(Received for publication, March 29, 1995; and in revised form, June 12, 1995)
From the
Three insect peptides showing high sequence similarity and
belonging to the same structural family incorporating a cysteine knot
and a short three-stranded antiparallel -sheet were studied. Their
inhibitory effect on two serine proteases (bovine
-chymotrypsin
and human leukocyte elastase) is reported. One of them, PMP-C, is a
strong
-chymotrypsin inhibitor (K
= 0.2 nM) and interacts with leukocyte
elastase with a K
of 0.12
µM. The other two peptides, PMP-D2 and HI, interact only
weakly with
-chymotrypsin and do not inhibit leukocyte elastase.
Synthetic variants of these peptides were prepared by solid-phase
synthesis, and their action toward serine proteases was evaluated. This
enabled us to locate the P1 residues within the reactive sites (Leu-30
for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants
of PMP-D2 and HI were converted into powerful inhibitors of both
-chymotrypsin and leukocyte elastase, the most potent elastase
inhibitor obtained in this study having a K
of 3 nM.
In the last decade, naturally occurring serine protease
inhibitors (1) have been the focus of many studies, mainly for
two reasons: first, the target proteins control functions in a variety
of fundamental proteolytic processes in humans and mammals (blood
clotting, digestion, inflammation, fibrinolysis), in invertebrates such
as insects (immune system, digestion, protection against their
predators) or worms (protection against their host), and plants
(protection against insect attack); second, low molecular weight
inhibitors of serine proteases have been attractive tools for studying
the general aspects of protein conformation and protein-protein
interactions(2) . In the present study, we report the
inhibitory properties of three homologous peptides (primary and
tertiary structures) toward -chymotrypsin, trypsin, and human
leukocyte elastase.
We have previously isolated two peptides, PMP-C and PMP-D2, from the brain and the fat body of the insect Locusta migratoria(3) . These peptides are composed of 36 and 35 residues, respectively, and are cross-linked by three disulfide bonds. The Thr-9 of PMP-C has an uncommon O-glycosidic linkage to a single fucose moiety. There is 40% strict identity between PMP-C and PMP-D2 with conservation of the Cys positions(3) . Moreover, they are located on the same peptidic precursor, and, by Northern blot analysis, it has been shown that the gene encoding this precursor is mainly transcribed in the fat body(4) . In this paper, we describe the isolation and characterization of HI, a novel locust peptide.
Because the isolation from insect extracts is
time-consuming and yields only small amounts of peptides, we have
prepared, at a reasonable scale (5-10 mg), by solid-phase
synthesis, PMP-D2 (5) and PMP-C with and without the fucose
moiety. ()Although they are small peptides with a high
disulfide content, no sequence similarities could be found when
comparing them with small toxins or small protease inhibitors. However,
the milligram quantities of PMP-D2 obtained by solid-phase synthesis
enabled us to study its tertiary structure by two-dimensional nuclear
magnetic resonance, which showed interesting similarities with the
tertiary fold of both
-conotoxin GVIA, a calcium channel blocker,
and the Ascaris chymotrypsin/elastase inhibitor(6) .
This prompted us to evaluate their protease inhibitory activity.
In
the present paper, we report on the inhibitory activity of PMP-C,
PMP-D2, and HI toward serine proteases (bovine -chymotrypsin,
human leukocyte elastase, and porcine trypsin) using the natural and
synthetic peptides. Since the P1 residue (7) within the
reactive site of serine protease inhibitors determines the specificity
for the cognate enzyme, mutational or synthetic changes of the P1
residue and/or replacement of active site residues should greatly
influence both the specificity and the potency of the inhibition. For
that reason, we have prepared by solid-phase synthesis variants (
)of PMP-C, PMP-D2, and HI, in which one or two residues
within the reactive site have been changed, and we have evaluated their
inhibitory properties toward HLE, (
)
-chymotrypsin and
trypsin.
The authenticity of the PMP-C and PMP-D2 was assessed by electrospray mass spectrometry on a VG BioTech BIO-Q mass spectrometer and by coelution with PMP-C and PMP-D2 obtained after isolation from the brain and the fat body of the same insect(3) .
