Cloning and Expression of the HumanN-Acetylneuraminic Acid Phosphate Synthase Gene with 2-Keto-3-deoxy-d-glycero- d-galacto-nononic Acid Biosynthetic Ability

Lawrence, Shawn M.; Huddleston, Kathleen A.; Pitts, Lee; Nguyen, Nam; Lee, Yuan C.; Vann, Willie F.; Coleman, Timothy A.; Betenbaugh, Michael J.

doi:10.1074/jbc.m000217200

Cited by 91 publications

(87 citation statements)

References 34 publications

(46 reference statements)

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“…The activity of UDP-GlcNAc 2-epimerase in Sf9 cells is considerably lower than that in rat liver cytosol (15). Further, no signiˆcant levels of SAS activity (16) and CMP-SAS activity (17) exist in Sf9 cells. In general, insect cells are not capable of producing su‹cient levels of Neu5Ac and CMP-Neu5Ac for sialylation to take place e‹ciently, due to the lack of these essential enzymes.…”

Section: B Insect Cell-produced N-glycansmentioning

confidence: 99%

“…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).…”

Section: E-4 Expression Of Sialylated N-glycansmentioning

confidence: 99%

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Humanization of recombinant glycoproteins expressed in insect cells

Tomiya¹

2009

TIGG

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Many of the most valuable recombinant DNA products, including monoclonal antibodies, are secreted glycoproteins, which contain N-glycans. The biochemical nature of these attached glycans depends on the characteristics of the host cell used, the protein synthesized, and the culture environment. The capacity to produce functional glycoproteins with desired glycosylation patterns depends on the presence of proper cellular enzymatic machinery required for the processing of N-glycans. It is well recongnized that expression of therapeutic proteins using insect cells in combination with the baculovirus-expression system has potential advantages in terms of the ease of large scale, low cost production of heterologous glycoproteins. However, the glycoforms expressed in several commonly used insect cell lines are markedly diŠerent from those expressed in human cells. As N-glycans often aŠect the in vivo biological activity, pharmacokinetics, and several other properties of glycoproteins, the glycoforms produced in this system have become a major concern in the last few years. Accordingly, considerable eŠort has been invested in the past decade in order to gain a better understanding of the processing of N-glycans in insect cells that generate diŠerent glycoforms, and also to overcome the bottlenecks to producing glycoproteins with humanized Nglycans that are more suitable for therapeutic use.

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Section: B Insect Cell-produced N-glycansmentioning

confidence: 99%

Section: E-4 Expression Of Sialylated N-glycansmentioning

confidence: 99%

Humanization of recombinant glycoproteins expressed in insect cells

Tomiya¹

2009

TIGG

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“…However, a significant difference is that the mammalian ortholog, NeuNAc phosphate synthase (E.C. 2.5.1.57) shows strong preference toward phosphorylated ManNAc ManNAc-6-P (4,5,8), and therefore the final product of the reaction after condensation with PEP is NeuNAc-9-P. Additionally, human NeuNAc phosphate synthase was found to possess 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid-9-P (KDN-9-P) synthetic activity (4). Contrarily, the mouse protein cannot produce deaminated NeuNAc at the level of fluorescent detection after 1,2-diamino-4,5-methylenedioxybenzene derivatization coupled with http://bmbreports.org BMB reports HPLC separation (5).…”

Section: Introductionmentioning

confidence: 99%

“…KDN-9-P and NeuNAc-9-P synthetic activities of human and chimeric enzyme were assayed as described (4,9). Briefly, 100 μl of the substrate solution (25 mM Man-6-P or 5 mM ManNAc-6-P, 25 mM MnCl2, and 16.6 mM PEP in 50 mM Tris HCl buffer pH 8.0) were added to 100 μl of enzyme solution.…”

Section: Enzyme Assaymentioning

confidence: 99%

Replacement of the antifreeze-like domain of human N-acetylneuraminic acid phosphate synthase with the mouse antifreeze-like domain impacts both N-acetylneuraminic acid 9-phosphate synthase and 2-keto-3-deoxy-D-glycero-Dgalacto- nonulosonic acid 9-phosphate synthase activities

Reaves

Lopez²,

Daskalova³

2008

BMB Reports

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Human NeuNAc-9-P synthase is a two-domain protein with ability to synthesize both NeuNAc-9-P and KDN-9-P. Its mouse counterpart differs by only 20 out of 359 amino acids but does not produce KDN-9-P. By replacing the AFL domain of the human NeuNAc-9-P synthase which accommodates 12 of these differences, with the mouse AFL domain we examined its importance for the secondary KDN-9-P synthetic activity. The chimeric protein retained almost half of the ability of the human enzyme for KDN-9-P synthesis while the NeuNAc-9-P production was reduced to less than 10%. Data from the homology modeling and the effect of divalent ions and temperature on the enzyme activities suggest conformational differences between the human and mouse AFL domains that alter the shape of the cavity accommodating the substrates. Therefore, although the AFL domain itself does not define the ability of the human enzyme for KDN-9-P synthesis, it is important for both activities by aiding optimal positioning of the substrates.

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“…One approach has been to use conventional baculovirus vectors to express genes encoding these functions under the transcriptional control of the polyhedrin (polh) promoter during the very late phase of infection. This approach has been used to introduce mammalian enzymes involved in CMPsialic acid biosynthesis and to show that this can induce the accumulation of high levels of CMP-sialic acid in the virus-infected insect cells (Lawrence et al, 2000Viswanathan et al, 2003Viswanathan et al, , 2005. This approach also has been used to express genes encoding mammalian glycosyltransferases and to obtain evidence that introduction of these enzymes can lead to sialylation of a co-expressed recombinant N-glycoprotein (Chang et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT‐1 cells cultured in serum‐free medium

Hill¹,

Aumiller²,

Shi³

et al. 2006

Biotech & Bioengineering

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The inability to sialylate recombinant glycoproteins is a critical limitation of the baculovirusinsect cell expression system. This limitation is due, at least in part, to the absence of detectable sialyltransferase activities and CMP-sialic acids in the insect cell lines routinely used as hosts in this system. SfSWT-1 is a transgenic insect cell line encoding five mammalian glycosyltransferases, including sialyltransferases, which can contribute to sialylation of recombinant glycoproteins expressed by baculovirus vectors. However, sialylation of recombinant glycoproteins requires culturing SfSWT-1 cells in the presence of fetal bovine serum or another exogenous source of sialic acid. To eliminate this requirement and extend the utility of SfSWT-1 cells, we have isolated a new baculovirus vector, AcSWT-7B, designed to express two mammalian enzymes that can convert N-acetylmannosamine to CMP-sialic acid during the early phase of infection. AcSWT-7B was also designed to express a model recombinant glycoprotein during the very late phase of infection. Characterization of this new baculovirus vector showed that it induced high levels of intracellular CMP-sialic acid and sialylation of the recombinant Nglycoprotein upon infection of SfSWT-1 cells cultured in serum-free medium supplemented with N-acetylmannosamine. In addition, co-infection of SfSWT-1 cells with AcSWT-7B plus a conventional baculovirus vector encoding human tissue plasminogen activator resulted in sialylation of this recombinant N-glycoprotein under the same culture conditions. These results demonstrate that AcSWT-7B can be used in two different ways to support recombinant Nglycoprotein sialylation by SfSWT-1 cells in serum-free medium. Thus, AcSWT-7B can be used to extend the utility of this previously described transgenic insect cell line for recombinant sialoglycoprotein production.

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Cloning and Expression of the HumanN-Acetylneuraminic Acid Phosphate Synthase Gene with 2-Keto-3-deoxy-d-glycero- d-galacto-nononic Acid Biosynthetic Ability

Cited by 91 publications

References 34 publications

Humanization of recombinant glycoproteins expressed in insect cells

Humanization of recombinant glycoproteins expressed in insect cells

Replacement of the antifreeze-like domain of human N-acetylneuraminic acid phosphate synthase with the mouse antifreeze-like domain impacts both N-acetylneuraminic acid 9-phosphate synthase and 2-keto-3-deoxy-D-glycero-Dgalacto- nonulosonic acid 9-phosphate synthase activities

Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT‐1 cells cultured in serum‐free medium

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