From the Division of Protein Engineering, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Taejon 305-600, Korea
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ABSTRACT |
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The metastability of inhibitory serpins (serine
proteinase inhibitors) is thought to play a key role in the facile
conformational switch and the insertion of the reactive center loop
into the central -sheet, A-sheet, during the formation of a stable
complex between a serpin and its target proteinase. We have examined
the folding and inhibitory activity of a very stable variant of human
1-antitrypsin, a prototype inhibitory serpin. A
combination of seven stabilizing single amino acid substitutions of
1-antitrypsin, designated Multi-7, increased the
midpoint of the unfolding transition to almost that of ovalbumin, a
non-inhibitory but more stable serpin. Compared with the wild-type
1-antitrypsin, Multi-7 retarded the opening of A-sheet
significantly, as revealed by the retarded unfolding and latency
conversion of the native state. Surprisingly, Multi-7
1-antitrypsin could form a stable complex with a target elastase with the same kinetic parameters and the stoichiometry of
inhibition as the wild type, indicating that enhanced A-sheet closure
conferred by Multi-7 does not affect the complex formation. It may be
that the stability increase of Multi-7
1-antitrypsin is
not sufficient to influence the rate of loop insertion during the
complex formation.
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INTRODUCTION |
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The serpin1 (serine
proteinase inhibitor) superfamily includes inhibitors such as
1-antitrypsin (
1AT), antithrombin,
1-antichymotrypsin, C1 inhibitor, and non-inhibitory
members such as ovalbumin and angiotensinogen (1). Serpins share a
common tertiary structure composed of three
-sheets and several
-helices (Fig. 1). One of the
intriguing aspects of serpin structure is that the native conformation
of the inhibitory serpins is strained (2-4). Proteolytic cleavage of
the reactive center loop or the conversion into the more stable latent
form (Fig. 1) accompanies a complete insertion of the reactive center
loop into the central
-sheet, A-sheet, with concomitant release of
the strain (5, 6). In a non-inhibitory but more stable serpin,
ovalbumin, the cleavage of the loop does not induce a drastic
conformational switch as in inhibitory serpins (7). The ability of loop
insertion of serpins thus appears to be very critical to the
conformational switch and inhibitory function. Upon binding a target
proteinase, the reactive center loop of a serpin is thought to be
inserted into the A-sheet to form a stable complex between the
proteinase and the inhibitor (8-10). It has been suggested that a
critical factor for the formation of a stable complex is the rate of
loop insertion (8, 11). The metastable structure of inhibitory serpins
has the advantage of a facile conversion into an alternative stable
conformation (12). It is possible that the native strain of inhibitory
serpins is utilized for the facile loop insertion during the complex
formation with a target proteinase (9, 12-14). However, the precise
mechanism underlying the complex formation is yet to be elucidated.
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Previously we have identified several hydrophobic core mutations of
1AT that increased the conformational stability,
presumably by enhancing the closure of the A-sheet (12). The mutations did not affect proteinase binding, as revealed by the unchanged association rate constants with porcine elastase. It was suspected, however, that these mutations would affect the formation of the stable
complex upon proteinase binding by retarding the loop insertion, resulting in substrate-like behavior. In the present study we tested
this assumption by analyzing a very stable
1AT carrying seven stabilizing mutations (Fig. 1: F51L, T59A, T68A, A70G, M374I, S381A, and K387R) identified previously (12). The mutant was designated
Multi-7. The mutational effects on the inhibitory activity as well as
other structural properties such as folding-unfolding transition were
also examined.
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MATERIALS AND METHODS |
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Recombinant 1AT Proteins and Plasmids--
The
plasmid for
1AT expression in Escherichia
coli (15), the purification of recombinant
1AT
protein (15), and the detailed method for in vitro
translation products of
1AT (16) were described previously. The plasmid for the in vitro translation of
ovalbumin was constructed by substituting
1AT cDNA
in the in vitro translation vector, pF(BLG)AT (16), with the
cDNA of ovalbumin from pYOV5 (gift from Dr. H. J. Kim).
Concentrations of
1AT were determined in 6 M
guanidine hydrochloride using a value of
A1 cm1 % = 4.3 at 280 nm,
calculated from the tyrosine and tryptophan content of the
1AT protein (17) and based upon
Mr = 44,250.
1AT activity was
measured as residual porcine pancreatic elastase activity employing 1 mM
N-succinyl-(Ala)3-p-nitroanilide as a chromogenic substrate (18). Individual thermostable mutations of
1AT were previously reported (12). Combination of the
mutations of
1AT and site-specific mutations of
ovalbumin were made by oligonucleotide-directed mutagenesis (19).
Chemicals--
Ultrapure urea was purchased from Schwarz/Mann.
[35S]Methionine was purchased from NEN Life Science
Products. Human plasma 1AT, porcine pancreatic elastase,
human leukocyte elastase, bovine pancreatic trypsin
N-succinyl-(Ala)3-p-nitroanilide, and
N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide were purchased from Sigma. All other chemicals were reagent grade.
Urea-induced Equilibrium Unfolding Transition--
Equilibrium
unfolding as a function of urea was monitored by fluorescence
spectroscopy and CD spectroscopy, details of which were described
previously (12, 15). The buffer used for folding experiments was 10 mM phosphate, 50 mM NaCl, 1 mM
EDTA, 1 mM -mercaptoethanol, pH 6.5, unless described
otherwise. The native protein was incubated in the folding buffer
containing various concentrations of urea at 25 °C. The protein
concentration for the unfolding transition was 20 µg/ml for
fluorescence spectroscopy and 50 µg/ml for CD spectroscopy.
Experimental data of the fluorescence measurement were fitted to a
two-state unfolding model, as described previously (12, 15).
Kinetics of Unfolding and Refolding--
The kinetic study of
unfolding and refolding of 1AT was performed by
measuring the change of fluorescence intensity (
ex = 280 nm and
em = 360 nm) of the native or unfolded proteins upon exposure to various concentrations of urea (12). The final protein
concentration was 5 µg/ml, and the experimental temperature was
25 °C. The major kinetic phase was examined, and the data were
fitted to a two-state (for unfolding) or three-state (for refolding)
model to obtain the relaxation time:
F(t)=
F(i)
e(
t/
i) + F
, where F(t) is the
fluorescence at time t, F(i) is the
fluorescence of phase i at zero time,
i is the
relaxation time for phase i, and F
is the fluorescence at infinite time (Sigma Plot, Jandel Corp.).
Transverse Urea Gradient Gel Electrophoresis-- Gels were prepared with a gradient of 0-8 M urea perpendicular to the direction of electrophoresis with an opposing gradient of acrylamide from 15 to 11% (20). Four slab gels (100 × 80 mm) were prepared simultaneously in a multigel caster (Hoeffer) by using a gradient maker and a single channel peristaltic pump. The electrode buffer was 50 mM Tris acetate, 1 mM EDTA, pH 7.5. The native protein (20 µg in 100 µl), the protein unfolded in 8 M urea (10 min at room temperature), or the in vitro translation product was applied across the top of the gel. The gels were run at a constant current of 6 mA for 3 h at a controlled temperature of 25 °C. The protein bands were visualized either by Coomassie Brilliant Blue staining or by autoradiography.
Conversion into the Latent Form--
Formation of the latent
1AT was examined with in vitro translation
products, as described (12). Conditions for the formation of latent
1AT have been reported previously (21). The translation products labeled with [35S]methionine were incubated at
60 °C for 4 h in 20 mM Tris, pH 7.4, which
contained 0.7 M sodium citrate. Various conformations of
1AT were analyzed by transverse urea gradient gel
electrophoresis.
Complex Formation with Proteinases--
Complex formation of
Multi-7 1AT with a proteinase was examined by monitoring
the SDS-resistant
1AT-proteinase complex (22, 23).
Purified
1AT was incubated in assay buffer (30 mM phosphate, 160 mM NaCl, 0.1% PEG 6000, 0.1% Triton X-100, pH 7.4) with porcine pancreatic elastase or human
leukocyte elastase at designated molar ratios of
1AT to
proteinase. Samples were incubated at 37 °C for 10 min and were
analyzed by 10% SDS-polyacrylamide gel electrophoresis. The protein
bands were visualized by Coomassie Brilliant Blue staining.
Concentrations of
1AT were determined by
A280 in 6 M guanidine hydrochloride
(17). The active site concentration of human leukocyte elastase was
determined as described previously (22) with trypsin-titrated human
plasma
1AT and a substrate,
N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. The active concentration of porcine pancreatic elastase was determined by measuring the initial rates of hydrolysis of 1 mM
N-succinyl-(Ala)3-p-nitroanilide by increasing the concentration of elastase, using a previously reported standard titration curve (24). The stoichiometry of inhibition
was determined by titration reactions as described (25). The reaction
mixture (50 µl volume) in the assay buffer contained 100 nM porcine or human elastase. After incubation with various
amounts of recombinant wild-type or Multi-7
1AT for 10 min at 37 °C, the reaction mixture was diluted 10-fold with the assay buffer, and residual enzyme activity was determined.
Inhibitory Parameters of 1AT--
Inhibition
kinetic studies for the interaction of
1AT with porcine
pancreatic elastase were performed by analyzing progress curve kinetic
experiments (26). The active concentration of porcine pancreatic
elastase was determined as described above. The active site titration
of
1AT was measured by employing 1 mM
N-succinyl-(Ala)3-p-nitroanilide and
a known activity of porcine pancreatic elastase (18). The assays were
performed at 25 °C in reaction buffer containing 50 mM
Tris, 50 mM NaCl, 0.1% PEG 4000, and 0.05% v/v Triton
X-100, pH 8.0, and started by the addition of elastase, at the final
concentration of 1.25 nM. A typical progress curve
experiment consisted of 6 assays (1 zero and 5 non-zero concentrations
of inhibitor) and the slow development of inhibition was determined by
continuously monitoring the appearance of p-nitroaniline at
410 nm. The amount of product formed was calculated by using a molar
absorption coefficient of 8,800 M
1
cm
1 for p-nitroaniline at 410 nm. The
inhibition of the enzyme (E) by
1AT (I) is
described in Scheme I, where S is the substrate, and P is
p-nitroaniline.
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(Eq. 1) |
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RESULTS |
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Equilibrium Stability of Multi-7
1AT--
Equilibrium unfolding transition of Multi-7
1AT was examined as a function of increasing urea
concentration at 25 °C. When the transition was monitored by
intrinsic fluorescence intensity (Fig.
2A), the midpoints of
transition of the wild-type and the mutant protein were 1.8 and 4.8 M urea, respectively, yielding
G of 8 kcal
mol
1. A similar shift in the transition midpoint was
observed by far UV CD signal (Fig. 2B) or by transverse urea
gradient gel electrophoresis (Fig. 2C). The unfolding
transition of the wild-type
1AT monitored by far UV CD
signal or urea gradient gel electrophoresis exhibited at least two
phases, indicative of at least one equilibrium folding intermediate.
Because of the midpoint shift of the first unfolding phase, the
equilibrium unfolding intermediate was not detected with the Multi-7
mutant protein. The unfolding transition was reversible for both the
wild-type and the mutant proteins, as revealed by refolding on urea
gradient gel electrophoresis (Fig. 2C, Refolding). The transition
of Multi-7 was rather abrupt at the transition midpoint.
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The Multi-7 1AT Is as Stable as Ovalbumin--
The
stability of Multi-7
1AT was compared with that of
ovalbumin with in vitro translation products on urea
gradient gel electrophoresis. Fig. 3 shows that ovalbumin is much more
stable than the wild-type
1AT, and its unfolding
transition follows a two-state process. The overall transition of
ovalbumin was very similar to that of Multi-7
1AT. The
in vitro translation product of the wild-type ovalbumin
yielded two species of the native form that exhibited a slight
difference in unfolding transition on urea gradient gel electrophoresis
(Fig. 3, Ovalbumin, Wt). Ovalbumin contains one disulfide bond between
Cys-73 and Cys-120, which is not present in
1AT. To
compare the stability of Multi-7
1AT with that of
ovalbumin without disulfide contribution, we constructed recombinant
versions of ovalbumin that cannot form the disulfide bond by
substituting cysteines into alanines. The mutant ovalbumin yielded only
a single species of the native form on urea gradient gel with the same
unfolding midpoint as Multi-7
1AT (Fig. 3, Ovalbumin,
C73A). The amino acid substitutions themselves do not appear to
contribute significantly to the stability because all three different
forms of mutant ovalbumin (C73A, C120A, and C73A/C120A) showed the
unfolding transition at a urea concentration similar to that of the
less stable wild-type species (data not shown). The stability of
Multi-7 was similar to that of the mutant ovalbumin which could not
form the disulfide. The unfolding transition of both Multi-7
1AT and ovalbumin were abrupt.
