©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Preparation and Characterization of Latent -Antitrypsin (*)

(Received for publication, September 28, 1994; and in revised form, December 21, 1994)

David A. Lomas (§) Peter R. Elliott Wun-Shaing W. Chang Mark R. Wardell Robin W. Carrell

From the Department of Haematology, University of Cambridge, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant five-membered A beta-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 alpha-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, alpha(1)-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 alpha-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.


INTRODUCTION

alpha(1)-Antitrypsin (or alpha(1)-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 beta-sheet(3, 4, 5, 6, 7, 8, 9, 10, 11) . The intact inhibitor has an exposed, mobile, reactive center loop with the P(1)-P(1)` 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(1)-P(1)` 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 beta-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)^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.


MATERIALS AND METHODS

Bovine alpha-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.

Purification of Native M Antitrypsin

M antitrypsin was freshly isolated from both fresh and stored plasma from confirmed M homozygotes by 50 and 75% ammonium sulfate fractionation followed by thiol exchange and Q-Sepharose chromatography as detailed previously (40) . The protein was stored in 50 mM Tris, 50 mM KCl, pH 7.4, with 0.1% (v/v) beta-mercaptoethanol, and total protein was determined from the A(41) . The resulting protein was always more than 75% active as an inhibitor of bovine alpha-chymotrypsin (40) and migrated as a single species on SDS and non-denaturing polyacrylamide gel electrophoresis (PAGE).

Preparation of Latent (L) Antitrypsin

Latent antitrypsin was prepared by incubating isolated antitrypsin (final concentration 0.05-0.75 mg/ml) at 65-68 °C for 6-24 h in 20 mM Tris, pH 7.4, and 0.7 M sodium citrate. The citrate was removed by dialysis against 20 mM Tris, pH 8.6 (3 times 5 liters), at 4 °C and the antitrypsin concentrated to 0.8-1.0 mg/ml. This material was then heated at 60 °C for 3 h to convert any residual active antitrypsin to polymer(40) . Monomeric antitrypsin was separated from polymer by anion-exchange chromatography on a fast protein liquid chromatography (FPLC) Mono-Q column. The protein was loaded in 20 mM Tris, pH 8.6 at 1 ml/min and latent antitrypsin separated from polymer and aggregates by a 0-1.0 M sodium chloride gradient in 20 mM Tris, pH 8.6 over 60 min. The peaks were pooled, protein concentration was determined by absorbence(41) , and activity by titration against bovine alpha-chymotrypsin(40) . The conformation of antitrypsin in each peak was assessed by SDS and non-denaturing PAGE and transverse urea gradient (TUG) gel electrophoresis.

Preparation of M Antitrypsin Polymers

Antitrypsin polymers were prepared by incubating M antitrypsin (final concentration 0.2 mg/ml) in 50 mM Tris, 50 mM KCl, pH 7.4, with 0.1% (v/v) beta-mercaptoethanol at 60 °C for 3 h. Polymers were confirmed by non-denaturing PAGE and a concomitant loss of inhibitory activity against bovine alpha-chymotrypsin.

SDS-Polyacrylamide Gel Electrophoresis

The molecular mass of proteins was assessed on a 7.5-15% (w/v) linear gradient gel in 190 mM glycine, 25 mM Tris pH 8.8 buffer with 1% (w/v) SDS. All samples were heated in boiling water for 2 min in 1% (w/v) SDS prior to electrophoresis.

Non-denaturing Polyacrylamide Gel Electrophoresis

This was performed on a 7.5-15% (w/v) linear gradient gel with a discontinuous buffer system containing 53 mM Tris, 68 mM glycine, pH 8.9, cathodic buffer in the upper chamber and 100 mM Tris pH 7.8 anodic buffer in the lower chamber(27, 42) .

Transverse Urea Gradient Gel Electrophoresis

7.5% (w/v) polyacrylamide gels were cast with a double lumen tube and a peristaltic pump to give a linear gradient from 0 to 8 M urea using the non-denaturing-PAGE buffer system(17, 42) . The gels were rotated through 90°, the stacking gel poured, and the gels run using the discontinuous buffer system described above. Electrophoresis was performed at room temperature with a constant current of 15 mA until the front reached the end of the gel (approximately 2 h). The proteins were visualized by staining with Coomassie Blue.

Proteolytic Cleavage of the Reactive Center Loop

Antitrypsin was cleaved at the reactive center loop by incubating with S. aureus V8 proteinase in 50 mM ammonium bicarbonate, pH 8.0, for 2 h at 37 °C. Samples that were mixed with native antitrypsin for TUG gel analysis were treated with the inhibitor 3,4-dichloroisocoumarin to a final concentration of 1 mM to prevent cleavage of the native protein. Cleavage with papain type IV was achieved by incubating with antitrypsin in 50 mM Tris, 20 mM imidazole, 100 mM NaCl, pH 6.5, for 2 h at 37 °C. In all cases complete reactive center loop cleavage was confirmed by a characteristic band shift on 7.5-15% (w/v) SDS-PAGE.

Isoelectric Focusing

Isoelectric focusing was performed on a 5% (w/v) polyacrylamide gel with a pH gradient of 3.5-10 as described previously(40) . The gels were prefocused at 500 V for 30 min before loading 15 µg of protein in each lane. The samples were run into the gel at 500 V for 30 min, and focusing was continued at 800 V for 2-3 h and then at 1200 V for 20 min. All samples were compared with an MM antitrypsin standard.

Electron Microscopy

Proteins in solution were adhered to a charged carbon membrane, washed with 50 mM Tris, 50 mM KCl, pH 7.4, and negatively stained with 1% (w/v) uranyl acetate. The individual molecules were visualized at times 57,000 magnification, with the images being subsequently magnified by photographic enlargement.

Heat Stability Assays

Native, latent, and reactive center loop cleaved antitrypsin were incubated at 0.2 mg/ml in 20 mM Tris pH 7.4, or 75 mM Tris, 75 mM glycine, 75 mM NaH(2)PO(4), pH 7.5, (40) at constant temperatures between 30 and 100 °C for 2 h. The solutions were rapidly cooled on ice and aliquots removed for analysis by non-denaturing PAGE. The remaining antitrypsin was then filtered through a 0.22-µm membrane and residual protein determined by rocket immunoelectrophoresis.

Insertion of a Synthetic Reactive Center Loop Peptide into the A Sheet of Native and Latent Antitrypsin

The patency of the A sheet of native and latent antitrypsin was probed with an 11 mer peptide (Ac-Ser-Glu-Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-Ile) representing the sequence P14-P4 of the reactive center loop of antithrombin. The synthetic peptide was purified by reverse phase HPLC on a C(18) column with a 30-50% (v/v) acetonitrile gradient in 50 mM ammonium acetate. The peptide was freeze-dried and dissolved in 50 mM Tris, 100 mM NaCl, pH 7.6. Fifty µg of native or latent antitrypsin (1 mg/ml in storage buffer) were incubated with an equal volume of 100-fold molar excess of the peptide in a water bath at 37 °C for 72 h(43) . Aliquots of protein were removed at various time points, snap frozen in liquid nitrogen, and stored at -80 °C until required. The rate of binary complex formation was then assessed by separating the proteins on 7.5-15% (w/v) non-denaturing PAGE as described above.


RESULTS

Preparation of Latent Antitrypsin

The optimum conditions for the formation of latent antitrypsin were assessed by incubating M antitrypsin (final concentration 0.25 mg/ml) in a range of concentrations of sodium citrate over a variety of temperatures for 4-24 h. The resulting material was then separated by non-denaturing-PAGE and residual activity assessed by titration against bovine alpha-chymotrypsin. Higher concentrations of citrate were required to prevent polymerization than described previously for antithrombin (10) . Incubation of antitrypsin in 0.7 M sodium citrate at 60 °C resulted in the loss of only 25% activity over a period of 24 h, a temperature of 70 °C favored polymerization rather than monomer formation; the optimum temperature was 65-68 °C. Heat treatment of native M antitrypsin at 67 °C for 4 h in the absence of citrate resulted in polymerization of all the protein and a complete loss of inhibitory activity against bovine alpha-chymotrypsin (data not shown). Further heating at this temperature favored elongation of the polymer chain as determined by non-denaturing-PAGE. The presence of 0.7 M sodium citrate resulted in the preservation of inhibitory activity against bovine alpha-chymotrypsin although this fell from a starting value of 80% to 13% after heating at 67 °C for 4 h, 9% after 8 h, 5% after 12 h and 4% after 24 h. In all cases there was demonstrable polymer and monomer on non-denaturing-PAGE, the latter having a more cathodal electrophoretic mobility than native antitrypsin (data not shown); electron micrographs confirmed that antitrypsin polymers were significantly shorter than those formed in the absence of citrate. For all further experiments latent antitrypsin was prepared by incubating M antitrypsin at a concentration of 0.25 mg/ml in 0.7 M sodium citrate at 67 °C for 12 h although protein concentrations of 0.05-0.75 mg/ml and incubations for 24 h gave similar results.

