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Published ahead of print on June 15, 2006, doi:10.1165/rcmb.2005-0207OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 540-548, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0207OC

Identification of a 4-mer Peptide Inhibitor that Effectively Blocks the Polymerization of Pathogenic Z {alpha}1-Antitrypsin

Yi-Pin Chang, Ravi Mahadeva, Wun-Shaing W. Chang, Anshuman Shukla, Tim R. Dafforn and Yen-Ho Chu

Department of Chemistry and Biochemistry, National Chung Cheng University, Chia-Yi; Institute of Cancer Research, National Health Research Institutes, Zhunan, Taiwan, R.O.C.; Department of Medicine, University of Cambridge, Cambridge; and Department of Biosciences, University of Birmingham, Birmingham, United Kingdom

Correspondence and requests for reprints should be addressed to Yen-Ho Chu, Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Rd., Min-Hsiung, Chia-Yi, Taiwan 62102, R.O.C. E-mail: cheyhc{at}ccu.edu.tw


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
{alpha}1-Antitrypsin (AT) is a major proteinase inhibitor within the lung. The Z variant of AT (E342K) polymerizes within the liver and lung, resulting in hepatic aggregation of AT and tissue deficiency, predisposing to early onset of cirrhosis and emphysema, respectively. Polymerization of the aberrant protein can be prevented in vitro by specific peptides such as FLEAIG. This peptide serves as a lead molecule to design a shorter peptide that may be effective as a therapeutic agent. In this study we employed a systematic chemical approach using alanine scanning of Ac-FLEAIG-OH and subsequent peptide shortening to study the binding of shorter peptides to Z-AT. While two additional 6-mer peptides Ac-FLAAIG-OH and Ac-FLEAAG-OH were found to bind to Z-AT, their daughter peptides Ac-FLEAA-NH2 and Ac-FLAA-NH2 also bound avidly to Z-AT and prevented polymerization of the protein. Further comparative studies revealed that the binding of Ac-FLAA-NH2 was more specific for Z-AT. The peptide–AT complex formation was enhanced by the presence of C-terminal amide group on the peptide, and circular dichroism analysis demonstrated that a random coil rather than a beta-helical conformation favored binding of the peptide to AT. In summary, this study has identified novel small peptides that inhibit Z-AT polymerization, and are a significant advance towards the treatment of Z-AT–related cirrhosis and emphysema.

Key Words: {alpha}1-antitrypsin • {alpha}1-proteinase inhibitor • alanine scanning • polymerization • serpin


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
{alpha}1-Antitrypsin (AT), also named {alpha}1-proteinase inhibitor, is the best-characterized member of the serpin (serine proteinase inhibitor) superfamily. Members of this superfamily have a common tertiary structure based on a dominant five-stranded beta-sheet A that supports an exposed 17-residue reactive loop (1–5). Interaction with the target proteinase results in cleavage of the serpin at its reactive loop (methionine-serine P1-P1' bond) (6, 7), followed by a marked conformational change within the molecule. The reactive loop inserts between strands 3 and 5 of beta-sheet A as strand 4 (s4A), translocating the proteinase to the lower pole of the molecule with the formation of a 1:1 enzyme:inhibitor complex (8–11). The normal AT protein is designated as M-AT according to its isoelectric point. One in 2,000 North Europeans are homozygous for the highly polymerogenic Z protein (12, 13). The Z mutation of {alpha}1-AT lies between the base of reactive loop and the head of strand 5 of beta-sheet A (P17, E342K), which may lead to the partial insertion of the reactive loop between strands 3 and 5 (14, 15). This process opens beta-sheet A and allows insertion of the reactive loop of a second molecule into beta-sheet A of the first to form a dimer and subsequently polymers (16, 17). Z {alpha}1-AT polymers are retained within the rough endoplasmic reticulum of the hepatocyte, as PAS-positive, diastase-resistant inclusion bodies that predispose Z-AT homozygotes to neonatal hepatitis, hepatocellular carcinoma, and juvenile cirrhosis (18–22). The subsequent secretory defect results in inadequate pulmonary defense against elastolytic enzymes, leading to early onset of panacinar emphysema (23). Polymers of Z-AT are also found in the emphysematous alveoli and are chemotactic to neutrophils (24–26).

Abnormal polymerization of other serpins such as antithrombin III, {alpha}1-antichymotrypsin, neuroserpin, and C1 inhibitor have also been reported to be associated with thrombosis, liver cirrhosis, early onset dementia, and angio-edema, respectively (15, 27–31). During the past decade, several approaches have been attempted to prevent serpin polymerization. For example, site-directed mutagenesis can inhibit polymerization in vitro and can restore secretion of polymerogenic AT mutants (32, 33), and the naturally occurring solute trimethylamine N-oxide can prevent polymerization (34). Another in vitro method, named synthetic reactive-loop peptide annealing, is also capable of blocking AT polymerization by binding various synthetic 12-14 mer peptides (homologous to the reactive loop of AT) to beta-sheet A (35–40). These synthetic peptides, however, are too large to be clinically relevant and do not bind specifically to the pathogenic Z-AT. Only a most recent study has demonstrated that a 6-mer peptide (Ac-FLEAIG-OH; P7–2) preferentially recognizes Z-AT, thereby fulfilling a critical requirement for an inhibitor of polymerization (14). Although this Ac-FLEAIG-OH peptide remains too large to be clinically useful as a therapeutic agent, its specific binding to Z-AT serves as a platform for further optimization studies.

