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

Department of Chemical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.
Glycoconjugate Journal (Impact Factor: 2.52). 04/2001; 18(3):205-13. DOI: 10.1023/A:1012452705349
Source: PubMed


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.

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    • "This limitation may be in the levels or amount of 2,6/2,3sialyltransferase, 1,4galactosyltransferase, or alternatively, at the level of nucleotide sugar substrate CMP-sialic acid. Indeed, our group has shown that such nucleotide sugar transporters can be prevalent in eukaryotic expression systems and we and others have shown that the limitation can be overcome by the expression of the relevant pathway enzymes responsible for conversion of UDP-GlcNAc to CMP-Neu5Ac (Bork et al., 2007; Jeong et al., 2008,2009; Lawrence et al., 2001; Tomiya et al., 2001; Viswanathan et al., 2003, 2005). Alternatively, Gu and Wang 1998 reported improved sialylation on IFN-g by feeding CHO cell line with N-acetylmannosamine, and showed it was possible to increase sialylation on glycoproteins by adding nucleotide sugar substrate precursors. "
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    ABSTRACT: Sialic acid, a terminal residue on complex N-glycans, and branching or antennarity can play key roles in both the biological activity and circulatory lifetime of recombinant glycoproteins of therapeutic interest. In order to examine the impact of glycosyltransferase expression on the N-glycosylation of recombinant erythropoietin (rEPO), a human α2,6-sialyltransferase (ST6Gal1) was expressed in Chinese hamster ovary (CHO-K1) cells. Sialylation increased on both EPO and CHO cellular proteins as observed by SNA lectin analysis, and HPLC profiling revealed that the sialic acid content of total glycans on EPO increased by 26%. The increase in sialic acid content was further verified by detailed profiling of the N-glycan structures using mass spectra (MS) analysis. In order to enhance antennarity/branching, UDP-N-acetylglucosamine: α-1,3-D-mannoside β1,4-N-acetylglucosaminyltransferase (GnTIV/Mgat4) and UDP-N-acetylglucosamine:α-1,6-D-mannoside β1,6-N-acetylglucosaminyltransferase (GnTV/Mgat5), was incorporated into CHO-K1 together with ST6Gal1. Tri- and tetraantennary N-glycans represented approximately 92% of the total N-glycans on the resulting EPO as measured using MS analysis. Furthermore, sialic acid content of rEPO from these engineered cells was increased ∼45% higher with tetra-sialylation accounting for ∼10% of total sugar chains compared to ∼3% for the wild-type parental CHO-K1. In this way, coordinated overexpression of these three glycosyltransferases for the first time in model CHO-K1 cell lines provides a mean for enhancing both N-glycan branching complexity and sialylation with opportunities to generate tailored complex N-glycan structures on therapeutic glycoproteins in the future. Biotechnol. Bioeng. 2015;9999: 1-8. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 07/2015; 112(11). DOI:10.1002/bit.25650 · 4.13 Impact Factor
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    • "In this study, we report the in planta expression of three enzymes required for the synthesis of CMP- Neu5Ac in mammals: mouse UDP-GlcNAc 2-epimerase/ N-acetylmannosamine kinase (GNE; Horstkorte et al., 1999), human N-acetylneuraminic acid phosphate synthase (NANS; Lawrence et al., 2000), and human CMP-sialic acid synthetase (CMAS; Lawrence et al., 2001). The genes were simultaneously expressed in Arabidopsis, which resulted in the generation of significant amounts of Neu5Ac and CMP-Neu5Ac from endogenous precursors. "
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    ABSTRACT: Previous studies have reported that plants contain negligible amounts of free or protein-bound N-acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use of plants as a biopharmaceutical expression system, since N-glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans. For the synthesis of Neu5Ac-containing N-glycans, plants have to acquire the ability to synthesize Neu5Ac and its nucleotide-activated derivative, cytidine monophospho-N-acetylneuraminic acid. In this study, we have generated transgenic Arabidopsis (Arabidopsis thaliana) plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-N-acetylneuraminic acid synthetase. Simultaneous expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase and N-acetylneuraminic acid phosphate synthase resulted in the generation of significant Neu5Ac amounts (1,275 nmol g(-1) fresh weight in leaves) in planta, which could be further converted to cytidine monophospho-N-acetylneuraminic acid (2.4 nmol g(-1) fresh weight in leaves) by coexpression of CMP-N-acetylneuraminic acid synthetase. These findings are a major step toward the production of Neu5Ac-containing glycoproteins in plants.
    Plant physiology 06/2008; 147(1):331-9. DOI:10.1104/pp.108.117572 · 6.84 Impact Factor
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    • "Based on this data the possibility that nuclear targeting occurs by chance, as a consequence of the basic structure of the active site, could not be ruled out. The isolation of the human (Lawrence et al. 2001) and rt CMP-Sia-syn (Nakata et al. 2001) cDNAs now permits the analysis of nuclear localization in these species. However, because the human cDNA encodes a protein with 94% identity to the murine homologue , in which all structural and functional motifs are perfectly conserved, the intracellular localization of the more distant fish enzyme was analysed. "
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    ABSTRACT: The terminal sugar sialic acid (Sia) plays a pivotal role in cell-cell interaction and recognition. A prerequisite for the biosynthesis of sialoglycoconjugates is the activation of Sia to cytidine monophosphate-Sia (CMP-Sia), by CMP-Sia synthetases (CMP-Sia-syn). CMP-Sia-syn are conserved from bacteria to man, and have been found to reside in the nucleus of all vertebrate species analysed to date. We previously cloned the CMP-Sia-syn from rainbow trout (rt) and identified three clusters of basic amino acids (BC) that might act as nuclear localization signals (NLS). Here, we utilised chimeric proteins and rt CMP-Sia-syn mutants in which putative NLS sequences were deleted, to identify the nuclear transport signal. Divergent from the mouse enzyme, where the crucial NLS is part of the enzyme's active site, in the rt CMP-Sia-syn the NLS and active site are disparate. The crucial NLS in the fish enzyme is bipartite and the functionality depends on a free N-terminus. Comparative analysis of all putative rt NLS in mouse and fish cells identified a second inferior motif (rtBC5-6), which was functional only in fish cells suggesting some differences in transport mechanism or folding variabilities in fish. Moreover, based on computational analyses of putative CMP-Sia-syn from distant deuterostomian organisms it was concluded that CMP-Sia-syn nuclear localization is a relatively recent invention, originating in echinoderms. In summary, our data describing structural differences in the NLS of vertebrate CMP-Sia-syn, and the independence of Sia activation from the subcellular localization of the enzyme, provide supporting evidence that nuclear localization is linked to a second yet unknown function.
    Glycobiology 10/2007; 17(9):945-54. DOI:10.1093/glycob/cwm064 · 3.15 Impact Factor
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