CHEMBIOCHEM
COMMUNICATIONS
DOI: 10.1002/cbic.201300207
Development of a Novel FRET Probe for the Real-Time
Determination of Ceramidase Activity
Krishna P. Bhabak,[a] Anett Hauser,[a] Susanne Redmer,[a] Sebastian Banhart,[b]
Dagmar Heuer,[b] and Christoph Arenz*[a]
Sphingolipids are major constituents of cellular membranes.
Besides their function in membrane structure integrity, some
of them also act as signalling molecules in various cellular pro-
cesses.[1] Among the various sphingolipids, ceramide is consid-
ered a central molecule; it is either biosynthesised de novo or
obtained through degradation of sphingomyelin or glyco-
sphingolipids.[2] The N-acyl moiety of ceramide is cleaved by
ceramidases to form the lysolipid sphingosine that undergoes
further phosphorylation to sphingosine-1-phosphate (S1P). Al-
though ceramide induces apoptosis, S1P triggers cell prolifera-
tion, thus indicating the importance of a proper balance in the
concentrations of these lipids for normal cell development.[3]
The concept that ceramide acts as a “second messenger”
has provoked vivid debate, not least because of the error-
prone methods for ceramide quantification.[4] In recent reports,
ceramide is usually referred to as a “signalling lipid”, because
the increase in its cellular concentration seems to be slower
than that of classical signal-transduction events. In addition,
two competing models for ceramide’s mode of action now
exist: one includes intracellular ceramide-binding proteins like
ceramide-activated protein kinases and cathepsin D, and the
other emphasises the formation of lipid micro domains as sig-
nalling platforms.[5] Its role as a signalling molecule is further
complicated by the fact that several distinct cellular pools of
ceramide exist and many enzymes are involved in its synthesis
and degradation; some of these display considerable chain-
length-dependent selectivity.[6] The direct cleavage of ceramide
to sphingosine is catalysed by a set of ceramidases (CDases)
that differ in pH optima and localisation. CDases can be classi-
fied into three major subtypes:[7] acid ceramidase (aCDase),[8]
neutral ceramidase (nCDase),[9] and alkaline ceramidase (al-
kCDase).[10–12] Members of the aCDase subtype are responsible
for the constitutive degradation of ceramide in lysosomes, and
deficiency in activity leads to massive lysosomal accumulation
of ceramide, thereby resulting in Farber disease.[13,14] On the
other hand, it has been shown that aCDase is up-regulated in
prostate cancer[15] and melanomas,[16] thereby leading to the
marker.[15,16] Moreover, aCDase in tumour cells confers resist-
ance to chemo- and radiotherapy, and inhibition of this
enzyme has been proposed as a target in cancer therapy.[17] It
has been shown that a small portion of aCDase can be secret-
ed to the outer leaflet of the plasma membrane;[18] however,
functional relevance of this secretion is unlikely because of the
relatively narrow pH optimum of this enzyme.
The nCDase subtype is a ubiquitously expressed enzyme; it
has been found secreted from or firmly attached to the extra-
cellular side of the plasma membrane, as well as in endosome-
like structures and mitochondria. The enzyme can be stimulat-
ed by various cytokines and protects cells from cytokine-in-
duced apoptosis.[19] The alkCDase subgroup comprises three
different enzymes; these have been localised to the endoplas-
mic reticulum (ER) and Golgi apparatus. All alkCDases have
been implicated in cell proliferation in various tissue types.
Today, ceramide and its metabolites are conveniently quanti-
fied by MS, but during work-up, topological information and
time resolution are largely lost. Therefore, robust and conven-
ient real-time assays for enzymes involved in sphingolipid me-
tabolism would be highly beneficial for clarifying the identity,
topology, and kinetics of enzymes involved in sphingolipid-
mediated signalling.
Conventionally, ceramidase activity is determined by radio-
activity-based methods with 14C- or 3H-labelled ceramides as
substrates.[12] However, fluorescent spectroscopic methods
have recently attracted much research attention because of
their high sensitivity, specificity, and signal-to-noise ratio. Over
recent years, different singly labelled fluorescent ceramide ana-
logues have been developed, with NBD,[12] BODIPY,[20] coumar-
in,[21] or diphenylhexatriene (DPH)[22] as fluorescent tags, at-
tached at either the fatty acid or sphingosine part of ceramide
unit. Moreover, quantification of ceramidase activity with
singly-labelled ceramide analogues is more complicated and
tedious, because of the postreaction separation of ceramide
and sphingosine parts, thus limiting their use in high-through-
put ceramidase assays.[23] As an alternative to monolabelled
probes, a fluorescence-quenched ceramide analogue was de-
veloped with pyrene and 3,5-dinitrobenzyl moieties.[24] Howev-
er, this probe had limited application due to very hydrophobic
nature of pyrene, relatively low quantum yield, and the lack of
an inbuilt normalising standard. Thus, we sought a quick and
reliable real-time assay to determine ceramidase activity in
homogenous systems.
hypothesis that aCDase level could serve as
a tumour
[a] Dr. K. P. Bhabak, A. Hauser, S. Redmer, Prof. Dr. C. Arenz
Department of Organic & Bioorganic Chemistry
Humboldt Universitꢀt zu Berlin, Institute for Chemistry
Brook-Taylor Str-2, 12489 Berlin (Germany)
[b] Dr. S. Banhart, Prof. Dr. D. Heuer
Robert Koch Institute
In recent reports, Schultz and co-workers have developed
doubly fluorescent-labelled phosphoglycerolipids as FRET
probes to selectively monitor phospholipase A2 activity.[25–27]
The selection of donor–acceptor fluorescent pairs such as 7-ni-
Nordufer 20, 13353 Berlin (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300207.
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 1049 – 1052 1049