Establishment of N-acetylmannosamine (ManNAc) analogue-resistant cell lines as improved hosts for sialic acid engineering applications
Establishment of N-acetylmannosamine (ManNAc) analogue-resistant cell lines as improved hosts for sialic acid engineering applications
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DOI：
10.1021/bp049841q
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发表时间：
2004-11-01
影响因子：
2.9
通讯作者：
Yarema, KJ
中科院分区：
文献类型：
--
作者：
Kim, EJ;Jones, MB;Yarema, KJ
基于代谢底物的唾液酸工程技术，其中外源提供的N-乙酰甘露糖胺（ManNAc）类似物被唾液酸生物合成途径利用，允许细胞表面被赋予新的物理和化学性质，并显示出提高重组糖蛋白质量的前景。然而，许多ManNAc类似物的体外毒性阻碍了该技术的大规模采用。在这项研究中，我们使用了一种选择策略，其中细胞受到逐步更高水平的ManNAc类似物，以建立新的细胞系，显示出降低敏感性类似物诱导的体外毒性。通过Annexin V-FITC检测方法和DNA片段化分析证明，对糖类似物诱导的细胞凋亡的敏感性降低对应于抗性细胞系中唾液酸产生的增加。ManNAc类似物抗性细胞系对星形孢菌素和凋亡激活Fas抗体诱导的凋亡表现出交叉抗性。我们建议，用于开发这些新型细胞系的选择策略，这些细胞系作为基于底物的唾液酸工程应用的上级宿主，通常适用于生物技术应用的宿主细胞系的开发。
Metabolic substrate-based sialic acid engineering techniques, where exogenously supplied N-acetylmannosamine (ManNAc) analogues are utilized by the sialic acid biosynthetic pathway, allow the cell surface to be endowed with novel physical and chemical properties and show promise for increasing the quality of recombinant glycoproteins. The in vitro toxicity of many ManNAc analogues, however, hinders the large-scale adoption of this technology. In this study, we used a selection strategy where cells were subjected to progressively higher levels of ManNAc analogues to establish novel cell lines that showed decreased sensitivity to analogue-induced in vitro toxicity. The decreased sensitivity to sugar analogue-induced apoptosis, demonstrated by the Annexin V-FITC detection method and DNA fragmentation assays, corresponded to increased sialic acid production in the resistant cell lines. The ManNAc analogue-resistant cell lines exhibited cross-resistance to apoptosis induced by staurosporine and an apoptosis-activating Fas antibody. We propose that the selection strategy employed to develop these novel cell lines, which serve as superior hosts for substrate-based sialic acid engineering applications, will generally apply to the development of host cell lines for biotechnology applications.