Labeling and enrichment of Arabidopsis thaliana matrix metalloproteases using an active-site directed, marimastat-based photoreactive probe

Article abstract of DOI:10.1016/j.bmc.2011.06.068

Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed a photoreactive probe based on the MMP inhibitor marima

Full text of DOI:10.1016/j.bmc.2011.06.068

Bioorganic & Medicinal Chemistry 20 (2012) 592–596
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Labeling and enrichment of Arabidopsis thaliana matrix metalloproteases using
an active-site directed, marimastat-based photoreactive probe
Janina Lenger a,b, Farnusch Kaschani b, Thomas Lenz c, Christian Dalhoff c, Joji Grace Villamor b,
b,
Hubert Köster c, Norbert Sewald a, , Renier A. L. van der Hoorn
⇑
⇑
a Bioorganic and Organic Chemistry, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany
b Plant Chemetics Lab, Chemical Genomics Centre of the Max Planck Society, Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany
c caprotec bioanalytics GmbH, Volmerstrasse 5, 12489 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 29 June 2011
Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play
diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed
a
photoreactive probe based on the MMP inhibitor marimastat, conjugated to
a
4-azi-
Keywords:
Matrix metalloprotease (MMP)
Arabidopsis thaliana
Marimastat
Photoreactive probe
Activity-based protein profiling (ABPP)
Capture Compound Mass Spectrometry
(CCMS)
dotetrafluorobenzoyl moiety as photoreactive group and biotin as detection or sorting function. The
probe labels At2-MMP, At4-MMP, At5-MMP, and likely other plant MMPs in leaf extracts, as shown by
transient At-MMP expression in Nicotiana benthamiana, protein blot, and LC–MS/MS analysis. This
MMP probe is a valuable tool to study the post-translational status of MMPs during plant immunity
and other MMP-regulated processes.
Ó 2011 Published by Elsevier Ltd.
1. Introduction
the MMPs in N. benthamiana enhances susceptibility to infections
by Pseudomonas syringae pv. tabaci.2h Furthermore, MtMMPL1 from
Matrix metalloproteases (MMPs) are zinc-containing endopep-
tidases that reside in the extracellular matrix either as soluble,
transmembrane or membrane-associated proteins.1a The Merops
database classifies MMPs in clan MA, family M10.1b MMPs are ex-
pressed as zymogens and activated by inter- and intra-proteolytic
cleavage of the prodomain, thereby displacing the conserved cys-
teine residue that coordinates the active site zinc.1c Human MMPs
have important functions in tissue remodeling and an imbalance in
their tight post-translational regulation causes various pathologi-
cal conditions.1d
M. truncatula is induced early during nodulation with symbiotic
bacteria, and silencing of this gene disturbs the nodulation process.
However, this MMP-like protein contains a mutation of an active
site residue which may render it proteolytically inactive.2e
The A. thaliana genome encodes for five MMPs (At1-MMP–At5-
MMP),2c which have high homology to human MMP-7. At1-, At2-,
At3-, and At5-MMPs carry a cleavable signal peptide and a C-termi-
nal transmembrane domain.2c At3-MMP has an additional putative
GPI anchor motif.2c At4-MMP has an uncleavable signal peptide
and no transmembrane domain or GPI anchor motif.2c Similar to
the activation of human MMPs,1c incubation of At1-MMP with an
organomercury compound leads to the formation of a truncated
protein of 27 kDa.2c It can be assumed that At2-MMP–At5-MMP
also require processing before they gain activity. Once activated,
animal MMPs are regulated by endogenous proteinaceous inhibi-
tors,1b but no functional inhibitor homolog has been identified in
plants yet. These studies2 make clear that plant MMPs are interest-
ing research subjects involved in diverse biological processes.
However, their post-translational regulation hampers a prediction
of MMP activity based on transcript or protein levels. A direct read-
out of MMP activity in complex proteomes would therefore repre-
sent a valuable tool to provide functional information on MMPs.
