From the Department of Genetics, University of
Cambridge, Downing Street, Cambridge CB2 3EH, United Kingdom, the
Departments of ** Haematology and ¶ Medicine, University
of Cambridge, Cambridge Institute for Medical Research, Wellcome
Trust/Medical Research Council Building, Hills Road,
Cambridge, CB2 2XY, United Kingdom, and
Cambridge Centre for
Proteomics, Department of Biochemistry, University of Cambridge,
Downing Street, Cambridge CB2 1QW United Kingdom
Received for publication, September 10, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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Necrotic (Nec) is an important component of
the proteolytic cascade that activates the Toll-mediated immune
response in Drosophila. The Nec protein is a member
of the serpin (SERine Protease
INhibitor) superfamily and is thought to regulate the
cascade by inhibiting the serine protease Persephone. Nec was
expressed in Escherichia coli, and the purified protein
folded to the active native conformation required for protease
inhibitory activity. Biochemical analysis showed that Nec had a broad
inhibitory specificity and inhibited elastase, thrombin, and
chymotrypsin-like proteases. It did not inhibit trypsin or kallikrein.
These data show that Necrotic is likely to inhibit a wide range of
proteases in Drosophila and that Nec has the specificity
requirements to act as the physiological inhibitor of Persephone
in vivo.
The mammalian and insect innate immune responses are conducted
through highly conserved intracellular signaling cascades that allow
the host to deliver an efficient, rapid, and potent counterattack against invading microorganisms (1-5). However, while mammalian systems are primarily activated through Toll-like receptor 4 (TLR4),1 which directly
recognizes and binds microbial molecules (see Fig. 1a), the
Drosophila innate immune response is initiated through pattern recognition proteins circulating in the Drosophila
hemolymph (see Fig. 1b) (6). Recognition of molecules such
as peptidoglycan and lipopolysaccharide in the hemolymph
initiates an extracellular serine protease cascade, resulting in the
cleavage and activation of the Toll ligand, Spätzle (Spz) (6).
This serine protease cascade is tightly regulated by the protease
inhibitor, Necrotic (Nec) (also known as Spn43Ac) (7, 8).
Nec is a member of the serpin (SERine Protease
INhibitor) superfamily (7, 8). nec null mutants
result in constitutive expression of the antifungal Toll target gene,
Drosomycin, similar to that seen in Toll gain of function
alleles, and show Spz predominantly present in its cleaved form (8).
Furthermore, in flies containing nec;Tl or
nec;spz double mutations, Drosomycin is not
induced in the absence of immune challenge (8). Recently, mutations in
the catalytic triad of Persephone (Psh), a member of the serine protease family, were discovered to suppress the nec mutant
phenotype and activate the Toll pathway (9), confirming that Toll
activation is mediated through at least one serine protease, which is
in turn regulated by Nec.
Members of the serpin superfamily of proteins are involved in the
regulation of a large number of processes including coagulation (antithrombin), the acute phase response ( Sequence alignments against serpins of known structure and function
show that Nec has an Ala-rich hinge region and a predicted P1-P1'
Leu-Ser active site, suggesting it has an inhibitory profile similar to
that of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin
(
1AT) and
1-antichymotrypsin
(
1ACT)), and tissue repair (plasminogen activator
inhibitor-1 (PAI-1)) (10). The mechanism of
serpin-dependent protease inhibition requires that the
serpin reactive center loop (RCL) be presented as an ideal substrate
for its target protease (11). The protease binds and cleaves a peptide
bond at the P1-P1' position of the RCL (12). Cleavage results in an
SèR (stressed to relaxed) conformational change caused by the
rapid insertion of the RCL into a central 5-stranded
-sheet (Sheet
A). This rapid loop-sheet insertion takes place before the deacylation
step can be completed, thereby drawing the covalently attached protease to the opposite pole of the serpin molecule (13-15). The result is a
distortion of the catalytic site of the protease and its entrapment in
a covalently linked serpin-protease complex. Besides cleavage of the
RCL, a stable 6-stranded A-sheet can be formed by two other
conformational changes in the serpins. These forms are denoted the
latent (16) and the polymeric conformations (17-19). The RCL of the latent serpin is inserted into the
A sheet without cleavage, whereas the polymeric is
characterized by the sequential incorporation of the RCL of one serpin
into the A sheet of another. Both forms are inactive as inhibitors, and
mutations that favor these conformations are associated with a wide
variety of diseases (20).
1-antichymotrypsin (Fig.
