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*
Center for Blood Research, Boston, MA 02115; and
Department of Veterans Affairs Medical Center, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106
| Abstract |
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Val) indicated a role for P2 in specificity
determination. To define this role and that of other reactive center
loop residues, a number of different amino acids were introduced at P2,
as well as at P6 (Ala439) and P8'/9'
(Gln452Gln453). Ala439
Val is a
naturally occurring mutant observed in a patient with hereditary
angioedema. Previous data suggested that
Gln452Gln453 might be a contact site for C1s.
Reactivity of the inhibitors toward target (C1s, C1r, kallikrein,
factor XIIa, and plasmin) and nontarget proteases (
-thrombin and
trypsin) were studied. Substitution of P2 with bulky or charged
residues resulted in decreased reactivity with all target proteases.
Substitution with residues with hydrophobic or polar side chains
resulted in decreased reactivity with some proteases, but in unaltered
or increased reactivity with others. Second order rate constants for
the reaction with C1s were determined for the mutants with activities
most similar to the wild-type protein. The three P2 mutants showed
reductions in rate from 3.35 x 105
M-1s-1 for the wild type to 1.61, 1.29, and
0.63 x 105 for the Ser, Thr, and Val mutants,
respectively. In contrast, the Ala439
Val and the
Gln452Gln453
Ala mutants showed little
difference in association rates with C1s, in comparison with the
wild-type inhibitor. The data confirm the importance of P2 in
specificity determination. However, the P6 position appears to be of
little, if any, importance. Furthermore, it appears unlikely that
Gln452Gln453 comprise a portion of a protease
contact site within the inhibitor. | Introduction |
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sheets and eight or nine
helices
(7). Serpins act as suicide substrates in which the amino
acid sequence of the reactive center loop mimics that of an ideal
natural substrate of target proteases (8). Reaction with
target protease results in formation of an initial Michaelis complex.
After this initial interaction, an intermediate complex forms that
either develops into a stable complex or dissociates to release the
inactive cleaved serpin and the active enzyme (8). The
potency of an inhibitor depends on the relative rates of these two
reactions. The rate of dissociation of the stable complex is extremely
slow (9).
The primary recognition element for serpin-protease association is a 15
aa residue exposed segment known as the reactive center loop. This loop
varies in sequence and length; it is mobile and can adopt different
conformations. Cleavage of the reactive center loop by nontarget
proteases results in complete insertion of the loop into the
five-stranded
sheet A, thereby converting it to a six-stranded
sheet (10). In the complex with protease, the reactive
center loop is similarly inserted; the protease is thereby displaced
from the site of initial interaction at one pole of the inhibitor
molecule to the other pole (11, 12, 13, 14). The amino acid
sequence of the reactive center loop from approximately P5 through P5'
varies among the serpins and, to a great extent, determines the
specificity of the inhibitor (15, 16, 17, 18, 19, 20, 21). The P1 residue is
the major determinant of serpin specificity, as has been illustrated by
analysis of both naturally occurring serpins and serpins with new
unique engineered mutations (15, 20). Because a serpin
with a single P1 residue may inhibit a variety of target proteases and
because different serpins with the same P1 residue inhibit different
proteases, other residues must be involved in specificity
determination. Analyses of mutant serpins, which indicate that the P1
residue is not the sole determinant of specificity, support this
assumption (16, 17, 18, 19, 20, 22, 23, 24). Other residues within the
reactive center region also play a significant role. However, the roles
of different residues appear to vary with different serpin-protease
combinations.
We described a variant C1INH in which the P2 residue (Ala443) was substituted with Val (25, 26). This mutant had a decreased rate of reactivity toward C1s and C1r, and had acquired weak inhibitory activity toward trypsin, but retained normal activity against the target proteases factor XIIa and plasma kallikrein. A patient with hereditary angioedema (HAE) has been described in which the P6 residue (Ala439) was substituted with a Val (27). It was not clear whether this resulted in absolute deficiency of C1INH or in a dysfunctional inhibitor. An identical mutation at the P6 position of antithrombin, also without functional analysis, previously had been reported (28). Analysis of autoantibodies to C1INH from patients with acquired angioedema led to the identification of the P8'/P9' (Gln452Gln453) region as a potential contact site with C1s during complex formation. To further characterize the roles of these reactive center region residues in complex formation and in specificity determination, we have created mutant proteins with substitutions at each of these positions. In the case of P2, a variety of substitutions varying in side chain size, charge, and hydrophobicity have been constructed. We have analyzed the reactivity of these mutant proteins with target proteases, and have determined their sensitivity to heat denaturation as a measure of conformation. In addition, we have analyzed the reactivity of the mutant inhibitors with plasmin and thrombin.
