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Biol. Chem., Vol. 389, pp. 91–98, January 2008 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.006
Cloning, expression and characterization of insulin-degrading
enzyme from tomato (Solanum lycopersicum)
Yoann Huet1,a, Jochen Strassner2 and Andreas
Schaller1,*
Institute of Plant Physiology and Biotechnology,
University of Hohenheim, D-70593 Stuttgart, Germany
2
Altana Pharma AG, D-78467 Konstanz, Germany
1
* Corresponding author
e-mail: [email protected]
Abstract
A cDNA encoding insulin-degrading enzyme (IDE) was
cloned from tomato (Solanum lycopersicum) and
expressed in Escherichia coli in N-terminal fusion with
glutathione S-transferase. GST-SlIDE was characterized
as a neutral thiol-dependent metallopeptidase with insulinase activity: the recombinant enzyme cleaved the oxidized insulin B chain at eight peptide bonds, six of which
are also targets of human IDE. Despite a certain preference for proline in the vicinity of the cleavage site, synthetic peptides were cleaved at apparently stochastic
positions indicating that SlIDE, similar to IDEs from other
organisms, does not recognize any particular amino acid
motif in the primary structure of its substrates. Under
steady-state conditions, an apparent Km of 62"7 mM and
a catalytic efficiency (kcat/Km) of 62"15 mM-1 s-1 were
determined for Abz-SKRDPPKMQTDLY(NO3)-NH2 as the
substrate. GST-SlIDE was effectively inhibited by ATP
at physiological concentrations, suggesting regulation of
its activity in response to the energy status of the cell.
While mammalian and plant IDEs share many of their biochemical properties, this similarity does not extend to
their function in vivo, because insulin and the b-amyloid
peptide, well-established substrates of mammalian IDEs,
as well as insulin-related signaling appear to be absent
from plant systems.
Keywords: insulinase; insulysin; metalloprotease; plant;
proteolysis.
Introduction
Insulin-degrading enzyme (IDE) is an evolutionary well
conserved zinc-dependent metalloendopeptidase of the
pitrilysin family and belongs to the M16A family of peptidases, according to the MEROPS classification (Rawlings et al., 2004). M16 peptidases are characterized by
the conserved HXXEH motif for zinc binding, which is a
functional inversion of the more common HEXXH motif
of other metalloproteases, and they have thus been
Present address: Plant Biology and Pest Control, Jules Verne
University of Picardie, F-80000 Amiens, France.
a
named inverzincins (Ding et al., 1992). It was demonstrated by site-directed mutagenesis that the two histidine
residues are involved in the coordination of the zinc ion,
while the glutamate is required for catalysis (Perlman et
al., 1993; Becker and Roth, 1995).
Inverzincins generally accept a wide variety of peptide
substrates and appear to lack selectivity for specific
sequence motives in the primary structure of their substrates. Indeed, IDE was shown to cleave many different
peptides, including insulin and glucagon (Kirschner and
Goldberg, 1983), natriuretic peptides (Muller et al., 1992),
transforming growth factor a (Garcia et al., 1989; Gehm
and Rosner, 1991), b-endorphin and dynorphins (Safavi
et al., 1996), growth hormone releasing factor (Garcia
et al., 1989), amylin (Bennett et al., 2000) and amyloid
b peptide (Qiu et al., 1998; Vekrellis et al., 2000) at unrelated sites. However, despite the degeneracy of cleavage
sites, these peptide substrates are recognized by IDE
and cleaved with high affinity, while other biologically
important peptides are not cleaved at all. IDE can therefore not be considered as a general peptidase for nonspecific peptide turnover (Duckworth, 1990; Safavi et al.,
1996). Similarly, mitochondrial processing peptidase
(MPP) and plant stromal processing peptidase (SPP) are
specific inverzincins, which process mitochondrial and
plastid precursor proteins to release the N-terminal targeting peptides after import into the respective organelle
(Vander Vere et al., 1995; Richter and Lamppa, 1998;
Gakh et al., 2002). The targeting peptides are variable in
length and primary structure, and there is little sequence
conservation at MPP cleavage sites in yeast and plant
systems (Branda and Isaya, 1995; Glaser et al., 1998).
