在Glycoblasts、Cartoonist和SimGlycan软件的帮助下确定碳水化合物结构。还原和固相全甲基化聚糖的基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）分析导致在1000至4500 m/z离子范围内检测到高甘露糖、杂合和复合型N-连接聚糖。GGTA 1/CMAH敲除猪样本中高甘露糖、不完全和木糖基化N-连接聚糖的相对量增加。所有猪样本的核心岩藻糖基化和触角岩藻糖基化的量均显著较高。我们首次比较了人、家猪和转基因猪的血清蛋白糖组，这对异种移植很重要。
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 to 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.