Abstract
Manipulations of cell surface glycosylation or glycan decoration of selected proteins hold immense potential for exploring structure-activity relations or increasing glycoprotein quality. Metabolic glycoengineering describes the strategy where exogenously supplied sugar analogues intercept biosynthetic pathways and are incorporated into glycoconjugates. Low membrane permeability, which so far limited the large-scale adaption of this technology, can be addressed by the introduction of acylated monosaccharides. In this work, we investigated tetra-O-acetylated, -propanoylated and -polyethylene glycol (PEG)ylated fucoses. Concentrations of up to 500 µM had no substantial effects on viability and recombinant glycoprotein production of human embryonic kidney (HEK)-293T cells. Analogues applied to an engineered Chinese hamster ovary (CHO) cell line with blocked fucose de novo synthesis revealed an increase in cell surface and recombinant antibody fucosylation as proved by lectin blotting, mass spectrometry and monosaccharide analysis. Significant fucose incorporation was achieved for tetra-O-acetylated and -propanoylated fucoses already at 20 µM. Sequential fucosylation of the recombinant glycoprotein, achieved by the application of increasing concentrations of PEGylated fucose up to 70 µM, correlated with a reduced antibody’s binding activity in a Fcγ receptor IIIa (FcγRIIIa) binding assay. Our results provide further insights to modulate fucosylation by exploiting the salvage pathway via metabolic glycoengineering.
Highlights
Glycosylation is a highly versatile, complex and ubiquitous form of post-translational modification.Glycans, covalently attached carbohydrate moieties, participate in a variety of intracellular processes or dictate basic protein characteristics such as biological activity, protein folding or proteolytic stability [1]
We aimed to investigate if the application of fucose analogues to CHO RMD cells may quantitatively reach the trastuzumab fucosylation level of the CHO reference cell line, and, if this level can be further increased by metabolic glycoengineering
We reported the first characterization of novel fucose analogues for metabolic glycoengineering of recombinant glycoproteins as a proof-of-principle
Summary
Glycosylation is a highly versatile, complex and ubiquitous form of post-translational modification. Since the sialic acid metabolism served as a workhorse for methodology development and pioneered this technique [5], the scope of metabolic glycoengineering has been broadened to further glycosylation pathways In this regard, the deoxyhexose fucose was addressed as the first non-amino sugar. Fucosylation is known to crucially affect one of the antibody’s effector functions: antibodies lacking core fucose in their Fc N-glycan exhibit significantly increased antibody-dependent cellular toxicity (ADCC) [23] In this regard, we aimed at a sequential antibody fucosylation by applying controlled concentrations of fucose analogues in our metabolic glycoengineering experiments. Biosynthesis of L-fucose (red triangle) occurs mainly via de novo synthesis and, to a minor extent, through the salvage pathway that is exploited here in the course of metabolic glycoengineering; Acyl moieties (shown in B) are introduced to fucose to facilitate cellular uptake across the plasma membrane and are thought to be cleaved intracellularly by cytosolic esterases.
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