The peptide referred to as HI was reduced and alkylated by 4-vinylpyridine (as described in (3) ) and was then subjected to automated Edman degradation on an Applied Biosystems Sequencer, model 471A in the liquid pulse mode, which yielded a 35-amino acid sequence (Fig. 1). The molecular mass of HI, as determined by electrospray mass spectrometry, was 3716.43 ± 0.18 Da, which is in excellent agreement with the sequencing data (deduced mass M = 3722.28 Da minus 6 for 3 disulfide bridges).
Figure 1: A, amino acid sequence of the peptide named HI and comparison with the sequence of PMP-D2 (72% identity); B, sequence comparison between PMP-C and PMP-D2 (a gap is introduced to maximize the homology between the two sequences).
Peptide purity was estimated to be 95% by analytical RP-HPLC using a stepwise gradient as for the purification of HI. Overall yields, based on resin, were usually between 2 and 3%. The authenticity of the synthetic peptides was checked by electrospray mass spectrometry. Table 1shows the deduced mass from Edman degradation and the measured mass of the different variants (the 6-Da difference between the mass deduced from amino acid sequence and that measured by electrospray mass spectrometry is due to the involvement of the 6 Cys in 3 disulfide bonds).
Trypsin inhibition was assessed by reacting 4 µM enzyme with 16 µM peptide for 30 min at 25 °C and measuring the enzyme activity with 1 mM benzoyl-Arg-pNA.
Fig. 2shows the effect of increasing quantities of synthetic
PMP-D2 on constant quantities of chymotrypsin. Substrate was added to
equilibrium mixtures of enzyme and inhibitor. The release of p-nitroanilide versus time was stable after
20-30 s indicating that E, I, S (enzyme, inhibitor and
substrate, respectively), and their complexes have reached their
equilibrium. Calculation of the best estimate of the
substrate-dependent equilibrium constant K was performed by nonlinear
regression analysis of the experimental data based on (13) :
Figure 2:
Inhibition of -chymotrypsin by
PMP-D2. A constant
-chymotrypsin concentration (6 µM)
was incubated at 25 °C for 10 min with increasing concentrations of
PMP-D2 (5.2 to 52 µM). After addition of
glutaryl-Phe-pNA (1.4 mM, final concentration), the
release of p-nitroanilide was followed at 410 nm. Fractional
activity (a) is defined in the text. Experimental points
(+) and theoretical line(-) generated using and
the best estimate of K
.
where a, the enzymic fractional activity, is the ratio
of the velocity in the presence of inhibitor to that in its absence.
The true K (Table 2) was deduced from K
using the following
relationship: K
= K
/(1 +
[S]
/K
),
where[S]
is the initial substrate concentration.
The K
value for natural PMP-D2 was shown to be
identical with that of the synthetic peptide (Table 2).
Equilibrium dissociation constants governing the interactions between chymotrypsin and synthetic HI and between HLE and PMP-C (natural and synthetic nonfucosylated) were determined using similar equilibrium titration experiments (Table 2).
Since PMP-C
(natural and synthetic nonfucosylated) binds chymotrypsin very tightly, K was obtained through k
and k
. These parameters were determined
using the progress curves method(13, 14) . A typical
curve illustrating chymotrypsin inhibition by nonfucosylated PMP-C is
shown in the inset of Fig. 3A. The curve is
biphasic, i.e. the pre-steady state release of product is
followed by a steady state, confirming that PMP-C reversibly interacts
with chymotrypsin. Since no significant decrease of the initial
substrate concentration occurred during the progress of the reaction
and, since I
10
E
,
product accumulation versus time can be described by the
following equation:
Figure 3:
A, effect of increasing concentrations
of synthetic nonfucosylated PMP-C (0.14 to 0.72 µM) on the
apparent first order rate constant (k). The inset shows a progress curve for the inhibition of -chymotrypsin:
the substrate Suc-Ala
-Pro-Phe-pNA (0.25
mM) and PMP-C (0.43 µM) were allowed to
equilibrate in the cuvette, and the reaction was initiated by the
addition of
-chymotrypsin (10 nM, final concentration),
the release of p-nitroanilide was followed for 6 min. The
theoretical curve is generated using and the best estimate
of k. B, effect of increasing concentrations of
Suc-Ala
-Pro-Phe-pNA (1/F) on the apparent
first order rate constant (k) of the
-chymotrypsin
PMP-C complex (F = 1 +
[S]
/K
).
