Engineering intracellular CMP-sialic acid metabolism into insect cells and methods to enhance its generation.
Previous studies have reported that insect cell lines lack the capacity to generate endogenously the nucleotide sugar, CMP-Neu5Ac, required for sialylation of glycoconjugates. In this study, the biosynthesis of this activated form of sialic acid completely from endogenous metabolites is demonstrated for the first time in insect cells by expressing the mammalian genes required for the multistep conversion of endogenous UDP-GlcNAc to CMP-Neu5Ac. The genes for UDP-GlcNAc-2-epimerase/ManNAc kinase (EK), sialic acid 9-phosphate synthase (SAS), and CMP-sialic acid synthetase (CSAS) were coexpressed in insect cells using baculovirus expression vectors, but the CMP-Neu5Ac and precursor Neu5Ac levels synthesized were found to be lower than those achieved with ManNAc supplementation due to feedback inhibition of the EK enzyme by CMP-Neu5Ac. When sialuria-like mutant EK genes, in which the site for feedback regulation has been mutated, were used, CMP-Neu5Ac was synthesized at levels more than 4 times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAc feeding. Addition of N-acetylglucosamine (GlcNAc), a precursor for UDP-GlcNAc, to the media increased the levels of CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAc supplementation, creating a bottleneck in the conversion of Neu5Ac to CMP-Neu5Ac at higher levels of UDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance the levels of the sialylation donor molecule, CMP-Neu5Ac, a critical limiting substrate for the generation of complex glycoproteins in insect cells and other cell culture systems.
Viswanathan
Karthik
K
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Narang
Someet
S
Hinderlich
Stephan
S
Lee
Yuan C
YC
Betenbaugh
Michael J
MJ
D000818:Animals; D001120:Arginine / Q000235:genetics; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000737:chemistry* / Q000378:metabolism*; D006595:Hexosamines / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry* / Q000378:metabolism*; D007930:Leucine / Q000235:genetics; D008360:Mannosephosphates; D009036:Moths / Q000821:virology; D016297:Mutagenesis, Site-Directed; D017349:N-Acetylhexosaminyltransferases / Q000096:biosynthesis / Q000235:genetics; D000216:N-Acylneuraminate Cytidylyltransferase / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D029461:Sialic Acid Storage Disease / Q000235:genetics; D018411:Spodoptera / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics
C430285:2-acetamido-2-deoxy-mannose-6-phosphate; D006595:Hexosamines; D008360:Mannosephosphates; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D001120:Arginine; D017349:N-Acetylhexosaminyltransferases; C059053:N-acetylmannosaminyltransferase; D000216:N-Acylneuraminate Cytidylyltransferase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; D007930:Leucine; C002022:N-acetylmannosamine