1c) (7). Nec also has an
88-100-residue-long N-terminal extension that is unusual among
serpins. The extension contains regions rich in glutamines and
prolines, including a 24-residue polyglutamine repeat of unknown function (7).
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Fig. 1.
The mammalian (a) and
Drosophila (b) Toll-mediated innate immune
response are conserved intracellularly but are activated through
different mechanisms. In mammalian systems, the microbial sugar
lipopolysaccharide (LPS) binds directly and activates the
Toll-like receptor 4 (TLR4) in conjunction with the
co-receptor CD14. Activation of TLR4 initiates a secondary messenger
system involving the adapter proteins MyD88 and IRAK, resulting in the
degradation of I B and allowing the NF-
B transactivator to enter
the nucleus and activate expression of anti-microbial peptide
(AMP) genes. In Drosophila, fungal and
Gram-positive bacterial infection results in activation of pattern
recognition molecules (PRM), such as peptidoglycan
recognition protein (PGRP), circulating in the hemolymph.
These activated molecules activate a serine protease(s), such as Psh,
which result in the cleavage and activation of the Toll ligand, Spz.
Activated Spz (Spz*) signals through the Toll
(Tl) receptor, initiating an intracellular secondary
messenger cascade very similar to that of the mammalian system and
involving the MyD88 homologues Tube (Tub) and dMyD88, the
IRAK homologue Pelle (Pll), the I
B homologue Cactus
(Cact), and the NF-
B homologue (DIF).
Degradation of Cact allows the DIF transactivator to then enter the
nucleus, where it can induce the expression of target antimicrobial
peptides. c, a model of the native conformation of Necrotic
showing the 5-stranded
-sheet A (green) and
-sheet B
(yellow) that are required for structural stability, the RCL
(red), and the N-terminal extension (cyan)
leading into the
-helix A (blue) of the core serpin
structure. The Leu-Ser P1-P1' residues of the RCL are indicated.
The purpose of this study is to define the conformation and inhibitory
profile of Nec. To achieve this we have expressed and characterized the
recombinant protein. Using this material we have been able to show that
Nec acts as a potent elastase inhibitor but also strongly inhibits the
activity of thrombin and chymotrypsin. Nec does not inhibit trypsin and
kallikrein. Thus, Nec is likely to have a broad inhibitory specificity
in the fly and is potentially responsible for the inhibition of
multiple proteases. Additionally, Psh, which has a thrombin-like S1
architecture, is likely to be inhibited by Nec in vivo.
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MATERIALS AND METHODS |
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Materials--
Restriction enzymes were purchased from Roche
Molecular Biochemicals, T4 DNA-ligase was purchased from New
England Biolabs (Hitchin, Hertfordshire, UK), and oligonucleotides were
synthesized at Sigma. The IMPACT-NT protein purification system was
purchased from New England Biolabs. Porcine pancreatic trypsin, bovine
pancreatic -chymotrypsin, porcine pancreatic kallikrein, porcine
pancreatic elastase (PPE), and bovine serum thrombin were purchased
from Sigma. Human neutrophil elastase (HNE) was purchased from Athens Research and Technology (Athens, GA). The chymotrypsin substrate N-succinyl-Ala-Ala-Pro-Phe-para-nitroanilide, the
trypsin and kallikrein substrate
N-benzoyl-DL-arginine-para-nitroanilide, and the PPE/HNE substrate
N-methoxy-Ala-Ala-Pro-Val-para-nitroanilide were
purchased from Sigma. The thrombin substrate, S-2238
(H-D-Phe-pipecolyl-Arg-para-nitroanilide) was from Chromogenix (Quadratech, Epsom, UK). The rabbit anti-nec antibody was a kind gift from Dr. J.-M. Reichhart, Université Louis Pasteur, Strasbourg, France.
Protein Purification--
Expression and purification of Nec was
achieved by fusion with an N-terminal tag consisting of a
chitin binding domain and a protein splicing element (Intein). The
Intein sequence undergoes specific self-cleavage in the presence of
thiols, such as cysteine or DTT, allowing separation of the target
protein from the chitin-bound affinity tag. The Nec sequence with (Nec)
and without (Nec-N) the N-terminal extension was amplified using PCR
from nec cDNA (7) and cloned into the INTEIN
pTYB12 expression vector using SpeI and NotI
restriction sites. Escherichia coli BL21(DE3) cells containing the pTYB12-Nec-
N construct were induced at an OD of 0.5-0.6 with 0.1 mM
isopropyl-1-thio-
-d-galactopyranoside and grown overnight at
23 °C. Cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM EDTA, and lysed by sonication in the presence of a
mixture of protease inhibitors (Sigma). Lysis extracts were isolated by
high speed centrifugation and then applied to a column containing the
IMPACT-NT chitin-resin. On-column self-cleavage of the INTEIN sequence
was carried out by overnight incubation with 50 mM DTT at
4 °C. The protein was further purified through a Superdex 200 gel
filtration column in 100 mM Tris-HCl, pH 7.4 (Amersham Biosciences).
Circular Dichroism (CD)-- CD experiments were performed using a JASCO J-810 spectropolarimeter in 100 mM Tris-HCl, pH 7.4, at 25 °C. Thermal unfolding experiments were performed by monitoring the CD signal at 216 or 222 nm between 25 and 95 °C using a heating rate of 1 °C/min at a concentration of 0.2 mg/ml in a 0.05-cm pathlength cuvette. The TM was calculated through regression analysis as detailed previously (21). The results were the mean of three independent experiments.
Complex Formation Assays-- Recombinant Nec protein was incubated with varying amounts of serine proteases for 15 min at room temperature in 50 mM Hepes, 150 mM NaCl, 0.01% w/v dodecyl-maltoside, pH 7.4. The reactions were stopped through incubation with 1% w/v SDS-PAGE loading buffer and boiling for 3 min. The samples were pulse-centrifuged and then immediately run on a 10% w/v SDS gel, and the protein was visualized by staining with Coomassie Blue.
Determination of Reaction Parameters Describing
Protease Inhibition--
The inhibition rate constant
(ka) for Nec was determined for all serine
proteases except thrombin under pseudo-first order conditions
(i.e. [I] 10[E]0) using the progress-curve
method (22, 23). Rate constants of inhibition were measured at 25 °C
in 50 mM Hepes, 150 mM NaCl, 0.01% w/v
dodecyl-maltoside, pH 7.4, by adding the enzyme (20 nM
final) to a mixture of Nec protein (from 200 to 800 nM) and the appropriate substrate (1 mM) and recording the release
of product as a function of time. The progress curves were then
analyzed according to Equation 1 (22, 23).
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
Transverse Urea Gradient (TUG) PAGE-- TUG PAGE was carried out using 7.5% w/v polyacrylamide gels as detailed previously. Twenty-five µg of Nec protein was loaded on each gel and visualized with silver staining.2
Protein Identification by Mass Spectrometry--
Proteins within
excised gel pieces were digested to peptide fragments by modified
trypsin (Promega) using a Micromass MassPrep Station. 6.4 µl (~50%
of total) of the digestion supernatant was applied to a Micromass
quadrupole-time-of-flight/capillary liquid chromatography system for
liquid chromatography/tandem mass spectrometry analysis. Peptide
separation occurred using a PepMapC18 180 µm internal diameter, 15-cm
length capillary liquid chromatography column (LC Packings/Dionex San
Francisco, CA). Identification of the peptides analyzed was carried out
searching with uninterpreted fragmentation data using MASCOT
(Matrix Science Ltd., London, UK).
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RESULTS |
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Expression and Purification of Necrotic Protein--
Initial
attempts to express full-length Nec resulted in no detectable protein,
possibly due to secondary structure within the GC-rich mRNA region
coding for the poly-Q tract. A new truncated gene was constructed
containing the serpin core structure but without the first 99 residues
of the N terminus (Nec-N). The residues "Ala-Gly-His-Met-Thr"
were added to the N terminus of the Nec-
N protein as a result of
fusion to the chitin-binding protein so that the N terminus of the
recombinant protein is AGHMTPPPVF (Fig.
2a). Nec-
N eluted from the
chitin column at ~90% purity after overnight cleavage with DTT as
determined by SDS-PAGE (Fig. 2b). The protein was further
purified using Superdex 200 gel filtration to remove low molecular mass
contaminants and protein aggregates (Fig. 2c), producing a
final yield of 3 mg of pure monomeric Nec-
N per 6 liters of culture.
The identity of Nec-
N was confirmed by Western blot analysis using
an antibody to Nec protein (not shown). Moreover, matrix-assisted laser
desorption ionization mass spectrometric analysis produced 47 peptide
matches covering 45% of the Nec sequence.
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Characterization of Nec-N--
Stability studies conducted on
the purified protein revealed that Nec-
N was folded and contained
-helix and
-sheet secondary structures characteristic of serpins
(Fig. 3a). Protein stability measurements made using circular dichroism revealed a loss of secondary
structure at 222 nm at 48.4 ± 0.7 °C (Fig. 3b). To
test whether RCL cleavage resulted in a stable, relaxed species,
trypsin-cleaved Nec-
N was analyzed by TUG electrophoresis. Nec-
N
was incubated with porcine pancreatic trypsin at an enzyme to serpin
molar ratio of 1:500 in buffer A (50 mM Hepes, 150 mM NaCl, 0.01% w/v dodecyl-maltoside, pH 7.4) for 15 min
at 22 °C. The resulting protein had a migration profile on 10% w/v
SDS-PAGE characteristic of a RCL-cleaved serpin. Assessment by TUG-PAGE
revealed two different profiles: a classic "S" curve,
representative of the gradual unfolding that is characteristic of a
native serpin, and a species that was stable at higher urea concentrations, indicative of partial or complete loop insertion into
-sheet A (Fig. 3c). These results demonstrate the
stressed to relaxed transition required for the serpin
inhibitory mechanism. Taken together, these data are typical of serpins
in their native monomeric conformation as opposed to the latent,
RCL-cleaved or loop-sheet serpin polymers and suggest proper folding of
recombinant Nec-
N.
|
Protease Inhibition by Nec-N--
Serpin inhibition results in
a stable serpin-protease complex joined through an ester linkage
between the P1 residue from the serpin RCL and the catalytic serine
from the protease. To test for potential inhibitory activity, Nec-
N
was incubated with a select panel of serine proteases: bovine
pancreatic
-chymotrypsin, HNE, PPE, bovine serum thrombin, porcine
pancreatic trypsin, and porcine pancreatic kallikrein. Formation of a
covalently linked serpin-protease complex was observed as a cathodal
band shift on a 10% w/v SDS-PAGE. Nec-
N forms complexes with the
thrombin, neutrophil elastase, pancreatic elastase, and chymotrypsin
after a 15-min incubation at 22 °C (Fig.
4). Increasing serpin concentration while
the protease concentration remained constant showed an increase in the
complex band at 60-70 kDa and the cleaved serpin at 35-40 kDa coupled
with a decrease of native Nec-
N. The identity of these bands was
confirmed for the Nec-thrombin reaction using mass spectrometric
analysis with the complex band yielding 37 peptides matching 34% of
the Nec sequence and 19 peptides matching 21% of the thrombin
sequence. The cleaved band produced 36 peptides covering 34% of the
Nec sequence. The other minor bands observed are most likely due to
degradation of major species by uninhibited protease.
|
In contrast, Nec-N failed to form a complex with trypsin and
kallikrein. Comparison of 15-min incubations using 1:1 and 1:4 enzyme
to serpin molar ratios showed an increase in Nec-
N degradation as
more serpin was added and no complex formation. Experiments in which
the trypsin and kallikrein concentration was increased and Nec-
N
kept constant (enzyme to serpin molar ratios of 1:0.02, 1:0.16, 1:0.8,
1:4, and 1:20) resulted in increased Nec degradation in association
with increased enzyme and no complex formation. Finally, an incubation
of 1:0.16 Nec to enzyme molar ratio at 22 °C that was stopped at 10, 15, and 20 min by boiling in 1% w/v SDS buffer showed an increase in
Nec degradation over time and no complex formation. This repeated
failure to form an inhibitory complex between Nec-
N and the
proteases trypsin and kallikrein show that Nec-
N is not an active
inhibitor of these serine proteases.
Inhibition Kinetics of Nec-N--
The kinetics of Nec-
N
inhibition was further examined through the determination of both the
association rate constant (ka) and the
stoichiometry of inhibition (SI) values for each protease from the
panel. Each reaction was conducted at room temperature (22 °C) in
buffer A. ka values for all proteases except
thrombin were determined under pseudo-first order conditions using the progress curve method and were repeated at least three times over a
range of concentrations of Nec-
N (between 100 and 800 nM) and 20 nM enzyme in the presence of excess
substrate (1 mM) (Fig. 5).
The thrombin association rate constant was determined under pseudo-first order conditions using the discontinuous method, where
enzyme and inhibitor were preincubated for varying amounts of time
before substrate is added using time points of 0, 3, 5, 10, 15, 25, and
35 min. The results showed that the interaction between Nec-
N and
elastase-like proteases was the most rapid (Table I), with
ka values of 1.1 × 105
M
1 s
1 for HNE and 5.7 × 105 M
1 s
1 for PPE.
