RESUMO
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.
Assuntos
Células CHO/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Sialiltransferases/metabolismo , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Elementos Antissenso (Genética)/genética , Elementos Antissenso (Genética)/metabolismo , Sequência de Bases , Células CHO/efeitos dos fármacos , Cricetinae , Regulação para Baixo , Glicosilação , Humanos , Antígenos CD15/genética , Antígenos CD15/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Sialiltransferases/genética , Tetraciclina/farmacologia , Regulação para Cima , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Genetic engineering of oligosaccharide biosynthesis pathways in mammalian cells makes possible generation of new recombinant glycoproteins of potential importance in the biopharmaceutical industry. Most prior investigations of glycosylation engineering of secreted heterologous glycoproteins involve terminal steps of oligosaccharide biosynthesis. In particular, increasing the frequency of bisected structures within the glycoform distribution has not before been considered. A Chinese hamster ovary (CHO) cell line capable of producing bisected oligosaccharides on glycoproteins was created by overexpression of a recombinant N-acetylglucosaminyltransferase III (GnT-III). Interferon beta (IFN-beta) was chosen as a model and potential therapeutic secreted heterologous protein to demonstrate the effect of recombinant GnT-III-expression on product glycosylation. IFN-beta with bisected oligosaccharides was produced by the GnT-III-engineered CHO cells but not by the unmodified parental cell line.
Assuntos
Glicoproteínas/biossíntese , Interferon beta/biossíntese , N-Acetilglucosaminiltransferases/biossíntese , Engenharia de Proteínas , Animais , Células CHO , Cricetinae , Meios de Cultura , Immunoblotting , Proteínas Recombinantes/biossínteseRESUMO
Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities.