Activity based subcellular resolution imaging of lipases

Article abstract of DOI:10.1016/j.bmc.2011.04.018

Lipases play a key role in whole body energy homeostasis. Dysregulation of lipolytic activities affects lipid absorption, mobilization, and transport, and is causative for lipid-related diseases. Regulation of enzymes involved in lipid metabolism is gover

Full text of DOI:10.1016/j.bmc.2011.04.018

Bioorganic & Medicinal Chemistry 20 (2012) 628–632
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Activity based subcellular resolution imaging of lipases
⇑
Martin Viertler , Matthias Schittmayer , Ruth Birner-Gruenberger
Proteomics Core Facility, Center for Medical Research and Institute of Pathology, Medical University of Graz, Stiftingtalstrasse 24, A-8010 Graz, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 19 April 2011
Lipases play a key role in whole body energy homeostasis. Dysregulation of lipolytic activities affects lipid
absorption, mobilization, and transport, and is causative for lipid-related diseases. Regulation of enzymes
involved in lipid metabolism is governed by a complex network of protein–protein and protein–small
molecule interactions. Thus these enzymes have to be studied under the physiologically most relevant
conditions, that is, in vivo. Our latest generation of activity based probes designed for capturing of lipases
employs bioorthogonal chemical linker groups, which are membrane permeable and thus allow studying
protein activity in living cells. Another advantage is the virtually unlimited choice of reporter tags. Here
we report on a novel method combining in vivo activity based labeling of lipases with in situ detection of
lipolytic activities by on slide click chemistry and imaging by fluorescence microscopy. We demonstrate
that cytosolic as well as organelle resident lipases are specifically labeled in intact living cells. This
method will shed light on the (sub)cellular localization of lipolytic proteomes of cells and tissues in
health and disease directly at enzymatic activity level without the need of prior knowledge of the iden-
tities of the responsible enzymes or dependence on the availability of specific antibodies.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Activity based proteomics
On-slide click chemistry
Activity based cell imaging
Subcellular lipase localization
1. Introduction
bioorthogonal linker group.4,5 These groups are small and biologi-
cally inert yet they can be targeted with high specificity in a sub-
Lipases are key players in whole body energy homeostasis.1
Control of lipolysis of triacylglycerols and cholesteryl esters stored
in lipid droplets of adipose tissue plays an important role in energy
partitioning and balance and maintains the size of fat depots in the
body. Dysregulation of these lipolytic activities affects lipid absorp-
tion, mobilization and transport, and is causative for lipid-related
diseases. As many enzymes, lipases are strongly regulated at the
posttranslational level.
Activity based probes (ABP) are ideal tools to observe changes in
enzyme activities, providing a readout of enzymatic activity rather
than mere protein abundance. These probes typically consist of a
reactive group, a binding group and a reporter tag. While the reac-
tive group covalently binds the probe to the target protein based
on the target’s enzymatic mechanism, the binding group ensures
the probe is recognized by the target protein as a potential sub-
strate in the first place. The reporter tag can either be an affinity
tag,2 a fluorophore,2,3 or, in the latest generation of ABP, a so called
sequent step, for example by a click chemistry reaction. Major
advantages of this approach are the superior membrane perme-
ability of the probes and no adverse effect of the reporter tag on
probe recognition, both frequently observed for fluorophore carry-
ing ABP.6 One possibility to couple the bioorthogonal linker group
and the reporter tag is copper catalyzed azide alkyne [3+2]
cycloaddition (CuAAC).7 In recent years several click chemistry
reactions, including Staudinger ligation,8 strain promoted azide
alkyne cycloaddition9,10 and CuAAC11 have been successfully em-
ployed to visualize small molecules8,10,12–14 as well as proteins15,16
in metabolic labeling experiments.