Activity-based protein profiling (ABPP) is an efficient strategy to
detect proteins in their active isoforms. This approach is based on
the use of fluorescent or biotinylated probes that react covalently
Plant MMPs have been described for soybean,2a,b Arabidopsis
thaliana,2c cucumber,2d Medicago trancatula,2e pine,2f tobacco,2g
and Nicotiana benthamiana.2h Plant MMPs are encoded by intron-
less genes and consist of a signal peptide, a prodomain, a protease
domain carrying the zinc-binding motif HExGHxxGxxH and often a
C-terminal transmembrane domain. Plant MMPs play versatile bio-
logical roles in growth and development2d,e,j as well as in pathogen
and symbiont infections.2g–i Transcript levels of MMP-encoding
genes in soybean, tobacco and N. benthamiana are strongly induced
during infection with pathogenic bacteria,2g–i and silencing one of
⇑
Corresponding authors. Tel.: +49 521 106 2051; fax: +49 521 106 8094 (N.S.);
tel.: +49 221 5062 245; fax: +49 221 5062 207 (R.A.L.v.d.H.).
E-mail addresses: norbert.sewald@uni-bielefeld.de (N. Sewald), hoorn@mpipz.
mpg.de (R.A.L. van der Hoorn).
0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.06.068

J. Lenger et al. / Bioorg. Med. Chem. 20 (2012) 592–596
593
with active site residues of target enzymes in an activity-depen-
dent manner.3 ABPP has been used in plants to detect activities
of papain-like Cys proteases,4a,b the proteasome4c,d and Ser hydro-
lases.4e Probes for these enzymes are based on irreversible inhibi-
tors that trap the enzyme in the covalent intermediate state.
However, similar irreversible inhibitors are not available for metal-
loproteases since their catalytic mechanism does not involve a
covalent intermediate with the substrate. Hence, an additional
chemical step, for example, involving a photoreaction, is required.
Therefore, probes for metalloproteases are typically based on
reversible inhibitors, equipped with a photoreactive group. The
feasibility of this concept was proven, for example, for kinases5
and other protein classes, including metalloproteases.6 Photoreac-
tive probes that target the active site display the accessibility of the
active site, which is a hallmark for enzyme activity.4f Capture
compounds, the basis of a technology termed Capture Compound
Mass Spectrometry (CCMS), are trifunctional small molecules
containing a selectivity, photo-reactivity and sorting function in a
defined and optimized geometry, with which a covalent bond is
formed outside of the binding site of the protein upon irradiation.7
Proteins that have affinity to the reversible inhibitor are covalently
attached upon photo-activation and the cross-linked proteins are
isolated using streptavidin-coated magnetic beads. In contrast to
ABPP, CCMS is not restricted to (active) enzymes but addresses
all kinds of proteins interacting with small molecules, including
inhibitors, substrate(analog)s, and drugs.
published probes are peptidomimetics of succinyl hydroxa-
mate6b–d which do not contain the hydroxyl group in position R1
and often employ different amino acids to target the S02 pocket. An-
other group of hydroxamate-based peptide probes carries a C-ter-
minal hydroxamate.6e Some hydroxamate-based probes contain
the photoreactive group (probe type A,6a Fig. 1) or various amino
acid side chain residues (probe type B,6b Fig. 1) at the R2 position
to target the S02 pocket. Probes of type B6b are similar to our probes
1 and 2, however, the original marimastat (R2 = tert-butyl) has not
been employed for labeling MMPs before.
The position6d and nature6e of the photoreactive group is an
importantparameter for metalloprotease probes. The most common
and best characterized photoreactive groups are aryl azides, fluori-
nated aryl azides, diazirines and benzophenones. The carbene gen-
erated from the trifluoromethyl diazirine shows a high reactivity
and good C–H and O–H insertion rates.10a However, the increased
reactivity and long half life of azo-intermediates can lead to false-
positive labeling reactions, especially in biological matrices relevant
for MMP investigations.10a The slightly less reactive aryl azides may
undergo isomerization to 1,2-didehydroazepines. The fluorinated
aryl azide derivatives maintain the broader reactivity of the primar-
ily generated nitrene.10b,c Triplet benzophenones show a decrease in
reactivitycompared to nitrenesgenerated from aryl azides and carb-
enes from diazirines, but their ability to revert to the singlet ground
state and to be reactivated several times10d,e is considered beneficial.
We therefore chose to incorporate two different photocrosslinkers
into our probes; an azidotetrafluorobenzoyl moiety for probe 1
and benzophenone for probe 2 (Fig. 2).
Hydroxamates are a well-studied group of reversible inhibitors
suitable for metalloprotease probes. They are bidendate chelators
that bind zinc in the active site and are potent broad-spectrum
inhibitors of MMPs. Marimastat and batimastat (Fig. 1) are two
important examples of this compound class.8 Plant MMPs are sen-
sitive to hydroxamate inhibitors since both At1-MMP2c and
cucumber MMP2d are efficiently inhibited by batimastat. Here,
we developed high-affinity, marimastat-based photoreactive
probes and tested them for labeling plant MMPs.
The IC50 value for the inhibition of active recombinant human
MMP-2 was determined for probe 1 to be 4.7 0.9 nM. For marim-
astat itself we determined an IC50 value of 3.2 1.0 nM which com-
pares well with the reported value of 6 nM for human MMP-2.8 The
affinity of probe 1 to human MMP-2 is relatively high compared to
previous metalloprotease probes which were reported with IC50
values in the range of low nM to high
l
M.6a–e We validated the
functionality of probe 1 by capturing human recombinant MMP-
2 and showed by SDS–PAGE that active MMP-2 is captured as
full-length protein, lacking the pre- and pro-domains (see Supple-
mentary data for details).16
We cloned At2-, At4-, and At5-MMP into binary vectors for
expression in planta to demonstrate labeling of plant MMPs (see
Supplementary data for expressed protein sequences). The cloned
MMPs were transiently overexpressed as C-terminally HA-tagged
proteins in N. benthamiana by means of transient Agrobacterium
tumefaciens-mediated transformation (agroinfiltration).11 Protein
extracts of leaves transiently overexpressing At-MMPs were ana-
2. Results and discussion
We employed the marimastat moiety as the targeting group of
our probe since it inhibits a wide range of human MMPs in the low
nM concentration range and shares similar inhibition profiles with
batimastat,8 which inhibits one of our target proteins, At1-MMP.2c
For probe synthesis, our previously described marimastat-linker
construct9 was coupled to two different conjugates containing bio-
tin and a photoreactive moiety to yield probes 1 and 2 (Fig. 2, see
Supplementary data for synthesis). Our probes differ from previ-
ously described hydroxamate-based probes.6a–e The majority of
lyzed by western blotting with
a-HA antibodies. Only At4- and
At5-MMP were detected with sizes of 40 and 50 kDa, respectively,
consistent with the expected molecular mass of glycosylated full-
length proteases (with aglycon masses of 34 and 37 kDa, respec-
tively, Fig. 3B; lanes 1 and 2). MMP overexpression does not affect
the protein profile when compared to the p19 control, indicating
that MMPs do not degrade abundant proteins and that MMPs do
not accumulate to very high levels (data not shown).
Extracts of leaves expressing At4- and At5-MMP were incubated
and crosslinked with probe 1 as detailed in Figure 3A. Preincuba-
tion with an excess of marimastat prior to labeling with probe 1
was used as a control for labeling specificity. Extracts of leaves
not overexpressing At-MMPs served as a control for the detection
of endogenously biotinylated proteins. Detection of biotinylated
proteins after labeling revealed specific, marimastat-sensitive sig-
nals of 25 kDa in At4-MMP and 35 kDa for At5-MMP samples
(Fig. 3B, lanes 6 and 8). These signals did not appear in extracts
of leaves not overexpressing At-MMPs (Fig. 3B, lane 4). No signals
of 25 or 35 kDa appeared on the anti-HA western blot of the la-
beled proteins (Supplementary data, Fig. S1), indicating that the
Figure 1. General structure of hydroxamate-based MMP inhibitors and probes. S10
and S02 refer to the substrate binding pockets that bind amino acids at the P10 and P02
positions of the substrate, respectively.

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J. Lenger et al. / Bioorg. Med. Chem. 20 (2012) 592–596
Figure 2. Structures of the marimastat-based probes 1 and 2 as used for labeling experiments of Arabidopsis thaliana MMPs.
HA tag is not present on the labeled MMP proteins. Meanwhile, the
precursor At4-MMP and At5-MMP were detected to similar levels
on anti-HA western blots, irrespective of UV treatment or probe
presence (Supplementary data, Fig. S1). A 30 kDa signal is detected
in all lanes and represents an endogenously biotinylated protein,
presumably biotin carboxyl carrier protein, BCCP (Fig. 3B, lanes
3–8). The molecular mass of the labeled proteins is 15 kDa lower
than that of the full-length proprotease.