Kinetic Analysis of Unfolding and Refolding of Multi-7
1AT--
Kinetic unfolding and refolding of Multi-7
1AT were performed by monitoring the intrinsic
fluorescence change as a probe. Fig. 4
shows that the unfolding of
1AT occurs in an all-or-none process at all urea concentrations, and the unfolding of Multi-7
1AT was retarded significantly. Refolding of
1AT exhibited multiple kinetic states. There were two
major refolding phases in which the intensity of fluorescence
decreased: a fast decrease (
= 200-500 s), and a further slow
decrease (
= 1,000-3,000 s). The amplitudes of the two phases were
about same at near zero urea concentration, but the amplitude of slow
phase increased as a function of urea. Refolding of Multi-7
1AT was facilitated slightly in both phases, but urea
dependence of the refolding rate at lower urea concentrations
disappeared in such a way that the difference in refolding rate was
minimized when extrapolated to 0 M urea (the relaxation
time of 339 and 106 s at 0 M urea for the fast phase
of wild-type and Multi-7, respectively). In particular, the rate of the
slow refolding phase, which intersects with the unfolding kinetics at
the same urea concentration (1.9 and 4.8 M for the
wild-type and Multi-7, respectively) as the equilibrium midpoint (Fig.
2A), was not significantly altered in the Multi-7 mutant
(1072 s at 0 M urea for both). The amplitude of both phases was not changed by the mutation. These results indicate that the major
effect of the Multi-7 mutation in the unfolding-refolding transition is
the retardation of the unfolding rate.
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Conversion of Multi-7 1AT into the Latent
Form--
The native structure of inhibitory serpins is considered as
a kinetically trapped folding intermediate because the intact native
form can convert into a more stable latent form. It was expected that
the mutations that stabilize the native state of serpin will retard the
conversion into the latent state. This was the case with Multi-7
1AT, as shown in Fig. 5.
Unlike the native form of
1AT that undergoes unfolding
transition in urea (Fig. 2C), the latent form does not
unfold even in the presence of 8 M urea. When heat
denaturation was performed under conditions where the production of the
latent
1AT was favored (about 50% of the wild-type
1AT converted into the latent form), the Multi-7 mutant
converted into the latent form much less readily than the wild-type
protein. The results showed that the Multi-7 mutation retarded the
insertion of the reactive center loop into the A-sheet, although the
accessibility of the loop insertion was not affected.
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Inhibitory Activity of Multi-7 1AT--
To
investigate the effect of the Multi-7 mutation in
1AT on
the inhibitory activity, complex formation of wild-type and Multi-7
1AT with various proteinases was examined. Both
wild-type and the mutant inhibitor formed a tight SDS-resistant
proteinase-inhibitor complex with porcine and human elastases (Fig.
6A). In addition, the mutation
did not alter the partitioning between the inhibitory and substrate
pathways, as revealed by the unchanged values of stoichiometry of
inhibition in the titration of elastase by
1AT (Fig.
6B: 1.1 and 1.7 for human and porcine elastase,
respectively). This was also confirmed by densitometric scanning of the
SDS-resistant complex formation shown in Fig. 6A. Inhibition
kinetic studies showed that the association rate constant
(ka) of the wild type and the Multi-7 mutant
1AT for porcine pancreatic elastase was 5.11 ± 0.7 × 105 M
1
s
1 and 5.03 ± 1.0 × 105
M
1 s
1, respectively. The
dissociation constant (Ki) was 95 ± 24 pM and 117 ± 29 pM for the wild type and
the Multi-7, respectively. The results clearly showed that the ability
of
1AT to form a complex with a target proteinase was
not affected by the Multi-7 mutation.