| Materials and Methods |
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Mutations at the C1INH P2 position were introduced using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Primers corresponding to nucleotides 16,773 through 16,802 of the C1INH genomic sequence that varied in the nucleotides at positions 16,785 through 16,787 were designed. These substitutions led to replacement of Ala443 with Asn, Gly, Leu, and Thr. The same approach was used to replace the P6 Ala residue with Val and both the P8' and P9' residues (Gln452 and Gln453) with Ala. Mutated and wild-type proteins then were expressed transiently in COS-1 cells. For some experiments, newly synthesized proteins were metabolically radiolabeled with [35S]Met (Amersham Life Sciences, Arlington Heights, IL). Medium was harvested 72 h after transfection and dialyzed into PBS.
Thermal stability
COS-1 cell culture supernatants (100 µl) containing recombinant inhibitors were incubated with and without trypsin (5 µg) at 40, 50, 60, 70, and 80°C for 120 min, following which they were subjected to centrifugation at 14,000 x g for 30 min. Quantitation of residual Ag in the supernatant was performed using an ELISA as described by Aulak et al. (29).
Complex formation between C1INH proteins and proteases
Radiolabeled recombinant wild-type or mutant C1INHs were
incubated for 1 h at 37°C with C1s (8.4 µg), C1r (6.4 µg)
(Advanced Research Technologies, San Diego, CA), kallikrein (15 µg),
factor XIIa (3.5 µg) (Enzyme Research Laboratories, South Bend,
IN), plasmin (0.2, 0.5, 1, 2, 5, and 10 µg/µl) and
-thrombin
(0.2, 0.5, 1, 2, 5, and 10 µg/µl) (plasmin and thrombin provided by
Dr. V. Donaldson, Childrens Hospital Research Foundation, Cincinnati,
OH), or trypsin (0.5 µg; Sigma, St. Louis, MO). Each protease was
incubated in molar excess relative to recombinant C1INH. After
incubation, PMSF (final concentration, 1 mM) was added, and Triton
X-100 (0.5%), deoxycholic acid (0.25%), SDS (0.5%), and EDTA (5 mM)
were added to each sample. The IgG fraction of rabbit anti-human
C1INH (3 µl) then was added, and samples were incubated at 4°C
overnight. A suspension of fixed Staphylococcus aureus (6
µl, IgG sorb; The Enzyme Center, Boston, MA), sonicated and washed
three times (with 1% Triton X-100, 1% SDS, 0.5% deoxycholate, and 5
mg/ml BSA in 0.02 M sodium phosphate and 0.15 M NaCl, pH 7.4), was
added to each sample and incubated at 4°C for 1 h. The pellets
(14,000 x g for 3 min) were washed once with the above
washing solution and three times with a washing solution lacking BSA.
Each precipitate then was dissolved in SDS sample buffer without
reducing agent (15 µl; 4.2% SDS, 20% glycerol, 0.1 M Tris-HCl, pH
6.5, and 0.01% bromophenol blue), vortexed, boiled for 3 min, and
subjected to brief centrifugation and electrophoresis on SDS-7.5%
polyacrylamide gel. Gels were fixed, dried, and exposed to x-ray film
(Kodak XAR-5; Eastman Kodak, Rochester, NY) at -70°C.
Inhibitor assay and calculation of second order rate constants
COS-1 cell supernatants (40 ml) containing recombinant C1INH
proteins were collected and concentrated to 25 ml. The C1INH
concentrations of the samples were measured by ELISA (29).
The concentrations of the recombinant mutant C1INH proteins after
concentration were: wild type (5.5 µg/ml),
Ala443
Val mutant (5 µg/ml),
Ala443
Ser mutant (6.5 µg/ml),
Ala443
Thr mutant (5.5 µg/ml),
Ala439
Val mutant (2.5 µg/ml), and
Gln452Gln453
Ala mutant
(1.6 µg/ml). The amount of active inhibitor in each C1INH preparation
was determined by incubation of varying quantities of mutant inhibitor
with a fixed known quantity of fully active C1s for a prolonged period,
following which residual C1s esterolytic activity was determined
by hydrolysis of the chromogenic substrate S-2314 (Chromogenix,
Molndal, Sweden). The active concentration of C1s was determined by
incubation of varying molar ratios of C1s and plasma-derived C1INH of
known concentration (determined using an extinction coefficient of 3.7)
(30) followed by determination of the amount of complex
formation by SDS-PAGE.
