Engineering of coordinated up- and down-regulation of two glycosyltransferases of the o-glycosylation pathway in Chinese hamster ovary (CHO) cells
Article first published online: 31 MAR 2000
Copyright © 2000 John Wiley & Sons, Inc.
Biotechnology and Bioengineering
Volume 68, Issue 3, pages 239–244, 5 May 2000
How to Cite
Prati, E. G.P., Matasci, M., Suter, T. B., Dinter, A., Sburlati, A. R. and Bailey, J. E. (2000), Engineering of coordinated up- and down-regulation of two glycosyltransferases of the o-glycosylation pathway in Chinese hamster ovary (CHO) cells. Biotechnol. Bioeng., 68: 239–244. doi: 10.1002/(SICI)1097-0290(20000505)68:3<239::AID-BIT1>3.0.CO;2-7
- Issue published online: 31 MAR 2000
- Article first published online: 31 MAR 2000
- Manuscript Received: 21 OCT 1999
- Manuscript Accepted: 9 JAN 1999
- Swiss Priority Program in Biotechnology (SPP BioTech)
- CHO cells;
- ST3Gal I inhibition;
- C2GnT overexpression
Production of O-linked oligosaccharides that interact with selectins to mediate cell–cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required.
We report here the partial cloning of the CMP-sialic acid:Galβ1,3GalNAcα2,3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galβ1,3GalNAc-R β1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed “one-step” transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express α1,3-fucosyltransferase VI (α1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 239–244, 2000.