To verify the identity and of the labeled proteins, we enriched
biotinylated proteins on streptavidin beads. The samples were di-
gested on-bead with trypsin and eluted peptides were analyzed by
LC–MS/MS (Fig. 3A). At4-MMP was unambiguously identified from
the At4-MMP-containing sample, with 21 spectral counts covering
48% of the protein sequence (Fig. 3C, Supplementary data, Tables
3–5). From the At2-MMP containing samples, three At2-MMP pep-
tides were identified with moderate scores (Fig. 3C, Supplementary
data, Tables 3–5). Two peptides identified from the At5-MMP sam-
ple cover 8% of the At5-MMP sequence (Fig. 3C, Supplementary data,
Tables 3–5). Importantly, no peptides of At2-MMP, At5-MMP, and
only two peptide spectral counts for At4-MMP were found if the
samples were preincubated with an excess of marimastat (Fig. 3C
and Supplementary data, Tables 3–5), confirming that labeling can
be competed by addition of the original MMP inhibitor.
The same labeling workflow (Fig. 3A) was applied to the lysates
using probe 2. However, no At-MMP labeling was detected by
streptavidin blotting or LC–MS/MS analysis (data not shown).
The inferior labeling performance of the benzophenone-bearing
probe 2 is supported by the literature. In a comparative study of
the labeling efficiencies of metalloprotease probes with benzophe-
none and trifluoromethyldiazirine as reactive groups, no labeling
of spiked metalloprotease in crude lysates was observed for the
benzophenone probe in contrast to a clear signal for the diazirine
probe.6e
for example, is active as a 27 kDa protein.2c Furthermore, Soy-
bean MMP (SMEP1) was isolated as a 19 kDa active protease2a
;
cucumber MMP is active as 22 and 18 kDa isoforms,2d and
tobacco MMP is active as 36 and 22 kDa isoforms.2k Similarly,
the 62 kDa human MMP-2 produces a 43 kDa isoform that lacks
part of the protease domain after the Zinc binding domain12a
and the 82 kDa human MMP-9 produces 35 kDa mature prote-
ases.12b The mechanisms of post-translational regulation in
MMPs are not completely understood yet and the physiological
function of the various active isoforms are still under discussion.
C-terminal truncation may alter substrate specificity as the C-
terminal domain is important for substrate binding.1,12 These as-
pects should be further investigated by time-dependent labeling
of lysates and addition of various protease inhibitors to deter-
mine when cleavage occurs as well as the class of protease
which is responsible for MMP truncation.
The peptide coverage indicates that the labeled At4-MMP
retained most of its prodomain. The presence of a prodomain
in a labeled MMP implies that it was not inhibiting protease
activity during labeling. Otherwise, the active site zinc would
be coordinated by the Cys switch motif of the prodomain, and
not be available to bind the hydroxamate moiety in probe
1. The presence of a prodomain in active MMPs is not unprece-
dented. The active isoform of human MMP-9 also contains the
prodomain with the cysteine switch.13 Furthermore, inhibition
of human MMPs by TIMP-1 traps the protease in a prodomain-
containing isoform.1b Therefore, the presence of part of the
prodomain does not exclude MMP labeling.
The labeling procedure can be further improved to expand the
range of MMPs detected by our approach. We detected overexpres-
sed At4-MMP, At5-MMP, and At2-MMP from total leaf extracts.
Since most MMPs are transmembrane proteins, their signals can
be increased, simply by isolating membrane proteins before or
after labeling. Further adjustments of the extraction procedure
are needed to detect the other MMPs more efficiently.
The small size of labeled MMPs is consistent with the fact
that plant MMPs are active as 20–35 kDa isoforms. At1-MMP,

J. Lenger et al. / Bioorg. Med. Chem. 20 (2012) 592–596
595
MMPs in a marimastat-sensitive manner. Further optimization and
application of this methodology will contribute to the elucidation
of the role and post-translational regulation of plant MMPs during
plant–pathogen interactions and other biological processes.
4. Experimental
4.1. Protein expression
At-MMPs were amplified from cDNA isolated from different
developmental stages of A. thaliana Col0 by RT-PCR and cloned into
pFK26.14 Cloning details and primer sequences are given in Supple-
mentary Tables 1 and 2, respectively. The reverse primer intro-
duces a HA epitope tag at the C-terminus. Clones were verified
by sequencing and correct inserts were shuttled into binary vector
pTP5 using EcoRI and HindIII restriction sites. The binary plasmids
were transformed into A. tumefaciens GV3101 by electroporation.
