Endo-ß-Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism-Based Probes and Activity Measurement.

ABSTRACT

ß-Glucoside-configured cyclophellitols are activity-based probes (ABPs) that allow sensitive detection of ß-glucosidases. Their applicability to detect proteins fused with ß-glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M-777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4-methylumbelliferyl ß-d-lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre-blocking with conduritol ß-epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous ß-glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high-resolution detection moieties) should assist further research in living cells and organisms.