FRET probes for measuring sphingolipid metabolizing enzyme activity

Accepted Manuscript Title: FRET probes for measuring sphingolipid metabolizing enzyme activity Authors: Zainelabdeen H Mohamed, Cosima Rhein, Essa M. Saied, Johannes Kornhuber, Christoph Arenz PII: DOI: Reference:

S0009-3084(18)30165-8 https://doi.org/10.1016/j.chemphyslip.2018.09.014 CPL 4690

To appear in:

Chemistry and Physics of Lipids

Received date: Accepted date:

24-8-2018 21-9-2018

Please cite this article as: Mohamed ZH, Rhein C, Saied EM, Kornhuber J, Arenz C, FRET probes for measuring sphingolipid metabolizing enzyme activity, Chemistry and Physics of Lipids (2018), https://doi.org/10.1016/j.chemphyslip.2018.09.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

FRET probes for measuring sphingolipid metabolizing enzyme activity

Zainelabdeen H. Mohamed,1 Cosima Rhein,2 Essa M. Saied,1,3 Johannes Kornhuber2

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and Christoph Arenz1,*

1 Institute

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for Chemistry, Humboldt Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany 2Department of Psychiatry and Psychotherapy, Friedrich-Alexander Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany 3Chemistry Department, Faculty of Science, Suez Canal University, Ismailia, Egypt

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*Correspondence should be submitted: [email protected]

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Abstract

Förster resonance energy transfer (FRET) probes are unique tools in biology, as they allow for a non-destructive monitoring of a certain state of a biomolecule or of an

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artificial substrate within living cells in real time. FRET substrates indicate their relative cleavage rate and thus the in situ activity of a given enzyme. In contrast to quenched

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probes or turn-on probes, one of the two separate signals of the FRET probes can be used as internal reference, which makes ratio-imaging and quantitation of cleavage

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events independent of cellular delivery possible. In this review, we describe the first examples of sphingolipid FRET probes in comparison to different alternative probes.

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Finally, we give an outlook on future probes and their potential application.

1. Introduction

The mammalian cell hosts an enormous and unseizable arsenal of chemical reactions. Most of these reactions are highly chemoselective and stereospecific and produce thousands of different primary and secondary metabolites in a highly controlled 1

fashion. The control of cellular events is mediated by cascades of specific enzymes that reside in different compartments of an individual cell. Attempts at characterizing one specific component of this entangled network traditionally include either lysis or fixation of the cell. Enzymes can be isolated or enriched based on their biophysical properties and characterized in vitro by means of radioactively or fluorescently labeled substrates. (Schwarzmann et al., 2013) Alternatively, in vivo metabolic events can be assessed by pulse-chase experiments, which include the feeding of cells with a labeled

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substrate. Typically, such procedures include cell lysis and analysis of the labeled

metabolites. Naturally, all the topological information is lost under such conditions.

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Especially in lipid science, where virtually all metabolites reside in asymmetric membranes, spatial information including the orientation of a lipid in a distinct membrane (e.g. cytosolic or extracellular) is key for a comprehensive understanding of cellular events. Indeed, spatial differentiation might be even desirable between

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different domains within the same orientation of a given membrane, as suggested in

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the model of “lipid rafts” or liquid ordered vs liquid disordered phases.(Chiantia and London, 2013; Simons and Ikonen, 1997) Normally, probes enabling the investigation

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in spatial distribution of metabolites include pulse-chase labeling, and often also

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fixation of cells. Under such conditions, not only time-resolution is low, but also the identity of a metabolite residing in a given topology is hard to assess. In the present

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review, we want to highlight the advantages and limitations of FRET probes enabling a real time monitoring of metabolic events in living cells and discuss the first examples

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in the field of sphingolipids (SL).

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2. Fluorescence-based probes for assessment of SLs localization and trafficking Fluorescent molecules provide unique opportunities as they allow for an assessment of biological events within the natural environment of living cells in real time. Due to the high sensitivity and high spatial resolution of fluorescence microscopy, fluorescent

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substrates in biologically relevant amounts can be followed throughout the cell. Traditionally, according to Abbe’s diffraction limit, the resolution of microscopy is limited by the wavelength of light and is practically limited to 200 nm to 500 nm. With the development of super resolution microscopy, resolutions between 20 nm to 70 nm have been achieved,(Hell, 2015) which is believed to be roughly the size of membrane microdomains.(Eggeling et al., 2009).