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 alpha-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 (^1EDPQGDAAQK- and ^6DAAQK-) 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.



Storage and Activity

The specific activity of latent antitrypsin against bovine alpha-chymotrypsin remained at <1% when stored at 4 °C for 25 days. There was no change in the electrophoretic mobility on non-denaturing PAGE.

Cleavage of the Reactive Center Loop

Latent antitrypsin was inactive as an inhibitor of bovine alpha-chymotrypsin suggesting that the reactive center loop is inaccessible for complex formation. The reactive center loop was inaccessible to cleavage with Staphylococcus aureus V8 proteinase, which cleaves at the P4-P5 bond of the reactive center loop, and papain which cleaves at P6-P7 and P1-P1` residues (17) (Fig. 5a and b).


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.



Thermal Stability

Native and cleaved antitrypsin showed a heat stability profile (Fig. 6) similar to those described previously (24, 40) whether performed in 20 mM Tris pH 7.4 or 75 mM Tris, 75 mM glycine, 75 mM NaH(2)PO(4), pH 7.5. Latent antitrypsin was stable to 70 °C but then precipitated resulting in a fall in rocket height (Fig. 6). As loop-sheet polymerization accounts for the thermal precipitation of antitrypsin (40) all fractions were separated by non-denaturing-PAGE. Native antitrypsin formed polymers after heating at 60 °C for 2 h, cleaved antitrypsin did not polymerize even after heating at 100 °C for 2 h. Latent antitrypsin remained monomeric until 70 °C; heating for 2 h at higher temperatures resulted in some polymerization (80 °C) and then aggregation (90 and 100 °C).


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.



Reactive Center Loop Peptide Insertion

The antithrombin reactive center loop peptide BC11 annealed with native antitrypsin with a half-time of 30-60 min. Latent antitrypsin did not accept the peptide even after incubation for 72 h at 37 °C (Fig. 7).


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.



Formation of Latent Antitrypsin by Treatment with Guanidinium Chloride

Previous work has suggested that serpins may be induced to adopt a latent conformation by treatment with mild denaturants at 4 °C(38) . It is now apparent that such treatment favors the formation of loop-sheet polymers in antithrombin(10) . Antitrypsin was therefore incubated at 1 mg/ml with 0, 1.1 and 1.5 M guanidinium chloride at 4 °C for 16 h as described previously(40) . The protein was dialyzed into 5 liters 20 mM Tris pH 7.4 with 3 changes and heated at 60 °C for 3 h to convert any active monomer to polymer. Analysis of the preparations by non-denaturing-PAGE showed that incubation with both 1.1 and 1.5 M guanidinium chloride resulted in the formation of dimer as well as monomer. There was no residual monomeric band after heating at 60 °C for 3 h suggesting that, as with antithrombin, this treatment does not induce thermostable latency in antitrypsin. The partial loss of activity that was previously attributed to formation of the latent conformation is likely to represent a combination of partial denaturation and loop-sheet dimerization, the latter being undetected by gel filtration (40) .

Refolding of Latent Antitrypsin with Guanidinium Chloride

100 µg native and latent M antitrypsin (specific activities 97% and 0.7% respectively) were incubated in 6 M guanidinium chloride (final concentration 0.33 mg/ml) for 10 min before being refolded by twenty fold dilution with 200 mM Tris, 10 mM EDTA pH 8.0 as described by Powell and Pain(25) . Aliquots were taken 0, 20, 40, 60, 90, 120 and 180 min after dilution and incubated with 5 pmol bovine alpha-chymotrypsin to assess activity. M antitrypsin regained approximately 70% of inhibitory activity against bovine alpha-chymotrypsin (Fig. 8). Refolding latent antitrypsin increased the specific activity from <1% to a value that differed between batches but ranged from 10-35%. The refolding curves shown in Fig. 8are duplicates from the same preparation of latent antitrypsin and show an increase in inhibitory activity from 0.7% to approximately 35%.


Figure 8: Refolding of native and latent antitrypsin with 6 M guanidinium chloride. Native (up triangle and times) and latent (box 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 alpha-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.




DISCUSSION

Alpha(1)-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 alpha-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.


FOOTNOTES

*
This work was supported by the Medical Research Council (United Kingdom) and the Wellcome trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
MRC Clinician Scientist Fellow. To whom correspondence should be addressed.


ACKNOWLEDGEMENTS

We thank Dr. J. T. Finch, Laboratory of Molecular Biology, MRC center, Cambridge for the electron micrographs and Dr. L. Packman, Department of Biochemistry, University of Cambridge for peptide synthesis, amino acid analysis, and N-terminal sequence analysis.


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