Pre-sequence proteases (PrePs), which also belong to
the M16 family and lack specificity towards particular
peptide bonds are responsible for the further degradation
of targeting peptides after release from protein precursors (Ståhl et al., 2002; Bhushan et al., 2003; Moberg
et al., 2003). A recent report on the structure of human
IDE in complex with several peptide substrates (Shen et
al., 2006) sheds light on the mechanisms underlying substrate recognition by M16 peptidases. The four domains
of the enzyme fold to enclose a chamber, just large
enough to accommodate peptides of up to about 50
amino acids. Additional selectivity is provided by favorable interactions between the enzyme and the N-terminus as well as the cleavage sites within the substrate
peptides. Moreover, the charge distribution in the catalytic cavity explains why peptides with positive charges
in proximity of their C-termini are usually poor substrates
(Shen et al., 2006).
IDE, as the name suggests, has first been implicated
in the regulation of insulin signaling, and experimental
evidence confirming its role in the intracellular degradation of this hormone continues to cumulate since its
activity was first described 50 years ago (Duckworth
2008/236
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92 Y. Huet et al.
et al., 1988, and references therein). More recently, attention has focused on the capacity of IDE to degrade bamyloid peptides involved in the neurodegenerative
Alzheimer disease (Kurochkin, 2001; and references
therein). Indeed, IDE-deficient rodents suffer not only
from hyperinsulinemia and glucose intolerance, but also
accumulate high levels of amyloid peptides in the brain
(Farris et al., 2003; Miller et al., 2003). A broader role for
IDE is also supported by the fact that IDE cleaves many
biologically important peptides other than insulin, by its
localization in cell types that do not bind or internalize
insulin (Duckworth, 1988). Moreover, IDE homologs are
found in organisms lacking insulin-related peptides and
insulin signaling, including plants. We report here the biochemical characterization of IDE from tomato which is
the first report on a M16A peptidase from any plant
source.
Results
A partial cDNA showing similarity to human IDE was
cloned from a tomato shoot cDNA library in a functional
screen for proteases cleaving the peptide AVQSKPPSKR
DPPKMQTD (systemin), and its 59end was completed by
RACE-PCR. The full-length cDNA of 3358 bp (accession
number AJ308542) encompassed an open reading frame
of 2913 bp coding for a protein of 971 amino acids with
a calculated molecular mass of 112 kDa. The tomato
(Solanum lycopersicum) sequence, called SlIDE, shared
the highest degree of amino acid identity with plant
orthologs, including two Arabidopsis sequences (68 and
62% identity with At2g41790 and At3g57470, respectively), and is 39% identical to human IDE (Figure 1). The
predicted domain organization of SlIDE resembles that of
human IDE. In both enzymes, the catalytic M16 domain
(Pfam domain PF00675; http://www.sanger.ac.uk/Software/Pfam/) is located close to the amino-terminus. The
M16 domain (amino acids 34–172 in SlIDE) includes the
conserved HXXEH motif, which is characteristic for the
inverzincin family of metalloproteases (Figure 1). It is followed by two M16-C domains (PF05193; amino acids
197–378 and 665–853, respectively), which have been
implicated not in catalysis but rather in substrate binding
and/or the interaction with other proteins (Taylor et al.,
2001). Similar to human IDE, the primary structure of
SlIDE comprises a potential peroxisomal targeting signal
at its extreme carboxy-terminus (VRL).
For functional expression in Escherichia coli, the open
reading frame of SlIDE was cloned into the expression
vector pGEX-G to obtain the recombinant protein in
N-terminal fusion with glutathione S-transferase (GST).