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Not all fluorescent probes provide the same amount of information. The probably first example of a fluorescent substrate of sphingolipid metabolism is NBD-ceramide 1 (Figure 1). In cells, NBD-ceramide was reported to be a Golgi marker.(Lipsky and Pagano, 1985) It later became clear that the compound was apparently rapidly metabolized to sphingomyelin by sphingomyelin synthases. Since metabolism does not change the fluorescent properties of this and related probes per se, the reaction products must be quantified due to their relative fluorescence intensities after

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separation by thin layer chromatography (TLC) or by high performance liquid

chromatography (HPLC), respectively. Moreover, the labelling of lipids with fluorescent

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dyes is likely to change their biophysical properties, transport and metabolism.

Although NBD is rather small, it is rather polar and some reports have suggested that the dye upon incorporation into longer lipid chains can indeed induce loop formation etc.(van Meer and Liskamp, 2005) To circumvent this kind of non-natural behavior, a

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group of polyene lipids like the sphingomyelin derivative 2 has been developed. These

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lipids comprise five conjugated (mostly E-configured) double bonds in otherwise unmodified acyl chains and allow for cellular localization studies using two-photon

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excitation (2PE) microscopy (Kuerschner et al., 2005; Kuerschner and Thiele, 2014;

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Nieves et al., 2015).

Figure 1: Different sphingolipids derivatives used for cell-based imaging.

“Clickable” sphingolipids as more sophisticated versions of such reporters have been reported recently (Gaebler et al., 2013). In one prominent study, sphingosine derivative 3 (Figure 1) with a terminal alkyne group has been incubated with cells and 3

metabolized to various more complex sphingolipids.(Haberkant et al., 2016) After different time intervals, the probe can be reacted with an azide-version of a fluorescent dye in a highly selective click reaction in situ and the localization of the metabolites can be identified by microscopy. After cell lysis, the chemical nature of the metabolites can be assessed and in principle also be correlated to the previously reported distribution within a cell, which allows linking SL metabolism with topological information. The most interesting feature of the above probe however was its diazirine group that allows light

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induced cross-linking and thus identification of novel lipid binding proteins.(Haberkant et al., 2016; Hoglinger et al., 2017) Since the reaction of alkynes with azides requires

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significant and toxic amounts of copper ions (Copper(I)-catalyzed azide-alkyne cycloaddition, CuAAC),(Kolb et al., 2001) fixation of the cells prior to staining is necessary. Therefore, if selective staining of a precursor and its downstream

metabolites is the primary focus, the use of azide-labeled precursors like the

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sphingosine derivative 4 is suggested (Figure 1).(Collenburg et al., 2016) For this type

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of label, a number of biorthogonal click reactions that do not rely on copper catalysis is available.(Baskin et al., 2007) Bioorthogonal ligations in principle allow for specific

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labeling of suitably labeled metabolites in live cells. However, in many cases, the

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excess dye has to be removed in order to have sufficient contrast, which limits the applicability of such approaches.(Lapinsky and Johnson, 2015) A highly interesting

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combination of biorthogonal ligation with super-resolution microscopy was recently reported by Erdmann et al who used the ‘’tetrazine-clickable’’ ceramide probe (Cer-

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TCO) 5 (Figure 1) to visualize Golgi structure and dynamics in living cells (Erdmann et al., 2014). A more detailed review on the use of sphingolipid derivatives in ligation

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chemistries can be found in this issue (Izquierdo and Delgado, 2018). 3. Activity-based probes

Activity based probes (ABPs) are irreversible inhibitors of a given enzyme that

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covalently react with the enzyme of interest, thereby providing it with a label suitable for quantitation. In an ideal case, the number of labeled enzyme molecules is equal to its active fraction. With this technique, enzymes like glucocerebrosidase (GBA) (Witte et al., 2010) or the acid ceramidase (Ouairy et al., 2015) have been labeled in situ with fluorescent dyes. (Izquierdo and Delgado, 2018)

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Figure 2: Activity-based probe 6 and mode of action (right side): the probe covalently reacts with

glucocerebrosidase (GBA) involving general acid-base catalysis and a nucleophilc attack. Only active enzyme is stained. Enzymes that are dysfunctional due to mislocation are not stained.

