(Received for publication, September 28, 1994; and in revised form, December 21, 1994)
From the
Members of the serine proteinase inhibitor or serpin superfamily
have a common molecular architecture based on a dominant five-membered
A -pleated sheet and a mobile reactive center loop. The reactive
center loop has been shown to adopt a range of conformations from the
three turn
-helix of ovalbumin to the cleaved or latent inhibitor
in which the reactive center loop is fully inserted into the A sheet of
the molecule. While the cleaved state can be achieved in all inhibitory
serpins only plasminogen activator inhibitor-1 and, more recently,
antithrombin have been shown to adopt the latent conformation. We show
here that the archetypal serpin,
-antitrypsin, can
also be induced to adopt the latent conformation by heating at high
temperatures in 0.7 M citrate for 12 h. The resulting species
elutes at a lower sodium chloride concentration on an anion-exchange
column and has a more cathodal electrophoretic mobility on
non-denaturing polyacrylamide gel electrophoresis and isoelectric
focusing than native M antitrypsin. Latent antitrypsin is inactive as
an inhibitor of bovine
-chymotrypsin, is stable to unfolding with
8 M urea, and is more resistant to heat-induced loop-sheet
polymerization than native but less resistant than cleaved antitrypsin.
The reactive center loop of latent antitrypsin is inaccessible to
proteolytic cleavage, and its occupancy of the A sheet prevents the
molecule accepting an exogenous reactive center loop peptide. The
activity of latent antitrypsin may be increased from <1% to
approximately 35% by refolding from 6 M guanidinium chloride.
-Antitrypsin (or
-proteinase
inhibitor) is the most abundant circulating proteinase inhibitor and
the archetypal member of the serine proteinase inhibitor or serpin
superfamily(1) . Members of this family are important in the
control of serine proteinases involved in phagocytosis, coagulation,
complement activation, and fibrinolysis (see (2) for review).
They have a unique inhibitory specificity, but crystallographic data
show that they share a common molecular architecture based on a
dominant five-stranded A
-sheet(3, 4, 5, 6, 7, 8, 9, 10, 11) .
The intact inhibitor has an exposed, mobile, reactive center loop with
the P
-P
` residues acting as a ``peptide
bait'' for the cognate proteinase(12, 13) . The
precise conformation of the reactive center loop in the active
inhibitor when it docks with an enzyme is unknown but is likely to lie
between the three-turn helical conformation of ovalbumin (4, 11) and the canonical structure of the small
inhibitors(14, 15) . In order for serpins to adopt a
canonical conformation, the helical reactive center loop must unfold
with its N-terminal stalk inserted into the gap between strands 3 and 5
of the A sheet(16, 17) , such reactive loop insertion,
however, may not be essential for proteinase recognition (18) .
After docking with the enzyme, the loop is presumed to insert further
into the A sheet to form an extended canonical structure that fits the
substrate binding site of the cognate proteinase, the locked
conformation(19) . Dissociation of the enzyme-inhibitor complex
may result in the release of the intact serpin (20) or cleavage
of the loop at the P
-P
` residues(21) .
Reactive center loop cleavage results in a profound conformational
rearrangement in which the loop is fully inserted into the A sheet of
the molecule(3, 22, 23) . The cleaved species
is inactive, has increased thermostability (24) , and is more
resistant to unfolding with denaturants (25) when compared to
the native protein. It is clear that the sequence of events involved in
proteinase inhibition requires the loop to be mobile and to adopt a
variety of conformations.
A consequence of the mobility of the loop
is the formation of loop-sheet polymers in which the reactive center
loop of one molecule is inserted into a -pleated sheet of a
second. This interaction accounts for the biologically important
retention of Z (26) and Siiyama (27) antitrypsin in
the endoplasmic reticulum of the liver and hence the accompanying
plasma deficiency. More recently plasminogen activator inhibitor-2 (28) and mutants of C1-inhibitor(29, 30) ,
antithrombin (31) , and antichymotrypsin (32) have also
been shown to spontaneously polymerize in vivo and a
loop-sheet dimer has been identified in the crystal structure of
antithrombin(9, 10) .
A second consequence of the
mobile reactive center loop is the latent conformation seen in the
crystal structures of plasminogen activator inhibitor I (PAI-1)(7) and antithrombin(10) . In both cases the
uncleaved loop of the inhibitor is stably incorporated into the A sheet
of the inhibitor to form an inactive, thermostable molecule that shares
immunological epitopes with the cleaved species(33) . This
conformation occurs spontaneously in plasma PAI-1, but inhibitory
activity may be restored by denaturant unfolding and refolding (34, 35, 36, 37) . The
conformational change in PAI-1 had been predicted prior to its
structural confirmation. There was also an accompanying prediction (38) that the same latent conformation could be induced in
other serpins by denaturant stress. However, the first attempts to
demonstrate this with antithrombin and antitrypsin were later shown to
be negated by the predominant formation of short chain
polymers(39) . It was subsequently shown that latent
antithrombin could be quantitatively prepared and isolated by
pasteurization with 0.25 M citrate at 60
°C(10, 31) .
We report here the preparation, purification, and characterization of latent antitrypsin prepared in vitro using a similar protocol. As for PAI-1, latent antitrypsin is inactive as an inhibitor and has a reactive center loop that is inaccessible to proteolytic cleavage. The latent form is more thermostable and resistant to unfolding in urea than the native protein suggesting that the molecule has been stabilized by the incorporation of the reactive center loop into the A sheet of the molecule.
Bovine -chymotrypsin,
succinyl-L-alanyl-L-alanyl-prolyl-L-phenylanalyl-p-nitroanilide
(Suc-Ala-Ala-Pro-Phe-p-nitroanilide), Staphylococcus
aureus V8 proteinase, 3,4-dichloroisocoumarin, papain type IV, and
trisodium citrate were from Sigma, Dorset, U.K.
Citrate was removed by extensive dialysis into 20 mM Tris,
pH 8.6 and the protein concentrated to approximately 1.0 mg/ml and
reheated at 60 °C for 3 h to convert any residual active
antitrypsin to polymer(40) . The inactive monomer was separated
from polymer by ion exchange on a mono-Q column in 20 mM Tris
pH 8.6. Higher concentrations of Tris, such as those used to purify
native M and Z antitrypsin(40) , reduced monomer binding to the
resin. Pasteurized antitrypsin was separated into four fractions by
anion exchange as shown in Fig. 1; the specific activity of each
fraction against bovine -chymotrypsin was 0.3%, 0.5%, 0.3% and 0%
for peaks 1, 2, 3 and 4 respectively. Omission of the reheating step
resulted in an increase in the specific activity of peak 2,
representing residual active monomeric antitrypsin. Antitrypsin
polymers and aggregates were converted to monomer by boiling for 2 min
with 1% w/v SDS. No fraction contained reactive center loop cleaved
antitrypsin when assessed by SDS-PAGE and, in particular, N-terminal
sequencing of fraction 1 confirmed only N-terminal, but not reactive
center loop cleavage (
EDPQGDAAQK- and
DAAQK-)
as described previously(40) . The fraction that eluted before
peak 1 was concentrated and 5 µg loaded onto SDS and
non-denaturing-PAGE; no antitrypsin was visualized on either gel.
Figure 1: Elution profile of antitrypsin heated at 0.25 mg/ml at 67 °C for 12 h in 0.7 M sodium citrate. The citrate was removed by dialysis and the protein concentrated to 1 mg/ml and reheated at 60 °C for 3 h. The protein was loaded onto a mono-Q column in 20 mM Tris pH 8.6 and eluted with a 0-1.0 M sodium chloride gradient (dotted line). The fractions were pooled as indicated.
Non-denaturing-PAGE analysis revealed fraction 1 to be monomeric, fraction 2 was a mixture of monomer and polymer and fraction 3 was short chain polymers, fraction 4 represented antitrypsin aggregates (Fig. 2). As fractions 1 and 3 were homogeneous these were further characterized by transverse urea gradient (TUG) gel electrophoresis and isoelectric focusing. Fraction 1 had a TUG gel profile similar to cleaved antitrypsin and significantly different from that of native M antitrypsin (Fig. 3); this represented the latent conformation. Isoelectric focusing confirmed the more cathodal migration seen on native-PAGE along with a new band pattern when compared to native M antitrypsin (Fig. 4). Fraction 3 had the characteristic profile of polymers on a TUG gel although these were significantly different from M antitrypsin polymers formed by incubating antitrypsin at 67 °C for 12 h in the absence of 0.7 M sodium citrate. In the latter case the polymers were much longer with relatively few entering the separating gel and most remaining in, or on top of, the stacking gel. The polymers shown, for comparative purposes, in Fig. 3were prepared by heating M antitrypsin (0.2 mg/ml) at 60 °C for 3 h as described in the Methods section. Although latent antitrypsin represented approximately 30% of the pasteurized material when visualized by non-denaturing PAGE, the yield following chromatography was typically 200-900 µg from 8 mg starting M antitrypsin.
Figure 2: 7.5-15% w/v non-denaturing-PAGE of the fractions eluted from the mono-Q column. Lanes 1 and 2 contain 10 µg protein and lanes 3 to 6 5 µg protein. Lane 1, antitrypsin heated at 67 °C for 12 h in 0.7 M sodium citrate; lane 2, as for lane 1 following the removal of citrate by dialysis and reheating at 60 °C for 3 h; lane 3, peak 1; lane 4, peak 2; lane 5, peak 3; lane 6, peak 4.
Figure 3: 7.5% w/v transverse urea gradient gel electrophoresis of native and latent antitrypsin. All gels contain 50 µg protein unless otherwise indicated, the left of each gel represents 0 M urea and the right 8 M urea. Upper left, native antitrypsin; upper right, antitrypsin cleaved at the reactive center loop with Staph. aureus V8 proteinase; middle left, latent antitrypsin (peak 1); middle right, native antitrypsin (40 µg) mixed with latent antitrypsin (40 µg); lower left, fraction 3 from the anion exchange column: antitrypsin polymers formed by heating antitrypsin (0.25 mg/ml) at 67 °C with 0.7 M citrate for 12 h; lower right, antitrypsin polymers formed by heating antitrypsin (0.2 mg/ml) at 60 °C for 3 h.
Figure 4: Isoelectric focusing of native and latent M antitrypsin. All lanes contain 15 µg protein. The top represents the anode and the bottom the cathode. Lane 1, native antitrypsin; lane 2, latent antitrypsin; lane 3, reactive loop cleaved antitrypsin; lane 4, native antitrypsin.
Figure 5: A (above), cleavage of native and latent antitrypsin with Staph aureus V8 proteinase. Native and latent antitrypsin were incubated with enzyme in 50% v/v reaction buffer at 37 °C for 2 h before being loaded onto a 7.5-15% w/v SDS-PAGE. All lanes contain 5 µg protein and the ratios of inhibitor:enzyme are based on w/w. Native represents the uncleaved native or latent antitrypsin, cleaved is antitrypsin cleaved at the reactive center loop and peptide is the 4 kDa peptide that is released following reactive center loop cleavage. Lane 1, markers (kDa); lane 2, native antitrypsin; lane 3, native antitrypsin:V8 proteinase 800:1; lane 4, native antitrypsin:V8 proteinase 400:1; lane 5, native antitrypsin:V8 proteinase 100:1; lane 6, native antitrypsin:V8 proteinase 25:1; lane 7, latent antitrypsin; lane 8, latent antitrypsin:V8 proteinase 800:1; lane 9, latent antitrypsin:V8 proteinase 400:1; lane 10, latent antitrypsin:V8 proteinase 100:1; lane 11, latent antitrypsin:V8 proteinase 25:1. B (below), cleavage of native and latent antitrypsin with papain. Native and latent antitrypsin were incubated with enzyme in 50% v/v reaction buffer at 37 °C for 2 h before being loaded onto a 7.5-15% w/v SDS-PAGE. All lanes contain 5 µg protein and the ratios of inhibitor:enzyme are based on w/w. Native represents the uncleaved native or latent antitrypsin, cleaved is antitrypsin cleaved at the reactive center loop and peptide is the 4 kDa peptide that is released following reactive center loop cleavage. Lane 1, markers (kDa); lane 2, native antitrypsin; lane 3, native antitrypsin:papain 800:1; lane 4, native antitrypsin:papain 400:1; lane 5, native antitrypsin:papain 100:1; lane 6, native antitrypsin:papain 25:1; lane 7, latent antitrypsin; lane 8, latent antitrypsin:papain 800:1; lane 9, latent antitrypsin:papain 400:1; lane 10, latent antitrypsin:papain 100:1; lane 11, latent antitrypsin:papain 25:1.
Figure 6: Heat stability of native, latent and cleaved antitrypsin. All samples were heated at 30-100 °C for 2 h before being separated by 7.5-15% w/v non-denaturing-PAGE or being filtered through a 0.22 µm filter and assayed by rocket immunoelectrophoresis. Upper, middle and lower gels represent native, latent and reactive center loop cleaved antitrypsin (with Staph. aureus V8 proteinase) respectively, all lanes contain 5 µg protein. Lane 1, sample heated at 30 °C; lane 2, 40 °C; lane 3, 50 °C; lane 4, 60 °C; lane 5, 70 °C; lane 6, 80 °C; lane 7, 90 °C; lane 8, 100 °C. Upper, middle and lower rocket immunoelectrophoresis from native, latent and cleaved antitrypsin respectively showing the same samples following filtration, all rockets are run in duplicate with the temperature shown below each pair.
Figure 7: 7.5-15% w/v native-PAGE to show the rate of insertion of the antithrombin reactive center loop peptide BC11 into native and latent antitrypsin. The incubations were performed at 37 °C with the BC11 peptide in 100 fold molar excess over antitrypsin. Native (above) and latent (below) antitrypsin at time points: lane 1, 0 h; lane 2, 0.5 h; lane 3, 1 h; lane 4, 2 h; lane 5, 4 h; lane 6, 10 h; lane 7, 24 h; lane 8, 48 h; lane 9, 72 h. Each lane contains 5 µg protein, N represents native, L latent antitrypsin and BC the protein containing the synthetic reactive center loop peptide.
Figure 8:
Refolding of native and latent antitrypsin
with 6 M guanidinium chloride. Native ( and
) and
latent (
and +) M antitrypsin (100 µg) were incubated
with guanidinium chloride for 10 min before refolding by twenty fold
dilution with 200 mM Tris, 10 mM EDTA, pH 8.0.
Samples were taken at 0, 20, 40, 60, 90, 120 and 180 min after
refolding by dilution (R) and were immediately incubated with bovine
-chymotrypsin to assess inhibitory activity. As antitrypsin
continues to refold during the kinetic assay each point is plotted at
the time at which the assay was stopped by the addition of substrate
(approximately 10 min after the sample was taken). The dotted lines
represent the hypothetical loss and restoration of inhibitory activity
following treatment with guanidinium chloride. The graph shows the
results of duplicate refolding experiments on the same preparation of
latent antitrypsin.
Alpha-antitrypsin is the archetypal member of the
serine proteinase inhibitor or serpin superfamily. Crystallographic and
biochemical data suggest that members of this family are able to adopt
a variety of conformations as a result of a mobile reactive center
loop: native, cleaved at the reactive center loop, complexed with
enzymes, polymerized and latent(44) . We report here the
preparation and characterization of a stable conformation of
antitrypsin which has the same properties as the prototypic latent
serpin plasminogen activator
inhibitor-1(7, 33, 34, 36) . This
latent form of antitrypsin, like latent antithrombin(10) , can
be prepared by heating at high temperatures in stabilizing sodium
citrate for 12-24 h. The conformation is induced less readily in
antitrypsin and requires higher temperatures and higher concentrations
of sodium citrate. The resulting mixture contains thermostable monomer
and short chain polymers that are strikingly different from those of
antitrypsin heated for the same length of time in the absence of
citrate. The short length of citrate buffered polymers most likely
reflects the incorporation of a latent species which terminates polymer
chain extension. Latent antitrypsin can be purified from the short
chain polymers by anion exchange. It elutes from the column at a lower
sodium chloride concentration than native M antitrypsin and has a more
cathodal electrophoretic mobility on non-denaturing PAGE and
isoelectric focusing.
The latent conformation is inactive as an
inhibitor of bovine -chymotrypsin indicating that the loop is
inaccessible for enzyme:inhibitor interaction. This is confirmed by
limited proteolysis with Staph. aureus V8 proteinase and
papain type IV which are unable to cleave latent antitrypsin at the
P4-P5 and P6-P7/P1-P1` bonds respectively(17) . Latent
antitrypsin is resistant to unfolding with 8 M urea suggesting
that, as with the cleaved species, the reactive center loop is inserted
into the A sheet to stabilize the antitrypsin molecule. Moreover the
occupancy of the A sheet by the reactive center loop accounts for the
abolition of peptide annealing even after incubation at 37 °C for
72 h.