where P is the product concentration, v is the rate of substrate hydrolysis at t = 0, v
is the steady state velocity. k, the
apparent first order rate constant governing the pre-steady state was
calculated from the experimental data by nonlinear regression analysis
using . Chymotrypsin inhibition was studied using synthetic
nonfucosylated PMP-C concentrations varying from 0.14 to 0.72
µM. Fig. 3A shows the effect of
[I]
, the initial inhibitor concentration on the
apparent rate constant k; the linear increase of k strongly suggests that no reaction intermediate accumulates
(within the range of inhibitor concentrations used) and that E and I interact according to a simple bimolecular and reversible
mechanism.
Fig. 3B shows the effect of the initial
substrate concentration on k; the linear increase of k with 1/F (F = 1 +
[S]/K
) indicates that
inhibitor and substrate compete for the binding to the enzyme. Hence, k and I
are related as follows(15) :
where K is the Michaelis constant. k
, the second order rate constant was calculated
from the slope of the linear curve shown in Fig. 3A using K
= 23 µM. k
, the first order dissociation rate constant,
is given by the intercept of the curve with the ordinate. The values of k
, k
, and K
(K
= k
/k
) of fucosylated and
nonfucosylated PMP-C are shown in Table 2; these kinetic
constants are similar, indicating that the fucose moiety does not
affect chymotrypsin inhibition.
The inhibition properties (toward chymotrypsin and HLE) of the variants of PMP-C, PMP-D2, and HI were examined using similar methods, and the results are given in Table 2.
As shown in Table 2, the L30V
variant lost most of its ability to inhibit -chymotrypsin.
However, it retained its weak activity toward HLE (Table 2). The
spectacular change in the inhibition of
-chymotrypsin observed
with the L30V variant indicated that it was possible to lose
specifically the inhibitory activity toward chymotrypsin, while
retaining the anti-elastase property. Therefore, we concluded that the
changed amino acid Leu was the P1 residue, the decrease in affinity
being due to an inappropriate P1 residue and not to the misfolding of
the PMP-C variant.
Thus, the most probable
reactive sites P1-P`1 in PMP-D2 and HI are Arg-29-Lys-30. To confirm
this hypothesis, the presumed P1 residues (Arg) were replaced by Leu in
PMP-D2 and HI in order to have the same reactive site Leu-Lys as in
PMP-C. The potency of both R29L variants toward -chymotrypsin
inhibition was increased significantly, and they were both converted
into HLE inhibitors. The PMP-D2 variant was found to be the strongest
inhibitor of HLE obtained in this study (Table 2).
When
comparing the amino acids within the reactive site P3-P`3 of PMP-C and
the R29L variants of HI and PMP-D2, one can notice the strict identity
of the recognition site between PMP-C and the HI variant (CTLKAC),
whereas PMP-D2 variant has a Gly in the P`2 position (CTLKGC) instead
of Ala; these results are indicative of an Ala in position P`2 which
may be preferable to Gly for -chymotrypsin inhibition, while the
contrary is true for elastase inhibition.
To investigate the role of the residues beyond the P1-P`1 bond, we
have prepared a double variant of PMP-C: K31M/A32G (which has the same
P3-P`3, CTLMGC, as the most powerful elastase inhibitor R29L/K30M
variant of PMP-D2) and examined its inhibitory activity; this variant
is still 7- to 8-fold weaker HLE inhibitor than the double variant of
PMP-D2 (a Met as P`1, in this case, does not increase the potency
toward HLE). In contrast, the double variant of PMP-C is more effective
toward -chymotrypsin than the PMP-D2 double variant. Thus, the
residues beyond the P1-P`1 bond seem to have an effect on the
specificity toward proteases, and variants of PMP-D2 seem to be more
specific toward elastases than the variants of PMP-C.
The results of this study clearly show that PMP-C and PMP-D2
differ significantly with respect to their selectivity toward serine
proteases, even though they exhibit a high sequence homology (45%) and
are structurally related. ()It seems reasonable to
hypothesize that a common ancestor might have adapted for specific and
diverse biological functions by punctual mutations that do not affect
the overall three-dimensional structure. In that respect, it is
remarkable that a unique substitution in the reactive site of PMP-D2
(from Arg to Leu) is sufficient to restore the serine protease
inhibitory activity.