The bulk of the expressed protein accumulated in inclusion bodies. However, a fraction could be purified from
bacterial extracts by affinity chromatography on immobilized glutathione. The progress of purification, yielding
approximately 100 mg of GST-SlIDE per liter of culture,
is shown in Figure 2. The apparent molecular mass after
SDS-PAGE analysis of the purified protein (approximately
150 kDa) is consistent with the mass expected for the
fusion protein (26 kDa for GST and 112 kDa for SlIDE).
Small amounts of lower molecular weight species are
present in the purified fraction and likely represent degradation or arrested translation products as they immunoreact with an antibody directed against the N-terminus
of the fusion protein, including the GST moiety and the
first 142 N-terminal residues of SlIDE (not shown). The
presence of such low-molecular weight fragments during
bacterial expression appears to be a recurring problem,
as it was also reported for the recombinant human IDE
(Chesneau and Rosner, 2000).
GST-SlIDE was confirmed as a bona fide IDE using
MALDI-TOF mass spectrometry to characterize its activity in vitro. The oxidized insulin B chain was rapidly processed at the Glu13-Ala14, Ala14-Leu15 and Tyr16-Leu17 peptide bonds (complete processing in less than 5 min under
the chosen reaction conditions), while cleavage at His10Leu11, Leu15-Tyr16, Phe24-Phe25, Phe25-Tyr26 and Pro28Lys29 occurred at a slower rate (complete processing
required more than 30 min; Figure 3A). Six of these cleavage sites are identical with those generated by human
IDE (Duckworth et al., 1989; Figure 3A). In contrast, E.
coli pitrilysin, also an inverzincin from the M16A family,
cleaves the insulin B chain exclusively at the Tyr16-Leu17
bond (Anastasi and Barrett, 1995; Figure 3A). To rule out
the possibility that the observed activity is due not to
SlIDE but rather to a contaminating E. coli protease, a
mock purification was performed using a bacterial culture
expressing the anti-sense SlIDE cDNA, which resulted in
a protein preparation devoid of any proteolytic activity
(not shown).
Further characterization of SlIDE activity using synthetic peptide substrates revealed a certain preference
for proline one or two residues amino-terminal of the
hydrolyzed bond wi.e., the P1 and P2 sites, according to
the terminology of Schechter and Berger (1967)x. However, this is not an absolute requirement as glucagon, a
29-amino acid peptide lacking proline residues, was
nevertheless cleaved at two unrelated sites (Figure 3B).
Consistent with the identification of SlIDE in a screen for
proteases active against systemin, this peptide was
processed efficiently at the Lys14-Met15 bond (Figure 3B).
There are no common sequence elements at the cleavage sites in different synthetic peptide substrates (Figure
3B), and therefore SlIDE does not seem to require any
specific amino acid motif for substrate recognition. Consistently, the individual replacement of each of the amino
acids of the systemin peptide with alanine did not prevent recognition by SlIDE. However, it did affect the rate
of hydrolysis. The stability of alanine-substituted systemin analogs in the presence of SlIDE was compared in
MALDI-TOF MS experiments (data not shown). The rate
of hydrolysis was increased when residues in positions 4
(Ser), 12 (Pro), 13 (Pro) and 17 (Thr) were substituted with
alanine. All other substitutions rendered the peptide more
stable with respect to degradation by SlIDE. The data
suggest that SlIDE does not recognize any amino acid
sequence per se, but rather higher-order structural features of its substrates, a feature that is shared with
human IDE (Shen et al., 2006) and other M16 proteases
(Moberg et al., 2003).
A continuous fluorescence assay was used to further
characterize the activity of GST-SlIDE. The internally
quenched substrate Abz-SKRDPPKMQTDLY(NO3)-NH2
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Insulin-degrading enzyme from tomato 93
Figure 1 Comparison of human and tomato IDE sequences.
