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Plant Physiology 135:1170-1178 (2004) © 2004 American Society of Plant Biologists Activity Profiling of Papain-Like Cysteine Proteases in Plants1Laboratory of Phytopathology, Wageningen University, 6709 PD, Wageningen, The Netherlands (R.A.L.v.d.H., M.H.A.J.J.); Sainsbury Laboratory, John Innes Centre, NR4–7UH, Norwich, United Kingdom (R.A.L.v.d.H., S.C.P.); Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA, Leiden, The Netherlands (M.A.L.); and Department of Pathology, Stanford University Medical School, Stanford, California 94305–5324 (M.B.)
Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanism-based manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of biotinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B- and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.
Plant genomes encode hundreds of proteases, but little is known about what roles they play in the life of a plant. Functions for only a few of the more than 550 proteases of Arabidopsis (http://merops.sanger.ac.uk) have been determined genetically (for review, see Adam and Clarke, 2002  ; Beers et al., 2004). In general, proteases are thought to be involved in a range of processes, including senescence and defense responses (Beers et al., 2000; Van der Hoorn and Jones, 2004), as indicated by studies with protease inhibitors (e.g. Solomon et al., 1999; Chichkova et al., 2004). In many cases, proposed functions for proteases have been inferred from the observed differential expression of their mRNAs (e.g. Zhao et al., 2000; Gepstein et al., 2003). The progress in assigning roles for proteases, however, is significantly impeded by their redundancy and posttranslational regulation.
Typically, proteases contain an autoinhibitory prodomain that must be removed to activate the enzyme (Bryan, 2002
Proteases are classified based on their catalytic mechanisms into Ser, Cys, aspartic, and metallo proteases (Powers et al., 2002
To cleave a peptide bond, Ser and Cys proteases contain a Ser or Cys residue, respectively, in their active site that acts as a nucleophile in the first step of proteolysis (Powers et al., 2002
Protease activity profiling is based on biotinylated (or otherwise labeled) mechanism-based protease inhibitors that covalently react with proteases in an activity-dependent manner (Campbell and Szardenings, 2003
The simultaneous visualization of multiple protease activities will become an essential tool to unravel functions of the hundreds of proteases encoded by plant genomes. To date, however, protease activity profiling has only been used with mammalian and Drosophila proteases (Jessani et al., 2002 ; Kocks et al., 2003). In this study, we show that protease activity profiling can also be applied on plant extracts. After establishing parameters for labeling, we identified activities of different Cys proteases in a whole-plant extract and show an example where induction of a protease gene during senescence is not correlated with the activity of the encoded enzyme. Our data demonstrate the versatility of the technology and indicate that its application will provide important data on activities of many plant proteases.
Optimization of Protease Activity Profiling on Arabidopsis Leaf Extracts To establish protease activity profiling in plants, we optimized a protocol for profiling protease activities in extracts of Arabidopsis leaves (Fig. 1C, and "Material and Methods"). Labeling Arabidopsis leaf extracts typically results in signals in three major regions: 40, 30, and 25 kD (Fig. 1C, indicated with black, white, and gray dots, respectively). Each of these signals was competed with an excess of E-64, indicating specific labeling. Although only three prominent signals are visible, protein blots of higher resolution indicated that the upper and lower signals are composed of multiple bands (see later, e.g. Figs. 2 and 6), indicating that multiple active proteases of similar sizes are visualized.
Numerous parameters needed to be optimized for sensitive and reproducible labeling. Usually, about 0.7 mg of soluble protein was taken for each profiling reaction (one leaf half). However, dilution experiments showed that the same signals could be detected with 100-fold less input proteins, demonstrating the sensitivity of the procedure (data not shown). Specific labeling was detected within 5 min incubation (Fig. 2A, left), but a maximum was reached after 5 h labeling (Fig. 2A, right). Optimal labeling also required the presence of reducing agents (Fig. 2B). This observation is explained by inactivation of Cys proteases by oxidation of the active site Cys during protein extraction (Beynon and Bond, 2000 ). Finally, labeling was usually performed at pH 6, but when labeling was done at different pH values, profiles changed significantly (Fig. 2C). This result indicates that the choice of pH is crucial for the types of proteases that are intended to be displayed. The changes in relative intensities at different pH also indicate that different proteases are displayed, rather than one with different molecular masses. For all subsequent experiments, we chose the optimum labeling conditions, meaning high concentrations of soluble protein and 5 h labeling time at pH 6 in the presence of the reducing agent L-Cys.
