Angewandte
Chemie
DOI: 10.1002/anie.201411747
Fluorescent Probes Hot Paper
A FRET Probe for Cell-Based Imaging of Ganglioside-Processing
Enzyme Activity and High-Throughput Screening**
Guang-Yu Yang, Caishun Li, Michael Fischer, Christopher W. Cairo, Yan Feng, and
Stephen G. Withers*
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 quanti-
tation 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 local-
ize 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.
brates, the ganglioside degradation system involves numerous
enzymes, including neuraminidase on the plasma membrane
and acid ceramidase in the lumen of endosomes and
[
2]
lysosomes. Unsurprisingly, given their importance, disrup-
[3]
tion of these enzymes can result in disease states in humans.
Radiolabeled gangliosides have proved to be useful as tools
for elucidating glycosphingolipid metabolism and as sub-
strates for the assay of ganglioside-degrading enzymes.
However, such assays, which are usually based on TLC
separations of radioisotopically labeled compounds, are
laborious, poorly reproducible, and cannot be used for real-
[
4]
time measurements. New probes that overcome these
limitations would therefore be of great value for understand-
ing the cellular roles of this key class of molecules.
Over the past two decades, fluorescent probes have
become valuable tools in cell biology for the imaging of
[5]
enzymatic reactions and the localization of biomolecules.
For example, 4-methylumbelliferyl glycosides are used to
measure the activity of a range of glycosidases, including that
of ganglioside-degrading beta-hexosaminidase A for the
G
angliosides are a group of glycosphingolipids bearing one
or more sialic acid residues on their termini. They are often
found associated with other sphingolipids, cholesterol, and
some membrane proteins to form so-called “lipid raft”
structures in the cell membrane. In this way, gangliosides
can influence cell structure and interactions with the extra-
[
6]
analysis of GM2 gangliosidoses. Of particular value have
been fluorescence resonance energy transfer (FRET) probes
that allow ratiometric measurement, thereby providing
[7]
greater precision than measurement at a single wavelength.
The design of probes to interrogate membrane-associated
[
1]
cellular environment. As such, they play extremely impor-
tant roles in biochemical signaling, pathogen entry, mem-
brane transport, and intracellular protein sorting. In verte-
[
8]
proteins presents specific challenges, with only a handful of
successful examples having been used for the assay of
[
2]
[
9]
[10]
phospholipase A2, ceramidase,
GM2-activator protein,
[11]
and glucocerebrosidase.
Herein, we present the design,
[
*] Dr. G. Y. Yang, Dr. M. Fischer, Prof. S. G. Withers
Department of Chemistry, University of British Columbia
Vancouver, British Columbia V6T 1Z1 (Canada)
E-mail: withers@chem.ubc.ca
synthesis, and biological application of a small-molecule
FRET probe that can be used in the detection and quanti-
tation of a range of ganglioside-degrading enzymes, both in
cell lysates and in living cells. The utility of this reagent in the
high-throughput assay of two ganglioside-degrading enzymes
in crude cell lysates is demonstrated, as well as in the
ratiometric imaging of neuraminidase activity on the plasma
membrane of living cells.
Probe 1 is based on the structure of ganglioside GM3 and
carries two fluorophores, 7-hydroxycoumarin and BODIPY,
which are attached on either end of the molecule to serve as
FRET donor and acceptor, respectively (Figure 1). Placement
of the fluorophores at the extreme ends of the molecule yields
a reagent that can be recognized and cleaved by several
different ganglioside-degrading enzymes, namely sphingoli-
pid ceramide N-deacylase (Enz1), endoglycoceramidase
Dr. G. Y. Yang, Prof. Y. Feng
State Key Laboratory of Microbial Metabolism
School of Life Sciences and Biotechnology
Shanghai Jiao Tong University, Shanghai, 200240 (China)
Dr. C. Li, Prof. C. W. Cairo
Alberta Glycomics Centre, Department of Chemistry
University of Alberta, Edmonton, Alberta T6G 2G2 (Canada)
[
**] We thank the Canadian Institutes for Health Research and the
Natural Sciences and Engineering Research Council of Canada,
along with the National Basic Research Program of China
(
2011CBA00800 and 2012CB721000) and the National Natural
Science Foundation of China (No. 31100611) for financial support.
S.G.W. thanks the Canada Research chairs program for salary
support and M.F. thanks the Austrian Science Fund (FWF) (J3293-
B21) for an Erwin Schrçdinger postdoctoral fellowship. We also
thank Mark Okon and Lawrence McIntosh for access to NMR
facilities and technical help with the NMR characterization.
(Enz2), and neuraminidase (Enz3). Cleavage of the probe
by any of these enzymes will yield a large decrease in FRET
efficiency, thereby giving a detectable ratiometric fluorescent
signal.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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