Compared with the reactive loop sequences of various human serpins, no apparent sequence conservation is found in the P7–2 region (41). This unique P7–2 region in human serpins is likely to play a pivotal role in both binding affinity and recognition specificity for each protease inhibitor system. In this study, we used Ac-FLEAIG-OH as a starting point and employed the chemical method of alanine scanning together with peptide shortening to systematically study the binding of peptides to both M and the pathogenic Z-AT.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Preparation of Human {alpha}1-AT
M-AT was purchased from Calbiochem (La Jolla, CA). The purified Z-AT was prepared from the plasma of known homozygotes by 75% and 50% ammonium sulphate fractionation, followed by glutathione sepharose and Mono Q-sepharose column chromatography as previously described (40). The proteins migrated as a single band (> 95% purity) on SDS-PAGE and were in the native conformation on nondenaturing PAGE. The proteins were aliquoted, snap frozen, and stored at –80°C in 50 mM Tris, 50 mM KCl, pH 7.4.

Peptide Synthesis
Solid-phase peptide synthesis was carried out on PAL (Advanced ChemTech, Inc., Louisville, KY) or Wang resin (AnaSpec, Inc., San, Jose, CA) using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a homemade peptide synthesis apparatus. Each peptide sequence was elaborated in the C-to-N direction by iterative de-protection with 20% (vol/vol) piperidine in dimethylformamide followed by HBTU/HOBT-mediated coupling cycles with Fmoc-protected amino acids (Chem-Impex International, Inc., Wood Dale, IL). All peptides were assessed by standard ninhydrin test to ensure the completion of peptide bond formation. Final side chain de-protection and cleavage from the solid support were achieved with a cocktail of trifluoroacetic acid (TFA). The peptide solution was drained into a glass vial, and the solvent was evaporated with N2. The resulting syrup was redissolved in H2O and lyophilized on at least three occasions to eventually give the desired peptide. The purity and authenticity of the product was verified by HPLC (Hitachi L7420/L7100; Hitachi Ltd., Tokyo, Japan) using a Mightysil C18 column (25 cm x 4.6 mm, 5 µm; Kanto Chemical Co., Ltd., Tokyo, Japan). Products were assessed at 220 nm during a 30-min linear gradient of 0–100% acetonitrile (0.1% TFA) in water (0.1% TFA) at a flow rate of 1 ml/min. Molecular weight was determined on a Finnigan LCQ Advantage MAX spectrometer (Finnigan Ltd., San Jose, CA).

Assessment of Synthetic Reactive Loop Peptide Annealing to {alpha}1-AT
Synthetic peptides were incubated with Z and M-AT at 37°C with a 100-fold molar excess of peptides at a final concentration of 0.5 mg/ml in 50 mM Tris, 50 mM KCl, pH 7.4 for up to 14 d. Stock solutions of peptides were prepared in dimethyl sulfoxide (DMSO), and subsequent dilutions were made in 2% (vol/vol) DMSO/buffer to achieve the final concentration for assay. Binding of the peptides to M or Z-AT and the prevention of polymer formation were assessed on an 8% (wt/vol) nondenaturing PAGE containing 8 M urea (14). Proteins that associated with or without peptides were visualized by staining the gels with Coomassie Brilliant Blue R-250.

Assessment of Inhibition of Polymerization of {alpha}1-AT
Polymerization of AT is usually assessed by nondenaturing PAGE. It is also possible to assess the formation of polymers by nondenaturing PAGE containing urea (14). Although this method is used routinely in our laboratory, the appearances of polymers in relation to the native protein on the nondenaturing gel containing urea (Figure 1B) are quite different from the appearances of polymers on a nondenaturing PAGE without urea (Figure 1A). In nondenaturing gel containing urea, short chain polymers (S) are present below the native band (Figure 1B, lanes 2 and 3) and long chain polymers (L) appear above the native band (Figure 1B, lane 3). These differences are shown in Figures 1A and 1B, where native AT (lane 1) is shown in comparison with M-AT heated at 58°C for 1 h designed to produce predominantly short chain polymers (lane 2), and for 4 h designed to produce longer chain polymers (lane 3) on both nondenaturing PAGE without (Figure 1A) and with (Figure 1B) urea.


Figure 1
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  		Figure 1. (A) Native PAGE: 8% (wt/vol) nondenaturing PAGE demonstrating the pattern of heat-induced polymerization of M-AT. (B) 8% (wt/vol) nondenaturing PAGE containing 8 M urea showing that short chain polymers (S) migrate more anodal than native M-AT (N), while long chain polymers (L) migrate slower and appear above native protein. M-AT (4 µg) were heated at 58°C for 0 (lane 1), 1 (lane 2), and 4 (lane 3) h.

 
Densitometric Calculation of Rate of Binary Complex Formation
Gel patterns were digitized with a scanner and acquired by a personal computer. The amounts of binary complex were calculated as the band intensity and band area by Gel-Pro analyzer computer software (Media Cybernetic, Silver Spring, MD).

Circular Dichroism
Circular dichroism (CD) spectra were measured using a JASCO (Tokyo, Japan) J-715 spectropolarimeter in 50 mM phosphate (pH 7.6) using a 0.05-cm quartz cuvette. All experiments were carried out at ambient temperature, and spectra were an average of five scans (response time 1 s).

Assessment of Peptide Binding to Refolding {alpha}1-AT
M or Z-AT was incubated with 6 M urea for 10 min before dialysis against 50-fold molar excess of Ac-FLAA-NH2 solution at 37°C for 48 h. The dialysis buffer was exchanged with peptide solution three times to achieve an estimated concentration of 6 nM urea. Assessment of peptide binding to the refolding protein was judged by 8 M urea gel as aforementioned.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
In this study, alanine scanning of the lead compound Ac-FLEAIG-OH was first employed to assess the role of each residue in binding to M and Z-AT. Next, the most promising candidates were shortened to define the minimal length for specific binding. Furthermore, the peptide with shortest length was then analyzed by D-scanning experiment (substitution of partial or all residues to D-amino acid) to investigate the stereochemistry required for binding. Peptide binding was further characterized by varying the AT:peptide ratio and time course experiments. Finally, the most potent peptide was incubated with denatured M and Z-AT to investigate the binding affinity during protein refolding.

Alanine Scanning of the 6-mer Peptide Ac-FLEAIG-OH
Binding to M {alpha}1-AT. The peptides Ac-FAEAIG-OH (L2A), Ac-FLAAIG-OH (E3A), and Ac-FLEAAG-OH (I5A) were able to bind M-AT better than the previously reported Ac-FLEAIG-OH (Table 1; Figure 2A, lanes 4, 5, and 7), whereas Ac-ALEAIG-OH (F1A), Ac-FLEGIG-OH (A4G), and Ac-FLEAIA-OH (G6A) demonstrated poor binding to M-AT. There was insignificant binding of Ac-FLEAIG-OH to M-AT as described previously (14).


Figure 2
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  		Figure 2. Eight percent (wt/vol) nondenaturing PAGE containing 8 M urea demonstrating the effect of alanine-scanning of Ac-FLEAIG-OH on its binding to M- and Z {alpha}1-AT. M-AT (A) or Z-AT (B) were incubated with a 100-fold molar excess of Ac-FLEAIG-OH and its analogs that derived from alanine-scanning at 37°C for 3 d. The more anodal migration of the binary complexes (BC) than peptide-free protein (N) is due to the acquisition of extra conformation stability from the fully antiparallel beta-sheet A. All lanes contain 2.5 µg of AT.

 

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  		TABLE 1. THE EFFECT OF ALA SCANNING OF AC-FLEAIG-OH ON THE BINDING TO M- AND Z-AT AND ITS SEQUENCE ALIGNMENT TO THE P8–1 REGION OF THE REACTIVE LOOP

 
Binding to Z {alpha}1-AT. The peptides Ac-FLEAIG-OH, Ac-FLAAIG-OH (E3A), Ac-FLEAAG-OH (I5A), and Ac-FLEAIA-OH (G6A) all bound to Z-AT (Table 1; Figure 2B, lanes 2, 5, 7, and 8). Only two peptides—Ac-FLEAIG-OH and Ac-FLEAIA-OH (G6A), having their N-terminal phenylalanine and the negatively charged glutamate—remained unchanged bound with greater specificity to Z-AT.

In this series of peptides, Ac-FLAAIG-OH (E3A) and Ac-FLEAAG-OH (I5A) bound very avidly to Z-AT. As a result, these two peptides were systematically shortened from either the C- or N-terminus and the corresponding daughter peptides were individually synthesized for further studies.

The Effect of Shortening the Peptide Ac-FLAAIG-OH (E3A)
The peptide acid Ac-FLAAIG-OH (E3A) was shortened to 6 daughter peptide amides of various lengths. Shortening of the E3A peptide to Ac-LAAIG-NH2 and Ac-FLAA-NH2 did not affect the ability to form a binary complex with M- or Z-AT assessed after incubation for 3 d (Table 2 and Figure 3). Furthermore, both peptides were able to bind to Z-AT and to inhibit polymerization. The 4-mer Ac-AAIG-NH2 peptide, however, preferentially bound to M-AT but not Z-AT (Figure 3A, lane 7).


Figure 3
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  		Figure 3. Eight percent (wt/vol) nondenaturing PAGE containing 8 M urea demonstrating the effect of shortening the E3A peptide (Ac-FLAAIG-OH) on its binding to M- and Z {alpha}1-AT. M-AT (A) or Z-AT (B) were incubated with a 100-fold molar excess of a series of the N- and C-terminal truncated peptides of E3A (Ac-FLAAIG-OH) at 37°C for 3 d. Peptides capable of binding Z-AT prevent the formation of short chain (S) and long chain (L) polymers (B, lanes 2, 4, and 6). Each lane contains 2.5 µg of AT.

 

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  		TABLE 2. THE EFFECT OF SHORTENING OF THE E3A (AC-FLAAIG-OH) PEPTIDE ON ITS ABILITY TO BIND TO M- AND Z-AT AND ITS SEQUENCE ALIGNMENT TO THE P8–1 REGION OF THE REACTIVE LOOP

 
The Effect of Shortening the Peptide Ac-FLEAAG-OH (I5A)
The peptide acid Ac-FLEAAG-OH (I5A) was shortened to 6 daughter peptide amides of different lengths from both termini. Shortening of the I5A peptide to the peptides Ac-FLEAA-NH2 and Ac-LEAAG-NH2 did not affect binding to Z-AT (Figure 4B, lanes 2, 3, and 6). Both peptides annealed to Z-AT and significantly inhibited the formation of polymers (Figure 4B, lanes 3 and 6). The peptide Ac-LEAAG-NH2 bound to M-AT (Table 3; Figure 4A, lane 6); but in contrast, there was no significant annealing of the peptide Ac-FLEAA-NH2 to M-AT (Table 3; Figure 4A, lane 3).


Figure 4
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  		Figure 4. Eight percent (wt/vol) nondenaturing PAGE containing 8 M urea demonstrating the effect of shortening the I5A peptide (Ac-FLEAAG-OH) on its binding to M- and Z {alpha}1-AT. M-AT (A) or Z-AT (B) were incubated with a 100-fold molar excess of a series of the N- and C-terminal truncated peptides derived from I5A (Ac-FLEAAG-OH) at 37°C for 3 d. Peptides capable of binding Z-AT prevent the formation of short chain (S) and long chain (L) polymers (B, lanes 2, 3, and 6). Each lane contains 2.5 µg of AT.

 

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  		TABLE 3. THE EFFECT OF SHORTENING OF THE I5A (AC-FLEAAG-OH) PEPTIDE ON THE ABILITY TO BIND TO M- AND Z-AT AND ITS SEQUENCE ALIGNMENT TO THE P8–1 REGION OF THE REACTIVE LOOP

 
D-Scanning of Ac-FLAA-NH2
D-Scanning of the peptide Ac-FLAA-NH2 with M- and Z-AT impaired the ability of the peptide to anneal to AT (Figure 5). Only the C-terminally modified peptide Ac-FLADA-NH2 retained its ability to bind to M-AT (Figure 5A, lane 4). In the case of Z-AT, only incubation of the parent peptide Ac-FLAA-NH2 completely prevented the development of Z-AT polymers (Figure 5B, lane 2).


Figure 5
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  		Figure 5. Eight percent (wt/vol) nondenaturing PAGE containing 8 M urea demonstrating the effect of D-scanning of Ac-FLAA-NH2 on its binding to M- and Z {alpha}1-AT. M-AT (A) or Z-AT (B) were incubated with a 100-fold molar excess of Ac-FLAA-NH2 and its stereoisomers derived from D-scanning at 37°C for 14 d. All lanes contain 2.5 µg of AT.

 
Assessment of the Binding of Ac-FLAA-NH2 and Ac-FLEAA-NH2 at Different Molar Ratios
The efficacy of the peptide Ac-FLAA-NH2 was further assessed by incubating the peptide in different molar ratios with Z-AT (Figure 6A). A significant amount of binary complex was formed when lowering the molar ratio of peptide:AT to 10:1, respectively. At a 25-molar excess of Ac-FLAA-NH2, virtually all the native Z-AT had been converted into binary complex (Figure 6A, lane 3). The Ac-FLEAA-NH2 peptide, in contrast, bound more avidly to Z-AT (Figure 6B). At a molar ratio of 10:1 (peptide:AT), most of the Z-AT had been converted to binary complex (Figure 6B, lane 2).


Figure 6
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  		Figure 6. Eight percent (wt/vol) nondenaturing PAGE containing 8 M urea demonstrating the effect of the Ac-FLAA-NH2 and Ac-FLEAA-NH2 at different molar ratios on its binding to Z-AT. M- or Z-AT was incubated with Ac-FLAA-NH2 (A) or Ac-FLEAA-NH2 (B) in different molar ratio as indicated in the figure at 37°C for 3 d. All lanes contain 2.5 µg of AT.

 
Time Course for the Binding of Ac-FLAA-NH2 and Ac-FLEAA-NH2
To assess the specificity and binding characteristics further, binding of the Ac-FLAA-NH2 and Ac-FLEAA-NH2 peptides to M- and Z-AT was investigated (Figure 7). Within 24 h of incubation, both peptides had bound to Z-AT to form a binary complex (> 90%) and virtually no Z-AT was left in the native form (Figure 7B). Compared with binding to Z-AT, there was significantly reduced binding of both peptides to M-AT after 24 h (Figure 7A, lanes 2 and 6). The rate of formation of binary was obtained by densitometric analysis from the nondenaturing PAGE gels containing 8 M urea. (Figure 8 and Table 4). This indicated that both Ac-FLAA-NH2 and Ac-FLEAA-NH2 had an insertion rate similar to that of Z-AT, taking 13–15 h to convert 50% of the native Z protein to binary complex. Since Ac-FLAA-NH2 is shorter than Ac-FLEAA-NH2 and thus advantageous for potential clinical application, the 4-mer Ac-FLAA-NH2 peptide was therefore selected to investigate the requirement of N- and C-terminal group necessary for efficient AT annealing.


Figure 7
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  		Figure 7. Eight percent (wt/vol) nondenaturing PAGE containing 8 M urea demonstrating the effect of the Ac-FLAA-NH2 and Ac-FLEAA-NH2 with M- and Z-AT over time. M- or Z-AT was incubated with Ac-FLAA-NH2 (A) or Ac-FLEAA-NH2 (B) as indicated in the figure at 37°C for up to 3 d. All lanes contain 2.5 µg of AT.

 

Figure 8
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  		Figure 8. Peptide insertion rates of Ac-FLAA-NH2 and Ac-FLEAA-NH2 binding to M- or Z-AT. The ratio of binary complexes were extracted quantitatively from 8 M urea gels by densitometric analysis and plotted versus its incubation times to compare peptide insertion rate and binding specificity to M- and Z-AT. Solid circles: M-AT+Ac-FLEAA-NH2; open circles: M-AT+Ac-FLAA-NH2; solid squares: Z-AT+ Ac-FLEAA-NH2; open squares: Z-AT+ Ac-FLAA-NH2.

 

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  		TABLE 4. TIME TO ACHIEVE 50% OF BINARY COMPLEX FORMATION OF AC-FLAA-NH2 AND AC-FLEAA-NH2 BINDING TO M- AND Z-AT

 
Modification of the N- and C-termini of Ac-FLAA-NH2
FLAA peptides with and without N-terminal acetylation with either a C-terminal –NH2 or –OH group were generated and their ability to bind to AT was assessed. The presence of a C-terminal –NH2 or –OH group was found to affect the binding of FLAA to AT (Figure 9A). FLAA-NH2 was able to bind more avidly to AT compared with FLAA-OH. The presence of an N-terminal acetyl group on both FLAA-OH and FLAA-NH2 produced more peptide–AT complexes.


Figure 9
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  		Figure 9. (A) Eight percent (wt/vol) nondenaturing PAGE containing 8 M urea demonstrating the effect of modification to the N- and C-terminus of the peptide FLAA on binding to M-AT. M-AT was incubated with a 100-fold molar excess of each peptide at 37°C for 3 d. All lanes contain 2.5 µg of AT. (B) Comparison of the circular dichroic spectra of the peptides FLAA-NH2 (thick line) and FLAA-OH (thin line) dissolved in phosphate buffer. FLAA-OH demonstrates a positive signal at 220 nm, indicating a more beta-helical–like structure compared with FLAA-NH2. (C) Top: The FLAA peptides can adopt an extended (FLAA-NH2) or a beta-helical (FLAA-OH) conformation in solution. The beta-helical FLAA-OH is unable to bind between strands 3 and 5 of the A-sheet of {alpha}1-antitrypsin. Bottom: A stereogram showing the residues in {alpha}1-AT that are likely to form interactions with FLAA-NH2 (cyan) upon binding. For clarity, side chains of the protein are shown in black and backbone in grey.

 
Correlation between the Binding of Modified FLAA Peptides to AT and the Peptide CD Profile
CD spectra were obtained on the FLAA peptides to assess whether this difference in binding was related to differences in the structure of the peptides. Spectra obtained for both FLAA-NH2 and FLAA-OH showed a number of unusual features (Figure 9B). Most pronounced of these was a maximum at 220 nm and a minimum at 228 nm. It can also be seen that these features are somewhat more pronounced in the FLAA-OH peptide than in the FLAA-NH2 peptide. These spectral features are unusual but suggest that, in comparison with FLAA-NH2, FLAA-OH consists of a more beta-helical–like structure (Figure 9C) (42).