To confirm that the signals on the protein blots represent Cys proteases, labeling was performed in the presence of other inhibitors of Cys proteases (cystatin, antipain, leupeptin, and chymostatin) and inhibitors of Ser proteases (phenylmethylsulfonyl fluoride [PMSF]), metallo proteases (EDTA), and aspartic proteases (pepstatin). These experiments show that labeling is inhibited by other Cys protease inhibitors (Fig. 3A) but not by inhibitors of proteases from the other classes (Fig. 3B). The slightly reduced labeling at high PMSF concentrations is because PMSF also inhibits Cys proteases at high concentrations (Beynon and Bond, 2000
Protease Activity Profiles of Other Organs of Arabidopsis Having established labeling conditions of leaf extracts, we examined protease activity profiles of other Arabidopsis organs. Interestingly, although all signals still occur at the 25-, 30-, and 40-kD regions, the profiles differ significantly in molecular masses and in their relative intensities (Fig. 4). The profile obtained from a flower extract even displayed nine bands, illustrating that different tissues express unique patterns of protease activities.
Purification and Identification of Biotinylated Cys Proteases A large-scale labeling and purification was performed to identify the different biotinylated proteases. An extract from nine plants was labeled at pH 6, proteins were precipitated by acetone, and biotinylated proteases were purified on immobilized streptavidin. To monitor the purification procedure, samples were taken at each step. We noticed that much of the 40-kD signal was lost after dissolving the acetone pellet, apparently because these proteins do not redissolve well (Fig. 5A).
Biotinylated proteins isolated from the soluble extract were analyzed on a streptavidin-horseradish peroxidase (HRP)-stained protein blot and on a colloidal Coomassie-stained protein gel (Fig. 5B). Only four bands appeared on the protein gel, and each band was competed with an excess of E-64 during labeling (Fig. 5B, right). These signals matched the major biotinylated signals (Fig. 5B, left). The four bands were excised from the protein gel, and after in-gel digestion with trypsin, the resulting tryptic fragments were analyzed by matrix-assisted laser-desorption ionization time of flight (MALDI-TOF)-mass spectrometry (MS) and quadrupole (Q)-TOF-MS/MS.
In total, six different Cys proteases were identified unambiguously (Table I; Fig. 5C). Three of the four bands contained multiple proteases, and two proteases were identified twice, in bands 1 and 2 (Table I; Fig. 6C). Three of the proteases, Arabidopsis aleurain-like protease (AALP; Ahmed et al., 2000
All six protease genes are predicted to encode proteins containing a signal peptide, a prodomain, and the protease domain (Fig. 5C). Only RD21 has an additional C-terminal extension containing a granulin domain (Yamada et al., 2001 ). The identified tryptic fragments were derived only from the protease domain and not the prodomain (Fig. 5C), consistent with the inhibitory role of the prodomain that needs to be removed before the protease can react with DCG-04. Tryptic fragments carrying the active site Cys (Fig. 5C) were not identified, most likely because biotinylation of these fragments by DCG-04 yields peptides that are too large to be detected.
Of the identified Cys proteases, specific antibodies have been raised to XCP2 (E. Beers and E. Petzold, unpublished data), AALP (Ahmed et al., 2000
Intriguingly, the anti-RD21 antibody also cross-reacts with biotinylated proteins in the 40-kD region (Fig. 6A). This observation is consistent with previous studies. RD21 is known to exist in two active forms, a 38-kD intermediate form (iRD21) containing the C-terminal granulin domain, and a 33-kD mature form (mRD21) from which the granulin domain has been removed (Yamada et al., 2001
The AALP and RD21 transcripts have been shown to accumulate during leaf senescence (Gepstein et al., 2003
In principle, Cys protease activity profiling can be performed on any tissue containing Cys proteases that are inhibited by E-64. To demonstrate its wider applicability, we performed protease activity profiling on leaf extracts of various model plant species. The resulting protease activity profiles are highly polymorphic, with molecular masses ranging from 25 to 40 kD (Fig. 7), indicating that different Cys proteases are labeled in different plant species. Thus, this method can be applied in a broad spectrum of plant species.
