RESUMEN
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.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Manosidasas/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Colorantes Fluorescentes/química , Estructura Molecular , Rhodococcus/enzimologíaRESUMEN
Retaining ß-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining ß-glucosidases. Whereas ß-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining ß-glucosidases, ß-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining ß-glucosidases by the combined use of cyclophellitol ß-epoxide- and ß-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent ß-aziridine but not ß-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the ß-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining ß-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining ß-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.
Asunto(s)
Aminoácidos/metabolismo , Ciclohexanoles/metabolismo , Sondas Moleculares/metabolismo , beta-Glucosidasa/metabolismo , Sustitución de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Animales , Aziridinas/química , Aziridinas/metabolismo , Células COS , Dominio Catalítico , Chlorocebus aethiops , Ciclohexanoles/química , Compuestos Epoxi/química , Compuestos Epoxi/metabolismo , Humanos , Hidrólisis , Immunoblotting/métodos , Sondas Moleculares/química , Mutagénesis Sitio-Dirigida , Mutación Missense , Reproducibilidad de los Resultados , Azida Sódica/química , Azida Sódica/metabolismo , Especificidad por Sustrato , beta-Glucosidasa/química , beta-Glucosidasa/genéticaRESUMEN
A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian ß-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian ß-glucosidases (see picture). These probes offer new ways to study ß-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.