A Single Excitation-duplexed Imaging Strategy For Profiling Cell Surface Protein-specific Glycoforms

In eukaryotic cells, most of cell surface proteins are covalently attached to sugar chains, a repertoire of extremely diverse structures composed of several monosaccharide building blocks. The unique glycosylation pattern, a population of glycoforms that share the same polypeptide backbone, serves as an important determinant for protein conformation and activity, and in turn, these glycoforms are regulated by a particular set of signal pathways. Moreover, the variation of the glycan structures of a certain glycoprotein can be closely linked with specific changes of cell physiological states. Thus in situ visualization of glycoforms of a given glycoprotein on a cell surface can contribute to the understanding of glycosylation machinery and its pleiotropic regulation of biological functions, and the identification of new diagnostic biomarkers as well as therapeutic targets.Fluorescence resonance energy transfer (FRET)-based imaging technique has offered an attractive solution for imaging monosaccharides on specific cell surface proteins, such as EGFR, integrin αvβ3 and αxβ2, as well as intracellular proteins. These strategies rely on the respective labeling of protein and monosaccharide with donor and acceptor, and the emergence of FRET, thus allowing thedetection of only one kind of monosaccharide. Considering the diversity and complexity of the glycoforms, profiling multiple monosaccharides on a given cell surface protein is very significant for revealing complex glycan-regulated signal pathway machinery. Virtually no progress has been made in this challenging field owing to the difficulty in obtaining appropriate pairs of donors and acceptors without interference and to avoid the serious acceptor bleed-through resulting from the discrepancy between protein copy number and glycan abundance.These problems can be solved by using a donor for single excitation to simultaneously and effectively activate multiple acceptors of large amount. Owing to the unique polychromatic light emitting properties, upconversion luminescent nanoparticles (UNPs) provide an excellent solution. Multiple color emission from UNPs has been reported for nucleic acid assay and intracellular drug release monitoring. However, these systems were not designed for targeting a specific location, thus are not suitable for protein-specific glycoform study. This thesis focuses on to develop a site-specific duplexed luminescence resonance energy transfer system on cell surface for simultaneous imaging of two kinds of monosaccharides on a specific protein by single near-infrared excitation. The details of this work as follows:This work used UNPs to construct a site-specific duplexed luminescence resonance energy transfer (D-LRET) system on cell surface for simultaneous imaging of different protein-specific monosaccharides using mucinl as the model protein. The single excitation-duplexed imaging system utilizes aptamer modified upconversion luminescent nanoparticles as an energy donor to target the protein, and two fluorescent dye acceptors to tag two kinds of cell surface monosaccharides by a dual metabolic labeling technique. Upon excitation at 980 nm, only the dyes linked to protein-specific glycans can be lit up by the donor by two parallel energy transfer processes, for in situ duplexed imaging of glycoforms on specific protein. Using MUC1 as the model, this strategy can visualize distinct glycoforms of MUC1 on various cell types and quantitatively track terminal monosaccharide pattern. This approach provides a versatile platform for profiling protein-specific glycoforms, thus contributing to the study of the regulation mechanisms of protein functions by glycosylation.