While the lower abundance of proteins as compared to many
small molecules used for metabolic labeling is an issue, several
possible solutions for this problem have been established. First,
the intrinsic signal amplification capability of enzymes can be
exploited by using fluorogenic substrates to visualize enzymatic
activity.17,18 However, the released fluorescent products are often
soluble and limit spatial resolution to the cellular level, with cell
membranes acting as natural diffusion barrier. Another approach
by Blum et al.19 relied on reducing background by designing an
ABP carrying quenchers next to the fluorescent tag, which were re-
leased upon binding to the target enzymes. This approach was suc-
cessfully used for whole body imaging of mice.20 Recently, Yang
et al.21 employed alkyne modified versions of the antiobesity drug
Orlistat to identify potential side targets of the drug and also tested
the applicability of these probes for imaging applications. While
Abbreviations: ABP, activity based probe; ACN, acetonitrile; ATGL, adipose
triglyceride lipase; CuAAC, copper (I) catalyzed azide alkyne [3+2] cycloaddition;
DIPEA, diisopropylethylamine; FA, formic acid; HSL, hormone sensitive lipase; MGL,
monoglyceride lipase; TGH1, triacylglycerol hydrolase.
⇑
Corresponding author. Tel.: +43 31638572962; fax: +43 31638573009.
E-mail address: ruth.birner-gruenberger@medunigraz.at (R. Birner-Gruenber-
ger).
These authors contributed equally to the study and should therefore be regarded
as co-first authors.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.018

M. Viertler et al. / Bioorg. Med. Chem. 20 (2012) 628–632
629
they succeeded to label the major Orlistat target, namely fatty acid
synthase, as well as several other proteins in gel based experi-
ments, fluorescence microscopy experiments showed an ER local-
ized activity based stain, in contrast to the cytosolic stain
observed for immuno-histochemical detection of fatty acid
synthase.
Here we report the application of activity based click probes for
in vivo labeling of lipases with on slide click chemistry and confo-
cal fluorescence microscopy to realize in situ visualization of lipo-
lytic activities in the cell (Fig. 1). Since one of the major
mechanisms regulating lipolytic activity is localization of lipases
to or from lipid droplets, we followed published fixation proce-
dures optimized to ensure lipid droplet structural integrity.22
Moreover, we established a quadruple staining method to allow
staining of nuclei and lipid droplets, as well as protein immunode-
tection next to activity based labeling of the lipolytic activities.
Proteins tagged included the known intracellular lipases in-
volved in mobilization of intracellular triacylglycerols from lipid
droplets, namely adipose triglyceride lipase (ATGL),23 hormone
sensitive lipase (HSL)24 and monoglyceride lipase (MGL),25 as well
as the ER luminal triacylglycerol hydrolase 1 (TGH1).26
Scheme 1. Synthesis of activity based click probe. 5-hexyn-1-ol diluted (2) was
added drop-wise to hexylphosphonic acid dichloride (1). Subsequently, intermedi-
ate product (3) was reacted with 4-nitrophenyl (4) to yield hexynyl-4-nitrophenyl-
hexylphosphonate (5).
to fatty acid free bovine serum albumin (PAA laboratories, Pas-
ching, Austria) in a 3:1 molar ratio, as indicated in the text. For
4-nitrophenyl butyrate hydrolase assays, transfection was per-
2. Material and methods
formed with 6 l
g DNA in 25 cm2 cell culture flasks.
If not stated otherwise, reagents were purchased from
Sigma–Aldrich, Vienna, Austria.