Assessment of Ac-FLAA-NH2 Binding to Refolding {alpha}1-AT
M- and Z-AT were denatured by 6 M urea and dialysed against a 50-fold molar excess of peptide to assess binding of refolding protein. This revealed that both refolding M- and Z-AT were recognized by Ac-FLAA-NH2 but not Ac-FLEA-NH2 (Figure 10), as indicated by binary complex formation.


Figure 10
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  		Figure 10. Eight percent (wt/vol) nondenaturing PAGE containing 8 M urea demonstrating that Ac-FLAA-NH2 binds to refolding M-AT (A) or Z-AT (B). M- or Z-AT was treated with 6 M urea before dialysis against 50 fold molar excess of Ac-FLEA-NH2 (lane 2) or Ac-FLAA-NH2 (lane 3) peptide solutions at 37°C for 2 d. Native M or Z-AT and binary complex with Ac-FLAA-NH2 are in lane 1 and lane 4, respectively. All lanes contain 3.0 µg of AT.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
{alpha}1-AT is the physiologic inhibitor of neutrophil elastase, with a circulating plasma concentration of 1–2 mg/ml (12, 13). It is synthesized primarily by hepatocytes, from where it is secreted into the plasma and diffuses to the tissues. The crystal structure of the molecule is characterized by a dominant beta-sheet A and a reactive loop containing P1-P1' that lies away from the molecule (4–7). One in 2,000 individuals of North European descent are homozygous for the Z (E342K) variant. This variant is associated with retention of the protein within the endoplasmic reticulum of hepatocytes, which results in neonatal hepatitis, juvenile cirrhosis, and hepatoma (18–22). The associated failure of secretion of Z-AT leaves the lungs exposed to the elastolytic effects of neutrophil elastase, predisposing to premature emphysema (43, 44). Biochemical, biophysical, and crystallographic studies have elucidated the molecular pathophysiology underlying the aggregation of the Z variant in the liver (31). Polymers of Z-AT have recently been found in bronchoalveolar lavage fluid and are localized to the walls of emphysematous alveoli (25, 26). The retention of polymers within the lung is co-localized with an excess of neutrophils and have been shown to be chemotactic in vivo (24). Thus polymers of Z-AT are thought to directly contribute to the liver and lung disease in Z-AT homozygotes.

Polymerization of Z-AT can be inhibited by synthetic peptides with homology to the reactive loop which can bind to beta-sheet A (16, 36–39). The initial peptides were 12–14 amino acids in length, but too long to be considered as therapeutic agents. These longer peptides were also nonspecific binding to several members of the serpin superfamily. The development of the 6-mer peptide Ac-FLEAIG-OH (P7–2) was a significant step forward, as it was shorter and it bound specifically to Z-AT to inhibit polymer formation (14). A recent study further reported the identification of small peptides selected from commercial sources that readily anneal to the P8–3 region of both antithrombin and AT (45). In the present study, we have probed the minimal structural requirements for binding to Z-AT and M-AT by a systematic chemical approach: alanine scanning of Ac-FLEAIG-OH at first, then a systematic shortening of the resulting identified sequences, and finally the D-scanning and termini modification of the lead peptides.

Comparison of the reactive loop sequences of human serpins reveals a preference for threonine at P14 and P8, a tendency for the negatively charged glutamic acid at P13, and an 80% conservation of alanine at P12–9 (41). However, in the region of P7–2, no obvious pattern of sequence or residue conservation can be found. Furthermore, it may be critical for the insertion of this second half of the reactive loop, as specific residues may be necessary to compensate the energy required for the temporary displacement of helix F (46, 47). The residues of the previously identified peptide Ac-FLEAIG-OH are a combination of aromatic, hydrophobic, and negatively charged amino acids. To probe the specific residues that are involved in binding, alanine scanning of Ac-FLEAIG-OH was employed to identify essential residues required for binding. The amino acid alanine was chosen as the replacement residue because it maintains the chirality and eliminates the side chain beyond the beta carbon, yet does not alter the main-chain conformation. Moreover, it does not impose extreme electrostatic or steric effects. These studies revealed that a total of four 6-mer peptides Ac-FLAAIG-OH (E3A), Ac-FLEAAG-OH (I5A), Ac-FLEAIG-OH, and Ac-FLEAIA-OH (G6A) bound to Z-AT. Of these, Ac-FLAAIG-OH (E3A) and Ac-FLEAAG-OH (I5A) bound with greatest affinity to Z-AT, and Ac-FLEAIG-OH and Ac-FLEAIA-OH (G6A) were more specific for Z-AT (Figure 2). The substitution of an alanine for a glutamate (E3A and its daughter peptides) could be seen as a drastic change. Examination of the position of the equivalent glutamate side chain in the crystal structure of the antitrypsin–trypsin complex (PDB ID code, 1EZX) shows a close interaction between side chains of the glutamate 354 and lysine 331. This interaction is abolished in the Ac-FLAA-NH2 and Ac-FLAAIG-NH2 peptides and yet does not appear to affect its affinity. This may indicate that the interaction between the lysine and glutamate is not essential in the formation of binary complex. It is noted that the hydrophobic residues at P6 (Leu or Ala) and P4 (Ala) site of s4A appear to be critical to achieve optimal binding interaction with the nearby hydrophobic pockets, thus correlating with a recent report (45). The Ac-FLEGIG-OH (A4G) peptide failed to recognize M- and Z-AT, suggesting that a fully extended backbone is essential for peptides to be incorporated into beta-sheet A. In addition, we also noted that if the bulky side chains of Ac-FLEAIG-OH at P3 (Ile) and P5 (Glu) were replaced by the sterically less hindered alanine, the resulting peptides in turn exhibited improved binding to M- and Z-AT (Table 1).

Systematic shortening of the peptides having strong affinity to Z-AT (E3A and I5A) was performed not only to assess whether the properties of binding to Z-AT and inhibition of polymerization could be retained, but also to ultimately define the minimal length for binding. Truncation of these peptides to Ac-FLAA-NH2 from Ac-FLAAIG-OH (E3A) and Ac-FLEAA-NH2 derived from Ac-FLEAAG-OH (I5A) resulted in peptides that bound more selectively to Z-AT and inhibited its polymerization (Figures 3 and 4). Compared with previously reported peptides, only a 10:1 rather than a 100:1 molar excess of peptide was required for the conversion of the majority of the native protein into binary complex after 24 h of incubation (Figure 6). Moreover, time course experiments indicate that Ac-FLAA-NH2 and Ac-FLEAA-NH2 bind more avidly to Z-AT than M-AT (Figure 7). Note that both peptides formed at least 90% of binary complexes with Z-AT within 24 h, while Ac-FLAA-NH2 and Ac-FLEAA-NH2 took 72 h to convert M-AT to nearly 90% and 50% of binary complexes, respectively. Further quantitative measurement of binary complex formation by densitometry reveals that Ac-FLAA-NH2 and Ac-FLEAA-NH2 exhibit similar insertion rate to Z-AT (Figure 8 and Table 4). In addition, the binding specificity of Ac-FLEAA-NH2 to Z-AT was clearly demonstrated by comparison of binary complex formation with Ac-FLAA-NH2 at 24 h, as shown by the filled and open squares (95.5% versus 10.4%) in Figure 8. Unfortunately, despite extensive experiments it was not possible to quantify the rate of peptide insertion by changes in intrinsic tryptophan fluorescence as previously described (14). This was most likely due to the fact that the insertion of small peptides does not perturb the structure sufficiently to induce detectable changes in intrinsic tryptophan fluorescence.

The sequence alignment of Ac-FLAA-NH2 and Ac-FLEAA-NH2 with the reactive loop of AT reveals that the hydrophobic P6 (Leu) and P4 (Ala) sites are in keeping with the results of alanine scanning (Tables 2 and 3). However, there is an unexpected result from this study that remains unresolved: why did Ac-FLAAI-NH2 fulfill all experimental observations discussed above, yet fail to bind M- or Z-AT? One possible explanation is that the population of Ac-FLAAI-NH2 conformations in solution is incompatible with the topology between strands 3A and 5A. It is also likely that small peptides including Ac-FLAAI-NH2 may shuffle along the s4A region and make the binding unpredictable.

Under physiologic conditions, peptides can be degraded within the gastrointestinal tract before absorption into the systemic circulation. D-amino acid substitution has previously been incorporated in order to resist the proteolytic degradation by peptidases (48, 49). Hence, we prepared Ac-FLAA-NH2 and systematically substituted each L-residue with a D-residue to assess the effect of stereochemistry in the binding of this peptide to AT. As shown in Figure 5, none of the D-substituted peptides were capable of forming binary complexes with Z-AT and hence could not inhibit polymerization of Z-AT. These findings confirm the binding specificity of Z-AT with all-L Ac-FLAA-NH2 peptide, suggesting a remarkable stereochemical constraint of the Z-AT-peptide interface. Apparent differences in the appearances of the gels between M- and Z-AT are likely to be due to the polymerogenic tendency of Z-AT rather than reflect a differential effect in the binding of the different D-forms of Ac-FLAA-NH2 to M or Z-AT.

Modification of the FLAA peptide by N-terminal acetylation or a C-terminal –OH or –NH2 group altered the binding properties of FLAA (Figure 9A). In keeping with our previous work, more binary complex was apparent after incubation of AT with the N-terminal acetylated peptide (50). This finding is most likely to be due to enhanced dissociation of the nonacetylated peptide from the binary complex and may be therapeutically advantageous (50).

