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Published ahead of print on March 11, 2004, doi:10.1165/rcmb.2003-0276OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 133-139, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0276OC

Inhibiting Polymerization

New Therapeutic Strategies for Z {alpha}1-Antitrypsin–Related Emphysema

Helen Parfrey, Timothy R. Dafforn, Didier Belorgey, David A. Lomas and Ravi Mahadeva

Division of Respiratory Medicine, Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom

Address correspondence to: Helen Parfrey, Division of Respiratory Medicine, Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, MRC/Wellcome Trust Building, Hills Road, Cambridge CB2 2XY, UK. E-mail: hp226{at}cam.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Z variant of {alpha}1-antitrypsin (Z-AT) is present in 4% of Northern Europeans and is associated with liver cirrhosis and emphysema. Polymers accumulate within the hepatocyte and the subsequent plasma deficiency of AT renders the lungs susceptible to proteolysis and early onset emphysema. We have previously demonstrated that the Phe-Leu-Glu-Ala-Ile-Gly (6 mer) peptide specifically binds to Z-AT and inhibits polymerization. Here we present the first detailed biochemical study of the purified Z-AT-6 mer binary complex. Biochemical studies indicated that this complex was inactive as a proteinase inhibitor and the peptide annealed to ß-sheet A of Z-AT. Removal of the N-acetyl terminus of the 6 mer peptide did not affect the peptide's ability to prevent polymer formation. However, the nonacetylated 6 mer–Z-AT complex dissociated at a rate 2.75x faster than the acetylated 6 mer–Z-AT complex to yield an active inhibitor; Koff 5.5 ± 1.07 versus 2.0 ± 0.25 106 s–1, respectively. These biochemical data indicate a potential therapeutic approach whereby polymerization is prevented in the liver, with the gradual release of the peptide from the binary complex restoring proteinase inhibitory function within the tissues. Thus, it raises the novel prospect of ameliorating both the cirrhosis and the emphysema associated with Z-AT.

Abbreviations: {alpha}1-antitrypsin, AT • acetylated 6 mer peptide, Ac-6 mer • nonacetylated 6 mer peptide, non-Ac-6 mer • acetylated 12 mer peptide, Ac-12 mer • binary complex, BC • Z variant of {alpha}1-antitrypsin, Z-AT


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Serine proteinase inhibitors (serpins) regulate a variety of important physiologic pathways such as inflammation, coagulation, fibrinolysis, apoptosis, and extracellular matrix remodelling (1). Members of this family have a common tertiary structure based on a dominant five-stranded ß-sheet A that supports an exposed 17-residue reactive loop. Interaction with the target proteinase results in cleavage of the serpin at its reactive loop P1-P1' bond followed by a profound conformational change within the molecule. The reactive loop inserts between strands three and five of ß-sheet A, translocating the proteinase to the lower pole of the molecule and inactivating it via disruption of the catalytic site (2–6). In many serpins this elegant inhibitory mechanism can be subverted by point mutations that render ß-sheet A receptive to the reactive-loop peptide of another molecule. This can lead to sequential intermolecular reactive loop-ß-sheet A linkage to form polymers (7, 8) (Figure 1A). These loop-sheet polymers aggregate within their cell of synthesis, which in turn causes a plasma deficiency of the serpin. Both of these effects can manifest as disease. The accumulation of neuroserpin polymers leads to early onset familial inclusion body dementia (9), while the plasma deficiency of antithrombin, C1 inhibitor, and {alpha}1-antichymotrypsin gives rise to thrombosis, angioedema, and emphysema, respectively (10–12). However, polymerizing mutants of {alpha}1-antitrypsin (AT) are the most common serpins associated with disease (8, 13).