For the kinetic assays, equimolar concentrations of C1s and recombinant wild-type or mutant C1INH were incubated at 37°C in PBS. At different times (030 min), aliquots of the reaction mix were removed and the reaction stopped by dilution in the chromogenic substrate S2314 (0.5 mM in PBS). Residual enzyme activity was quantitated by monitoring hydrolysis of the substrate (increase in absorption over time at a wavelength of 405 nm). The second order rate constants were determined using the half-time of the reaction (t1/2), which is calculated by plotting the inverse of the enzymatic rate vs time; this is related to the (Ka) through the equation t1/2 = (Ka x E0)-1, where E0 is the total enzyme concentration.
| Results |
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Thermal stability provides a simple, but reliable measure of the
conformation of serpins (31). Normal intact serpins
multimerize at temperatures of 5060°C. This results in a decrease
in epitopes detected with polyclonal anti-C1INH antisera.
Therefore, with multimerization the apparent concentration of C1INH
decreases. Serpins that have been cleaved near the reactive center are
more stable and remain monomeric at temperatures as high as 8090°C.
Thermal denaturation curves of all the P2, the P6 Ala
Val, and the
P8'/P9' Gln
Ala mutant C1INHs were indistinguishable from that of the
wild-type protein (Fig. 1
). After
cleavage by trypsin, all of the recombinant proteins were stable at
elevated temperatures (5090°C). These data suggest that the
mutations did not induce any major conformational changes, and that
each was able to undergo the conformational rearrangement with reactive
center loop insertion that takes place in normal inhibitor serpins
following reactive center cleavage or complex formation (10, 11). Therefore, it is likely that each of the mutants would be
capable of complex formation with an appropriate protease.
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C1s.
The reactions of each of the recombinant inhibitors with the different
proteases in excess were assessed by SDS-PAGE following incubation for
1 h at 37°C. Recombinant wild-type C1INH formed a stable complex
with C1s, accompanied by the appearance of a relatively small amount of
cleaved C1INH, similar to previous observations (Fig. 2
A) (25, 26).
Compared with the wild type, recombinant C1INH with an Asn at the P2
position showed less complex formation with C1s in the 60-min
incubation period (Fig. 1
A). An appreciable quantity of the
native form of the inhibitor was present even after 1 h of
incubation. Furthermore, no cleavage of the
Ala443
Asn mutant was observed. It should be pointed
out that even with the same inhibitor-protease pair, in experiments
performed at different times, the amount of cleaved vs complexed
inhibitor, as assessed by SDS-PAGE, may vary somewhat. This likely is a
result of subtle differences in experimental conditions. Therefore,
over-interpretation of moderate changes in the amounts of cleaved
inhibitor should be avoided. However, as seen in Fig. 2
A,
the complete absence of cleavage of this mutant clearly differs from
the amount observed with the recombinant wild-type protein. The results
with the Ala443
Asn mutant are similar to those
observed previously with the P2 Asp mutant (26). The
results described here, together with previously published data
(26), are summarized in Table I
. Substitution of P2 with Gly, Leu, or
Thr did not significantly affect the amount of complex formation with
C1s or the amount of resulting cleaved inhibitor, in comparison with
the wild-type inhibitor (Fig. 2
, A and B). The
reactivities of these mutants were similar to those of the wild type
and the previously described P2 Ser substitution mutant
(26). Neither the P6 Ala
Val nor the P8'/P9' Gln
Ala
substitutions showed any significant alteration in complex formation
with C1s in comparison with the wild-type protein.
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Val,
Ala443
Ser, Ala443
Thr,
Ala439
Val, and
Gln452Gln453
AlaAla)
(Table II
Val mutant was much less active than the
wild-type inhibitor (25, 26). The wild-type inhibitor was
fully complexed within 0.5 min, whereas the mutant required
5 min
before complex formation was complete. As expected from these data, the
association rate for the Ala443
Val mutant with
C1s was substantially lower than that of the wild-type protein
(p = 0.04). In addition, the association rate
constants of the Ser and Thr mutants were reduced in comparison with
the wild-type inhibitor, although to a lesser extent than that of
the Ala443
Val mutant (5060% reduction in
rate as opposed to an 80% reduction for
Ala443
Val).
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Thr mutant), which may suggest a small
effect on the rate of complex formation with C1r (Fig. 3
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factor XIIa, recombinant wild-type C1INH
showed nearly complete complex formation with minimal amounts of
cleaved inhibitor or of remaining uncleaved inhibitor. However, a
significant quantity of cleaved inhibitor is released during the
reaction with plasma kallikrein (Fig. 4
Asp mutant with these proteases
(26) (Table I
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Val mutant complexed
with plasmin to an extent similar to that with the wild-type C1INH
(Fig. 5
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Trypsin.
We previously demonstrated that the Ala443
Val
mutant protein was relatively resistant to cleavage by trypsin in
comparison with the wild-type protein and that it had acquired the
ability to complex with trypsin at a very slow rate (26).
The reactivities of each of the other mutants appeared much more
similar to that of the wild-type protein in that they were readily
cleaved and showed little or no complex formation with trypsin (data
not shown).
| Discussion |
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1-antichymotrypsin, in which
reactivities with factor Xa and activated protein C were markedly
altered whereas that with trypsin was relatively unaffected
(18). Several characteristics of the reactivities of these
mutants are notable. As might be expected, bulky and/or charged amino
acids were poorly tolerated at this position, and likely
interfered with access of protease to the reactive center. The
mutants with P2 Ala
Asp and Asn substitutions reacted extremely
poorly with all of the proteases tested (Table I
Virtually any change in side chain from the wild-type Ala resulted in
decreased inhibitory activity against C1s (and C1r), whereas reactivity
with kallikrein and factor XIIa was less sensitive to these
substitutions. The sensitivity of reactivity with C1s to changes at P2
was apparent on SDS-PAGE for the
Ala443
Asn and Asp mutants, and for the
Ser, Thr, and Val mutants on kinetic analysis. Unfortunately, there
were insufficient quantities of the
Ala443
Gly and Leu variants for
kinetic studies with C1s and of any of the recombinant inhibitors for
kinetic analyses with proteases other than C1s. The slower rate of
reactivity of each of these variants with C1r was apparent on SDS-PAGE.
This high degree of specificity in the reactivity with C1s (and C1r) is
consistent with data from the recently reported crystal structure of
the catalytic domain of C1s (39). This demonstrated that
access to the C1s binding subsites, particularly S2, is partially
obstructed in comparison with many other proteases, such as trypsin.
This restricted access, as suggested by Gaboriaud et al.
(39), very likely explains the high degree of substrate
specificity of C1s and the fact that its only inhibitor in plasma is
C1INH.
The observations related to the reactivity of the P2 substitution mutants with thrombin are also of interest in relation to specificity determination. The increased reactivity of the P2 Val and Leu mutants with thrombin, as demonstrated by both increased complex formation and increased cleavage in comparison with wild type, correlates with thrombin specificity toward fibrinogen. Fibrinopeptide B, which has an Ala at the P2 position, is released more slowly than is fibrinopeptide A, which has a Val at the P2 position (40). In general, it was observed that more hydrophobic residues in the P2-P4 positions resulted in substrates that were better recognized by thrombin (41). The somewhat increased complex formation with, and cleavage of, the P2 Val and Leu mutants are consistent with these earlier observations. However, based on the previous data (41), it might have been expected that the P2 Gly mutant also would have been recognized, but it neither was complexed with nor cleaved by thrombin.
The Ala439
Val (P6) substitution mutant,
identified originally in a family with HAE (27), revealed
only a moderate decrease in activity against C1s in comparison
with the wild-type protein
(Ka 2.14 vs 3.35 x
105
M-1s-1). Its reactivity
with all of the proteases, including C1s (after a 1-h incubation), was
indistinguishable from normal on SDS-PAGE. Current information suggests
that angioedema is mediated by bradykinin (25, 42, 43).
