Labeling And Imaging Of Protein-Specific Glycosylation And Glycans-based Immunotherapy For Cancer Cells

Protein glycosylation is widespread and important in living organisms. Glycosylated proteins account for more than 70% of total proteins in cells. Glycosylation is closely related with the structure and function of proteins. The study of glycobiology has generally focused on understanding the role of global glycan structures in biological processes, but few studies paid attention to the protein-specific glycosylation. In fact, increasing evidences have suggested that glycosylation on specific target proteins, rather than overall cellular glycosylation status, governs the biological outcome. However, tools to probe protein-specific glycosylation in live cells are largely lacking. Two outstanding issues have hampered such an investigation: 1. Compared to other protein posttranslational modifications such as phosphorylation and acetylation, glycosylation is structurally heterogeneous; 2. Relatively low immunogenicity of glycans makes it difficult to develop high affinity antibody against site-specific glycosylation. To overcome these limitations, we combining metabolic labeling and in situ proximity ligation(METPLA) to label and image protein specific glycosylation, In this strategy, oligonucleotides were installed onto the glycan and the target protein, respectively. Close proximity (< 40 nm) of the two oligonucleotides enables the rolling-circle amplification. This technology has provided a novel way to visualize different types of glycosylation in situ, and permits the imaging of membrane receptor dimerization in dynamic cellular context.Mobilizing the immune system to target and eliminate tumor cells is a promising therapeutic strategy with exceptional specificity and efficacy. By recruiting pre-existing natural antibodies in human serum to the surfaces of tumor cells, this strategy could lead to complement dependent cytotoxicity(CDC), antibody dependent cellular cytotoxicity(ADCC) and antibody dependent cytophagy(ADCP), for destructing of cancer cells. Traditional approach has some intrinsic limitations. For instance, the non-covalent binding affinity between the ligand and the cell-surface receptor is generally weaker and less stable than covalent interactions, compromising the efficiency of immune response activation. Secondly, the defined number of cell-surface receptors could limit the number of binding molecules, resulting in saturation and insufficient targeting. Moreover, the interaction between the binding molecule and the receptor could induce cellular internalization of the complex, thus damping the magnitude of immune response. Here, we report a novel strategy by combining glycan metabolic engineering and bioorthogonal chemical ligation to artificially install different amounts of Rha epitopes onto the cell surface in a controlled manner. Cell-surface display of Rha epitopes promoted the recruitment of anti-Rha antibodies and induced significant complement-mediated cell cytotoxicity...