Since PMP-C is a tight-binding reversible inhibitor and has a small and compact shape and an exposed binding loop, we propose to include it in the large group of the ``small canonical tight-binding serine protease inhibitors.'' This group of protease inhibitors consists of 16 different families and includes peptides ranging from 29 to 120 amino acids(2) . Interestingly, the peptides of this group are structurally unrelated, but share some properties such as hydrophobic cores (often maintained by disulfide bonds), stability toward unfolding, and, more remarkably, an exposed binding loop (containing the scissile P1-P`1 bond) that fits into the active site cleft of the serine protease. The specificity of serine protease inhibitors is significantly, but not exclusively, determined by the nature of the P1 residue in the reactive site. Although in most families of proteins, the active regions are highly conserved, in the serine protease inhibitors, there is no consensus sequence for the reactive site emerging yet. Indeed, retention of activity in these proteins exists even though the P1 residue has been changed(1) . In some cases, substitutions lead to a predictable change in the inhibitory specificity(17, 19, 20, 21) .
Taking into account the variability of the P1 region, we have designed ``variants'' of PMP-C, PMP-D2, and HI by targeting precisely the amino acid replacement. The aim of this study was to determine the P1 residue of the peptides and to increase the affinity toward HLE, since a variety of elastase inhibitors have been shown to be effective in animal models of emphysema, acute respiratory distress syndrome, rheumatoid arthritis, cystic fibrosis, bronchitis, or acute pancreatitis.
We have proved that Leu-30 is actually the P1 position
by modulating successfully the inhibitory properties of PMP-C
(maintaining its activity toward HLE and decreasing it toward
-chymotrypsin) with the L30V variant. This result is unambiguously
confirmed in the case of PMP-D2 and HI, where Arg-29 (equivalent to
Leu-30 in PMP-C) was replaced by a Leu; the R29L variants are potent
-chymotrypsin inhibitors and they present a reasonable affinity
toward HLE.
Surprisingly, the replacement of the P1 = Leu-30 (in PMP-C) by Val increased the affinity toward HLE by a factor of 3 only. Indeed, several novel elastase inhibitors were obtained by replacing the P1 for Val(19, 20, 21) . Our results highlight the significant role of the nature of the amino acids within the reactive site other than the P1 side chain for elastase inhibition. We therefore evaluated the effect of the P`1 residue (large and basic side chain) by replacing the Lys-30 in the R29L variant of PMP-D2 (which was presently the most effective HLE inhibitor) by a Met (often found in P`1 position of HLE inhibitors), and the increased affinity of this double variant for HLE confirmed our previous observation. Up to now, the most appropriate P3-P`3 sequence for elastase inhibition is CTLMGC; however, it should be noticed that the double variant of PMP-D2 is more powerful than the equivalent double variant of PMP-C K31M/A32G; this is indicative of a sequence and/or conformational effect which influences the reactive site binding loop.
It is noteworthy that although PMP-C, PMP-D2, and HI have several Lys residues in their sequences, and PMP-D2 and HI have an additional Arg, none of these peptides inhibits porcine trypsin. This result is surprising, considering that many natural trypsin inhibitors have a Lys as P1 residue and that most synthetic substrates of trypsin have an Arg as P1 residue.
While this work was in progress, Boigegrain et
al. (22) have isolated from the hemolymph of L.
migratoria two peptides with sequences identical with PMP-C and
PMP-D2. They have shown that both PMP-C and PMP-D2 are powerful
inhibitors of -chymotrypsin (K
of 0.25 nM and 0.12 nM, respectively) and weak to medium inhibitors
of HLE (K
> 0.1 µM and of 18
nM, respectively). These values are significantly different
from ours. We have shown in this work that PMP-D2 interacts only weakly
with
-chymotrypsin (K
of 1.5 µM)
and does not inhibit HLE, and, similarly, PMP-C has a K
of 0.12 µM instead of a K
>
0.1 µM toward HLE. We have no explanation for this
discrepancy.
It will be interesting to further characterize which amino acid(s) outside the reactive site and which part(s) of the framework are essential for the protease inhibitory activity. This should help to design more affine and smaller peptidic analogues or peptidomimetics, which are required for a therapeutic use of these peptides.