Hong Yeongjin, Kang Ji Young, Kim Youn Uck, Shin Dong-Jun, Choy Hyon E, Maeda Yusuke, Kinoshita Taroh
Genomic Research Center for Enteropathogenic Bacteria and Department of Microbiology, Chonnam National University Medical School, Gwangju, Republic of Korea.
Biochem Biophys Res Commun. 2005 Oct 7;335(4):1060-9. doi: 10.1016/j.bbrc.2005.07.177.
Aerolysin, a secreted bacterial toxin from Aeromonas hydrophila, binds to glycosylphosphatidylinositol (GPI)-anchored protein and kills the cells by forming pores. Both GPI and N-glycan moieties of GPI-anchored proteins are involved in efficient binding of aerolysin. We isolated various Chinese hamster ovary (CHO) mutant cells resistant to aerolysin. Among them, CHOPA41.3 mutant cells showed several-fold decreased expression of GPI-anchored proteins. After transfection of N-acetylglucosamine transferase I (GnT1) cDNA, aerolysin was efficiently bound to the cells, indicating that the resistance against aerolysin in this cells was mainly ascribed to the defect of N-glycan maturation. CHOPA41.3 cells also accumulated GPI intermediates lacking ethanolamine phosphate modification on the first mannose. After stable transfection of PIG-N cDNA encoding GPI-ethanolamine phosphate transferase1, a profile of accumulated GPI intermediates became similar to that of GPI transamidase mutant cells. It indicated, therefore, that CHOPA41.3 cells are defective in GnT1, ethanolamine phosphate modification of the first mannose, and attachment of GPI to proteins. The GPI accumulation in CHOPA41.3 cells carrying PIG-N cDNA was not normalized after transfection with cDNAs of all known components in GPI transamidase complex. Microsomes from CHOPA41.3 cells had normal GPI transamidase activity. Taken together, there is an unknown gene required for efficient attachment of GPI to proteins.
气单胞菌溶素是嗜水气单胞菌分泌的一种细菌毒素，它与糖基磷脂酰肌醇（GPI）锚定蛋白结合，并通过形成孔道杀死细胞。GPI锚定蛋白的GPI和N-聚糖部分都参与气单胞菌溶素的有效结合。我们分离出了多种对气单胞菌溶素具有抗性的中国仓鼠卵巢（CHO）突变细胞。其中，CHOPA41.3突变细胞显示GPI锚定蛋白的表达下降了几倍。转染N-乙酰葡糖胺转移酶I（GnT1）cDNA后，气单胞菌溶素能有效地与细胞结合，这表明该细胞对气单胞菌溶素的抗性主要归因于N-聚糖成熟缺陷。CHOPA41.3细胞还积累了在第一个甘露糖上缺乏磷酸乙醇胺修饰的GPI中间体。稳定转染编码GPI-磷酸乙醇胺转移酶1的PIG-N cDNA后，积累的GPI中间体的谱与GPI转酰胺酶突变细胞的谱相似。因此，这表明CHOPA41.3细胞在GnT1、第一个甘露糖的磷酸乙醇胺修饰以及GPI与蛋白质的连接方面存在缺陷。携带PIG-N cDNA的CHOPA41.3细胞中的GPI积累在用GPI转酰胺酶复合物中所有已知成分的cDNA转染后并未恢复正常。CHOPA41.3细胞的微粒体具有正常的GPI转酰胺酶活性。综上所述，存在一个GPI与蛋白质有效连接所需的未知基因。