Rapid association constants of chymotrypsin and thrombin with Nec-
N
were also observed, with ka values of 4.4 × 104 M
1 s
1 and
1.0 × 104 M
1
s
1, respectively. The SI values showed that a 1:1
Nec-
N to enzyme ratio is sufficient for complete inhibition of HNE,
while higher concentrations of the serpin are needed for total
inhibition of PPE (SI = 2.2), chymotrypsin (SI = 2.0), and
thrombin (SI = 4.4). In contrast the ka and
SI values for the interaction between trypsin and Nec-
N, and
kallikrein and Nec-
N, could not be measured as no inhibition of
protease was observed.
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DISCUSSION |
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The aim of this study was to determine the biochemical
characteristics of the serine protease inhibitor Nec. Nec plays a
central role in the control of a proteolytic cascade responsible for
modulating the innate immune response in Drosophila. Nec was
identified as a regulator of the cascade through analysis of
necrotic gene knockouts (8). Nec is a member of the serpin
superfamily but differs from other serpins in having a novel N-terminal
extension containing a glutamine-rich sequence. This extension
abolished protein expression in E. coli, resulting in the
need to prepare a shortened construct (Nec-N) that contained only
the common serpin core. The design of the construct was based on
homology with antithrombin (25) and
1-antichymotrypsin
(13). A few other inhibitory serpins contain an N-terminal extension,
but none are as extensive as that found in Necrotic. These include
antithrombin and heparin cofactor II; in the former case the extension
is involved in forming a heparin binding site (26), whereas in the
later it provides an additional protease binding site (27). The N
terminus of Nec has little sequence homology with either of these
proteins making it difficult to assign a function. However, our study
of the shortened construct shows clearly that the N terminus is not required for inhibitory activity.
Members of the serpin superfamily inhibit proteases by undergoing a
striking conformational change with transition of the proteinase from
the upper to the lower pole of the inhibitor. The same transition may
also occur in association with point mutations and chaotropic
conditions to form inactive latent and polymeric species (16-19). It
is thus important to ensure that a conformationally pure sample of the
serpin has been obtained. SDS-PAGE of our purified sample showed that
Nec migrated as a single band at ~42 kDa. It was confirmed to be Nec
by Western blot and mass spectrometric analysis. The final step of the
purification process was gel filtration, which resulted in a single
peak, demonstrating that our purified product was monomeric. Circular
dichroism spectra of our recombinant protein gave a characteristic
signature for a sample containing both -helix and
-sheet
secondary structure. This is similar to the profile of other inhibitory
serpins such as
1-antitrypsin (21) and suggests the
correct folding of purified Nec-
N. Circular dichroism readings at
222 nm with temperatures increasing from 20 to 95 °C resulted in a
loss of
-helical secondary structure at 48.4 ± 0.7 °C,
showing that Nec-
N is in its stressed, native form. TUG analysis of
our sample showed typical unfolding of native Nec-
N between 1 and 3 molar urea. Cleavage of the RCL with trypsin resulted in a species with
greater stability at higher urea concentrations as a result of loop
insertion. Taken together, these results demonstrate successful
purification of active Nec-
N in its monomeric, native conformation.
The physiological role of an inhibitory serpin is defined by its
protease specificity. It is thus important that a study of the spectrum
of specificity for Necrotic be carried out. A panel of mammalian
proteases was chosen by virtue of each being an easily available, well
characterized member of a specific serine protease family. Results from
this study show that Necrotic has a relatively promiscuous specificity
being able to inhibit members of the elastase, thrombin, and
chymotrypsin families. We have shown that Nec-N successfully forms
inhibitory complexes with each protease and has significant association
rate constants in excess of 104
M
1 s
1 for each. Furthermore,
Nec-
N failed to form inhibitory complexes with both trypsin and
kallikrein, demonstrating that it retained some selectivity. Thus,
Necrotic may act in vivo on a number of serine proteases
from select protease families, performing a role as a broad-spectrum
protease inhibitor.
Recent studies have revealed that mutations of the gene encoding for
the serine protease, Psh, repress the nec
phenotype. However, it remains unclear from genetic analysis whether
Nec acts as a direct inhibitor of Psh or whether it inhibits an
upstream protease which in turn activates Psh or a combination of both.
The specificity of chymotrypsin-class serine proteases can be predicted
in part based on the composition of the S1 specificity pocket (28).