Here, we have shown that protease activity profiling can be used to monitor protease activities in plant tissues. We have identified six different Cys proteases form Arabidopsis, three of which were known only from the genome sequence. To our knowledge, for all proteases, including those previously described (XCP2, AALP, and RD21), activity had not been demonstrated. An essential feature of the technology is that it detects the activity of proteases because E-64 acts as a suicide substrate, locking the cleavage mechanism in the covalent intermediate state. Indeed, labeling is pH dependent, can be stimulated with reducing agents, and is inhibited by Cys protease inhibitors but not by inhibitors of other catalytic classes, all of which are characteristics that typify the activity of Cys proteases (Beynon and Bond, 2000 ). In addition, as expected for the broad specificity for the E-64-based probe, the six proteases that we identified in this study represent five of the eight groups that have been distinguished within 30 members of Arabidopsis papain-like proteases (Beers et al., 2004). Protease activity profiling has two primary applications. One is to search for differentially activated proteases in a particular biological system, as we showed for different Arabidopsis organs (Fig. 4). The procedure to separate the different biotinylated proteases, however, may still need to be optimized to deal with the overlapping complexity, for example using two-dimensional gel electrophoresis or multidimensional liquid chromatography-mass spectrometry. The other application is to use protease activity profiling when a particular enzyme is under investigation. Combined with protease-specific antibodies, protease activity profiling can be used to study the activity of a particular protease without the need to purify it from its native environment. For example, we examined activities of RD21 and AALP in extracts of senescing tissues (Fig. 6C). A similar procedure can be used to determine the optimum pH for the activity of a certain protease or to screen for inhibitors and determine a relative inhibition constant.
A number of variations can be exploited to gain additional information from protease activity profiling. First, conditions for protease labeling can be chosen (e.g. pH), offering opportunities to display subsets of protease activities. Second, protease activity profiling can be performed not only with total extracts of plants but also with subproteomes, such as proteins from the extracellular space, membranes, or isolated organelles. This approach may reveal low-abundant protease activities and also reduce the chances of accidentally activated proteases that may result from cellular disruption. Third, use of probes different from DCG-04 (used in this study) can reveal activities of other proteases. There are probes with a broad specificity described for Ser proteases (Liu et al., 1999 Finally, it must be acknowledged that the activity detected in extracts may not reflect the in planta activity in all cases. Detected activities may change as a result of extraction, where normally compartmentalized proteases are mixed, possibly leading to activation. This problem could be circumvented by including inhibitors of other protease classes and by using subproteomes. With the development of membrane-permeable probes, it may even be possible to bring the probe into living systems without disrupting the cellular structure.
The proteases identified in this study are of unknown function, but their proposed roles can be of great interest. XCP2, for example, is hypothesized to be involved in programmed cell death of tracheary elements (Funk et al., 2002
Plant Materials, Reagents, and Antibodies Arabidopsis ecotype Columbia was grown in a growth chamber at 22°C under 16-h light regime. Tissues were taken from 6-week-old plants that just started flowering. Model plant species were grown in a normal greenhouse.
DCG-04 was synthesized and purified as described previously (Greenbaum et al., 2000 Antibodies for detection of XCP2, AALP, and RD21 were kindly provided by Dr. Eric Beers and Dr. Earl Petzold, Dr. Natasha Raikhel, and Dr. Ikuko Hara-Nishimura, respectively. Anti-rabbit IgG antibodies, conjugated with HRP, are from Amersham Pharmacia Biotech (Bucks, UK).