2.3. Synthesis of activity based click probe hexynyl-4-
nitrophenyl-hexylphosphonate (5) (Scheme 1)
2.1. Cell culture
Two millimolars of hexylphosphonic acid dichloride (1) was
dissolved in a mixture of 5 mL absolute CH2Cl2, 10 mmol diisopro-
pylethylamine (DIPEA) and 0.2 mmol tetrazole (0.45 M in acetoni-
trile (ACN)). Two millimolars of 5-hexyn-1-ol diluted (2) in 20 mL
absolute CH2Cl2 was added drop-wise to the stirred solution. After
complete addition, the resulting mixture was stirred another 4 h at
room temperature. Reaction was monitored by TLC. After comple-
tion, 5 mmol 4-nitrophenyl (4) and 5 mmol DIPEA was added to
the product (3), the reaction was stirred for 16 h at room temper-
ature. Reaction was monitored by TLC and after completion prod-
uct (5) was confirmed by LC–MS (reverse phase C18; H2O/
ACN + 0.1% formic acid (FA); linear gradient from 0 min 40% ACN
to 10 min 100% ACN, positive full scan MS m/z 150–1000). All vol-
atile components were removed under reduced pressure on the ro-
tary evaporator. The residue was dissolved in 10 mL CH2Cl2 and
washed twice with 1 M aqueous K2CO3 (20 mL), twice with water
(20 mL) and once with brine (20 mL) before drying over sodium
sulfate. Sodium sulfate was removed by filtration and volatiles
were removed under reduced pressure on the rotary evaporator.
Residue was dissolved in 5 mL of H2O/ACN (60:40) and purified
by HPLC (reverse phase C18; H2O/ACN + 0.1% FA; linear gradient
All cell culture reagents were purchased from PAA laboratories,
Pasching, Austria. African green monkey kidney cells (COS-7; ATCC
Number CRL-1651) were propagated in full media (Dulbecco’s
Modified Eagle’s Medium (DMEM); 10% fetal bovine serum; peni-
cillin/streptomycin) at 37 °C in a humidified atmosphere, 5% CO2.
2.2. Transfection
N-Terminal HIS-tag fusion constructs of beta-galactosidase
(LacZ), murine hormone sensitive lipase (HSL), adipose triglyceride
lipase (ATGL) and monoglyceride lipase (MGL)27,28 as well as the
N-terminal GFP fusion construct of MGL29 were kindly provided
by Rudolf Zechner, University of Graz, Austria. The TGH1-eGFP30
fusion construct used in this study was a kind gift of Richard Leh-
ner, University of Alberta, Canada. 5 Â 104 COS-7 cells were seeded
on chamber slides (BD Biosciences, Vienna, Austria) the day before
transfection. Turbofectene™ in vitro reagent (Fermentas, St. Leon-
Rot, Germany) was used to transfect 0.5 lg of plasmid DNA per
chamber according to the manufacturers instructions. Stains were
performed 48 h post transfection. HIS-MGL and GFP-MGL transfec-
ted cells were loaded overnight with 150 lM oleic acid complexed
Figure 1. Workflow for activity based imaging of lipases. A schematic overview on the procedure is given. First activity based labeling of lipases is achieved in living cells.
Before click chemistry is applied on the specimen, the cells are fixed and permeabilized to achieve efficient translocation of click components into the cells.

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M. Viertler et al. / Bioorg. Med. Chem. 20 (2012) 628–632
from 0 min 40% ACN to 10 min 100% ACN). Fractions containing the
click probe were dried and the probe was subjected to LC–MS and
NMR analysis. m/z = 368.3 (expected [M+H]+ = 368.4); 1H NMR
(CDCl3): d 8.23 (m, 2H), 7.35 (apparent d, 2H), 4.13 (t, 2H,
J = 8 Hz), 2.20 (m, 1H), 2.10 (apparent s, 4H), 1.95–2.04 (m, 2H),
1.39–1.83 (m, 4H), 1.26 (apparent t, 6H), 0.87 (t, 3H, J = 7 Hz). 13C
NMR (CDCl3): d 155.3, 141.3, 126.5, 126.1, 121.4, 83.6, 69.4, 61.1,
31.4, 30.3, 29.5, 24.6, 22.5, 22.3, 21.4, 18.1, 14.3, 14.2.
based stains, Dylight647/GFP signals and counterstains were per-
formed in separate acquisition tracks. Montages of images were
prepared in ImageJ33 and contrast and brightness were adjusted.
