Establishment of N-acetylmannosamine (ManNAc) analogue-resistant cell lines as improved hosts for sialic acid engineering applications.
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.
Kim
Eun Jeong
EJ
Whitaker Biomedical Engineering Institute, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Jones
Mark B
MB
Rhee
Jun Kyu
JK
Sampathkumar
Srinivasa-Gopalan
SG
Yarema
Kevin J
KJ
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects*; D024861:Genetic Enhancement / Q000379:methods*; D006595:Hexosamines / Q000008:administration & dosage*; D006801:Humans; D019169:Jurkat Cells; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis* / Q000235:genetics; D013045:Species Specificity
D006595:Hexosamines; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine