Cloning and expression of human sialic acid pathway genes to generate CMP-sialic acids in insect cells

The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory ha lf-life. For sialylation to occur, the donor sugar nucleotide cytidine mono phospho-sialic acid (CMP-SA) must be generated and enzymatically transferre d to an acceptor oligosaccharide. However, examination of insect cells grow n in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac). To incre ase substrate levels, the enzymes of the metabolic pathway for CMP-SA synth esis have been engineered into insect cells using the baculovirus expressio n system. In this study, a human CMP-sialic acid synthase cDNA was identifi ed and found to encode a protein with 94% identity to the murine homologue. The human CMP-sialic acid synthase (Cmp-Sas) is ubiquitously expressed in human cells from multiple tissues. When expressed in insect cells using the baculovirus vector, the encoded protein is functional and localizes to the nucleus as in mammalian cells. In addition, co-expression of Cmp-Sas with the recently cloned sialic acid phosphate synthase with N-acetylmannosamine feeding yields intracellular CMP-Neu5Ac levels 30 times higher than those observed in unsupplemented CHO cells. The absence of any one of these three components abolishes CMP-Neu5Ac production in vivo. However, when N-acetyl mannosamine feeding is omitted, the sugar nucleotide form of deaminated Neu 5Ac, CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (CMP-KDN), is prod uced instead, indicating that alternative sialic acid glycoforms may eventu ally be possible in insect cells. The human CMP-SAS enzyme is also capable of CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) synthesis when provided with the proper substrate. Engineering the CMP-SA metabolic pathway may be benef icial in various cell lines in which CMP-Neu5Ac production limits sialylati on of glycoproteins or other glycans.