Therefore, prevention of attacks of angioedema very likely is dependent
on the inhibition of plasma kallikrein (which catalyzes the cleavage of
high m.w. kininogen to release bradykinin) by C1INH. Determination of
rate constants for this mutant (when available in larger quantities)
with kallikrein will be required to determine whether a relatively mild
dysfunction of this inhibitor is responsible for the development of
angioedema in this family. In addition, this family reportedly
presented with laboratory values consistent with type 1 HAE (decreased
C1INH antigenic and functional levels) as opposed to type 2 (decreased
C1INH functional levels with relatively normal antigenic levels). This
could be explained if the mutation resulted in either a decrease in
secretion of newly synthesized protein or in enhanced intracellular
degradation. The amount of recombinant mutant protein synthesized by
COS cells did not dramatically differ from the amount of wild-type
inhibitor synthesized, which argues against this explanation.
Unfortunately, these hypotheses cannot be tested directly because this
family is not currently available. In the original analysis of this
family, this substitution was the only mutation identified within the
coding region or at intron-exon junctions (27). In
addition, the argument that this mutation is sufficient to result in
HAE is supported by the observation that an identical mutation in
antithrombin was described in a family with recurrent venous thromboses
(28).
The Gln452Gln453
Ala
substitution was analyzed to test the hypothesis that these residues
make up part of a contact site for C1s. This was based on the
observations that autoantibodies to C1INH react with a synthetic
peptide corresponding to aa 448459 and that the epitope included
residues Gln452, Gln453,
Pro454, and Phe455
(44). These authors then went on to report experiments
that indicated that this peptide bound directly to C1s and prevented
its complex formation with C1INH (45). Substitution of
Gln452, Gln453, or
Phe455 in this peptide abrogated C1s binding
(46). This led to the conclusion that this region at the
distal end of the reactive center loop constitutes a C1s contact site.
The results of the experiments reported here show that substitution of
both Gln452 and Gln453 with
Ala residues has only a relatively marginal effect on the inhibition of
C1s (or, of the other proteases, with the exception of plasmin) by
C1INH. It seems unlikely that a contact site dependent on the side
chains of two adjacent Gln residues would be adequately replaced by Ala
residues. Therefore, it seems likely that these residues, at least, are
not part of a major contact site for C1s in its recognition of C1INH.
Recently, it was demonstrated that C1INH can be cross-linked to fibrin
by tissue transglutaminase via Gln453, and that
this immobilized C1INH retained its inhibitory activity toward
kallikrein and C1s (47). The
Gln452Gln453 mutant may
prove to be valuable to analyze the potential biologic role of this
cross-linking reaction.
In summary, these studies have further characterized the roles of amino acids within the reactive center loop of C1INH in specificity determination. In particular, the results emphasize the importance of the amino acid at the P2 position in recognition by the highly specific proteases C1r and C1s. Conversely, plasma kallikrein and factor XIIa are much more tolerant of variation at this position.
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| Footnotes |
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2 Current address: Lawrence Berkeley National Laboratories, 1 Cyclotron Road, Mail Stop 83-109, Berkeley, CA 94720. ![]()
3 Address correspondence and reprint requests to Dr. Alvin E. Davis III, Center for Blood Research, 800 Huntington Avenue, Boston, MA 02115. E-mail address: aldavis{at}cbr.med.harvard.edu ![]()
4 Abbreviations used in this paper: C1INH, C1 inhibitor; HAE, hereditary angioedema. ![]()
Received for publication March 21, 2001. Accepted for publication May 30, 2001.
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1-proteinase inhibitor: crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. J. Mol. Biol. 177:531.[Medline]
-1-proteinase inhibitor. Biochem. Biophsy. Res. Commun. 159:271.[Medline]
1-antitrypsin mutants against proteolytic enzymes of the kallikrein-kinin, complement and fibrinolytic systems. J. Biol. Chem. 265:10786.
1-proteinase inhibitor mechanism. J. Biol. Chem. 273:4569.This article has been cited by other articles:
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D. Liu, S. Cai, X. Gu, J. Scafidi, X. Wu, and A. E. Davis III C1 Inhibitor Prevents Endotoxin Shock Via a Direct Interaction with Lipopolysaccharide J. Immunol., September 1, 2003; 171(5): 2594 - 2601. [Abstract] [Full Text] [PDF] |
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T. Sulikowski, B. A. Bauer, and P. A. Patston {alpha}1-Proteinase inhibitor mutants with specificity for plasma kallikrein and C1s but not C1 Protein Sci., September 1, 2002; 11(9): 2230 - 2236. [Abstract] [Full Text] [PDF] |
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