From the Departments of Medicine and Haematology, University of Cambridge, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom
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ABSTRACT |
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1-Antichymotrypsin is an
acute phase protein that protects the tissues from damage by
proteolytic enzymes, but previous studies have shown that
1-antichymotrypsin within the lungs of patients with
chronic bronchitis and emphysema is intact but inactive as an
inhibitor. Ammonium sulfate fractionation followed by blue Sepharose
and DNA-Sepharose chromatography was used to isolate small amounts of
intact, monomeric but inactive
1-antichymotrypsin from
the plasma of 30 healthy blood donors. This species had a higher DNA
binding affinity with more anodal electrophoretic mobility than native
1-antichymotrypsin and was conformationally stable against thermal denaturation, 8 M urea, and 7 M
guanidinium chloride. The protein was unable to accept synthetic
reactive loop peptides, and the reactive loop was resistant to
proteolytic cleavage at the P5-P4 bond but
could be cleaved between P1
and P3
. These data suggest that this new
1-antichymotrypsin species
was in a conformation similar to those of the crystallographically
determined latent serpins, plasminogen activator inhibitor-1 and
antithrombin.
1-Antichymotrypsin from lung lavage
migrated with the same electrophoretic mobility as the putative latent
1-antichymotrypsin, suggesting that this is the inactive
conformation described previously in the lungs of patients with chronic
bronchitis and emphysema. This conformational transition of
1-antichymotrypsin, from an active to an inactive state,
within the lung may play an important role in the pathogenesis of
chronic lung disease.
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INTRODUCTION |
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Neutrophils migrate into the lungs by chemotaxis in response to an
inflammatory stimulus. They then degranulate and release destructive
proteolytic enzymes, which, if uncontrolled, cause tissue destruction
and lung damage (1). In healthy individuals, the lung is protected by
proteinase inhibitors of which the most important are members of the
serpin1 superfamily typified
by 1-antitrypsin and
1-antichymotrypsin (2-4).
1-Antichymotrypsin is an acute phase,
DNA-binding glycoprotein whose plasma concentration can increase
rapidly within hours of tissue damage (5). It functions by forming
stable equimolar complexes with several proteinases including cathepsin
G, chymotrypsin and mast cell chymases (6-8).
Like other inhibitory serpins, the crystal structure of active
recombinant 1-antichymotrypsin (9) revealed a dominant A
-sheet together with B and C
-sheets, eight well defined
-helices, and a mobile solvent-exposed reactive
loop2 containing the scissile
P1-P1
reactive bond, which interacts with and
inhibits the cognate proteinase. The mobility of the reactive loop of
1-antichymotrypsin and other inhibitory serpins is
illustrated by proteolytically modified structures in which cleavage at
the reactive site results in a marked and irreversible conformational
change, with the N-terminal portion of the loop being completely
inserted into the middle of the A-sheet as strand 4A. The once
connected P1 and P1
residues in this
conformation are widely separated by approximately 70 Å (11, 12). In
some other serpins, the intact reactive loop can also be stably
incorporated into the A-sheet in the absence of proteolytic cleavage to
form a latent conformation that shares high thermostability and
immunological epitopes with the cleaved species (13). This conformation
is the structural basis for the latency of plasminogen activator inhibitor-1 (14), and we have recently demonstrated the existence of a
similar conformation in antithrombin and
1-antitrypsin
(15-17). Initially, the term "latency" was used, since the
inhibitory activity of these intact inactive serpins could be regained
through a cycle of denaturation and renaturation (17-21). However, it
is now broadly used to represent intact serpin species that are
structurally similar to those of latent plasminogen activator
inhibitor-1 (14) and antithrombin (15). The biological significance of
the latent species is unclear, but the loss of activity due to the
deformation of their reactive loop would obviously damage the
physiological balance between the inhibitors and their target
proteinases.
Over a decade ago, studies on bronchoalveolar lavage fluid from
patients with chronic obstructive bronchitis and emphysema suggested
that the majority of 1-antichymotrypsin in the human lung is intact but inactive as an inhibitor of chymotrypsin-like enzymes (22). The cause for this has remained unclear. We report here
the isolation of this unusual, inactive
1-antichymotrypsin species as a minor component of
healthy human plasma. The novel
1-antichymotrypsin has
high DNA binding affinity and physical properties similar to the latent
form of other inhibitory serpins. This species had an electrophoretic
mobility similar to that of a conformation of
1-antichymotrypsin in bronchoalveolar lavage, and it
provides a molecular explanation for the inactivation of
1-antichymotrypsin in chronic bronchitis and
emphysema.
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EXPERIMENTAL PROCEDURES |
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Materials--
Rabbit anti-human
1-antichymotrypsin antibody and alkaline
phosphatase-conjugated goat anti-rabbit immunoglobulins were from Dako
A/S (Glostrup, Denmark). Sepharose CL-4B, blue Sepharose CL-6B,
chromatography columns, and fraction collectors were from Pharmacia
Biotech (St. Albans, UK). Amicon ultrafiltration YM-30 membranes,
stirred cells, and Centricon-30 were from Millipore Ltd. (Watford, UK).
The synthetic Ac-Ser-Glu-Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-Ile-Ala-OH peptide was prepared by the Protein and Nucleic Acid Chemistry Facility, Department of Biochemistry, University of Cambridge on an
Applied Biosystems peptide synthesizer (Warrington, UK). The modular
mini-PROTEAN II electrophoresis system was from Bio-Rad Laboratories
Ltd. (Hemel Hempstead, UK), the luminescence spectrometer was from
Perkin-Elmer (Beaconsfield, UK), and all other reagents were from
either Sigma Chemical Co. (Poole, UK) or BDH Chemicals Ltd. (Bristol,
UK).
Preparation of the DNA-Sepharose Chromatography Column-- DNA-Sepharose resin was prepared by dissolving 0.3 g of highly polymerized DNA from calf thymus in 150 ml of distilled H2O and incubating overnight on a rotating wheel with 200 ml of packed volume of ice-cold CNBr-activated Sepharose CL-4B in 10 mM potassium phosphate, 500 mM KCl, pH 8, at 4 °C. Fifteen ml of ethanolamine hydrochloride was then added to DNA-Sepharose resin, and the mixture was incubated on a rotating wheel for at least 4 h at 4 °C. The resin was then transferred to a Hartley funnel and washed with 1 liter of 10 mM potassium phosphate, pH 8, followed by 1 liter of 1 M potassium phosphate, pH 8, 1 liter of 1 M KCl, 1 liter of distilled H2O, and finally 1 liter of 10 mM potassium phosphate, 50 mM KCl, pH 6.8, before packing into a Pharmacia XK16/40 chromatography column. The DNA-Sepharose column (70 ml of resin) was equilibrated with 300 ml of 10 mM potassium phosphate, 50 mM KCl, 5 mM EDTA, pH 6.8, at a flow rate of 1 ml/min.
Isolation of 1-Antichymotrypsin from
Plasma--
The preparation of human
1-antichymotrypsin
described here is a modification of the original method described by
Travis et al. (23, 24) with all purification steps being
performed at 4 °C. Two hundred-ml samples of plasma from 30 healthy
blood donors were each mixed with 200 ml of saturated ammonium sulfate
for 30 min and then centrifuged at 1500 × g for 30 min. The supernatant was pooled, and 160 g of solid ammonium
sulfate per liter of supernatant was added, adjusted to pH 6.0 with
concentrated H2SO4, and left to stir for 30 min. The solution was centrifuged at 1500 × g for 50 min, the supernatant was discarded, and the pellet was resuspended in a
minimum volume of 30 mM sodium phosphate, pH 6.8, and
dialyzed against 5 mM EDTA, pH 6.8 (3 × 5 liters)
followed by 30 mM sodium phosphate, pH 6.8 (3 × 5 liters). The dialyzed solution was then applied to a blue Sepharose
chromatography column (Pharmacia XK50/30 column, 900 ml of resin) at a
flow rate of 3 ml/min. The column was washed with 30 mM
sodium phosphate, pH 6.8, and
1-antichymotrypsin was
eluted with 30 mM sodium phosphate, 0.1 M NaCl,
pH 6.8, and confirmed by rocket immunoelectrophoresis (25). The
fractions containing
1-antichymotrypsin were dialyzed
against 10 mM potassium phosphate, 50 mM KCl,
pH 6.8, and concentrated to approximately 10 ml before loading onto the
DNA-Sepharose column at 1 ml/min.
1-Antichymotrypsin was
then eluted with a 600-ml linear gradient from 50-500 mM
KCl. The fractions containing
1-antichymotrypsin were
again identified by rocket immunoelectrophoresis and dialyzed against
10 mM potassium phosphate, 50 mM KCl, 5 mM of EDTA, pH 6.8.
1-Antichymotrypsin
concentration was determined using the Lowry protein assay, and samples
were snap frozen in liquid nitrogen and stored at
80 °C until
use.
Polyacrylamide Gel Electrophoresis (PAGE)-- SDS-PAGE was performed using either 10% (w/v) or 7.5-15% (w/v) linear gradient acrylamide gels in 190 mM glycine, 25 mM Tris, pH 8.8, buffer with 0.1% (w/v) SDS (26). All samples were heated in boiling water for 5 min prior to electrophoresis. Nondenaturing PAGE was a modification of the method of Goldenberg (27) and was carried out on 7.5-15% (w/v) linear gradient gels using 53 mM Tris, 68 mM glycine, pH 8.9, as the cathode buffer and 100 mM Tris-HCl, pH 7.8, as the anode buffer.
Measurement of Conformational Stability-- The 20 mM Tris-HCl, pH 7.4, and 20 mM Tris-HCl, 7 M guanidinium chloride (GdmCl), pH 7.4, solutions were filtered through a 0.2-µm membrane prior to fluorescence assays. Each protein sample was incubated at 23 °C for 2 h at a final concentration of 10 µg/ml in 0.5 ml of 20 mM Tris-HCl, pH 7.4, with a range of denaturant concentrations between 0 and 7 M GdmCl. Denaturation curves were then determined by luminescence spectrometry at a wave length of 300-360 nm with slit widths of 10 nm. The unfolded fractions were analyzed as described by Pace et al. (28). Transverse urea gradient gels with a 0-8 M urea gradient were prepared according to the method of Goldenberg (27) and Mast et al. (29). Forty µg of each protein sample was loaded in a single well, and electrophoresis was performed at 15 mA until the dye front reached the base of the gel. The proteins were visualized by staining with Coomassie Blue.
Measurement of Thermal Stability--
The thermostability of
both active and inactive 1-antichymotrypsin were
determined by incubating the protein at 0.5 mg/ml in 10 mM
potassium phosphate, 50 mM KCl, pH 6.8, at constant
temperatures between 37 and 100 °C for 2 h followed by brief
centrifugation and careful deceleration without braking (17). The
solutions were then rapidly cooled on ice, and aliquots were removed
for analysis by nondenaturing PAGE.
Immunoblotting--
Purified plasma
1-antichymotrypsin species or lung lavage fluid were
separated by SDS-PAGE or nondenaturing PAGE and then electroblotted
onto nitrocellulose membrane.
1-Antichymotrypsin was
detected using a rabbit anti-human
1-antichymotrypsin
antibody and a second alkaline phosphatase-conjugated goat anti-rabbit antibody. The immunoblots were developed using the bromochloroindolyl phosphate/nitro blue tetrazolium substrate according to the method of
Harlow and Lane (30).
Active Site Titration and Association Kinetics of
1-Antichymotrypsin--
The activity of
1-antichymotrypsin was determined against bovine
-chymotrypsin of known active site, and kinetic parameters for the
interaction with bovine
-chymotrypsin and cathepsin G were
determined at 37 °C as detailed previously (31, 32). All buffers for
the cathepsin G kinetics contained 0.1% (v/v) Triton X-100 and the
substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was
present at a concentration of 400 µM and 6 mM
for bovine
-chymotrypsin and cathepsin G, respectively. The
Km values for bovine
-chymotrypsin and cathepsin
G with succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were
0.087 ± 0.007 (31) and 1.9 ± 0.23 mM,
respectively (32).
Formation of Binary Complex of 1-Antichymotrypsin
with a Synthetic Reactive Loop Peptide--
Thirty µg of active or
inactive
1-antichymotrypsin was incubated at a final
concentration of 0.5 mg/ml in 50 mM Tris, 50 mM
NaCl, pH 7.4, with a 100-fold molar excess of the synthetic 12-mer
antithrombin reactive loop peptide (Ac-SEAAASTAVVIA) at 37 °C for up
to 48 h. Five µg of protein was removed at each time point, snap
frozen in liquid nitrogen, and stored at
80 °C until required. The
formation of protein-peptide binary complexes was then assessed by
separating the proteins on nondenaturing PAGE as described above.
Analysis of the Conformation of the Reactive Loop by Limited
Proteolysis--
Cleavage of 1-antichymotrypsin by
porcine pancreatic trypsin (33) was performed at a 100:1 (w/w) ratio of
serpin to enzyme in 20 mM Tris, 20 mM
CaCl2, pH 8.3, for 0, 30, 60, and 90 min at 37 °C.
1-Antichymotrypsin was cleaved by Bitis
arietans snake venom (33, 34) by incubation at a 20:1 (w/w) ratio
of serpin to enzyme in 20 mM Tris-HCl, pH 7.4, for 0, 30, 60, and 90 min at 37 °C. The final concentration of
1-antichymotrypsin in each reaction was 0.2 mg/ml. At
each time point, 4 µg of
1-antichymotrypsin was taken,
and the cleavage reaction was terminated by heating at 100 °C for 10 min before separating the proteins on SDS-PAGE. At the end of the
cleavage reaction, an aliquot of the cleaved
1-antichymotrypsin was immediately snap frozen in liquid
nitrogen and subjected to N-terminal sequencing.
Bronchoalveolar Lavage--
Lavage samples were obtained from 10 consecutive patients undergoing bronchoscopy for investigation of
bronchogenic carcinoma. One hundred ml of normal saline was instilled
in 20-ml aliquots into the lung opposite of that under investigation.
The fluid was aspirated and stored on ice before being filtered through cotton gauze to remove mucus and centrifugation at 1500 × g for 10 min to remove the cell pellet. The supernatant was
concentrated and then assayed by SDS-PAGE, nondenaturing PAGE, and
immunoblotting to detect 1-antichymotrypsin. This study
was approved by the Local Research Ethics Committee, and all patients
gave informed consent.
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RESULTS |
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1-Antichymotrypsin was isolated from the plasma of
healthy blood donors by ammonium sulfate fractionation followed by blue Sepharose and DNA-Sepharose chromatography. Purification using the
DNA-Sepharose column yielded less than 2 mg of protein but reproducibly
(in 30 separate experiments) fractionated
1-antichymotrypsin into two peaks, denoted peak 1 and
peak 2, which eluted at KCl concentrations of approximately 185 and 255 mM, respectively (Fig. 1a). The protein yield from
peak 2 was typically only 50-100 µg of protein, and multiple
preparations were therefore required to fully characterize this
conformation. Peak 1 eluted at a salt concentration similar to that of
native
1-antichymotrypsin, and peak 2 eluted at the same
point as reactive loop-cleaved
1-antichymotrypsin. The
cleaved
1-antichymotrypsin control was prepared by
incubating native protein with B. arietans snake venom. HPLC
purification and amino-terminal sequencing of the cleavage fragments
confirmed that the venom cleaved at P1
-P2
(Ser-Ala) and P2
-P3
(Ala-Leu) of the
reactive loop and the Asn8-Leu9 bond of the
amino terminus.
1-Antichymotrypsin polymers formed by
heating native protein at 0.2 mg/ml at 60 °C for 2 h (Fig. 1b) did not bind to the column, suggesting that
polymerization resulted in the obfuscation of some or all of the lysine
residues at positions 210-212, 391, and 396 that constitute the DNA
binding domain (35). Nondenaturing PAGE showed that both peak 1 and peak 2
1-antichymotrypsin species were monomeric and
that peak 2 migrated more anodally than peak 1 with the same
electrophoretic mobility as reactive loop-cleaved
1-antichymotrypsin (Fig. 1b). Such a
difference in electrophoretic mobility between peak 1 and peak 2
1-antichymotrypsin on nondenaturing PAGE did not result from amino-terminal heterogeneity, since both species had mixed N-terminal sequences of
His
2-Pro
1-Asn1-Ser2-Pro3
or Asn1-Ser2-Pro3, in keeping with
the findings of others (36, 37). The integrity of both peak 1 and peak
2
1-antichymotrypsin was confirmed for all preparations
by SDS-PAGE in comparison with a reactive loop-cleaved control (Fig.
1c). In no case did peak 2
1-antichymotrypsin
migrate with similar electrophoretic mobility to the cleaved protein. Finally, this conformation was not an artifact of plasma storage, since
similar results were also obtained with fresh, nonfrozen plasma.
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Assessment of the Conformation of Peak 1 and 2 1-Antichymotrypsin--
Active site titration showed
that peak 1
1-antichymotrypsin had a specific activity
of 57-77% against bovine
-chymotrypsin, whereas the peak 2 species
consistently showed <1% inhibition. Ultraviolet scans of peak 2
1-antichymotrypsin were similar to those of peak 1 protein, indicating that the loss of activity did not result from
residual DNA binding from the purification protocol. Kinetic analysis
of native
1-antichymotrypsin, identical to that of peak
1 but purified on a higher capacity DNA-cellulose (32) rather than a
DNA-Sepharose column, revealed an association rate constant
(ka) of 5.0 ± 0.02 × 105
M
1 s
1 and a
Ki value of 42.8 ± 0.2 pM with
bovine
-chymotrypsin (n = 4 experiments) and a
ka of 1.7 ± 0.2 × 105
M
1 s
1 and a
Ki of 809 ± 39 pM against
cathepsin G (n = 3 experiments). These results are
similar to the values reported previously for recombinant
1-antichymotrypsin (32, 38) with the association rate
constant of plasma
1-antichymotrypsin with cathepsin G
being over 100-fold less than that determined by Beatty et
al. (7). The assessment of the thermal stability of the two
1-antichymotrypsin conformations from the DNA-Sepharose
column showed that peak 1
1-antichymotrypsin formed high
molecular mass polymers between 50 and 60 °C, but peak 2 and
venom-cleaved
1-antichymotrypsin remained stable and
monomeric at temperatures up to 100 °C (Fig. 2). Similarly, peak 1
1-antichymotrypsin displayed the characteristic unfolding transition of the serpins (29) with increasing concentration of urea (Fig. 3a), whereas
peak 2 and venom-cleaved
1-antichymotrypsin were
resistant to unfolding in up to 8 M urea (Fig. 3,
b and c). The significant difference in stability
between the active peak 1 and inactive peak 2
1-antichymotrypsin was clearly evident when an equal
mixture of the two conformations was assessed on the same transverse
urea gradient gel (Fig. 3d). This resistance to unfolding in
high concentrations of urea, in the absence of reactive-loop cleavage,
is characteristic of latent serpins (16, 17). One of the determinants
of latency is the restoration of activity following denaturation with
and refolding from GdmCl (17, 18), although the latent form of
antithrombin appears to be resistant to refolding (16). Refolding of
peak 2
1-antichymotrypsin with 6 M GdmCl did
not restore inhibitory activity against bovine
-chymotrypsin, and
fluorescence analysis was used to determine if this concentration of
GdmCl unfolded the peak 2 protein. Peak 1
1-antichymotrypsin unfolded at approximately 3 M GdmCl, but peak 2
1-antichymotrypsin was
resistant to unfolding in 7 M GdmCl (Fig.
4), which explains the finding that
higher concentrations of denaturant could not be used to refold and
reactivate this thermostable conformation.
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Bronchoalveolar Lavage--
There were similarities between the
characteristics of peak 2 1-antichymotrypsin and the
observation by Berman et al. (22) that
1-antichymotrypsin in lung lavage was monomeric and
inactive. Bronchoalveolar lavage was obtained from 10 consecutive
patients undergoing fiberoptic bronchoscopy for the investigation of
bronchogenic carcinoma. The lavage was concentrated in an Amicon cell,
and the conformation of
1-antichymotrypsin was assessed
by SDS- and nondenaturing PAGE followed by Western blot analysis (Fig.
7). Immunoblotting of SDS-PAGE for
1-antichymotrypsin showed that
1-antichymotrypsin from bronchoalveolar lavage was not
cleaved at the reactive loop (Fig. 7b). Analysis of
1-antichymotrypsin by nondenaturing PAGE revealed a band
that migrated with the same electrophoretic mobility as peak 2
1-antichymotrypsin (Fig. 7a), making it
likely that the conformation of
1-antichymotrypsin in
peak 2 is the same as the inactive species described previously (22) in
the lungs of patients with chronic bronchitis and emphysema. The
concentration of
1-antichymotrypsin in lavage was too
low to allow the isolation of peak 2
1-antichymotrypsin
for further analysis.
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DISCUSSION |
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1-Antichymotrypsin was first isolated from human
serum in 1965, but the purification procedure was lengthy and
complicated (40). Since then, several groups have attempted to simplify the isolation procedure using a variety of chromatographic techniques (6, 23, 24, 37, 38, 41, 42). The yield and quality of
1-antichymotrypsin was diverse, and two groups reported
unexplained additional
1-antichymotrypsin bands. Laine
and Hayem (42) consistently observed two bands of pure
1-antichymotrypsin on analytical PAGE, especially if the
protein had been lyophilized. Travis et al. (23) detected
two well separated lines when they examined whole human plasma using an
antiserum against
1-antichymotrypsin. There was no
explanation for these observations, but the authors speculated that a
form of desialylated
1-antichymotrypsin might exist in whole plasma. Berman et al. (22) found that
1-antichymotrypsin in lung lavage from patients with
chronic bronchitis and emphysema was intact but inactive against
cognate proteinases. The mechanism of this inactivation was unclear. It
was these studies and our recent description of latent
1-antitrypsin and antithrombin (16, 17) that raised the
possibility that
1-antichymotrypsin may adopt a latent
conformation in vivo.
Ammonium sulfate fractionation followed by blue Sepharose and
DNA-Sepharose chromatography was used to purify two species of
1-antichymotrypsin from human plasma. Sequence analysis
revealed that both
1-antichymotrypsin species contained
the
His
2-Pro
1-Asn1-Ser2-Pro3
and Asn1-Ser2-Pro3 isoforms that
correspond to the amino-terminal microheterogeneity that has been
described previously (36, 37). The physiological importance of these
isoforms is unknown, but they are generally believed to be the result
of signal peptidase cleavage or post-translational proteolysis (37).
There are no differences in apparent molecular mass, charge
microheterogeneity, or inhibitory activity between the two isoforms
(37), and since they were found in both peaks from DNA-Sepharose
chromatography, it is unlikely that they account for any of the
differences between the two species of
1-antichymotrypsin. Both of the
1-antichymotrypsin species from DNA-Sepharose
chromatography were intact and monomeric (Fig. 1), but whereas
1-antichymotrypsin in the first peak was functional as
an inhibitor of proteinases, the
1-antichymotrypsin in
peak 2 had no inhibitory activity. The relatively modest association
rate constant for the interaction of
1-antichymotrypsin
with cathepsin G and the high Ki raises the
possibility that cathepsin G may not be the primary target of this
proteinase inhibitor.
Peak 1 1-antichymotrypsin readily formed high molecular
mass polymers on heating at 50-60 °C (Fig. 2) and showed unfolding profiles in urea (Fig. 3) and GdmCl (Fig. 4) that are characteristic of
inhibitory members of the serpin superfamily. Similarly, this protein
accepted an exogenous reactive loop peptide, inferentially as s4A, to
form a binary complex with a distinctive band shift on nondenaturing
PAGE (Fig. 5).
1-Antichymotrypsin from peak 2, like
latent antithrombin and
1-antitrypsin (16, 17), was resistant to conformational transitions induced by heating to 100 °C, 8 M urea, and 7 M GdmCl and failed
to accept exogenous reactive loop peptides (Figs. 2-5). These results
strongly suggest that peak 2
1-antichymotrypsin has been
stabilized by the insertion of an extra strand into the A-sheet as s4A.
This is recognized in the serpin superfamily following reactive loop
cleavage (43), the formation of latent protein (15-17), and the
annealing of reactive loop peptides in the formation of high molecular
mass loop-sheet polymers (17, 44) or serpin-reactive loop peptide
binary complexes (39, 43). Peak 2
1-antichymotrypsin was
not polymeric as assessed by nondenaturing PAGE and was not cleaved at
the reactive loop as assessed by SDS-PAGE (Fig. 1c). The
protein migrated on the nondenaturing PAGE with the electrophoretic
mobility of cleaved protein, making it unlikely that it contained an
exogenous reactive loop peptide that had annealed to the A-sheet to
form a binary complex. Thus, the small quantity of
1-antichymotrypsin that was reproducibly isolated from
the plasma of healthy blood donors was likely to be in the latent
conformation.
The crystallographic structure of latent antithrombin (15) showed the
N-terminal portion of the reactive loop
(P14-P3) to be stably incorporated into the
A-sheet as s4A and the C-terminal portion of the loop
(P1-P12) to be externalized (Fig.
8). Porcine pancreatic trypsin cleaves
native
1-antichymotrypsin at the
P5-P4 residues (33). This enzyme cleaved peak
1
1-antichymotrypsin at the
P5-P4 bond, but no equivalent cleavage was
found in the peak 2 species (Fig. 6a), consistent with the
hypothesis that the N-terminal portion of its reactive loop is buried
in the A-sheet. Snake venom was able to cleave peak 1 and peak 2
1-antichymotrypsin at both the
P1
-P2
and P2
-P3
positions (Fig. 6b), in keeping with the model presented in
Fig. 8 showing that the C-terminal region of the loop in latent
1-antichymotrypsin is located on the outer aspect of the
molecule and therefore accessible to proteolytic attack. This provides
further evidence that the conformation of peak 2
1-antichymotrypsin differs significantly from the native species but closely resembles that of latent antithrombin and latent
plasminogen activator inhibitor-1 (14, 15, 45).