Latent antitrypsin is more thermostable than native antitrypsin but less so than the cleaved species. We have previously shown that the thermal lability of antitrypsin reflects loop-sheet polymerization (40) and for this reason the three conformations: native, latent and cleaved were assessed by non-denaturing PAGE after heating for 2 h at 30-100 °C. Native antitrypsin formed polymers at heating above 60 °C but formed aggregates at 90 and 100 °C, cleaved antitrypsin was stable after heating at 100 °C for 2 h. Latent antitrypsin was stable at temperatures up to 70 °C but at 80 °C there was evidence of polymer formation (based on the presence of precise bands on non-denaturing PAGE) and at 90 and 100 °C nonspecific aggregation. It is of interest that heating latent antitrypsin at 80 °C must expel the reactive center loop from the A sheet allowing the formation of loop-sheet polymers. Others have shown that latent bovine plasminogen activator inhibitor can be activated by high temperatures presumably by the same mechanism involving the expulsion of the reactive center loop from the A sheet allowing it to adopt an active conformation(45) .
The cardinal feature of latent PAI-1 is the restoration of activity following treatment with denaturants(34) . The activity of latent antitrypsin may be increased up to 50 fold by incubation with 6 M guanidinium chloride under conditions that unfold even cleaved antitrypsin(25) . The resulting material is, at most, 35% active and is therefore less susceptible to reactivation with denaturants than PAI-1(34) . This may indicate that despite treatment with high concentrations of guanidinium chloride latent antitrypsin retains some elements of secondary structure that affects its refolding following the dilution of guanidinium with buffer.
The biochemical data provide strong evidence that pasteurization of antitrypsin in high concentrations of citrate induces a latent conformation that has many similarities to the latent form of PAI-1 (33, 34) . Crystal analysis has shown that the structural basis of native latency in PAI-1 and induced latency in antithrombin is the incorporation of the whole reactive center loop, to P3, into the A sheet of the molecule(7, 10) . The degree of insertion of the reactive center loop into the A sheet in latent antitrypsin cannot be assessed precisely but is clearly sufficient to stabilize the molecule against denaturation with 8 M urea and render it more thermostable than native antitrypsin.
The recent crystal structure of an antithrombin dimer (10) in conjunction with the antithrombin variant Rouen VI (31) provides evidence for a link between the latent and polymerized species. The results indicate that opening of the A sheet may allow overinsertion of the reactive center loop to give the latent conformation, with an accompanying displacement of strand 1C. This allows the insertion of the reactive center loop of a second, active molecule to produce a loop-C sheet dimer. For the inhibitory serpins in general the partial insertion of the reactive center loop with release of strand 1C is likely to represent the conformational change that normally takes place to lock the inhibitor in the complex with its target proteinase. If this locked conformation is induced in the absence of the proteinase then it would predictably lead either to subsequent complete insertion of the reactive loop, to give the latent form, or to sequential linkage of the incompletely inserted loop of one molecule to the C sheet of the next to give loop-C sheet polymerization. This C sheet mechanism fits with the short chain polymers seen upon incubating antitrypsin with 0.7 M citrate at 67 °C for 12 h. Citrate appears to act as a stabilizing agent preventing polymerization and allowing the chaotropic effects of temperature to induce the stable incorporation of the reactive center loop into the A sheet of the antitrypsin molecule. The incorporation of a latent molecule into extending polymers would predictably lead to chain termination and so account for the short polymers visualized by electron microscopy and non-denaturing PAGE. Heating in the absence of citrate produces polymers of up to 20 antitrypsin molecules in length.
Although we have used the terminology latent antitrypsin there is, as yet, no evidence that this form occurs physiologically as does latent PAI-1. It is likely however that latent antitrypsin will be observed in vivo at least under the exceptional circumstances recently reported for latent antithrombin(31) . From a practical viewpoint the existence of the latent form of antitrypsin is of pharmaceutical significance as it forms under similar conditions to those used industrially to pasteurize plasma products for human administration.