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The activity-based in situ labeling is especially interesting in mild forms of

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sphingolipidoses, in which partially folded fractions of an enzyme do not reach the

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lysosomes. Such mislocated fractions of an enzyme might appear as active when provided with an appropriate buffer after cell lysis.(Witte et al., 2010) Noteworthy, the

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ABP 6 has been used to stain GBA in live cells, as monitored by time-lapse microscopy (Figure 2). In this example, an active separation of the unreacted probe from the

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labeled protein was apparently not necessary, probably due to its significant enrichment with the enzyme in the lysosomes. Although ABPs appear similar to

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quenched-fluorescence probes, it is necessary to underline that not more than one equivalent of probe molecule can be “metabolized” by the enzyme of interest.

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Therefore the scope of such probes usually differs from substrates allowing for multiple turnovers.

4. Probes changing fluorescent properties during metabolism

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A key prerequisite for monitoring of sphingolipid metabolism in living cells is the development of a homogeneous assay which does not require a physical separation of substrate and product. Some homogeneous assays, however, require the addition of toxic reagents, which restricts their use in living cells. Examples from the sphingolipid field include HMU-PC 7, which is a substrate for acid sphingomyelinase and requires pH of 9 to guarantee efficient deprotonation of the coumarine dye, which is liberated upon cleavage of the probe (Figure 3) (van Diggelen et al., 2005). Further examples 5

include the ceramidase probe 8 and the sphingosine-1-phosphate lyase probe 9 developed by the Fabrias group, which require the addition of hydroxide and peroxide for liberation of a highly fluorescent coumarine dye (Figure 3).(Bedia et al., 2010; Bedia et al., 2007; Sanllehi et al., 2017) Such probes are useful for high throughput assays or even cellular assay, but only after fixation with an end-point determination of

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substrate cleavage.

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Figure 3: The probes are first cleaved by either acid sphingomyelinase (7), ceramidases (8) or sphingosine-1-phosphate lyase (9), respectively. After addition of further reagents, highly fluorescent

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coumarine derivatives are formed.

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4. 1 Quenched probes

Quenched fluorescent probes are the prototype of turn-on probes. Usually, doubly labeled substrates are provided with a fluorescent dye whose fluorescence energy is

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transferred to another non-fluorescent dye, either by FRET or by collisional quenching. A cleavage reaction separating the quencher from the fluorescent dye is thus yielding an increase in fluorescence (Figure 4). An early example from the sphingolipid field is

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the quenched ceramide analogue 10 that was used for real-time monitoring of ceramidase activity in vitro.(Nieuwenhuizen et al., 2002). More recently, a very efficiently quenched glucocerebrosidase (GBA) probe 11 has been described. Due to the high quenching efficiency of ~99.9%, the probe could be utilized in live cells for imaging of GBA and for differentiation of wildtype cells from Morbus Gaucher cells depleted in GBA activity.(Yadav et al., 2015) 6

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Figure 4: Quenched probes 10 and 11 for assaying ceramidase and glucocerebrosidase activities,

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respectively. Probe 11 was used to monitor GBA activity in living cells.

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4.2 FRET substrates for ratio imaging in live cells

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In contrast to probes requiring an end-point treatment, FRET substrates change their fluorescent properties in the course of biological events and therefore do not need

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separation methods to indicate the process of interest. In contrast to simple quenched probes, the possibility of ratio imaging is provided, which facilitates the quantitation of

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the process of interest (Figure 5).

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cell culture dish

pulse/chase incubation

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NH2

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2) wash*

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Figure 5: Practical implications of the usage of different sphingolipid derivatives for cell-based imaging. A Direct incubation of cells with fluorescent lipids B Application of „clickable“ lipids. After pulse-chase

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incubation of the cells, the label is incorporated by different click chemistries. Usually, fixation and washing of the cells is necessary. C ABPs are incubated with cells for a certain time. Usually the unreacted probe is washed out. D FRET probes are incubated with the cells and can be used for live

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cell imaging without prior treatment. d: FRET donor (only visible when probe is cleaved) a: FRET acceptor (only visible when probe is intact).

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* = In some cases, washing is not necessary

FRET is the radiation free transfer of energy from one fluorescent dye to another. The main prerequisites for this transfer is the spectral overlap of the emission spectra of the donor dye with the excitation spectra of the acceptor dye and the spatial proximity of these dyes. Once a FRET pair has been defined, the first dye (FRET donor) with the shorter excitation wavelength is exited. If the FRET efficiency is high enough, the donor dye will transfer all its emission energy to the FRET acceptor, which is then excited 8

and emits fluorescent light at a wavelength higher than the emission wavelength of the donor dye. Therefore, in an ideal FRET pair, the FRET donor is excited, but only the FRET acceptor is emitting fluorescent light. In a setup in which the enzyme or process of interest will form a FRET pair or destroy an existing FRET setup, the change in fluorescence will affect both dyes in way that the emission of one color is increased, while the fluorescence intensity in the second color is decreasing. Since both effects are contrariwise, they both contribute to the responsiveness and thus to the sensitivity

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of the probe. Furthermore, the ratio of both fluorescence intensities is not dependent on the absolute but only of the relative concentrations of the intact FRET system on

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the one side and the free independent dyes on the other side. The latter feature also

known as “ratio-imaging” is a significant advantage over quenched probes or turn-on probes, in which the observed absolute fluorescence intensity is dependent on many factors like cellular delivery or export and thus not indicative of the cleavage (or

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formation) ratio (Figure 6). product

product

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t=0 wavelength / nm

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product

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t = end

intensity / AU

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wavelength / nm

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Figure 6: Illustration of the concept of ratio imaging. A Time response of a turn-on probe, often allowing only qualitative information B Increase in product fluorescence with a concomitant decrease in substrate florescence indicates the relative cleavage rate of probe molecules C Time response of a

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typical FRET probe cleavage, providing relative cleavage rates and thus quantitative information.

Some impressing examples of FRET pair formation exist in the field of small analyte determination like Ca2+, which can be sensed by a tandem fusion protein of calmodulin

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with two different fluorescent proteins.(Miyawaki et al., 1997) Small molecule sensors have been described for reactive specimen like cellular thiols, as exemplified in probe 12.(Yi et al., 2009). Examples of small molecule FRET substrates are rare. The first examples include probe 13 for indicating -lactamase activity (Zlokarnik et al., 1998) or probe 14 for monitoring phosphodiesterase activity.(Takakusa et al., 2002) The first example in the lipid field has been described by Wichmann et al, who synthesized a probe 15 for phospholipase A2 (PLA2).(Wichmann and Schultz, 2001) The molecule 9

mimicking phosphatidyl choline contained a NBD dye as FRET donor in the acyl portion, while a Nile Red dye was used as a FRET acceptor in the second lipid tail. This probe showed an impressive 70-fold FRET response upon cleavage and was used to monitor PLA2 activity in live cells.(Wichmann et al., 2006) The specificity of the probe for PLA2 was ensured by replacing the SN1 ester bond with an inert ether

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linkage, making cleavage of the probe by PLA1 impossible (Figure 7).

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4.3 Sphingolipid FRET probes 4.3.1 Ceramidase substrates 11

Recently we have developed several FRET-probes in order to monitor sphingolipid metabolism in live cells (Figure 8). Our motivation was to provide probes for monitoring sphingolipid-mediated signaling, catalyzed by sphingomyelinases and ceramidases, respectively. The ceramidases are important for cell fate and participate in suppression of cell death pathways.(Canals et al., 2011) Especially the acid ceramidase has been reported to be elevated in malignant disease like prostate cancers.(Camacho et al.,

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2013)

Figure 8: Sphingolipid derivatives as FRET substrates. Only 19 was successfully exploited to monitor

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enzyme activity in living cells.