Agroinfiltration in the presence of silencing inhibitor p19 was done
as described previously.14
4.2. At-MMP labeling and western blot analysis
N. benthamiana leaf lysates were prepared by grinding leaves in
0.4 M borate buffer (pH 7.6) [6.2 g boric acid in 250 mL water, ad-
justed to pH 7.6 using 10 M NaOH].16 The extract was cleared by
centrifugation and diluted with cross-linking buffer (25 mM Tris
pH 7.5, 60
tration of 1 mg/mL. 100
well plate and pretreated with marimastat in DMSO (500
concentration) or DMSO for 10 min. After addition of probe 1
(1 M final concentration), crosslinking was started by placing a
l
M ZnCl2, final concentrations) to give a protein concen-
L of the samples were transferred to a 96-
M final
l
l
l
hand-held UV lamp (254 nm (max) and 365 nm (max)) on top of
the 96-well plate. The samples were irradiated for 20–30 min on
ice. Afterwards, the reaction was stopped by adding 25
SDS–PAGE gel loading buffer and heating samples to 90 °C for
5 min. 10–15 L sample was separated by SDS–PAGE and blotted
lL 4ꢀ
l
onto PVDF membrane (Millipore).
Protein blots were incubated with antibodies or streptavidin-
horseradish peroxidase (Strep-HRP) in the presence of Tris-buf-
fered saline (50 mM Tris–HCl, pH 7.5; 150 mM NaCl) containing
0.1% Tween-20 and 4% BSA. HA-tagged proteins were detected
using mouse
a-HA antibody (Sigma) at 8:10000 and rabbit a-
mouse antibody conjugated with HRP (Pierce) at 6:10000. Biotinyl-
ated proteins were detected using Ultrasensitive Strep-HRP conju-
gates (Sigma) at 3:10000. Membranes were washed extensively
prior to detection of signals with Enhanced Chemiluminescence
(ECL, Pierce).
Figure 3. Labeling of leaf extracts overexpressing At-MMP. (A) Workflow of the
labeling experiment. (B) Detection of (labeled) MMPs in extracts of leaves
transiently overexpressing At4-MMP or At5-MMP together with silencing inhibitor
p19. The (ꢁ)control is from leaves agroinfiltrated with p19 alone. Proteins were
4.3. At-MMP labeling, enrichment and LC–MS/MS analysis
For enrichment of proteins labeled with probe 1, At-MMP-
expressing N. benthamiana leaf lysates in 50 mM Tris, pH 7.4,
extracted from agroinfiltrated leaves and detected on protein blots using
a-HA
antibody, or labeled with probe 1 and then detected with streptavidin-HRP (right).
(C) Identified peptides (red) covering the MMP prodomain (purple) and protease
domain (blue). Labeled proteins were purified from At-MMP containing extracts,
digested with trypsin and analyzed by LC–MS/MS. Characteristics of the MMP
protein sequences are indicated on top. Identified peptides are shown on dashed
lines with Xcorr scores indicated as red scales summarized on the bottom. +/ꢁ, with
or without marimastat competition, respectively. TM, transmembrane domain.
200 mM NaCl, 5 mM CaCl2 were incubated with 1
the presence or absence of 500 M marimastat in a final volume
of 100 L for 5 min on ice. The samples were UV-irradiated within
open tubes of a 200 L-PCR tube strip (recommended 0.2 mL Ther-
mo-Strip, Thermo Scientific, AB-1114) using the caproBox
(caprotec bioanalytics GmbH, Berlin) for 4 min at 2 °C. 25 L 5ꢀ
wash buffer (250 mM Tris–HCl, pH 7.5, 5 mM EDTA, 5 M NaCl,
42.5 M octyl-b- -glucopyranoside) was added to each of the
samples. After homogenization, 20 L 10 mg/mL streptavidin-
lM probe 1 in
l
l
l
l
l
D
3. Conclusion
l
coated magnetic beads (Dynabeads MyOne Streptavidin C1,
Invitrogen Dynal) were added and the samples were incubated
for 3 h at 4 °C keeping the beads in suspension by rotation. The
beads were collected using the caproMag magnetic device
Marimastat-based probe 1 is a high-affinity photoreactive
probe for MMPs. We have demonstrated by detecting labeled pro-
teins on streptavidin-HRP blots and by LC–MS/MS analysis that
probe 1 can label At4-MMPs and At5-MMP and likely several other

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(caprotec bioanalytics GmbH, Berlin), the supernatant was dis-
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luted from the 5ꢀ wash buffer), once with water, six times with
80% acetonitrile (ACN) and once with water (200 lL, respectively,
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mentary data.
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This work was financially supported by the Studienstiftung des
Deutschen Volkes (PhD fellowship to J.L.), the Max Planck Society,
and the Deutsche Forschungsgemeinschaft (DFG projects HO 3983
3-3 and HO 3983 4-1).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.068.
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