3. Results
3.1. Labeling with activity based probe
We transiently expressed the major cytosolic lipases (i.e., ATGL,
HSL, and MGL) fused to a HIS-tag in COS-7 cells. This cell line was
previously reported to show very low intrinsic lipase activity28 and
was therefore ideally suited to verify the specificity of our method.
To determine the degree of labeling of the ABP, we employed a
4-nitrophenyl butyrate hydrolase assay.31 Cells expressing
HIS-MGL incubated for 2 h with serum free media containing
2.4. Labeling of cells in culture
Stocks of click probe were dissolved in dichloromethane, di-
vided into aliquots of 30 or 100 nmol, dried in vacuo and stored
at À20 °C until use. Full media within chambers was exchanged
twice for phosphate buffered saline (PBS) to remove serum compo-
nents 48 h after transfection. Dried probe aliquots were taken up in
30 lM click probe showed no detectable residual lipolytic activity,
indicating a quantitative inhibition of lipases, while a control reac-
tion without probe showed an activity of 37 nmol minÀ1 mgÀ1 cell
lysate (Supplementary Fig. 1).
10
l
l DMSO and added to 1 mL DMEM containing Penicillin/Strep-
tomycin to a final concentration of 30 or 100
l
M (as indicated in
the text). Next PBS was exchanged for the labeling mix and cells
were incubated for 2 h (37 °C; humidified atmosphere; 5% CO2).
Efficiency of labeling was assessed by subjecting cell lysates to
4-nitrophenyl butyrate esterase activity assays as described by
Lehner and Verger.31
3.2. Activity based imaging of lipases
COS-7 cells expressing HIS-tagged versions of ATGL, HSL, and
MGL were incubated with activity based click probe in culture.
After cell fixation Atto532-azide was coupled to the probe by
CuAAC. We were able to observe a clear cytosolic signal (Fig. 2,
panels A) corresponding to successfully transfected cells as con-
trolled by an anti-HIS antibody stain (Fig. 2, panels B). Moreover,
the expression patterns within the cell showed an almost perfect
overlap of the two complementary stains (Fig. 2, panels C). To rule
out background staining caused by either activity based probe or
subsequent CuAAC, COS-7 cells expressing a HIS-LacZ fusion con-
structs were used as a mock control. In this case, only a low back-
ground signal was observed and it did not correlate to HIS-LacZ
expression (Fig. 2).
2.5. In situ detection of lipases
Fixation was performed as described by DiDonato et al.22 with
3% formaldehyde in PBS for 10 min at room temperature. Following
fixation, cells were rinsed three times by incubation in PBS for
5 min in total.
For HIS-tagged lipases, the cells were blocked and permeabili-
zed in blocking buffer (PBS containing 1% fatty acid free bovine ser-
um albumin (PAA laboratories, Pasching, Austria) and 0.1% Tween
20 (Bio-Rad Laboratories, Vienna, Austria)) for 30 min at room tem-
perature. Next, a mouse anti HIS-Dylight647 antibody (Tebu-bio,
We also observed that non lipid loaded COS-7 cells only exhib-
ited little to no lipid droplets, as shown by a BODIPY495/505 coun-
terstain (Fig. 2 C, white). Accordingly, the general appearance of
HIS-tagged lipases in COS-7 cells lacked lipid droplet like struc-
tures. Therefore, we loaded COS-7 cells expressing HIS-MGL with
oleic acid. As expected, a BODIPY 495/505 stain resulted in lipid
droplet like structures, but no accumulation of fluorescent signal
in the vicinity of these structures was observed for either activity
based or antibody stain.
While the spatial overlap of the activity based and antibody
stains was very good from the start, one of our major concerns
was variation of the intracellular signal intensities between the
two stains (non-uniform color distribution of overlays, Fig. 2, pan-
els C). We speculated that these intensity differences might be
caused by partial incompatibility of the immuno-histochemical
detection with the CuAAC and therefore switched our experimen-
tal set up to GFP-tagged lipases.
