Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation
Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation
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DOI：
10.1042/bj20090835
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发表时间：
2010-04-01
影响因子：
4.1
通讯作者：
Kim, Cheorl-Ho
中科院分区：
文献类型：
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作者：
Song, Kwon-Ho;Kang, Yun-Jeong;Kim, Cheorl-Ho
本研究从猪小肠中克隆了猪N-乙酰神经氨酸羟基酶基因(PCMAH)，该基因是合成N-羟基神经氨酸(NeuGc)的关键酶。PCMAH的开放阅读框(ORF)为1734bp，编码577个氨基酸，由14个外显子组成。器官表达谱分析表明，Ponah mRNA主要在猪的直肠、舌、脾和结肠组织中表达，其中以小肠表达最高。在pONAH的异位表达中，用克隆的pcMAH转染猪肾PK15细胞和人血管内皮细胞ECV304后，其NeuGc含量明显高于载体转染组。此外，在NeuGc的功能分析中，异位表达NeuGc的PCMAH细胞的HSMC(人血清介导的细胞毒)比对照细胞高。此外，人IgM与PCMAH细胞的结合显著增加，而与IgG的结合略有增加，表明人IgM型抗体是主要的抗NeuGc抗体。此外，通过shRNA(短发夹状RNA)沉默pcmah导致PK15中NeuGc含量和异种抗原性降低。结果表明，pcmah基因能够在人体内合成作为异种抗原的NeuGc，证实了NeuGc介导的猪-人异种移植排斥反应。
In the present study, the pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of NeuGc (N-glycolylneuraminic acid), was cloned from pig small intestine and characterized. The ORF (open reading frame) of pcmah was 1734 bp, encoding 577 amino acids and consisting of 14 exons. Organ expression pattern analysis reveals that ponah mRNA is mainly expressed in pig rectum, tongue, spleen and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of ponah, when pig kidney PK15 cells and human vascular endothelial ECV304 cells were transfected with the cloned pcmah, the NeuGc contents of these transfectants were greater in comparison with vector transfectants used as controls. In addition, in the functional analysis of NeuGc, HSMC (human-serum-mediated cytotoxicity) was elevated in the ectopic NeuGc-expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG was slightly increased, indicating that the human IgM type was a major anti-NeuGc antibody. Furthermore, pcmah silencing by shRNA (short hairpin RNA) resulted in a decrease in NeuGc content and xenoantigenicity in PK15. From the results, it was concluded that the pcmah gene was capable of synthesizing the NeuGc acting as a xenoantigen in humans, confirming the NeuGc-mediated rejection response in pig-human xenotransplantation.