Structural characterization of α1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies
Structural characterization of α1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies
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DOI：
10.1111/j.1399-3089.2009.00564.x
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发表时间：
2010-01-01
影响因子：
3.9
通讯作者：
Breimer, Michael E.
中科院分区：
文献类型：
--
作者：
Diswall, Mette;Angstrom, Jonas;Breimer, Michael E.
背景：α1,3-半乳糖基转移酶敲除（GalT-KO）猪的建立是为了避免 GalT-KO 猪对人异种移植中的超急性排斥反应。研究了 GalT-KO 猪心和肾糖脂，重点是消除 Gal 抗原以及是否会出现新抗原。非人灵长类动物被用作临床前移植实验模型。因此，将来自移植GalT-KO心脏的狒狒血清与人血清就与猪糖脂的反应性进行比较。方法：从GalT-KO和WT猪心脏和肾脏中分离出中性和酸性糖脂。使用人亲和纯化的抗 Gal Ig、抗血型单克隆抗体、凝集素、人血清以及 GalT-KO 猪心脏移植前后收集的狒狒血清在 TLC 板上测试糖脂免疫反应性。通过 HPLC 分离选定的糖脂级分，并通过质谱和质子 NMR 光谱进行结构表征。结果：GalT-KO 心脏和肾脏完全缺乏 α 3Gal 封端的糖脂。未封端的N-乙酰基乳糖胺前体化合物、H血型2型核心链化合物、P1抗原和x(2)抗原的水平增加。人血清抗体与 WT 器官中的 Gal 抗原和 N-羟乙酰神经氨酸 (NeuGc) 发生反应，而在 GalT-KO 组织中仅保留 NeuGc 反应性。发现狒狒抗体和人类抗体与猪糖脂的反应性存在明显差异。这对于 GalT-KO 器官中尚未鉴定的酸性化合物最为明显，这些化合物在相应的 WT 组织中含量较低或缺乏。结论：GalT-KO 猪心脏和肾脏完全缺乏 Gal 糖脂抗原，而通过竞争途径合成的糖脂增加。狒狒和人血清抗体对猪糖脂抗原表现出不同的反应模式，表明非人灵长类动物作为免疫排斥研究的人类临床前模型存在局限性。
Background:alpha 1,3-galactosyltranferase knockout (GalT-KO) pigs have been established to avoid hyperacute rejection in GalT-KO pig-to-human xenotransplantation. GalT-KO pig heart and kidney glycolipids were studied focusing on elimination of Gal-antigens and whether novel antigens would appear. Non-human primates are used as pre-clinical transplantation experimental models. Therefore, sera from baboons transplanted with GalT-KO hearts were compared with human serum regarding reactivity with pig glycolipids.Methods:Neutral and acidic glycolipids were isolated from GalT-KO and WT pig hearts and kidneys. Glycolipid immune reactivity was tested on TLC plates using human affinity-purified anti-Gal Ig, anti-blood group monoclonal antibodies, lectins, and human serum as well as baboon serum collected before and after GalT-KO pig heart transplantations. Selected glycolipid fractions, isolated by HPLC, were structurally characterized by mass spectrometry and proton NMR spectroscopy.Results:GalT-KO heart and kidney lacked alpha 3Gal-terminated glycolipids completely. Levels of uncapped N-acetyllactosamine precursor compounds, blood group H type 2 core chain compounds, the P1 antigen and the x(2) antigen were increased. Human serum antibodies reacted with Gal-antigens and N-glycolylneuraminic acid (NeuGc) in WT organs of which only the NeuGc reactivity remained in the GalT-KO tissues. A clear difference in reactivity between baboon and human antibodies with pig glycolipids was found. This was most pronounced for acidic, not yet identified, compounds in GalT-KO organs which were less abundant or lacking in the corresponding WT tissues.Conclusions:GalT-KO pig heart and kidney completely lacked Gal glycolipid antigens whilst glycolipids synthesized by competing pathways were increased. Baboon and human serum antibodies showed a different reactivity pattern to pig glycolipid antigens indicating that non-human primates have limitations as a human pre-clinical model for immune rejection studies.