Due to the less complicated synthesis, FRET substrates for ceramidases were the first

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target. The use of the NBD/Nile Red FRET pair was envisioned, due to its proven applicability for a lipid metabolizing enzyme.(Wichmann et al., 2006) In a preliminary work, a set of Nile Red- and NBD-labeled ceramides were synthesized and tested as

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potential substrates for ceramidases.(Bhabak et al., 2012) Since all modified ceramides were cleaved by neutral and acid ceramidases, the two doubly labeled ceramides 16 and 17 as potential FRET substrates for acid and neutral ceramidases were synthesized (Figure 8).(Bhabak et al., 2013) Evaluation of the excitation and emission spectra of the probe suggested a high FRET efficiency, but one probe was hardly cleaved at all, while the other probe was cleaved very slowly and a ~7.5-fold ration change upon cleavage was observed, which referred to a cleavage rate of about 12

15%. The reason for slow cleavage is most likely the bulky and hydrophobic Nile Red dye, which led to low Km, but also low kcat values, respectively. Incubation of HeLa cells with this probe led to an accumulation of the uncleaved probe in the Golgi apparatus, as observed previously for NBD-ceramide. This result suggests that transport of the substrate to the Golgi apparatus, where no significant ceramidase activity is expected, is faster than the cleavage of the probe within the endolysosomal compartment. Whether in the probes after localization to the Golgi were converted to

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sphingomyelin derivatives, has not been examined. Notably, the probes were delivered

as BSA complexes to make an endocytotic uptake more likely. Future efforts should

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be directed to synthesize ceramidase FRET substrates that are cleaved faster, provide a higher responsiveness and are delivered in a way that efficient lysosomal cleavage is facilitated.

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4.3.2 Sphingomyelinase substrates

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The acid sphingomyelinase is an enzyme involved in key events of sphingolipid signaling. Sphingomyelin is a very abundant constituent of mammalian plasma

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membranes and can be cleaved in one step to the apoptosis-inducing ceramide.

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Besides the constitutive lysosomal activity of the enzyme, an secreted version of the enzyme has been reported, but its activity under neutral conditions is questionable.

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Therefore, the existence of microenvironments of acidic pH at the outer surface of the plasma membrane has been discussed.(Kornhuber et al., 2015) Altered levels of acid sphingomyelinase activity are strongly associated with different types of inflammatory

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(Canals et al., 2011; Goggel et al., 2004) and malignant diseases.(Carpinteiro et al.,

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2015; Petersen et al., 2013)

To further investigate such processes, an acid sphingomyelinase FRET-probe was designed, using the previously described NBD/Nile Red pair. In contrast to ceramide however, where both dyes inherently had to be incorporated into both lipid tails, the

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choline head was modified with the relatively polar NBD and the membrane-resident part with Nile Red, respectively. Although the resulting sphingomyelin analogue 18 was readily cleaved by recombinant human acid sphingomyelinase (recASM), only a low ratio change was observed.(Pinkert et al., 2017) While the acceptor Nile Red fluorescence declined by a factor of 3 fold, a concomitant change in donor NBD fluorescence was not observed (Figure 9A), which was obviously due to the quenching of NBD fluorescence in aqueous environment. To make use of the NBD quenching 13

effect after probe cleavage, a second probe was synthesized in which NBD was the FRET acceptor. For this setup, a coumarine dye was identified as a suitable FRET donor. Indeed, upon cleavage of probe 19, an increase in coumarine fluorescence was accompanied by a very effective decline of the NBD fluorescence (Figure 9B) yielding in an about 80fold ratio change.(Pinkert et al., 2017) Before its application in cell-based assays, the probe was thoroughly tested. The probe allowed differentiation of different recASM concentrations over 3 orders of magnitude and incubation with different

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dose-dependent inhibition of the FRET change (Figure 9C).

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inhibitors of acid sphingomyelinase (Roth et al., 2009a; Roth et al., 2009b) showed a

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Figure 9: A fluorescence change of probe 18 upon incubation with recASM. The expected increase in NBD fluorescence at 550 nm did not occur. B fluorescence change of probe 19 in presence of recASM C NBD fluorescence of 19 in presence of recASM and different inhibitor concentrations.