The amino acid sequence deduced from the SlIDE cDNA is compared to human IDE (HsIDE, accession no. BC096339). Amino acid
sequences were aligned using ClustalW (http://www.ch.embnet.org). Identical amino acids and conservative replacements are shown
in black and gray shading, respectively (‘boxshade’ at http//www.ch.embnet.org/). Amino acids involved in active site Zn2q-binding
are indicated by asterisks. Open and closed circles indicate residues corresponding to the cytosolic fatty acid proteins signature
(Prosite access no. PS00214) present in HsIDE, and the cysteine residue previously suggested as responsible for NEM sensitivity of
animal IDEs, respectively. Potential peroxisomal targeting signals at the C-termini are underlined.
is derived from the systemin sequence and includes
the Lys14-Met15 peptide bond processed by GST-SlIDE.
Highest activity was observed at neutral pH, and 50% of
the activity were retained at pH 6.5 and 8 (Figure 4B).
Enzymatic activity was stimulated by KCl (or NaCl) and
full activity was observed at rather high concentrations
of )800 mM (Figure 4A). Divalent cations at mM concentrations (Ca2q, Zn2q, Mg2q and Fe2q) failed to stimulate
GST-SlIDE activity in the absence of KCl.
Under optimum steady-state reaction conditions
(pH 7.0; 800 mM KCl), an apparent Km of 62"7 mM was
determined for the fluorigenic peptide substrate and the
catalytic efficiency (kcat/Km) was found to be 62"15
mM-1 s-1 (Figure 4C). Consistent with SlIDE being a zincdependent metalloprotease, its activity was inhibited by
52% of the control in the presence of 1 mM EDTA, while
PMSF as a serine protease inhibitor was ineffective (Figure 4D). Resembling mammalian IDEs, but unlike E. coli
pitrilysin, the tomato enzyme was efficiently inhibited by
1 mM NEM (51% of control activity; Figure 4D). A more
physiological inhibitor and also the most potent in our
assay is ATP which, at 1 mM, reduced the activity of GST-
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94 Y. Huet et al.
Figure 2 Expression and purification of GST-SlIDE.
SlIDE was expressed in E. coli as an N-terminal fusion protein
with glutathione S-transferase (GST) and purified from bacterial
extracts. A Coomassie Blue-stained SDS-PAGE gel is shown
with crude extracts from control and induced cultures in lanes
1 and 2, respectively. The bulk of the GST-SlIDE fusion protein
was insoluble (3). However, some of it was present also in the
soluble fraction (4) and was purified by affinity chromatography
on immobilized glutathione (5).
Figure 3 Processing of the oxidized insulin B chain and other
unrelated peptides by GST-SlIDE.
Degradation of synthetic peptide substrates was monitored,
and cleavage products were identified by MALDI-TOF mass
spectrometry. Peptide substrates (2.5 nmol) were incubated with
1–2 pmol GST-SlIDE in a 50 ml reaction volume at room temperature, and aliquots of the reaction were taken at several time
intervals for MALDI-TOF analysis. (A) GST-SlIDE rapidly cleaves
insulin B chain at three sites in less than 5 min (line 1, long
arrows), while five other peptide bonds are cleaved at a slower
rate, requiring 30 min to 2 h for complete processing (line 1,
short arrows). Arrows in lines 2 and 3 indicate the cleavage patterns previously determined for human IDE (42) and E. coli pitrilysin (43), respectively. (B) GST-SlIDE cleaves (arrows) several
unrelated peptides without preference for any particular amino
acid sequence motif.
SlIDE down to 5% of the control. In contrast to rat IDE,
which is inhibited by long chain fatty acids with an IC50
ranging from 9 mM for linoleic acid (18:2) to 44 mM for
palmitic acid (16:0) (Hamel et al., 2003), GST-SlIDE activity was not affected by linoleic acid between 25 mM and
2.5 mM (Figure 4D). Consistent with the apparent insensitivity to fatty acid inhibition, the consensus sequence
of cytosolic fatty acid-binding proteins (Prosite access
Figure 4 Catalytic properties of GST-SlIDE.