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  		Figure 1. Mechanism of Z-AT polymer formation and inhibition. (A) The Z mutation (Glu342Lys) lies at the head of strand 5A and the base of the reactive center loop (blue circle) where it opens ß-sheet A (green) to allow partial reactive loop (red) insertion. The resultant polymerogenic conformation can readily accept the reactive loop of another AT molecule to form a dimer. This can then extend to form chains of polymers. Polymerization is regulated by both temperature and protein concentration. Insertion of the 6 mer peptide (yellow) into ß-sheet A, as strand 4, can prevent Z-AT polymer formation and lead to BC formation. (B) The BC was formed by incubation of monomeric Z-AT with 100-fold molar excess of either Ac-6 mer (left) or non-Ac-6 mer (right) peptides at 37°C for 24 h. Four micrograms of protein were removed at each timepoint and characterized by 7.5% (wt/vol) nondenaturing PAGE containing 8 M urea. Z-AT polymer formation was inhibited by both 6 mer peptides. (C) Monomeric Z-AT (0.5 mg/ml) (lane 1) was incubated at 37°C for 10 d alone (lane 2) or in the presence of 100-fold molar excess of either non-Ac-6 mer (lane 3) or Ac-6 mer (lane 4) peptide. All samples were assessed by 7.5% (wt/vol) nondenaturing PAGE and each lane contains 4 µg of protein. The Ac-6 mer BC migrates farther under nondenaturing conditions. Native represents monomeric Z-AT, Polymers the polymeric conformation, and BC the binary complex with either non-Ac-6 mer or Ac-6 mer.

 
AT is primarily synthesized in hepatocytes and is secreted into the plasma, reaching the lungs by passive diffusion (14). The normal protein is designated as M according to its isoelectric point. Up to 1 in 2,000 Northern Europeans are homozygous for the highly polymerogenic Z protein (Glu342Lys). This mutation lies at the head of strand five of ß-sheet A, at the base of the reactive center loop, and is likely to lead to partial reactive-loop insertion between strands three and five (15). This directly facilitates reactive-loop insertion of another molecule by opening ß-sheet A (15, 16) (Figure 1A). The Z variant of {alpha}1-antitrypsin (Z-AT) polymers are retained in the hepatocyte, as PAS positive-diastase–resistant inclusion bodies that predispose Z-AT homozygotes to neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma (8, 17–20). The subsequent secretory defect results in inadequate pulmonary defense against elastolytic enzymes, leading to early onset panacinar emphysema, which accounts for up to 2% of all cases of emphysema (17).

Preventing polymerization represents an important therapeutic goal as blocking this conformational transition restores the secretion of polymerogenic mutants of AT in the Xenopus oocyte model (18). Inhibition of polymerization in vitro can be achieved by the annealing of synthetic 12–14 mer peptides (homologous to the reactive center loop of AT) to ß-sheet A (19–21). However, these peptides are too large and do not bind specifically to the Z variant of AT, which makes them unsuitable as therapeutic agents (22, 23). Recently, we have demonstrated that the distinctive conformation adopted by the Z protein lends itself to the design of a 6 mer peptide (Phe-Leu-Glu-Ala-Ile-Gly; P7–2). This peptide preferentially recognizes the pathogenic Z protein, thereby fulfilling a critical requirement for an inhibitor of polymerization (15) (Figure 1A). However, by binding to ß-sheet A the peptide renders Z-AT inactive as a proteinase inhibitor. Therefore, although this therapy may prevent the liver disease, it would exacerbate any tendency to develop emphysema. One approach that would encompass these two seemingly incompatible properties of prevention of AT polymer formation in the liver with preservation of inhibitory function in the tissues would be to use an agent that bound to inhibit polymerization within the cell of synthesis, and then was slowly released to restore the inhibitory capacity of the protein within the tissues.

A detailed knowledge of the biochemical properties of the Z-AT–6 mer binary complex is essential to guide and interpret in vivo studies. Here we have biochemically characterized the purified complex. Furthermore, we present novel data demonstrating how chemical modification of the 6 mer peptide can significantly alter the stability of the binary complex such that after binding to inhibit polymerization the 6 mer peptide is gradually released to allow the Z-AT to function as a proteinase inhibitor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of Plasma M and Z-AT
M and Z-AT were purified from plasma of known homozygotes by 75% and 50% ammonium sulfate fractionation followed by glutathione Sepharose and Q-Sepharose column chromatography as detailed previously (20). The proteins migrated as a single band on sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide gel electrophoresis (PAGE). Both M and Z-AT had normal unfolding profiles on a 0–8 M transverse urea gradient gel (20). The proteins were aliquoted, snap frozen and stored at –80°C in 50 mM Tris, 50 mM KCl, pH 7.4.

Assessment of Proteinase Inhibitory Function
Although neutrophil elastase is the target proteinase for AT, bovine {alpha}-chymotrypsin produces more consistent and reliable results for determining proteinase inhibitory capacity (24). Inhibitory activity was calculated by incubating bovine {alpha}-chymotrypsin (5 pmols) of known active site (25) with increasing concentrations of AT (estimated active site concentration of 0.1 µM) in a total volume of 100 µl with 0.03 M sodium phosphate, 0.16 M NaCl, 0.1% (wt/vol) PEG 4000, pH 7.4 inhibition buffer. The reaction proceeded for 10 min at 25°C and the residual proteolytic activity was determined by the addition of the substrate succinyl-L-alanyl-L-alanyl-propyl-L-phenylanalyl-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) to a final concentration of 0.16 mM (26). The change in the OD405 over 3 min was observed. Active site values were obtained by plotting residual proteolytic activity against the amount of AT and extrapolating to the x-intercept (24). Both M and Z-AT were active as inhibitors against bovine {alpha}-chymotrypsin, 76 ± 0.2% and 62.7 ± 0.3%, respectively (mean ± SEM, n = 3).

The association rate constant (kass) for the inhibition of bovine {alpha}-chymotrypsin by the AT binary complexes was determined either under pseudo-first order conditions, i.e., [I] >= 10 [E]0, using the progress–curve method (27, 28) or by monitoring the time–dependent loss of activity over 5 h (29). The kass was measured at 25°C in the inhibition buffer (as above) with the substrate Suc-Ala-Ala-Pro-Phe-pNA. The Km of the substrate at 25°C in the inhibition buffer was 0.057 mM as measured independently.

Prevention of Z-AT Polymerization
Synthetic peptides corresponding to P7–2 of the reactive loop of AT (Phe-Leu-Glu-Ala-Ile-Gly-OH) (Sigma-Genosys Ltd., Cambridge, UK) were synthesized with (Ac-6 mer) and without (non-Ac-6 mer) amino-terminal acetylation to over 95% purity and were dissolved in water. Z-AT was incubated at 37°C alone or with a 100-fold molar excess of either 6 mer peptide at a final concentration of 0.5 mg/ml in 50 mM Tris, 50 mM KCl, pH 7.4 for 10 d. Prevention of polymer formation was assessed on a 7.5% (wt/vol) nondenaturing PAGE.

Preparation and Purification of M and Z-AT Binary Complexes
The 12 mer peptide (Ac-12 mer), comprising the P14–3 sequence of antithrombin (Ac-Ser-Glu-Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-Ile-Ala-OH) (Genosys Biotechnologies Inc., Cambridge, UK), was dissolved in 50 mM Tris, 50 mM NaCl, pH 7.4. All binary complexes were produced by incubating a 100-fold molar excess of either the 6 mer peptides with native Z-AT or the Ac-12 mer peptide with native M-AT, at a final concentration of 0.5 mg/ml in 50 mM Tris, 50 mM KCl, pH 7.4 at 37°C for 24 h. Formation of each binary complex (BC) was confirmed on a 7.5% (wt/vol) nondenaturing PAGE containing 8 M urea (15). To aid purification of the 6 mer BCs, the samples were heated under different conditions to polymerize any residual native protein. The Z-AT-Ac-6 mer BC sample was heated to 52°C for 24 h, while the Z-AT-nonacetylated BC sample was incubated at 37°C for a further 20 d. An amount of 7.5% (wt/vol) nondenaturing PAGE containing 8 M urea was used to confirm the presence of the BC. The BCs were then separated from Z-AT polymers and excess peptide by size-exclusion column chromatography using HiLoad 16/60 Superdex 200 (Pharmacia Biotech, Uppsala, Sweden) equilibrated in 20 mM Tris, pH 7.4.

The antithrombin-12 mer BC has been crystallized previously (21) and serves as a comparative control. All the M-AT was incorporated into a BC with the Ac-12 mer peptide after 24 h incubation at 37°C, thus no additional heating was required. The M-AT BC was purified from excess Ac-12 mer peptide by gel filtration as above. Fractions containing AT were identified by rocket immunoelectrophoresis (20) and pooled according to their molecular size before concentrating in a 30-kD Centriprep filter (Amicon Inc., Beverly, MA). Purity of the BC was assessed on a 7.5% (wt/vol) nondenaturing PAGE containing 8 M urea.

Biochemical Characterization of M and Z-AT BCs
The purified BCs were characterized on a 12% (wt/vol) SDS PAGE and 7.5% (wt/vol) nondenaturing PAGE. Inhibitory activity against bovine {alpha}-chymotrypsin was evaluated as above. The ability to form an SDS-stable complex with trypsin (Sigma, Poole, UK) was determined by incubating a 1:1 molar ratio of BC to proteinase in 0.16 M NaCl, 0.03 M sodium phosphate, 0.1% (wt/vol) PEG 4000, pH 7.4 inhibition buffer for 20 min at room temperature. The trypsin inhibitor, L-1-chloro-3-[4-tosylamido]-7-amino-2-hepatone.HCl (TLCK) (Sigma), was added to a final concentration of 2 mM and the samples were then heated at 98°C for 3 min in SDS sample-loading buffer before assessment on 12% (wt/vol) SDS PAGE. All proteins were visualized by Coomassie Brilliant Blue (BDH Ltd., Leicester, UK), silver staining, or by Western blot analysis. The latter was performed by transferring the proteins onto nitrocellulose membrane using standard techniques. AT was identified using a polyclonal rabbit antihuman AT primary antibody (Sigma) and a horseradish peroxidase-conjugated goat antirabbit immunoglobulin G secondary antibody (Sigma). The proteins were assessed after developing with an ECL chemiluminescence kit (Amersham Pharmacia Biotech, St. Albans, UK).

Circular Dichroism
Circular dichroism experiments were undertaken on a JASCO J-810 spectrapolarimeter as detailed previously (16).

Dissociation of M and Z-AT BCs
Dissociation of the BCs was assessed under physiologic conditions by incubating 0.5 mg/ml of purified BC at 37°C in 50 mM Tris, 50 mM KCl, pH 7.4. Aliquots were taken at time points up to 10 wk and examined by 7.5% (wt/vol) nondenaturing PAGE containing 8 M urea. Loss of intensity of the BC band was determined by densitometry (Quantity One BioRad Laboratories, Hercules, CA). The dissociation rate (Koff) and half-life were calculated from the semilog plot of the ln fractional loss of the BC against time in seconds.

To ascertain if dissociation of the BC had restored the native conformation of AT, the aliquots from specific timepoints were assessed for their inhibitory activity against bovine {alpha}-chymotrypsin and their ability to form SDS-stable complexes with trypsin as above.

N-Terminal Amino Acid Sequencing
Purified Z-AT-non-Ac-6 mer BC heated at 42°C for 24 h was electroblotted onto Immobilon-PSQ membrane (Millipore Corporation, Bedford, UK) and N-terminal sequencing was performed by the Department of Biochemistry, University of Cambridge.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Prevention of Z-AT Polymerization
The rate of formation of the Z-AT BC was similar for the non-Ac-6 mer and Ac-6 mer peptides at 37°C for 24 h (Figure 1B). Incubation of monomeric Z-AT with a 100-fold molar excess of either the non-Ac-6 mer or Ac-6 mer peptide at 37°C for 10 d completely inhibited Z-AT polymer formation (Figure 1C). This confirmed that the presence or absence of amino-terminal acetylation did not significantly impede the ability of the 6 mer peptide to anneal to ß-sheet A of Z-AT at physiologic temperature.

Binary Complex Formation and Purification
BCs were formed by incubating monomeric M or Z-AT with a 100-fold molar excess of Ac-12 mer or 6 mer peptides, respectively, at 37°C for 24 h. All the M-AT annealed to the Ac-12 mer peptide leaving no residual monomeric protein (data not shown). However, a small amount of monomeric Z-AT remained after incubation with the 6 mer peptides. To eliminate the monomeric protein, the samples were heated to polymerize the remaining native Z-AT before separation from the BC by size-exclusion chromatography. Several conditions were assessed. It was observed that when the Z-AT-Ac-6 mer sample was heated at 52°C for 24 h the residual monomeric protein polymerized, leaving a mixture of Z-AT-Ac-6 mer BC and polymers (data not shown). Under identical conditions the Z-AT-non-Ac-6 mer BC dissociated into native protein, which then polymerized. Hence, to achieve BC preservation and maximal polymerization of residual monomeric protein, the Z-AT-non-Ac-6 mer sample was heated at 37°C for a total of 21 d as the BC was stable at this temperature. Subsequent gel filtration column chromatography of the heated BC samples identified three distinct peaks for both the Ac-6 mer and non-Ac-6 mer peptide binary complexes (Figure 2A). Rocket immunoelectrophoresis showed that AT was present in peaks one and two only (data not shown). Peak three is likely to represent excess peptide. Nondenaturing PAGE demonstrated that peak one consisted of Z-AT polymers while the Z-AT BC was contained in peak two (Figures 2B and 2C). The Z-AT-non-Ac-6 mer BC fraction also contained less than 10% (by densitometry) of residual native protein that could not be separated further. The M-AT-Ac-12 mer BC was purified from excess peptide by size-exclusion chromatography as above. Two distinct peaks were produced equating to molecular sizes of 55 kD and 3 kD that corresponded to the BC and unbound Ac-12 mer peptide, respectively (data not shown).




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  		Figure 2. AT binary complex purification. The elution profile obtained for the purification of the Z-AT-non-Ac-6 mer BC from gel filtration column chromatography (see METHODS for details) shows three distinct peaks. Peaks 1 and 2 were found to contain AT by rocket immunoelectrophoresis (data not shown) while peak 3 represents unbound, excess peptide (A). The BC fraction separated from Z-AT polymers after size exclusion chromatography was characterized on 7.5% (wt/vol) nondenaturing PAGE without (B) and with (C) 8 M urea Lane 1: monomeric Z-AT; lane 2: Z-AT polymers (formed by heating Z-AT 1 mg/ml at 60°C for 20 min); lane 3: Z-AT BC control (formed by heating Z-AT at 0.5 mg/ml with 100-fold molar excess of Ac-6 mer peptide at 37°C for 24 h); lane 4: peak 1; lane 5: fractions from overlap of peaks 1 and 2; lane 6: peak 2. All lanes contain 4 µg of AT. Note the small amount of residual native protein in the BC fraction (10%) (lane 6).

 
Biochemical Characterization of the Purified BCs
All purified binary complexes migrated as a single band on SDS PAGE that was identical to intact, monomeric AT. The Z-AT BC with the Ac-6 mer peptide was more negatively charged than the non-Ac-6 mer BC, and thus migrated further into a nondenaturing gel (Figure 1C). Furthermore, it was the most stable in urea as it travelled furthest into the acrylamide gel.

The previously crystallized antithrombin-12 mer binary complex demonstrates that exogenous reactive-loop peptides are bound as strand four to ß-sheet A (21). This prevents inhibitory complex formation. In keeping with this, the purified Z-AT-non-Ac-6 mer BC, Z-AT-Ac-6 mer BC, and M-AT-Ac-12 mer BC all had lost inhibitory function as displayed by their inability to inhibit bovine {alpha}-chymotrypsin (13.3 ± 0.5%, 2.4 ± 0.4%, and 5.4 ± 1.2%, respectively) as measured by active site titration (mean ± SEM, n = 3). Furthermore, the association rate constant (kass) for each of the purified BCs was significantly reduced at <2 x 103 M–1s–1 compared with 2.5 ± 0.4 x 106 M–1s–1 for native Z protein (mean ± SEM, n = 3). Instead, they behaved as substrates for bovine {alpha}-chymotrypsin with the interaction resulting in cleavage of the BC (23). These findings confirm that the 6 mer peptides are bound to ß-sheet A of Z-AT, converting it from a five- to a six-stranded conformation, thus preventing serpin reactive-loop insertion as would occur during serpin–proteinase complex formation (19, 21, 30) (Figure 1A).

Circular Dichroism
Circular dichroism (CD) studies demonstrated a loss of {alpha} helical structure with increasing temperature for both monomeric M and Z-AT. This equated to a melting temperature of 63.6 ± 0.2°C and 61.9 ± 0.3°C for M and Z-AT, respectively (mean ± SEM, n = 3). These findings are in agreement with previous observations that Z-AT has a lower melting temperature and so will polymerize more readily than M-AT (16). The binary complexes of Z-AT with the acetylated and the nonacetylated 6 mer peptides had melting points above 95°C. Investigation of the secondary structure content of both the 6 mer peptides using CD revealed them to be in an identical extended conformation (data not shown).

Dissociation Rate of the BCs at 37°C
The rate of dissociation of the purified binary complexes was examined at physiologic temperature. Removal of the N-acetyl group from the 6 mer peptide increased the Koff by 2.75 fold, allowing this peptide to dissociate much faster from the BC (Table 1). Dissociation of both 6 mer BCs yielded a combination of Z-AT polymers, the native protein, and band I, which migrated to an intermediate position between that of native and loop-sheet dimer Z-AT on 8 M urea nondenaturing PAGE (Figures 3A and 3B). Attempts to promote the production of this species and to characterize it by N-terminal sequencing were unsuccessful. It is uncertain what this band represents, however we speculate that it may be a dimer of a binary complex with a native Z-AT molecule interacting through a reactive loop-C-sheet linkage (31–33).


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  		TABLE 1. Rates of dissociation of AT binary complexes

 


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  		Figure 3. AT binary complex dissociation. An amount of 7.5% (wt/vol) nondenaturing PAGE with 8 M urea showing the dissociation of Z-AT-Ac-6 mer BC (A) and Z-AT-non-Ac-6 mer BC (B) when incubated at 0.5 mg/ml (50 mM Tris, 50 mM KCl, pH 7.4) at 37°C for up to 11 d. Lane C: native Z-AT. All lanes contain 4 µg AT. The native protein, BC, short chain polymers (Polymer), and intermediate species (I) are indicated by arrows.

 
Assessment of Inhibitory Function of the Dissociated BCs
Although the native Z-AT starting material was 62.7 ± 0.3% active as an inhibitor against bovine {alpha}-chymotrypsin, the purified BCs were inactive as proteinase inhibitors as described previously. However, there was an increase in the proteinase inhibitory activity (as measured by active site titration) of the dissociated Z-AT-non-Ac-6 mer BC over the first three days, unlike the Z-AT-Ac-6 mer BC (Table 2). Thus after 3 d incubation at 37°C, the dissociated Z-AT-non-Ac-6 mer BC sample showed 28.6 ± 0.6% inhibitory activity against this proteinase, which indicates that nearly 50% of the original Z-AT activity had been restored. Furthermore, the material that dissociated from the Z-AT-non-Ac-6 mer BC was able to form a complex with porcine pancreatic trypsin (Figure 4, lanes 8–10). It was not possible to obtain an exact measurement for the kass of the dissociated BCs (all samples had association rate constants of <2 x 103 M–1s–1), though it is likely that the dissociated Z-AT-non-Ac-6 mer BC would have a kass similar to that for Z-AT. The kass can be precisely calculated for samples that contain pure proteinase inhibitor. This is clearly not the case for the dissociated BC samples, which consist of the BC, native Z-AT, and the 6 mer peptide. The capacity of the 6 mer peptide to dissociate and reassociate with the binary complex, the low concentration of native Z-AT, and the fact that we cannot correct for the active site all help to explain the disparity between active site titration and association rate constant measurements of proteinase inhibitory function. In addition, active site values were determined over minutes whereas assessment of kass required 5 h, which may allow reformation of the BC within the experimental confines. Hence, interpretation of the association rate constant is limited, as it does not reflect the in vivo situation. Thus active site titration and SDS-complex formation were the most accurate indices of proteinase inhibitory function under these circumstances.


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  		TABLE 2. Assessment of proteinase inhibitory function of the dissociated binary complexes

 


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  		Figure 4. Assessment of inhibitory function of the dissociated BCs. Complex formation with porcine pancreatic trypsin was assessed at a 1:1 molar ratio of trypsin to dissociated Z-AT-6 mer binary complex incubated for 20 min in 0.16 M NaCl, 0.03 M sodium phosphate, 0.1% (wt/vol) PEG 4000, pH 7.4 reaction buffer at 20°C. At the end of the reaction, excess trypsin was inhibited by the addition of 2 mM TLCK. The samples were heated at 98°C for three minutes and assessed by 12% (wt/vol) SDS PAGE. Lane 1: molecular mass markers (kD); lane 2: porcine pancreatic trypsin; lane 3: Staph aureus V8 protease cleaved M-AT; lane 4: Z-AT; lane 5: Z-AT + trypsin; lane 6: Z-AT-non-Ac-6 mer BC; lane 7: Z-AT-non-Ac-6 mer BC + trypsin; lanes 8–10: Z-AT-non-Ac-6 mer BC incubated at 0.5 mg/ml, 50 mM Tris, 50 mM KCl, pH 7.4, at 37°C for 1–3 d, respectively, and then incubated with trypsin for 20 min; lane 11: Z-AT-Ac-6 mer BC; lane 12: Z-AT-Ac-6 mer BC + trypsin; lanes 13–15: Z-AT-Ac-6 mer BC incubated at 0.5 mg/ml, 50 mM Tris, 50 mM KCl, pH 7.4, at 37°C for 1–3 d, respectively, and then incubated with trypsin for 20 min. There is a small amount of SDS-stable Z-AT-trypsin complex visible in lanes 7 and 12. This is due to the presence of residual native Z-AT present in the BCs that is capable of forming a complex when incubated with trypsin. All lanes contain 2 µg protein. The migration of reactive loop cleaved AT (Cleaved), trypsin-complexed AT (Complex) and free trypsin (Trypsin) are shown.

 
In contrast, the Z-AT-Ac-6 mer BC dissociated at a slower rate (Table 1 and Figure 3A) so that even after 8 d incubation the dissociated material was only 13.9 ± 1.9% active as an inhibitor of bovine {alpha}-chymotrypsin. Thus the Z-AT-non-Ac-6 mer BC not only dissociated faster, it also yielded an active inhibitor. The M-AT-Ac-12 mer BC did not dissociate under these conditions after 10 wk and was unable to form a complex with trypsin (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
AT is a 52-kD serine proteinase inhibitor produced primarily by hepatocytes and monocytes. It is secreted into plasma and reaches the lung by passive diffusion, where it acts as the major proteinase inhibitor against neutrophil elastase. The Z variant arises from a single point mutation of AT (Glu342Lys), which allows the protein to adopt a structure that renders it liable to polymerize. The accumulation of Z-AT polymers within hepatocytes is associated with liver cirrhosis and the accompanying plasma deficiency predisposes to uncontrolled proteolysis within the lungs, leading to early onset emphysema. Knowledge of the molecular basis of Z-AT polymerization has opened up a new therapeutic avenue for investigation. We have previously shown that exogenous peptides can bind to ß-sheet A of AT and inhibit polymerization (8, 20). These initial peptides were too large and nonspecific to be considered as therapeutic agents. However, we have recently designed a small 6 mer peptide that specifically binds to Z-AT to form a binary complex and in so doing, inhibited polymerization (15).

These studies present the first detailed biochemical assessment of the Z-AT-6 mer BC and reveal a hitherto unrecognized therapeutic application. The presence of an acetyl group at the N-terminus of the 6 mer peptide significantly enhanced stability of the binary complex with Z-AT, without altering the ability of the peptide to inhibit polymerization. CD studies demonstrated both peptides to be in an identical conformation, suggesting that the increased stability of the Z-AT-Ac-6 mer BC may be due to an additional hydrogen bond to strand three of ß-sheet A, created by the acetyl group (Figure 5).



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  		Figure 5. Molecular model of the Z-AT-6 mer binary complex. Top is a molecular model of Z-AT showing the predicted placement of the 6 mer peptide as the lower part of strand 4 of ß-sheet A. Bottom shows detailed molecular modelling of the nonacetylated 6 mer peptide (left) and the acetylated 6 mer peptide (right) between strands 3 and 5 of ß-sheet A. The N-terminal acetylated 6 mer peptide is likely to form an additional hydrogen bond with Val185 on strand 3A.

 
Furthermore, the nonacetylated peptide dissociated from the BC at an accelerated rate, releasing active proteinase inhibitor. The ability of the BC to dissociate accounts for the apparent failure of all the native Z-AT to accept the 6 mer peptides (Figure 1B). An equilibrium exists between native AT and the BC. When the peptide is in excess, any dissociated monomeric AT can reform a BC. However, when the additional peptide is removed, dissociation of the peptide from the BC becomes apparent, as indicated by the restoration of proteinase inhibitory activity. This behavior is not observed for the BCs containing the longer 12 mer peptide as the BC is stabilized by additional interactions so that the dissociation rate is almost zero.

Our data have important implications for potential therapy of Z-AT–related cirrhosis and emphysema. Within the hepatocyte endoplasmic reticulum there is a high concentration of Z-AT in a partially folded, pathologic state, which promotes polymer formation. We have previously demonstrated that such conformations more readily accept small peptides into ß-sheet A to prevent polymerization (15). The main drawback to this approach is the loss of function of Z-AT associated with binding of the peptide. Although the accumulation of polymers may be prevented, this strategy would further deplete the already low circulating levels of active AT, making the individual more vulnerable to emphysema. Thus, to prevent the pulmonary disease, replacement therapy with M-AT would be required.

Our biochemical data with the nonacetylated 6 mer peptide suggests that a novel therapeutic approach may be feasible. The combination of a molecule's ability to prevent polymer formation and to dissociate from the BC may be therapeutically advantageous in the context of Z-AT–related cirrhosis and emphysema. One would aim to deliver the therapeutic agent in excess to the endoplasmic reticulum of the hepatocyte, where it would bind to Z-AT and so prevent polymerization and the liver disease (34, 35). It would seem reasonable to propose that the annealing of a peptide mimetic to the pathogenic conformation would stabilize the protein and allow it to traverse the secretory apparatus of the cell safely, avoiding the degradative pathways. This would have two beneficial effects. First, increased amounts of Z-AT, as BC, would be released into the plasma and tissues compared with the 10–15% of Z-AT released from the liver cell of Z homozygotes. Second, in such an environment where there was no surplus peptide, dissociation of the BC would lead to release of an active inhibitor. This would significantly elevate levels of Z-AT in the tissues and may abrogate the need for replacement therapy with M-AT.

Currently there is no proven therapy for Z-AT–related disease other than organ transplantation. These exciting biochemical data demonstrate that peptide mimetics can be designed to selectively inhibit Z {alpha}1-antitrypsin polymerization and alleviate the associated plasma deficiency of the serpin. This innovative concept serves as an essential platform for the development of smaller, more specific peptide therapies. In addition, a number of important issues remain to be determined, including the most efficient way to deliver intact peptide to the endoplasmic reticulum of the hepatocyte and how the peptide and BC are processed within the cell. Future cellular and animal studies are necessary to address these questions. Nevertheless, this approach indicates that by investigating novel therapeutic avenues it may be possible to prevent both the cirrhosis and emphysema associated with Z-AT.


   Acknowledgments
 
This work is supported by the Wellcome Trust and the Medical Research Council (MRC). H.P. is an MRC Clinical Training Fellow and recipient of the Raymond and Beverly Sackler Award, T.R.D. is an MRC Career Development Fellow, and R.M. is a Wellcome Trust Advanced Clinical Fellow.

Received in original form July 24, 2003

Received in final form February 13, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Y.-P. Chang, R. Mahadeva, W.-S. W. Chang, A. Shukla, T. R. Dafforn, and Y.-H. Chu
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