Indeed, the activity based stain of COS-7 cells expressing YFP-
ATGL and GFP-MGL, respectively, did not only match the spatial
distribution of the GFP-signal but the two signals also exhibited
a highly uniform intensity distribution (Fig. 3A–C, Supplementary
Fig. 2). Interestingly, while we were unable to observe preferred
localization to lipid droplet like structures in oleate loaded cells
for HIS-tagged lipases, the GFP-tagged counterparts showed a
clearly increased signal intensity encircling these structures for
the activity based stain as well as the GFP-signal. A negative con-
trol CuAAC with unlabeled cells resulted in very low unspecific
background labeling (Supplementary Fig. 3).
Offenbach, Germany) diluted to 2 lg/mL in blocking buffer was
incubated for 1 h at room temperature. CuAAC was performed
using 0.1 mM tris-(benzyltriazolylmethyl)amine (TBTA)32 ligand,
5 mM copper(II)sulfate, 10 mM sodium ascorbate and 50
Atto532-azide (Atto-Tec, Siegen Germany). Nuclei were counter-
stained for 10 min with Hoechst 33422 (0.1 g/mL in PBS). Lipid
droplets were counterstained with BODIPY 495/505 (Invitrogen,
Vienna, Austria) (0.2 g/mL in PBS) for 10 min.
lM
l
l
For indirect labeling of GFP-MGL, the cells were permeabilized
with PBS containing 1% bovine serum albumin and 0.1% saponin
or 0.1% Tween 20 for 10 min. CuAAC coupling of biotin–azide
(Invitrogen, Vienna, Austria) to alkyne modified lipases was per-
formed using 0.5 mM bathocuproinsulfonate (BCS) as ligand,
2 mM copper(II)sulfate, 15 mM sodium ascorbate and 50 lM bio-
tin–azide (in 100 mM Tris–HCl, pH 8). CuAAC was performed for
2 h at 37 °C. Next, the slides were incubated with streptavidin–
Atto532 conjugate in blocking buffer for 1 h at room temperature.
For direct labeling of TGH-eGFP CuAAC reaction contained
0.5 mM BCS, 2 mM copper(II)sulfate, 15 mM sodium ascorbate
and 50
lM Atto532-azide and proceeded for 1 h at 37 °C. Cells
were counterstained with 0.1
l
g/mL Hoechst 33342 in PBS. In be-
tween all labeling steps, the slides were washed three times in PBS
for 5–10 min in total. In all experiments slides were mounted in
fluorescence mounting media (Dako, Vienna, Austria). Images were
acquired on a LSM510 Meta confocal laser scanning microscope
(Zeiss, Oberkochen, Germany) using a 63Â oil immersion objective
(NA 1.4). The following excitation laser wavelengths/emission
filter settings were used: 488 nm/505–530 nm band pass (GFP,
YFP, BODIPY); 543 nm/560–615 nm band pass (Atto532-azide,
Atto532–streptavidin, Rho6G-azide); 633/650 nm long pass
(HiLyte Fluor647, Atto647-azide). Image acquisition for activity
As biotin–streptavidin recognition serves as platform for a vari-
ety of signal amplification strategies in light and fluorescence
microscopy, we also tested the compatibility of our method to

M. Viertler et al. / Bioorg. Med. Chem. 20 (2012) 628–632
631
Figure 2. The activity based click probe detects cytosolic lipases in intact cells. COS-7 cells over-expressing HIS fusion constructs LacZ, HSL, ATGL, and MGL were labeled by
activity based probe (A) and by a Dylight647 coupled anti HIS antibody (B). As expected, all lipases were detected by the activity based click probe, while no specific
interaction of the probe with LacZ negative control was observed. In C, counterstains for nuclei (Hoechst 34422, blue false color) and lipid droplets (BODPIY 495/505, white)
are provided in an overlay. Only few endogenous lipid droplets were observed; to prove the feasibility of lipid droplet co-localization in our method MGL-HIS expressing cells
were loaded with oleic acid; scale-bars: 10 lm.