Unless otherwise indicated, GST-SlIDE activity was assayed in
20 mM potassium phosphate buffer at pH 7.0 in the presence
of 800 mM KCl using Abz-SKRDPPKMQTDLY(NO3)-NH2 (20 mM)
as the substrate. (A) GST-SlIDE activity is stimulated by KCl.
Proteolytic activity is expressed in arbitrary fluorescence units
and was measured in the presence of increasing concentration
of KCl up to 1.2 M. Data represent the mean"SD of three independent experiments. (B) GST-SlIDE activity as a function of pH.
GST-SlIDE activity was assayed in 20 mM MES, pH 5.0 to 6.5,
or 20 mM Tris-HCl, pH 7.0 to 10.0, in the presence of 800 mM
KCl. The activity is expressed in percent of the maximum activity
observed at pH 7.0. Data represent the mean"SD of three
experiments. (C) Substrate dependence of GST-SlIDE activity.
GST-SlIDE activity was assayed with increasing concentrations
of Abz-SKRDPPKMQTDLY(NO3)-NH2 and apparent catalytic
constants were derived from a double-reciprocal plot of the
data. The values represent the mean"SD of two replicates from
three independent experiments. (D) Residual activity was determined in the presence of 1 mM EDTA, NEM, PMSF, ATP, GTP or
25 mM linoleic acid. GST-SlIDE activity is expressed in percent
of the control. Data represent the mean"SD of three
experiments.
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Insulin-degrading enzyme from tomato 95
no. PS00214) previously detected in mammalian IDEs
(Hamel et al., 2003) is not present in the tomato
sequence. This signature sequence is defined by nine
residues, seven of which are conserved in human IDE,
but not in tomato (Figure 1).
Discussion
We have reported here the first cloning and biochemical
characterization of a plant IDE, a metalloproteinase from
the M16A subgroup in the inverzincin family of peptidases (http://merops.sanger.ac.uk; Rawlings et al.,
2004). The tomato (Solanum lycopersicum) cDNA for
SlIDE revealed 39% identity at the amino acid level to
the well-characterized human IDE, which is involved in
the intracellular degradation of insulin (reviewed by Duckworth et al., 1998). Expression in E. coli and biochemical
characterization of the recombinant tomato protein confirmed SlIDE as a true insulinase. Indeed, the tomato
enzyme cleaves the oxidized insulin B chain with a fragmentation pattern nearly identical to the one generated
by animal IDEs (Figure 3A; Duckworth et al., 1988). Moreover, its ability to cleave several unrelated short peptides
in vitro at apparently stochastic sites (Figure 3B) is a
characteristic feature of IDE (Duckworth et al., 1998),
pitrilysin (Cornista et al., 2004) and other M16 peptidases
of animal (Mzhavia et al., 1999; Falkevall et al., 2006) and
plant (Moberg et al., 2003) origin.
SlIDE behaves like a neutral thiol-dependent metalloprotease, with a pH optimum around 7 and ca. 50% of
its activity being inhibited by 1 mM EDTA or 1 mM NEM.
Sensitivity to thiol reagents (NEM) discriminates prokaryotic from higher eukaryotic IDE activities. While human
(Ding et al., 1992), Drosophila (Garcia et al., 1988) and
plant IDEs (this work) are efficiently inhibited, the E. coli
enzyme pitrilysin is not. Sensitivity to the thiol-modifying
compound has previously been attributed to the presence of a cysteine residue adjacent to the zinc-binding
histidines, which is conserved in human, rat and DroI EH), but absent from pitrilysin (Ding et
sophila IDEs (HXC
al., 1992; Perlman et al., 1993). In SlIDE, leucine is found
in the respective position which argues against a contribution of this residue to NEM sensitivity. This conclusion
is in agreement with the work of Perlman et al. (1993)
showing that substitution of the cysteine in human IDE
with either serine or glycine affected neither catalysis nor
inhibitor sensitivity.
