Imaging Glycosylation

摘要

Protein glycosylation comprises a large family of post-translational modifications in eukaryotic and prokaryotic organisms. Oligosaccharides play specific structural roles, maintaining and modulating effector functions that are physiologically relevant, and can be manipulated to optimize the properties of therapeutic antibodies. N-linked glycosylation pathways involve two ancient and highly conserved enzymatic steps that mediate the conversion of oligomannose glycans to hybrid-type and then to complex-type structures. Control and characterization of this process is essential in the production of antibody-based therapeutics, but conventional methods for characterizing glycosylation require significant amounts of material and do not report on the distribution of oligosaccharides on a molecule by molecule basis. Single molecule recognition imaging microscopy is a modification of atomic-force microscopy in which recognition events between a ligand bound to the probe and its target on the surface are mapped simultaneously with sample topography, producing a map of the distribution of target molecules on the surface. We apply it here to visually map the extent of the high-mannose modification on a human IgG_4 monoclonal antibody. In particular, we show that a biologically less effective variant of this antibody has a significant amount of high-mannose species whereas the functional molecule shows limited levels of high-mannose glycoform.