Tissue plasminogen activator coexpressed in Chinese hamster ovary cells with alpha(2,6)-sialyltransferase contains NeuAc alpha(2,6)Gal beta(1,4)Glc-N-AcR linkages.
Genetic alteration of the set of oligosaccharide biosynthesis enzymes expressed in a genetically engineered host cell line is a plausible strategy for manipulating the oligosaccharides on a cloned glycoprotein coexpressed in that cell line. This hypothesis was verified for the particular case of sialylation of recombinant human tissue plasminogen activator (tPA) expressed by an engineered Chinese hamster ovary (CHO) cell line. The gene for rat liver beta-galactoside alpha(2,6)-sialyltransferase (2,6-ST) was cloned behind the MMTV promoter in the vector pMSG and transfected into a tPA-expressing CHO cell line. Selected and screened transfectants exhibited significantly greater surface fluorescence than controls in flow cytometric analyses of cells labeled with Sambacus nigra agglutinin (SNA)-biotin and streptavidin-R-phycoerythrin; SNA specifically binds to NeuAc alpha(2,6)Gal beta(1,4)Glc-N-AcR linkages, which are synthesized by 2,6-ST and which are not normally found on CHO cells. SNA blots of partially purified tPA from the culture supernatant demonstrated that tPA synthesized in the 2,6-ST transfectants possessed terminal NeuAc alpha(2,6)Gal beta(1,4)Glc-N-AcR linkages, while tPA from the original recombinant CHO cell line did not. Besides possibly allowing the production of glycoproteins in cell culture with glycosylation more closely resembling that in humans, extensions of this strategy have the potential to tailor the pharmacokinetics, targeting, and antigenic properties of cloned glycoproteins.
Minch
S L
SL
Department of Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Kallio
P T
PT
Bailey
J E
JE
D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006224:Cricetinae; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012799:Sialyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D012799:Sialyltransferases; D010959:Tissue Plasminogen Activator; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase