Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

Abstract

The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2′-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems.

A new insect cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency

“…1). However, Sfie1SWT and Sf39KSWT achieved lower final densities than Sf9 cells, as observed previously with some other glycoengineered insect cell lines (Aumiller et al, 2003; Geisler and Jarvis, 2012a). Importantly, Sfie1SWT and Sf39KSWT cells had virtually identical growth curves, indicating that the observed difference in growth relative to the parental cell line was not due to constitutive expression of the nine glycogenes by Sfie1SWT cells.…”

“…The baculovirus ie1 promoter, which is constitutively active in uninfected insect cells, is most commonly used for insect cell transformation and was the promoter used for all of our previous insect cell glycoengineering efforts (Aumiller et al, 2003; Geisler and Jarvis, 2012a; Hollister et al, 2002; Hollister and Jarvis, 2001; Mabashi-Asazuma et al, 2013). In contrast, the baculovirus 39K promoter, a delayed early promoter, is not active in uninfected cells because it requires one or more virus-encoded transcription factors (Passarelli and Guarino, 2007).…”

“…B4GALT1 and ST6GAL1/ST3GAL3 activities were measured using samples containing 100 μg of total protein, as described previously (Aumiller et al, 2012), except the reactions were quenched by adding an equal volume of 10% trichloroacetic acid rather than cold buffer, and glass fiber filters were washed immediately after the addition of samples, without drying. Sialic acid determinations were performed using TBA assays, as described previously (Geisler and Jarvis, 2012a), except we added 5 mg of sample to each assay.…”

“…Our approaches involved incorporating foreign genes encoding glycosyltransferases, enzymes involved in sialic acid and CMP-sialic acid biosynthesis, and a CMP-sialic acid transporter into either baculovirus expression vectors (Hill et al, 2006; Jarvis and Finn, 1996; Jarvis et al, 2001; Seo et al, 2001; Tomiya et al, 2003) or insect cell lines (Aumiller et al, 2003; Aumiller et al, 2012; Breitbach and Jarvis, 2001; Geisler and Jarvis, 2012a; Hollister et al, 2002; Hollister and Jarvis, 2001; Hollister et al, 1998; Mabashi-Asazuma et al, 2013). The latter efforts yielded new, glycoengineered insect cell lines capable of producing biantennary, terminally sialylated N -glycans.…”

Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein

“…3B) Considering the lack of interaction of the ficolin-1 Y271F mutant, devoid of a sialic acid-binding capacity, and the requirement of the GP mucin-like domain for ficolin-1 binding, our data strongly suggest that ficolin-1 recognizes sialylated glycans of the mucin domain of GP. In addition, we did not detect any interaction between wild-type ficolin-1 and immobilized purified recombinant EBOV expressing GP lacking the TM domain (rEBOVGP⌬TM) expressed in Sf9 insect cells (data not shown), which is consistent with the absence of glycan sialylation of recombinant glycoproteins expressed in Sf9 cells (58).…”