Molecular and Cellular Glycobiology Unit, Department of Biological Science, Sungkyunkwan University, Chunchun-Dong, Suwon 440-746, South Korea.
Biochem J. 2010 Mar 15;427(1):179-88. doi: 10.1042/BJ20090835.
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 pcmah 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 pcmah, 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.
在本研究中，我们从猪小肠中克隆并鉴定了 CMP-N-乙酰神经氨酸羟化酶基因（pcmah），该基因是合成 NeuGc（N-羟乙酰神经氨酸）的关键酶。pcmah 的 ORF（开放阅读框）长 1734bp，编码 577 个氨基酸，由 14 个外显子组成。组织表达谱分析显示，pcmah mRNA 主要在猪直肠、舌、脾和结肠组织中表达，在小肠中表达水平最高。在 pcmah 的异位表达中，当猪肾 PK15 细胞和人血管内皮 ECV304 细胞转染克隆的 pcmah 时，与用作对照的载体转染体相比，这些转染体中的 NeuGc 含量更高。此外，在 NeuGc 的功能分析中，与对照相比，异位表达 NeuGc 的 pcmah 转染细胞中的人血清介导的细胞毒性（HSMC）升高。此外，与人 IgM 的结合在 pcmah 转染细胞中显著增加，而 IgG 的结合略有增加，表明人 IgM 型是主要的抗 NeuGc 抗体。此外，通过 shRNA（短发夹 RNA）沉默 pcmah 导致 PK15 中 NeuGc 含量和异种抗原性降低。结果表明，pcmah 基因能够合成作为人类异种抗原的 NeuGc，证实了在猪-人异种移植中 NeuGc 介导的排斥反应。