Physiological regulators of IDE include nucleotide triphosphates (Camberos et al., 2001; Song et al., 2004)
and long chain fatty acids (Hamel et al., 2003) allowing
for the metabolic control of enzyme activity. Linoleic acid
(18:2) was shown to be the most potent fatty acid inhibitor with an IC50 of 9 mM for rat IDE (Hamel et al., 2003).
Consistent with fatty acid inhibition, mammalian IDEs
contain an almost perfect match to the consensus of the
‘cytosolic fatty-acid binding proteins signature’ sequence
(Figure 1; Prosite access no. PS00214). In contrast, SlIDE
was neither sensitive to linoleic acid inhibition (Figure 4D)
up to a concentration of 2.5 mM, nor does it contain
the aforementioned sequence motif. Interestingly, the
‘cytosolic fatty acid binding proteins signature’ can be
recognized in the protein sequences of mouse and dog
IDEs, but not in Arabidopsis and Drosophila homologs,
suggesting that the regulation by long chain fatty acids
may occur in mammals specifically.
In addition to fatty acids, ATP appears to contribute
to the metabolic control of IDE activity in mammals.
The inhibition of insulin degradation by ATP was first
observed in vivo (Hashimoto et al., 1987), and physiological concentrations of ATP were later shown to inhibit the
degradation of insulin by purified IDE in vitro (Camberos
et al., 2001). In contrast, Song and coworkers reported
an enhanced rate of degradation for small fluorigenic
peptide substrates in the presence of ATP, due to allosteric activation of IDE (Song et al., 2004, 2005; Yao and
Hersh, 2006). SlIDE was found to be inhibited by ATP at
physiological concentrations, suggesting maximum
SlIDE activity in planta under conditions of low cellular
ATP concentration. Depletion of the cytosolic ATP pool
occurs when oxygen availability limits the rate of respiratory ATP production. IDE activity would thus increase
in response to hypoxia, and a role for SlIDE in stress
adaptation and the regulation of energy homeostasis
under low-oxygen conditions may be envisaged. In this
context, it is of particular interest to note that the expression of one of the two IDE paralogs in Arabidopsis
(At3g57470) is induced by more than two-fold following
anoxia treatment as the only one out of many different
stress regimes (Zimmermann et al., 2004; https://
www.genevestigator.ethz.ch).
SlIDE activity was found to increase with increasing
salt concentrations well into the molar range (Figure 4A).
In previous studies, the activity of mammalian IDE was
shown to depend on the state of oligomerization (Song
et al., 2003; Li et al., 2006), and high salt concentrations
used in our experiments were suspected to alter the
equilibrium between the monomer and the more active
oligomeric forms. However, gel filtration analysis in the
presence of low (150 mM) or high (2 M) potassium chloride did not reveal any significant changes in the quaternary structure of SlIDE. The GST-SlIDE monomer was
undetectable, likely due to the fact that GST forms homodimers by itself. Three oligomeric forms of approximately
280, 420 and 600 kDa were observed, which likely represent the dimer, trimer and tetramer at an estimated
ratio of 1:2:3. The relative abundance of oligomers was
the same at low and high salt concentrations (data not
shown).
SlIDE cleaves several proline-rich oligopeptides,
including systemin, substance P and bradykinin in vitro,
and a proline residue was frequently observed proximal
to the hydrolyzed bond. Proline plays an important
physiological role in protecting biologically active peptides against enzymatic degradation and, indeed, many
proteases are unable to cleave peptide bonds in the
vicinity of this imino acid. Hence, a stabilizing proline residue is frequently found and conserved in peptide hormones, neuropeptides and growth factors in animals
(Mentlein, 1988; Yaron and Naider, 1993; Vanhoof et al.,
1995) and also in plant peptides with signaling function
(Ryan and Pearce, 2003; Ito et al., 2006). The stability of
such peptides is thus controlled by peptidases evolved
to specifically hydrolyze proline-containing substrates
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96 Y. Huet et al.
