Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells.
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:Galbeta1,3GalNAcalpha2, 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:Galbeta1, 3GalNAc-R beta1,6-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 alpha1, 3-fucosyltransferase VI (alpha1,3-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.
Prati
E G
EG
Institute of Biotechnology, ETH Zürich, CH-8093 Zürich, Switzerland.
Matasci
M
M
Suter
T B
TB
Dinter
A
A
Sburlati
A R
AR
Bailey
J E
JE
D000469:Alkaline Phosphatase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016375:Antisense Elements (Genetics) / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D015536:Down-Regulation; D006031:Glycosylation; D006801:Humans; D016256:Lewis X Antigen / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology; D015854:Up-Regulation; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D016375:Antisense Elements (Genetics); D016256:Lewis X Antigen; D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase; D012799:Sialyltransferases; D000469:Alkaline Phosphatase; D013752:Tetracycline; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase