An O-GlcNAcase responsive fluorogenic probe for biological applications

Abstract

In the current study, we designed and prepared five fluorogenic probes (β-GlcNAc-FC, β-GlcNPr-FC, β-GlcNBu-FC, β-GlcNVa-FC and β-GlcNAc-Bn-FC) for the detection of O-GlcNAcase in live cells. Among these probes, β-GlcNAc-FC was found to be the best fluorogenic substrate for O-GlcNAcase. In addition, β-GlcNAc-FC was able to monitor cytosolic and nuclear O-GlcNAcase in cells. By using this probe, we found that O-GlcNAcase activity in AGS cells is higher than those in Capan-1 and HaCaT cells. Also, we observed that fluorescence signals arising from β-GlcNAc-FC are very low in cells cultured under glucose deprivation conditions and gradually increases when the concentration of glucose in culture media increases up to 10 mM. Furthermore, ER and mitochondrial stresses caused by respective treatment with tunicamycin and FCCP did not affect O-GlcNAcase activity, as judged from the observation that the fluorescence intensity arising from β-GlcNAc-FC in cells exposed to these stresses was quite similar to that in cells cultured under normal conditions. Taken together, the findings indicate that β-GlcNAc-FC is a useful fluorogenic probe to determine the level of O-GlcNAcase activity in live cells.