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DISCUSSION |
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It has been suggested that rapid insertion of the reactive center
loop into the A-sheet during the complex formation with a target
proteinase is critical for the inhibitory activity of serpins (8, 11).
In the present study, we examined the effect of Multi-7 mutation on the
inhibitory activity of human 1AT, which stabilized the
native state of the molecule and retarded opening of the A-sheet. The
mutant
1AT could form a stable complex with various
target proteinases, and the stoichiometry of inhibition was not
affected. These results indicate that the A-sheet closure conferred by
the stability increase of Multi-7
1-antitrypsin does not
affect the complex formation with target proteinases. The results
support the notion that, in addition to intramolecular interactions,
other interactions such as contact between the inhibitor and its target
proteinase contribute significantly to the conformational change needed
for the complex formation.
Conformational Properties of Multi-7 1AT--
The
equilibrium unfolding of the wild-type
1AT exhibited at
least two phases with one equilibrium intermediate, which is compact
(Fig. 2C) and retains approximately 70% of the native CD
signal (Fig. 2B) but most of the native fluorescence is
dequenched (Fig. 2A). Unfolding of the Multi-7
1AT molecule did not exhibit the equilibrium unfolding
intermediate (Fig. 2, B and C). Moreover, the
unfolding pattern of Multi-7
1AT on urea gradient gel
electrophoresis was very similar to that of ovalbumin (Fig. 3).
Ovalbumin, although sharing a common tertiary fold with inhibitory
serpins, is more stable and is not active as a proteinase inhibitor.
Molecular properties of ovalbumin are quite different from those of
inhibitory serpins. For instance, the equilibrium unfolding transition
of inhibitory serpins including
1AT (Fig. 2) is very
complex (5, 27-29), whereas the unfolding transition of ovalbumin is
more or less fitted to a two-state model when monitored either by far UV CD signal (5), intrinsic fluorescence (30), or by UV absorbency and
intrinsic viscosity (31). The unfolding midpoint of C73A mutant
ovalbumin, which could not form disulfide bonds, was very close to that
of Multi-7
1AT (Fig. 3). Previous studies on the reversible unfolding of disulfide-reduced authentic ovalbumin also
yielded the same value (4.8 M) of transition midpoint
(Cm) in urea as Multi-7
1AT at
25 °C (30). The difference in Cm values of the
wild-type and Multi-7
1AT yields
G of 8 kcal/mol, using the m (measure of dependence of
G on denaturant concentration) value of 2.6 determined
previously (12). It is interesting to note that neither the Multi-7
mutant
1AT nor ovalbumin exhibit a smooth unfolding
transition seen in regular globular proteins (Fig. 3). It appears that
the equilibrium unfolding of Multi-7
1AT resembles that
of ovalbumin.
Retarded A-sheet Opening of Multi-7 1AT--
The
x-ray crystal structure of Multi-7
1AT (Fig. 1) was
determined with 2.7-Å resolution recently (32). The uncleaved
wild-type structure has yet to be determined. Mutation sites of Multi-7
1AT appear to be well packed as in ovalbumin (33) and
the presumed native state of the wild-type around the mutation sites
would increase steric hindrance. The reactive center loop of Multi-7
1AT is not inserted into the A-sheet at all, as in the
crystal structure of intact
1-antichymotrypsin (34). A
peculiar feature of Multi-7
1AT is that strands 3 and 5 of the A-sheet are hydrogen-bonded all the way up to the top of the
sheet, which is not the case in other native structures of inhibitory
serpins (34-36) and ovalbumin (33).
