Abstract
Terminally sialylated N-glycoproteins are of great interest in therapeutic applications. Due to the inability of prokaryotes to carry out this post-translational modification, they are currently predominantly produced in eukaryotic host cells. In this study, we report a synthetic pathway to produce a terminally sialylated N-glycoprotein in the periplasm of Escherichia coli, mimicking the sialylated moiety (Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-) of human glycans. A sialylated pentasaccharide, Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-, was synthesized through the activity of co-expressed glycosyltransferases LsgCDEF from Haemophilus influenzae, Campylobacter jejuni NeuBCA enzymes, and Photobacterium leiognathi α-2,6-sialyltransferase in an engineered E. coli strain which produces CMP-Neu5Ac. C. jejuni oligosaccharyltransferase PglB was used to transfer the terminally sialylated glycan onto a glyco-recognition sequence in the tenth type III cell adhesion module of human fibronectin. Sialylation of the target protein was confirmed by lectin blotting and mass spectrometry. This proof-of-concept study demonstrates the successful production of terminally sialylated, homogeneous N-glycoproteins with α-2,6-linkages in the periplasm of E. coli and will facilitate the construction of E. coli strains capable of producing terminally sialylated N-glycoproteins in high yield.
Highlights
Escherichia coli is a commonly used host for the production of biotherapeutic and other highvalue proteins
We designed a pathway to produce a terminally sialylated, homogeneous N-glycoprotein with a short glycan in the E. coli periplasm (Figure 1)
The pathway involves six steps: (1) To provide CMP-Neu5Ac for sialylation, the CMP-Neu5Ac is synthesized by NeuBCA synthases, utilizing UDP-GlcNAc as substrate; (2) the precursor of lipid-linked oligosaccharides (LLOs) is synthesized by WecA to provide the starting sugar; (3) which is extended by LsgCDEF GTases at the inner surface of cytoplasmic membrane to synthesize the tetrasaccharide glycan Gal-β-1,4GlcNAc-β-1,3-Gal- β-1,3-GlcNAc-; (4) followed by transfer of Neu5Ac onto this tetrasaccharide glycan via an α-2,6-linkage by the use of the pl-ST6α-2,6-STase; (5) flipping of the sialylated glycan from the cytoplasmic to the periplasmic side of the membrane by PglK flippases; (6) and, the sialylated glycan is transferred to the glyco-tagged (DQNAT motif) (Lizak et al, 2011) recombinant protein using OTase PglB from C. jejuni
Summary
Escherichia coli is a commonly used host for the production of biotherapeutic and other highvalue proteins. Due to its strong electronegativity, sialic acids can increase the solubility or resistance to proteolytic degradation of a glycoprotein, as well as enhancing its residence time in blood and promoting transportation of drugs and ions into cells (Aquino et al, 1980; Raju et al, 2001; Bork et al, 2009; Meuris et al, 2014; Cuccui and Wren, 2015; Gupta and Shukla, 2018; Thi Sam et al, 2018) It is unsurprising, that extensive research efforts have focused on overcoming the traditional bottlenecks associated with recombinant production of terminally sialylated glycoproteins of biopharmaceutical importance
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