(Cunningham and O’Connor, 1997), and such a role may
be taken by SlIDE in planta.
Despite the low degree of sequence conservation, IDE
homologs in plants and in mammals appear to share
many of their biochemical features. However, the similarity does not seem to extend to physiological function:
insulin and b-amyloid peptide, two well-established substrates of human IDE, do not exist in plants. SlIDE was
identified in a functional screen for proteases able to
cleave the plant wound hormone systemin. While cleavage of systemin was confirmed for the recombinant
enzyme in vitro, this peptide is not likely to be a physiologically relevant substrate of SlIDE in vivo: SlIDE does
not co-localize with systemin in the leaf apoplast, plants
silenced for SlIDE expression were not affected in systemin-mediated wound signaling, nor did they show any
obvious growth defects under greenhouse conditions
(unpublished results). Therefore, the role of insulinases in
plants and their physiological substrates remain to be
characterized.
Materials and methods
Cloning of the SlIDE gene
A partial SlIDE cDNA was isolated in a genetic screen for proteases able to cleave the plant wound-signaling peptide systemin (AVQSKPPSKRDPPKMQTD). The screening procedure was
based on the loss of reporter gene expression in the yeast, Saccharomyces cerevisiae, as a result of the proteolytic inactivation of the modified GAL4 transcription factor and was adapted
from a method developed by B. Kohorn (Smith and Kohorn,
1991; Kohorn et al., 1992; Hauser et al., 2001). Briefly, a plasmid
carrying the yeast GAL4 gene was engineered for the expression
in yeast of a modified transcription factor, in which the systemin
sequence was inserted between its two functional domains. Proteolytic cleavage of systemin would then result in the separation
of the two domains and in the inactivation of GAL4. The plasmid
was introduced into a S. cerevisiae reporter strain carrying the
lacZ gene for b-galactosidase under control of the GAL1/10
promoter. A tomato cDNA library was constructed in pYES2
(Invitrogen, Groningen, Netherlands) with poly(A)q RNA isolated
from tomato (Solanum lycopersicum, cv. Castlemart II) shoot tissue (complexity of 3.5=106 independent cDNAs with an average
size of 1.4 kb) and used to transform the reporter strain. The
screening for cDNA clones conferring loss of GAL4 function was
performed as described previously (Smith and Kohorn, 1991;
Kohorn et al., 1992).
To obtain the full-length SlIDE cDNA, RACE (rapid amplification of cDNA ends)-PCR was performed using the SMART RACE
cDNA amplification kit (Clontech, Palo Alto, CA, USA), according
to the manufacturer’s instructions. In a first step, single stranded
cDNA was synthesized from total RNA of tomato leaves using
M-MLV reverse transcriptase (Promega, Madison, WI, USA) and
oligo(dT) as the primer. Subsequently, the full-length SlIDE cDNA
was amplified using a gene-specific primer (59-ACC TGT TGA
TAT GGC TAT TGG AAC TTG A-39) and the universal primer
provided with the kit. Three independent PCR products were
gel-purified, cloned into the pCR-XL-TOPO vector (Invitrogen)
and sequenced (accession no. of the full-length SlIDE cDNA is
AJ308542). The open reading frame of SlIDE was then amplified
by PCR using Pfu DNA polymerase (Stratagene, La Jolla, CA,
USA) and synthetic oligonucleotide primers (forward primer:
59-ATG GCA GTT GGT AAA AAG GA-39; and reverse primer: 59GGG GGG CTT CTC GAG ATG AT-39). The PCR product was
cloned into the StuI/SmaI sites of pGEX-G (Görlach and Schmid,
1996), a derivative of pGEX-3x (GE Healthcare, Uppsala,
Sweden), to yield pGEX-IDE. This construct allows expression
of SlIDE in N-terminal fusion with GST in E. coli. The identity of
all PCR-generated clones was confirmed by sequence analysis
of at least three independent PCR products using fluorescent
dideoxy chain terminators in the cycle sequencing reaction
(Perkin Elmer, Foster City, CA, USA) and the Applied Biosystems
model 373A DNA sequencer (Foster City, CA, USA).