Inhibitory Activity of Multi-7 1AT--
Various
biochemical and structural studies with the mutant forms of inhibitory
serpins suggest that the loop insertion is necessary for the formation
of a stable complex although not sufficient to confer inhibitory
activity (8-10). Alteration of inhibitory function by oversized or
charged amino acid substitutions at the P-even positions (P14, P12, and
P10) in the reactive center loop region was attributed to a retarded
loop insertion (39-43). It was expected therefore that mutations that
interfere with the loop insertion, like Multi-7 in this study, would
affect the formation of the stable complex by retarding the loop
insertion, thus inducing substrate-like behavior, even if binding
proteinase was not affected. The Multi-7
1AT did not
affect the inhibitory activity measured by inhibition kinetic
experiments, nor did the Multi-7 mutation significantly influence the
partitioning between the inhibitory pathway and the substrate pathway
(Fig. 6). In addition, the stability of the complexes was not
influenced by the Multi-7 mutation up to 72 h examined (data not
shown). It was reported that the partitioning of C1 inhibitor between
the inhibitory and substrate pathways are temperature-dependent; the
substrate pathway is more favorable at low temperatures (44). This
suggests that a kinetic step is involved in complex formation. We
examined the complex formation at a low temperature (20 °C) but
could not detect any mutational effects, although more of the cleaved
forms are produced at 20 than at 37 °C in all cases (data not
shown). These results suggest that enhanced A-sheet closure conferred
by the stability increase of the Multi-7 molecule is not sufficient to
influence the complex formation with target proteinases.
Implication on the Mechanism of the Complex Formation--
It is
very likely that the metastability of inhibitory serpins is utilized
for the facile loop insertion during the complex formation with a
target proteinase (9, 12-14). It was suggested that the drive toward a
more stable state due to metastability results in trapping the
proteinase-inhibitor complex as an acyl-enzyme inhibitor (13, 14),
possibly accompanying conformational change in proteinases also (45,
46), including a distortion of the active site (47). We have
constructed a variant 1AT that is as stable as ovalbumin
(
G > 8 kcal/mol), but the mutational effect on
the inhibitory activity is not comparable with the dramatic shift in
stability. How can one reconcile such a paradox? It is possible that
the drive stemmed from the hydrophobic core of the serpin molecule can
indeed influence the conformational switch during the complex
formation, but such a drive is only slightly diminished for Multi-7
1AT. The increase in stability by Multi-7 may be only a
small fraction of much greater binding energy of the complex between a
serpin inhibitor and a target proteinase, and consequently the
stability increase of Multi-7 is not sufficient to influence the rate
of loop insertion during the complex formation. Since the stoichiometry
of inhibition is defined by (1 + ksubstrate/kinhibitory), the value is not sensitive to the changes of partitioning when one of
the pathways is dominant (e.g. either when the value is close to 1 as with the interactions of many inhibitory serpins and
their cognate target proteinases or when the value is very large as
with ovalbumin). For instance, increasing the rate of the inhibitory
pathway of ovalbumin significantly by hinge region mutations does not
lead to detectable complex formation (48). There is, however, another
class of mutations that particularly affects the stoichiometry of
inhibition significantly. Many of the mutations in the reactive loop
region such as G349P (22) or T345R (40) of
1AT induce
the substrate pathway more effectively, even if they may not increase
the conformational stability as much as Multi-7. They may exert the
mutational effect by interfering with the loop insertion more directly
(e.g. blocking accessibility). None of the seven mutations
in Multi-7 is located in the region directly involved in the loop
insertion in the proposed model (10). It is worth examining if further
increases in the stability of
1AT by additional
substitutions at the residues not directly involved in the loop
insertion would affect the inhibitory activity.
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ACKNOWLEDGEMENTS |
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We are grateful to Ok Sun Hwang for technical assistance. We also thank to Dr. Hyo Joon Kim for providing pYOV5 plasmid.
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FOOTNOTES |
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* This work was supported by grants from the Korean Ministry of Science and Technology.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.
Present address: Laboratory of Cellular and Molecular
Immunology, NIAID, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892.
§ Present address: Laboratory of Cell Signaling, NHLBI, National Institutes of Health, 3 Center Dr., Bethesda, MD 20892.
¶ To whom correspondence should be addressed: Division of Protein Engineering, Korea Resarch Institute of Bioscience and Biotechnology, 52 Eh-eun-dong, Yu-song-ku, Taejon 305-333, Korea. Tel.: 82-42-860-4140; Fax: 82-42-860-4593; E-mail: mhyu{at}kribb4680.kribb.re.kr.
1
The abbreviations used are: serpin, serine
proteinase inhibitor; 1AT,
1-antitrypsin.
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REFERENCES |
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