Imaging Of Protein-specific Glycosylation By METPLA

Protein glycosylation is one of the most prevalent posttranslational modifications, and plays a critical role in regulating protein behaviors including stability, folding, trafficking and function. However, tools to probe protein-specific glycosylation in cells are largely lacking. Here we describe a highly sensitive fluorescence imaging strategy to visualize protein-specific glycosylation by combining glycan metabolic tagging and in situ proximity ligation (termed METPLA).This method provides a general way to visualize different types of glycosylation in situ, and permits the imaging of membrane receptor dimerization in a dynamic cellular context. We first demonstrated our strategy by imaging EGFR sialylation. Then we imaged the sialylation of glucose transporter type 4 (GLUT4) and its glycosylation-deficient mutant N57Q with the METPLA, and the results clarifies further that signals came from the same protein, and not from sialylation of other adjacent proteins. Thus, our METPLA strategy can be applied for imaging protein-specific glycosylation. We probed the subcellular localization of sialylated EGFR by labelling it with METPLA and simultaneously immnuo-staining different cellular organelle markers. The results showed that our METPLA strategy endows high spatial resolution to enable the visualization of subcellular localization of the glycosylated fraction of proteins. When EGFR predominantly exists in monomeric state, the use of antibody that recognizes EGFR extracellular domain (N-terminal) produced minimal METPLA fluorescence signals but the antibody that targets EGFR intracellular domain (C-terminal) yielded robust signals. To test our hypothesis, we subjected A549 lung cancer cells to serum starvation overnight followed by the treatment of EGF to induce EGFR dimerization. Subsequent performance of METPLA demonstrated increased fluorescence signals in a time-dependent and dose-dependent manner.In order to confirm that METPLA fluorescence signals only came from the dimerization and not from the kinase activation induced by the dimerization of EGFR, we inhibit the kinase activity of EGFR. Thus, our METPLA strategy is a novel tool for visualization of EGFR dimerization in situ. In addition, we visualized fucosylation and GalNAcylation on membrane proteins with METPLA, indicating the general applicability of our strategy.In conclusion, we have developed a highly sensitive fluorescence imaging strategy for protein-specific glycosylation detection-METPLA. Thus, this study will likely provide a powerful tool for in-depth investigation of the roles of glycosylation on protein structure and function.