In cell lysates from different cell types, 19 was rapidly cleaved at acidic pH, while cell

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lysates at pH 7.4 as well as lysates from ASM knock-out mouse embryonic fibroblasts (MEFs) at any pH did not induce a change in fluorescence.(Pinkert et al., 2017) Also, incubation with enriched neutral sphingomyelinase did not affect the FRET pair, suggesting that 19 is no substrate for this enzyme (unpublished data). After in vitro characterization, the FRET substrate 19 was converted into a BSA complex and its cleavage was monitored in different cell types. Since the short 14

excitation wavelength of the coumarine at 355 nm is not compatible with an unbiased observation of FRET probe metabolism, a two-photon excitation (2PE) microscope had to be used. Indeed 2PE excitation at 720 nm yielded as expected in a strong signal at the emission maximum for NBD at 550 nm, which was also observed, when the NBD was excited directly at 466 nm. All experiments showed an apparent probe cleavage, for at least 48 h.(Pinkert et al., 2017) Notably, also the ASM knock-out MEFs showed a slow background reaction, of unknown origin.(Pinkert et al., 2017) One explanation

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could be a reverse sphingomyelin synthase activity that has been reported

previously.(Huitema et al., 2004; van Helvoort et al., 1994) Overexpression of ASM in

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wildtype MEFs however significantly increased FRET change, showing that different ASM levels can be differentiated under live cell conditions. A similar experiment was

performed in human neuroglioma cells overexpressing an ASM-CherryN1 fusion protein (Figure 10, unpublished data). Also in this example, a faster conversion of the

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sphingomyelin substrate 19 into its ceramide cleavage product was observed, as

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anticipated by a markedly changed ratio of green/red fluorescence intensities. A partial co-localization of substrate, product and ASM-CherryN1 fusion protein suggests the

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topology of ASM activity and resembles previously published results (Pinkert et al.,

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2017).

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Figure 10. FRET substrate 19 in Human H4 neuroglioma cells with and without ASM over expression (ASM OE). ASM-cherry (left panel) or control vector plasmid DNA coding for CherryN1 only (right panel) were expressed for 24 hrs before BSA-complexed FRET probe 19 was added to the media for 2 hrs at a final concentration of 1 µM. Green: uncleaved probe. Red: cleaved probe (ceramide) Blue: cherry-tag (ASM in left panel). Scales indicate 10 µm.

The identity of cleavage product was confirmed after extraction of treated cells with organic solvent and subsequent HPLC analysis. HPLC also confirmed the increased product formation in cells overexpressing ASM. (Pinkert et al., 2017) 16

5. Outlook and further developments 5.1 Sphingomyelinase FRET probes The sphingomyelin analogue 19 is to our knowledge the first FRET substrate allowing for real time monitoring of a sphingolipid metabolizing enzyme in live cells. Nevertheless, this substrate still provides many possibilities for further improvement,

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which is discussed here as to underline the tremendous potential of this approach. Although the substrate shows a high responsiveness, its sensitivity or brightness can

very likely be optimized by incorporation of coumarines being especially sensitive for

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two photon excitation,(Wessig et al., 2016) as shown by Roger Tsien’s group.(Furuta et al., 1999)

Probe 19, similarly to natural sphingomyelin, is a charged molecule unable to permeate

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through membranes. Cellular entry proceeds very likely via endocytosis, which suggests that the majority of the probe is cleaved within the acid compartments of the

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cell. Therefore, it is very unlikely that intact probe molecules can reach other topologies

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within the cell, like Golgi apparatus or the inner leaflet of the plasma membrane.

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Addressing additional topologies within cells would require different delivery strategies or even structurally modified probe molecules.

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For sensing intracellular sphingomyelinase activities with non-lysosomal localization, membrane permeable and thus more lipophilic probes would be necessary. This might

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be achieved by transient masking of the negatively charged phosphodiester group in form of biologically labile phosphotriesters, as shown by Schultz et al for some

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phosphatidyl inositols (Mentel et al., 2011). As mentioned above, substrate 19 is no substrate for neutral sphingomyelinase 2. Though, minor structural modification of the substrate may change the nature of enzyme activities that can be monitored. Sphingomyelin derivatives displaying an

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analogue of the natural choline ammonia salt, should mimic the natural substrate much better. Such derivatives should be metabolized much faster (Gaudino et al., 1997; Sandbhor et al., 2009) and could even be substrates of the neutral sphingomyelinases. Finally, we are currently working on FRET substrates based on other fluorescent dyes working in the visible range of light which would make the use of 2PE microscopy dispensable. 17