The modification of peptide acid to peptide amide conveniently eliminates the negative charge on the C-terminus and therefore transforms the peptide to a conformation resembling that of internal peptide segments, rather than the protein termini. As anticipated, the FLAA-NH2 peptide was found to bind more avidly to AT compared with FLAA-OH. To further investigate whether this finding was due to differences in the structure of these peptides, CD spectra for these peptides were obtained. The most prominent feature of these spectra was a maximum between 218 and 220 nm. These features do not correspond to those expected from conventional protein secondary structure (222 nm and 208 nm corresponding to {alpha}-helix and 214 nm for beta-sheet), and only show a small similarity to the spectrum expected from a randomly ordered protein. Such spectral features have in fact only been observed on a couple of occasions, most notably in studies of the beta-helical transmembrane peptide gramicidin, suggesting the peptides in these studies may take up a similar conformation (42) (Figure 9B). The observation that these features are more pronounced in the FLAA-OH peptide than the FLAA-NH2 suggest that the FLAA-OH has a greater beta-helical–like content, whereas FLAA-NH2 contains more random structures. These data suggest that the presence of beta-helical–like structure impedes the binding of the FLAA peptides to AT (Figure 9C). In terms of structure this seems reasonable, because in order to bind to AT the peptide must form backbone hydrogen bonds with strands 3 and 5 of the A-sheet. It might be expected that the energy cost of breaking the intramolecular interactions that stabilize the beta-helical structure in order to form these intermolecular hydrogen bonds with AT would reduce the binding affinity. A close examination of the structures of the FLAA-OH and FLAA-NH2 peptides suggested that the most likely mechanism for the formation of the compact beta-helical–like conformation of the FLAA-OH would result from charge–charge interactions between the terminal groups. These interactions would be abolished by the conversion of the carboxylic acid at the C-terminus to an amide.

Eighty-five percent of Z-AT aggregates within the ER of the hepatocyte. These aggregates are unusual in that they retain almost all secondary structural elements of the correctly folded protein. They appear to be macro-polymers produced by the linkage of fully folded {alpha}1-AT (16, 51). For this sort of polymer to have occurred it is highly likely that the protein has fully folded before polymer formation, which was the reason for the use of fully folded Z-AT for the testing of peptide binding. However, aggregation of the protein could also occur during refolding of the protein in the endoplasmic reticulum. Therefore, the conformation of M- or Z-AT was loosened by the chaotropic agent urea, and then the disrupted structures were refolded by dialysis against serial changes of buffer which contained peptide of interest. The preliminary results revealed that both of the refolding M- and Z-AT were recognized by the 4-mer peptide Ac-FLAA-NH2, but not the control peptide Ac-FLEA-NH2 (Figure 10). In principle, a myriad of conformational changes occur as the chaotrope concentrations decrease gradually during the dialysis course. Structural changes of protein folding in vivo may not have identical or even similar intermediates and ground state. However, the recognition of reorganized scrambled protein by the peptide Ac-FLAA-NH2 lies behind the hope that it will eventually lead to the discovery of novel small molecule inhibitors.

There is currently no effective treatment for Z {alpha}1-AT–related cirrhosis and emphysema. Biochemical studies have demonstrated that the most potent peptide inhibitors of serpin polymerization are peptides with homology to the reactive loop (39). In this study, we have for the first time used a systematic approach combining alanine scanning and peptide shortening to study the structural requirements and the minimal peptide length required for binding. Our studies have demonstrated that a 4-mer peptide (Ac-FLAA-NH2) and two 5-mer peptides (Ac-LAAIG-NH2 and Ac-FLEAA-NH2) bind tightly with Z-AT and thus inhibit polymerization. These findings will be of substantial benefit for future combinatorial technology, as the characterization of lead compounds reduces redundancy and improves the efficiency of library screening. Finally, our data reported provide a significant advance toward a therapy for ultimately treating Z-AT–related emphysema and cirrhosis.


   Acknowledgments
 
The authors are grateful to Mr. Kai-Chun Lin and Mr. Chien-Hung Chen (the National Health Research Institutes, Taiwan, R.O.C.) for their assistance in protein purification. They also thank and Dr Alison Rodger (Warwick University, U.K.) for helpful discussions about the circular dichroism data. The authors appreciate the constructive comments from the reviewers.


   Footnotes
 
This work was supported by the National Science Council of Taiwan, R.O.C. (NSC93-2113-M-194-019, NSC92-2751-B-001-014, and NSC92-2218-E-194-015 to Y.-H.C.), and the National Health Research Institutes of Taiwan, R.O.C. (NHRI-PA-093-PP-07, NHRI-PA-093-CB-04, and NHRI93A1-NSCPP14-5 to W.-S.W.C.), the Alpha One Foundation and Action Medical Research (to R.M.), and the Medical Research Council, U.K. (to T.R.D.).

Originally Published in Press as DOI: 10.1165/rcmb.2005-0207OC on June 15, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 3, 2005

Accepted in final form May 27, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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