Proteins were extracted by grinding one rosette leaf in an Eppendorf tube, mixing with 0.5 mL of water, and centrifugation through a micro Biospin column (Bio-Rad, Hercules, CA). Usually, approximately 0.5 mg of protein was labeled in 0.5 mL of total volume, containing 50 mM sodium acetate buffer, pH 6, 10 mM L-Cys (Sigma), and 2 µM DCG-04. Control samples contained also 0.2 mM E-64. Labeling was done for 5 h at room temperature. Proteins were then precipitated by adding 1 mL ice-cold acetone, followed by centrifugation (1 min, 10,000g). The acetone pellet was washed with 70% acetone and dissolved in 500 µL of TBS (50 mM Tris, pH 7.5, 150 mM NaCl) containing a protease inhibitor cocktail (Complete tablet; Roche, Mannheim, Germany) and 10 µL of TBS-washed magnetic streptavidin beads (Promega, Southampton, UK). Solutions with streptavidin beads were gently shaken overnight at room temperature. The beads were then washed once with TBS using a magnetic holder and boiled in 50 µL of SDS sample buffer. More extensive washes with TBS were done for detection with protease-specific antibodies.
Biotinylated proteins were separated on 10% SDS gels (Laemmli, 1970 A time course of labeling was done by taking aliquots from a large-scale labeling and stopping the labeling by adding an excess of E-64 followed by acetone precipitation. Profiling at different pH was done using 50 mM buffers of sodium acetate (pH 4–6.5) or Tris (pH 7–10). Competition of protease labeling by protease inhibitors was done by preincubating the extracts at pH 6 for 10 min with 3-fold dilution series of various protease inhibitors.
Large-scale purification was done similarly as above and is detailed below. Complete 6-week-old plants (nine individuals) were ground with sand and water in a mortar. The extract was centrifuged (15 min, 10,000g), and the supernatant was taken for labeling. Labeling was done for 6 h at 19°C in 45 mL total volume containing 50 mM sodium acetate buffer, pH 6; 0.4 mM PMSF, 10 mM EDTA; 10 mM L-Cys; and 0.2 mM DCG-04 (and 10 mM E-64 only in the control). Proteins were precipitated with 90 mL of cold acetone, and the pellet was washed with 70% acetone. Proteins were dissolved from the acetone pellet into 45 mL of TBS and centrifuged to remove nondissolved precipitate. Before and after this centrifugation, samples were taken for small-scale purification as described above. Protease inhibitor cocktail and 100 µL of TBS-washed magnetic streptavidin beads were added to the supernatant. After overnight incubation at room temperature, streptavidin beads were collected in an Eppendorf tube using a magnetic holder and washed twice with TBS; twice with TBS containing 1 M NaCl; twice with TBS containing 1% Triton X-100; and once with water, and then boiled in 50 µL of SDS sample buffer. Proteins present in 3 µL of sample were separated on a large 10% SDS gel and transferred to polyvinylidene difluoride for detection with streptavidin-HRP, whereas the remainder of the sample was separated on the same large SDS-gel for staining with colloidal Coomassie Brilliant Blue.
Bands were excised from the Coomassie-stained gel and subjected to in-gel tryptic digestion (Shevchenko et al., 1996 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers At4g01610 (CatB-like), At1g20850 (XCP2), At1g47128 (RD21), At5g60360 (AALP), At3g45310 (aleurain-like), and At1g06260 (TPE4A-like).
We thank Dr. Hermen Overkleeft and Dr. Jules Beekwilder for useful suggestions, Dr. Eric Beers, Dr. Earl Petzold, Dr. Natasha Raikhel, and Dr. Ikuko Hara-Nishimura for providing the protease-specific antibodies, and the John Innes Centre Proteomics Facility for sample preparation and analysis. Received February 24, 2004; returned for revision April 13, 2004; accepted May 12, 2004.
1 This work was supported by the Dutch Organisation for Scientific Research (N.W.O.; Veni grant to R.A.L.v.d.H., Vidi grant to M.H.A.J.J., and Talent grant to R.A.L.v.d.H.) and by the Gatsby Charitable Foundation.  www.plantphysiol.org/cgi/doi/10.1104/pp.104.041467. * Corresponding author; e-mail renier.vanderhoorn{at}wur.nl; fax 31–317–483412.
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-mercaptoethanol (
