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Abstract

The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. For sialylation to occur, the donor sugar nucleotide cytidine monophospho-sialic acid (CMP-SA) must be generated and enzymatically transferred to an acceptor oligosaccharide. However, examination of insect cells grown in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac). To increase substrate levels, the enzymes of the metabolic pathway for CMP-SA synthesis have been engineered into insect cells using the baculovirus expression system. In this study, a human CMP-sialic acid synthase cDNA was identified 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-acetylmannosamine feeding is omitted, the sugar nucleotide form of deaminated Neu5Ac, CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (CMP-KDN), is produced instead, indicating that alternative sialic acid glycoforms may eventually 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 beneficial in various cell lines in which CMP-Neu5Ac production limits sialylation of glycoproteins or other glycans.

Expression of a Functional Drosophila melanogaster CMP-sialic Acid Synthetase

“…Localization of DmCSAS to the Secretory Compartment of the CellWhereas the mammalian sialic acid 9-phosphate synthase responsible for generating sialic acid is a soluble cytoplasmic enzyme (31), both human and murine CSAS, which activate the sialic acid with a nucleotide, have been reported to localize to the nucleus (22,23,33). As mentioned previously, murine CSAS possesses an N-terminal potential NLS, BC1, and an internal NLS, BC2 (22,29).…”

“…In Vitro CMP-sialic Acid Synthetase Assay-The in vitro CMP-sialic acid synthetase assay was performed as previously described (23). Briefly, 40 ml of cell lysate was added to the 100 l of substrate mixture (0.2 M Tris, 0.2 mM dithiothreitiol, 20 mM MgCl 2 , 5.5 mM CTP, and 2.8 mM Neu5Ac) and incubated for 40 min at 37°C.…”

N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism

“…The biosynthesis of N-acetylneuraminic acid (Neu5Ac) from ManNAc can be either achieved via phosphorylation by ManNAc 6-kinase and aldol addition of phosphoenolpyruvate (PEP) by sialic acid 9-P-synthase following an enzymatic dephosphorylation (Scheme 1, route A) [9,10], or the direct (reversible) aldol addition by sialic acid dephosphorylation (Scheme 1, route A) [9,10], or the direct (reversible) aldol addition by sialic acid aldolase (Scheme 1, route B) [11]. The resulting sialic acid is then converted into its activated precursor form CMP-N-acetylneuraminic acid and transported into the Golgi apparatus, where it is ultimately utilized by various sialyltransferases to decorate oligosaccharide chains [12]. As ManNAc is the sole carbohydrate precursor in the biosynthesis of N-acetylneuraminic acid, a better understanding of the ManNAc biosynthesis will therefore also lead to a more profound knowledge of the sialic acid metabolism in cells.…”

Humanization of recombinant glycoproteins expressed in insect cells

“…When insect cells are cultured in serum-free medium for the production of a recombinant glycoprotein, the inability to synthesize CMP-Neu5Ac becomes a key problem. In order to overcome this limitation, our group has cloned mammalian SAS (16) and CMP-SAS (17), and has introduced these into Sf9 cells. When cultured in serum-free medium supplemented with N-acetylmannosamine (ManNAc), a precursor of Neu5Ac, Sf9 cells expressing a human SAS produced a high level of Neu5Ac (16).…”

“…When cultured in serum-free medium supplemented with N-acetylmannosamine (ManNAc), a precursor of Neu5Ac, Sf9 cells expressing a human SAS produced a high level of Neu5Ac (16). Furthermore, a high level of CMPNeu5Ac production can be achieved by co-infecting two diŠerent baculoviruses, each encoding SAS or CMP-SAS, in Sf9 cells with ManNAc supplement in the culture medium (17). In a more recent study, our group has shown that ManNAc supplementation could be superseded by expressing UDP-GlcNAc 2-epimerase/ManNAc kinase in combination with SAS and CMP-SAS, with or without supplementation of less expensive GlcNAc (77).…”