title: A FRET probe for cell-based imaging of ganglioside-processing enzyme activity and high-throughput screening.
abstract: Gangliosides are important signaling molecules in the cell membrane and are processed by several enzymes. Deficiencies in these enzymes can cause human lysosomal storage diseases. Building an understanding of the pathways of glycosphingolipid catabolism requires methods for the analysis of these enzymatic activities A GM3-derived FRET probe was synthesized chemoenzymatically for the detection and quantitation of a range of ganglioside-degrading enzymes, both in cell lysates and in living cells. This is the first substrate that enables the ratiometric fluorogenic assay of sphingolipid ceramide N-deacylase and endoglycoceramidase and can detect and localize neuraminidase activity in living cells. It is therefore a valuable tool for building a better understanding of membrane-confined enzymology. It also enables the robust and reliable assay of ganglioside-degrading enzymes in a microtiter plate, thus opening the door to screening for novel or engineered biocatalysts or for new inhibitors.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1 (Canada); State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240 (China).
authors.forename: Guang-Yu
authors.initials: GY
authors.lastname: Yang
authors.forename: Caishun
authors.initials: C
authors.lastname: Li
authors.forename: Michael
authors.initials: M
authors.lastname: Fischer
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Cairo
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005732:Gangliosides / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000737:chemistry; D009439:Neuraminidase / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005732:Gangliosides; D015335:Molecular Probes; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase; D009439:Neuraminidase; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase
keywords: FRET; enzymes; fluorescent probes; gangliosides; high-throughput screening