5.2 Future sphingolipid FRET probes FRET probes rely on efficient transfer of energy between two fluorophores that are in closest proximity. Theoretically, biosynthetic enzymes like sphingomyelin synthase or glucosylceramide synthase could be used to build up a FRET system. Practically, such an approach would be hard to realize in vivo or even in vitro since its efficiency would be minimized by the presence of unlabeled substrates, as only doubly labeled reaction

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products can function as a FRET pair. In contrast, a change of an existing FRET signal can be much easier achieved by separation of two fluorophores of a pre-formed FRET system, by a catabolic enzyme like acid sphingomyelinase or glucocerebrosidase.

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Therefore, future sphingolipid FRET substrates will most likely be used to monitor enzymes involved sphingolipid degradation. Modified glycosphingolipids could be

synthesized to monitor respective lysosomal glycosphingolipid hydrolases. Further

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probes to monitor sphingomyelinases and ceramidases would be most worthwhile as these enzymes are often triggered by transient signals and stimuli (Hannun and Obeid,

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2018). Also, FRET substrates for sphingosine-1-phosphate lyases or phosphatases

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would be highly desirable. A direct FRET substrate these enzymes however would

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essentially need labeling of the phosphate, which would turn sphingosin-1-phosphate’s phosphomonoester into a phosphodiester. Whether such a molecule would be

6. Conclusion

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efficiently cleaved by one of the named enzymes is questionable.

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The recently developed ASM FRET substrate 19 is the first probe for a real-time measurement of sphingolipid metabolism in live cells. The specificity of the reaction

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was shown as well as its principle usefulness for a quantitative assessment of ASM activity. Work is in progress to further modify and optimize the probe. It will be interesting to use the probe for tackling key questions in the field, such as where the ASM activity relevant for lipid signaling events is located. Apart from its delivery as a

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BSA complex or in DMSO, methods to selectively label the plasma membrane with fluorescent sphingomyelin have recently been reported (Li et al., 2016) and might be amenable for the ASM FRET substrate or its future version as well. Whether the concept shown for the acid sphingomyelinase can be successfully transferred to further enzymes involved in sphingolipid metabolism is still an open question. While in vitro FRET assays are comparatively easy-to-achieve, a probe for applications in living cells depends on many different factors, including cellular uptake 18

and trafficking. Certainly, the FRET approach in the field of sphingolipids has not been fully exploited yet. 7. Experimental Human H4 neuroglioma cells were transfected with 1 µg of ASM-cherry or control vector plasmid DNA using X-tremeGENE DNA transfection reagent from Roche. (Newcomb et al., 2018; Rhein et al., 2014) After 24 hrs, the BSA-complexed FRET

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probe 19 was added to the media for 2 hrs at a final concentration of 1 µM. ASM

overexpression increased ASM activity 5-fold over control as measured in a parallel

experiment using thin layer chromatography.(Muhle and Kornhuber, 2017) Cells were

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washed with PBS and live cell microscopy was performed on a Zeiss LSM 880 NLO

equipped with a 680-1300 nm tunable and fixed 1040 nm 2-photon laser from Newport SpectraPhysics and the ZEN 2010 software (Carl Zeiss MicroImaging). Two-photon

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excitation at 730 nm resulted in the detection of the uncleaved probe that indicates

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sphingomyelin (NBD signal, green) and the cleaved probe that indicates ceramide

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tag that indicates ASM overexpression.

gratefully

acknowledge

funding

by

the

Deutsche

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authors

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Acknowledgements

The

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(MCC signal, red). Parallel excitation at 1040 nm resulted in the detection of the cherry-

Forschungsgemeinschaft, DFG (AR-376/12-2 to C.A.). Z.H.M is grateful for a

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fellowship provided by the SALSA graduate school (DFG excellency programme). The authors would like to thank Juliana Monti for excellent technical assistance. We thank Philipp Tripal and Benjamin Schmid, Optical Imaging Centre Erlangen, FriedrichAlexander Universität Erlangen-Nürnberg, Erlangen, Germany, for excellent technical

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support in imaging.

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References

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