Localized Chemical Remodeling For Live Cell Imaging Of Protein-Specific Glycoform

Protein glycosylation is a ubiquitous post-translational,diversity-generating covalent modification process.The glycosylation pattern of a protein,as characterized by the population of glycoforms,is subject to elaborate control by cellular synthetic machinery and in turn,defines protein's biological functions and cell's recognition and signaling properties.A hallmark of protein glycosylation is its highly dynamic and adaptive nature in response to the physiological state of a cell.Live cell imaging of protein-specific glycoform in an in situ fashion can therefore not only help to advance the understanding of glycosylation pathways and functions but also provide a potentially working channel for diagnostic and therapeutic usageMetabolic oligosaccharide engineering(MOE)has been the tour de force tool developed for glycoform imaging.This ingenious approach permits system-level incorporation of reactive unnatural carbohydrate moiety for subsequent bioorthogonal elaboration with one assay probe,and protein-specific tagging of the other probe is achieved through either protein fusion or affinity binding;a distance-dependent physical process between two probes,such as Forster resonance energy transfer,is then used to confine detection to the intra-glycoprotein spacing level and can therefore report on the glycoform associated with a particular protein.The ability to perturb,profile,and perceive glycans routinely has notably facilitated the decoding of glycomics information and identification of disease biomarkers.However,despite the exciting progress witnessed in this promising field,drawbacks are apparent when using MOE as a protein-specific glycoform imaging tool:MOE is a non-selective,global chemical remodeling(GCM)technology exerting unnecessary perturbation on the whole-cell scale,which creates essentially a cell in a different physiological state,and therefore,the originally targeted native cell is no longer available for examination;although tolerance of unnatural carbohydrate analog at the enzyme level is clear,metabolic delivery at the cellular level is a complex,poorly understood process,leading inevitably to unpredictable metabolic efficiency and installation percentage issues;this unpredictability renders it problematic to translate glycoform data acquired on a cell in its metabolically altered state to the corresponding information for the native cell and as a consequence,comparison among cells in different physiological states and across cell lines can become a formidable challenge.With these inadequacies in mind,we embark on an endeavor to develop a protein-specific imaging strategy that both minimizes perturbation on the cell and allows straightforward interpretation of the glycoform data.Ideally,the perturbation should be applied only at the to-be-probed carbohydrate site of interest,and glycoform information acquired through a well-defined chemical transformation can be representative of and proportionally extrapolated to the whole protein-specific target glycoform population.We communicate herein a localized chemical remodeling(LCM)methodology for live cell imaging of protein-specific glycoform.This methodology relies on a combination of affinity binding,off-on switchable catalytic activity,and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging.Instead of relying on a physical process,LCM essentially provides a molecular ruler that can report on intramolecular distance by using chemical reactivity as a reporter system.