Figure 3. The GFP-MGL fusion construct is detected by both direct and indirect labeling methods. COS-7 cells over-expressing GFP-MGL29 (B) were labeled for lipase activity
as described in Sections 2.4 and 2.5; BCS was used as Cu(I) ligand. Activity based click probe GFP-MGL was either detected directly with Atto532-azide (A, upper panel), or
indirectly by biotin–azide coupling and subsequent streptavidin–Atto532 recognition (A, lower panel). Both detection methods correlate to the GFP-MGL (see overlay in C)
signal. In D a transmission image is provided to depict cell boundaries. In contrast to the HIS-MGL construct, localization patterns indicative of lipid droplets were readily
observed; scale-bar: 10 lm; z-plane thickness: <1 lm.
biotin–streptavidin coupling. As depicted in Fig. 3, biotin labeled
GFP-MGL was readily detected employing a streptavidin–Atto532
conjugate after CuAAC.
their native subcellular compartments, ensuring optimal condi-
tions for each individual target enzyme. Moreover, membrane per-
meability of ABP is
a major issue for in vivo applications.
After demonstrating that our ABP was able to enter intact cells
by successfully staining three lipases located in the cytosol, we
were interested if we would also be able to target lipases within
subcellular compartments. To address this question, we employed
a GFP-tagged TGH, which was reported to reside within the lumen
of the ER.26 Also in this case, we observed highly similar signal dis-
tribution for activity based stain and GFP-signal (Fig. 4).
Surprisingly bulky and charged substrates and inhibitors have been
successfully employed for in vivo experiments, especially for the
visualization of proteases.17–20 Unfortunately, the exact mecha-
nism of cellular uptake of these compounds remains poorly under-
stood in most cases and it is generally assumed that endocytosis
plays a major role in the process.17,18 Since the fate of triacylglyce-
rols and cholesteryl esters undergoing endocytosis is hydrolysis by
lysosomal acid lipase34 this pathway is barred for activity based
probes mimicking lipids, underlining the importance of membrane
permeable ABP in this context.
4. Discussion
Here we successfully labeled three major cytosolic lipases and
one ER residing lipase with ABP in living cells. Activity assays re-
vealed that probe concentrations as low as 30 lM were sufficient
A major advantage of ABP with bioorthogonal linkers is their
ability to readily diffuse across cellular membranes. Therefore,
the probes can be employed to label their target enzymes within

632
M. Viertler et al. / Bioorg. Med. Chem. 20 (2012) 628–632
Figure 4. The activity based probe labels lipases within the ER in intact cells. An ER resident TGH-eGFP fusion construct30 was over-expressed in COS-7 cells (B). The cells
were stained for lipase activity as described in Sections 2.4 and 2.5 (A). CuAAC was performed with BCS as Cu(I) ligand. Nuclei were counterstained with Hoechst 333422 and
depicted in a merge of channels (C). The activity based stain (shown in red) co-localizes with the TGH-eGFP signal (shown in green). Scale-bar: 10
m.
lm; z-plane thickness:
2
l
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biotin–azide and subsequently stained with
a streptavidin–
Atto532 conjugate was comparable to that of direct azide–Atto532
coupling and no extensive staining of endogenous biotinylated
proteins was observed.
Conclusively, we expect that this study will open up new ways
for diagnostics and therapeutic intervention of lipid-related dis-
eases by shedding light on the (sub)cellular localization of lipolytic
proteomes of cells and tissues in health and disease directly at
enzymatic activity level.
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Acknowledgments
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This work was supported by the GOLD III (Genomics of Lipid
associated Disorders III; http://gold.uni-graz.at/) project in the
framework of GEN-AU (Genome research in Austria; http://
www.gen-au.at/) funded by the Bundesministerium für Wissen-
schaft und Forschung (BM.W_F).
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