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The formation of a latent serpin may be described as a two-step
process: an initial rapid transition, including partial incorporation of the N-terminal portion of the reactive loop into the A-sheet with
the concomitant release of strand 1C from the C-sheet (16), and a
second, much slower conformational change allowing full insertion of
the reactive loop into the A-sheet concurrent with sufficient position
exchange between the reactive loop and s3C/s4C to stabilize the
conformation (21, 46). This conformational transition is likely to be
facilitated in 1-antichymotrypsin, since it contains
four extra residues on the P
side of the reactive loop (47). The
crystal structure of latent and native antithrombin obtained from a
loop-sheet dimer (15, 45) and shown in Fig. 8 allows a direct
comparison of the structural perturbation that results from the
conformational transition to latency. In addition to the striking
changes in the conformation of the reactive loop, there are changes in
the "gate" region comprising the residues of s3C and s4C and the
turn between the two
-strands (16, 46). The positive charges of
Lys210-Lys211-Lys212 located on the
s3C/s4C turn contribute to the DNA binding domain of
1-antichymotrypsin (35), and it is plausible that
destabilizing this region in latent
1-antichymotrypsin
would allow increased lysine contact with DNA and hence the increased
binding affinity seen on elution from the DNA cellulose column.
Similarly, the formation of high molecular mass polymers must obscure
this region, abolishing the binding of this conformation to DNA. The
function of the DNA binding domain of
1-antichymotrypsin
in vivo is unclear, but any biological role may be modulated
by transition from the native to the latent or polymeric
conformation.
Despite evidence that 1-antichymotrypsin can inhibit
several proteinases in vitro and that it is selectively
concentrated and synthesized in the lung (48), its physiological role
remains unclear. The removal of
1-antichymotrypsin from
bronchoalveolar lavage had little effect on the total inhibitory
capacity, suggesting that the majority of lung
1-antichymotrypsin is inactive as a proteinase inhibitor
(22). Unlike
1-antitrypsin, which has a P1
methionine residue (49), the P1 leucine of
1-antichymotrypsin is resistant to oxidation and
inactivation. Moreover, our data demonstrate that this inactive
1-antichymotrypsin is intact and cannot be the product
of indiscriminate proteolytic attack by nontarget enzymes or
dissociation of proteinase-inhibitor complexes. Similarly the results
are unlikely to be explained by aberrant glycosylation (48), since peak
1 and peak 2
1-antichymotrypsins from plasma and lavage
have the same molecular mass on SDS-PAGE. The co-migration of
bronchoalveolar lavage
1-antichymotrypsin with latent
peak 2 protein on nondenaturing PAGE (Fig. 7a) provides strong support for our hypothesis that lung
1-antichymotrypsin may be inactivated by transition to
the latent conformation. The identification of this form in the lungs
of patients with chronic bronchitis and emphysema (22) implies that it
may contribute to the pathogenesis of this disease.
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. Robin Carrell (Department of Hematology, University of Cambridge) for helpful comments, Peter Elliott (Department of Hematology, University of Cambridge) for preparing Fig. 8, Dr. Stephen Bottomley (Department of Hematology, University of Cambridge) for advice on the fluorescence measurements, Dr. Len Packman (Department of Biochemistry, University of Cambridge) for peptide synthesis and N-terminal sequence analysis, and Dr. Clare LaRoche (Chest Medical Unit, Papworth NHS Trust) for providing the bronchoalveolar lavage.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust (United Kingdom).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.
To whom correspondence should be addressed: Dept. of Haematology,
University of Cambridge, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.
Tel.: 44 1223 336825; Fax: 44 1223 336827; E-mail: dal16{at}cam.ac.uk.
2
Residue numbers used for
1-antichymotrypsin in this report are counted from
asparagine. Thus, the normal amino-terminal sequence for
1-antichymotrypsin is either
His
2-Pro
1-Asn1-Ser2-Pro3
or Asn1-Ser2-Pro3. The reactive
loop amino acids are numbered from the
Leu358--Ser359 P1-P1
sessile bond, in keeping with the convention of Schechter and Berger
(10). Residues to the amino terminus of this bond are labeled
P2, P3, P4, etc., while those to
the carboxyl terminus are termed P2
, P3
,
P4
, etc.
1 The abbreviations used are: serpin(s), serine proteinase inhibitor(s); PAGE, polyacrylamide gel electrophoresis; GdmCl, guanidinium chloride; HPLC, high performance liquid chromatography.
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REFERENCES |
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