Abstract
To elucidate the biological and pathological functions of sialyltransferases (STs), intracellular ST activity evaluation is necessary. Focusing on the lack of noninvasive methods for obtaining the dynamic activity information, this work designs a sensing platform for in situ FRET imaging of intracellular ST activity and tracing of sialylation process. The system uses tetramethylrhodamine isothiocyanate labeled asialofetuin (TRITC-AF) as a ST substrate and fluorescein isothiocyanate labeled 3-aminophenylboronic acid (FITC-APBA) as the chemoselective recognition probe of sialylation product, both of which are encapsulated in a liposome vesicle for cellular delivery. The recognition of FITC-APBA to sialylated TRITC-AF leads to the FRET signal that is analyzed by FRET efficiency images. This strategy has been used to evaluate the correlation of ST activity with malignancy and cell surface sialylation, and the sialylation inhibition activity of inhibitors. This work provides a powerful noninvasive tool for glycan biosynthesis mechanism research, cancer diagnostics and drug development.
Highlights
Energy transfer (FRET) analysis[10], these separation-required methods cannot provide dynamic activity of STs in living cells, are difficult to verify the ST-related sialylation mechanisms
After the vesicle was delivered into cells, the intracellular ST could transfer the SA from cytidine-5’-monophospho-sialic acid (CMP-SA), which exists in Golgi apparatus, to the terminal position of glycan chains of the TRITC-AF
The unspecific adsorption did not interfere with the dynamic analysis of the intracellular ST activity since it could be considered as a stable background of FRET and eliminated by software wizard
Summary
Energy transfer (FRET) analysis[10], these separation-required methods cannot provide dynamic activity of STs in living cells, are difficult to verify the ST-related sialylation mechanisms. To improve glycan recognition avidity and avoid cross-binding, a chemoselective and bioorthogonal probe can be integrated into the system to track the sialylation product in living cells. This work encapsulated tetramethylrhodamine isothiocyanate labeled asialofetuin (TRITC-AF) as ST substrate and fluorescein isothiocyanate labeled APBA (FITC-APBA) as the chemoselective recognition probe of sialylation product in a liposome-based delivery vesicle to design a novel sensing vesicle for in situ analysis of ST activity (Fig. 1). The signal intensity depended on the formation of the sialylated TRITC-AF, could be used for assessment of ST activity This is the first report on the noninvasive analysis of ST activity in living cells
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