Analysis of the S1 specificity pocket architecture of Persephone, based
on key residues 188, 189, 216, 226, and 228 (chymotrypsin numbering)
(29, 30) and three-dimensional structure predictions, suggest that it
is similar to thrombin-like proteases. This matches well with the
inhibitory specificity determined here for Necrotic, strengthening
further the proposal of a direct interaction between these proteins
in vivo.
This study has shown that Nec exhibits strongest inhibition of
proteases with elastase-like specificity with an association rate
constant of >105
M1s
1 for both PPE and HNE
proteases. Elastase specificity pockets typically have a Val residue at
position 216 (chymotrypsin numbering) distinguishing them from other
chymotrypsin class serine proteases in which this position is
restricted to a glycine residue (31, 32). It is thereby likely that Nec
would have inhibitory activity against Drosophila proteases
containing this motif.
Studies of the TLR pathways in mammals have recently uncovered a number
of possible roles for serpins. It has also been understood for a long
time that antithrombin regulates the blood coagulation cascade by
inhibiting the serine proteases thrombin and the thrombin activator,
factor Xa. More recent studies have shown that thrombin can also
stimulate the release of nitric oxide via NF-B activation (33) and
that extracellular antithrombin can also inhibit
lipopolysaccharide-induced activation of NF-
B via the TLR4 receptor
in mammalian cell cultures (34, 35). These reports indicate that the
similarity of mammalian and Drosophila innate immune
responses may well extend to the extracellular cascades and are
strengthened by our finding that Nec can inhibit thrombin activity.
Several considerations should be taken in understanding the results of
this study. First, it is possible that Nec-N may lack some of the
specificity-determining regions that could be present in the N-terminal
extension. Alternatively, the N terminus may alter the rate of Nec
association or stoichiometry of inhibition with its target protease by
influencing the stability of either the native serpin or of the
serpin-protease complex. Resolution of this can only be achieved by
further study of the in vivo function of the protein in
combination with similar studies of the full-length protein. In
addition, because these experiments were conducted using mammalian
rather than Drosophila serine proteases, the true target of
Nec may have a higher ka value than the
association rate constants reported in this study. Moreover, the fly
microenvironment is more complex than that used in these in
vitro experiments, possibly generating high localized
concentrations of both enzyme and inhibitor. It remains likely,
however, that the physiological target(s) of Necrotic will have a
specificity that is included within the subset of those serine
proteases shown to interact in this study.
Overall this study has shown conclusively that Nec has the potential to
act as a competent serine protease inhibitor, with a relatively wide
range of specificities. This suggests that Nec may have multiple
targets in vivo and presents the opportunity to predict
physiological targets of Nec based on analysis of the specificity
pocket architecture of Drosophila proteases. It has also
been demonstrated that the N-terminal extension is not required for
inhibitory activity but does not exclude the possibility that it
functions to either modify specificity or influence the association rate through altering the stability of Nec. Finally, our data supports
the model that Nec inhibits the Toll-gated innate immune response
through the direct inhibition of the serine protease, Psh.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Aiwu Zhou of the University of Cambridge, Cambridge, UK, for his help with the TUG-PAGE experiments. We thank Dr. Jean-Marc Reichhart of the Université Louis Pasteur of Strasbourg, France, for the kind gift of the Nec antibody and nec cDNA, for many helpful scientific discussions, and for his review of this paper.
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FOOTNOTES |
---|
* This work is supported by the Wellcome Trust and the Medical Research Council (UK). The Cambridge Centre for Proteomics is a Drosophila proteomics facility funded by the Biotechnology and Biological Sciences Research Council Investigating Gene Function Initiative.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Gates Cambridge Fellow.
Medical Research Council Career Development Fellow. To whom
correspondence should be addressed: Dept. of Biological Sciences, University of Manchester, 2.205 Stopford Bldg., School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT,
UK. Tel.: 44-0-161-275-5538; Fax: 44-0-161-275-5082; E-mail: tim.dafforn@man.ac.uk.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M209277200
2 A. Zhou, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: TLR, Toll-like receptor; Spz, Spätzle; Nec, Necrotic; serpin, SERine Protease INhibitor; Psh, Persephone; RCL, reactive center loop; PPE, porcine pancreatic elastase; HNE, human neutrophil elastase; DTT, dithiothreitol; CD, circular dichroism; TUG, transverse urea gradient; LC, liquid chromatography; SI, stoichiometry of inhibition.
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