Expression and purification of SlIDE
For expression of the GST-SlIDE fusion protein in E. coli DH5a,
two 500-ml cultures were grown at 378C to an OD600 of 0.8.
Isopropyl-1-thio-b-D-galactopyranoside was added to a final
concentration of 0.1 mM, and cells were grown for another
4 days at 48C when they were harvested by centrifugation
(6000 g for 15 min at 48C). The cells were resuspended in 100 ml
of buffer A (50 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA)
containing 0.1 mg/ml DNAseI and 1 mg/ml lysozyme. After
20 min at room temperature, cells were lysed by sonication. The
cell debris was removed by centrifugation (10,000 g for 30 min
at 48C), and the supernatant was subjected to affinity chromatography on glutathione-sepharose 4B (GE Healthcare), according to the manufacturer’s instructions. The affinity matrix was
washed with buffer A and buffer B (50 mM Tris-HCl, pH 8.0), and
the fusion protein was eluted with 10 mM glutathione in buffer
B. The progress of purification was monitored by SDS-PAGE
using the Laemmli buffer system (Laemmli, 1970), and the purified protein was stored at -808C.
MALDI-TOF MS assay of SlIDE specificity
The degradation of synthetic peptide substrates (obtained from
Sigma or Enzyme Systems Products, Livermore, CA, USA) by
SlIDE was assayed using MALDI-TOF mass spectrometry (MS)
to detect and identify the generated peptide fragments. In a total
volume of 50 ml of reaction buffer (50 mM Tris-HCl, pH 7.5), the
assay contained 1–2 pmol of the protease and 2.5 nmol of the
respective peptide substrate. The reaction mixture was incubated at room temperature, 0.8 ml aliquots were taken at intervals
and mixed on the MALDI-TOF MS sample plate with an equal
volume of the crystallization matrix w2 parts of saturated 2,5dihydroxybenzoic acid in acetone, 1 part 0.1% (v/v) trifluoroacetic acidx. Crystals were washed repeatedly with cold
de-ionized water and air-dried before recording of the mass
spectra with a Voyager Elite mass spectrometer (Applied
Biosystems).
Steady-state kinetic analyses
For the continuous assay of SlIDE activity, AbzSKRDPPKMQTDLY(NO3)-NH2 (Jerini AG, Berlin, Germany) was
used as an internally quenched fluorogenic substrate. In a total
volume of 50 ml, the standard assay contained 2 pmol of the
GST-IDE fusion protein, 20 mM of the substrate and 800 mM KCl
in 20 mM potassium phosphate buffer at pH 7.0. The fluorescence was recorded continuously in a Cary Eclipse fluorimeter
(Varian, Darmstadt, Germany; lex: 320 nm, lem: 420 nm) at 258C.
To convert relative fluorescence units into molar quantities of
reaction product, defined quantities of the substrate peptide
were digested to completion using an excess of enzyme and the
resulting fluorescence recorded. The inhibitor profile of SlIDE
was determined by preincubating the enzyme for 5 min with
1 mM EDTA (ethylenediaminetetraacetic acid), NEM (N-ethylmaleimide), PMSF (phenylmethylsulfonyl fluoride), ATP, GTP, or
25 mM linoleic acid. Control reactions were performed in parallel
and contained an equivalent volume of the solvents of the
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Insulin-degrading enzyme from tomato 97
respective inhibitors, i.e., water for EDTA and the nucleotides, and ethanol for NEM, PMSF and linoleic acid.
Acknowledgments
We greatly appreciate valuable advice and the gift of plasmids
and yeast strains from Dr. Bruce Kohorn (Duke University, NC,
USA) allowing us to perform the functional screen for peptidases
in yeast.
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Received June 17, 2007; accepted September 20, 2007