Lung Polymers in Z

1-Antitrypsin

Deficiency-related Emphysema

Peter R. Elliott, Diana Bilton, and David A. Lomas

Departments of Medicine and Haematology, University of Cambridge, and Chest Medical Unit, Papworth Hospital, Cambridge, United Kingdom

Patients with 1-antitrypsin ( 1-AT) deficiency are at risk of developing early-onset panlobular basal emphysema, which has been attributed to uncontrolled proteolytic activity within the lung. Severe genetic deficiency of 1-AT is most commonly due to the Z mutation (342GluLys), which results in a block in 1-AT processing within the endoplasmic reticulum of hepatocytes. The retained 1-AT forms inclusions, which are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Our recent studies have shown that the accumulation of 1-AT is due to the Z mutation perturbing the structure of 1-AT to allow polymer formation, with a unique linkage between the reactive center loop of one 1-AT molecule and the A -pleated sheet of a second. The detection of loop-sheet polymers and other conformations of 1-AT in the lungs of patients with emphysema has been technically difficult. We show here that transverse urea-gradient-gel (TUG) electrophoresis and Western blot analysis may be used to characterize conformations of 1-AT in dilute samples of bronchoalveolar lavage fluid (BALF). This technique was used to demonstrate loop-sheet polymers in the lungs of patients with Z 1-AT-deficiency-related emphysema. Polymers were the predominant conformational form of 1-AT in BALF from the lungs of two of five Z homozygotes with emphysema, but were not detectable in any of 13 MM, MS, or MZ 1-AT controls. Because 1-AT loop-sheet polymers are inactive as proteinase inhibitors, this novel conformational transition will further reduce the levels of functional proteinase inhibitor in the lungs of the Z 1-AT homozygote, and so exacerbate tissue damage. Elliott, P. R., D. Bilton, and D. A. Lomas. 1998. Lung polymers in Z 1-antitrypsin deficiency-related emphysema. Am. J. Respir. Cell Mol. Biol. 18:670674.

Most Caucasians of North European descent are homozygous for the M variant of 1-antitrypsin ( 1-AT) (or 1proteinase inhibitor), but some 4% carry the Z deficiency allele (342GluLys), which results in plasma 1-AT levels that are 10 to 15% of normal. The low circulating levels of 1-AT expose the lungs to uncontrolled proteolytic attack, and predispose the Z homozygote to early-onset panlobular basal emphysema (1). The levels of plasma Z 1-AT are low because the protein is retained within the endoplasmic reticulum of the liver (2), where it forms periodic acid Schiff (PAS)-positive, diastase-resistant inclusions. These inclusions are associated with neonatal hepatitis (3), cirrhosis, and hepatocellular carcinoma (4). 1-AT is the archetypal member of the serine proteinase inhibitor or serpin superfamily (5), and is composed of a dominant A -sheet and a mobile reactive loop that acts as a pseudosubstrate for the cognate proteinase (Figure 1).
(Received in original form June 23, 1997 and in revised form October 8, 1997) Address correspondence to: Dr. D. A. Lomas, Department of Haematology, University of Cambridge, MRC Centre, Hills Road, Cambridge, CB2 2QH, UK. E-mail: dal16@cam.ac.uk Abbreviations: 1-antitrypsin, 1-AT; sodium dodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE; transverse urea gradient, TUG.
Am. J. Respir. Cell Mol. Biol. Vol. 18, pp. 670674, 1998

The Z mutation lies at the head of strand 5 of the A -sheet of the molecule and the base of the reactive center loop (6). It perturbs the folding (7) and structure of the protein (8), allowing a spontaneous conformational transition that results in the reactive center loop of one molecule inserting into the A -pleated sheet of a second to form chains of polymers (9, 10). Support for the loopsheet linkage comes from the recently delineated crystal structures of intact 1-AT, which show the reactive center loop to be in an extended -pleated conformation (11, 12) that is readily available for A -sheet insertion and polymer formation (Figure 1). It is these loopsheet polymers that then tangle to form hepatic inclusions and cause the concomitant plasma deficiency of 1-AT. The process of polymerization depends on both concentration and temperature (8, 9), and it is likely that inflammatory episodes, which exacerbate both of these factors, contribute to the accumulation of Z 1-AT within the hepatocyte and may account for the heterogeneity of the associated liver disease (13). Loopsheet polymerization accounts for the deficiency of two other mutants of 1-AT, Siiyama (53SerPhe) and Mmalton (52Phe deleted), that also form hepatic inclusions and produce severe plasma deficiency (14, 15). Loopsheet polymerization has also been described with mutants of C1inhibitor, antithrombin, and 1-antichymotrypsin, in association with angioedema, thrombosis, and emphysema, re-

Elliott, Bilton, and Lomas: Antitrypsin Polymers and Emphysema

671

Figure 1. Illustration of 1-AT, with the -pleated sheets and helices shown in gray. The cleaved reactive center loop (black) is fully inserted into the A -sheet of the 1-AT molecule (26) which stabilizes the protein, rendering it resistant to unfolding in urea (a). In the active inhibitor (b), the loop adopts an extended -sheet conformation that is readily available for docking with the cognate proteinase (11). This loop may insert into the A -sheet of a second 1AT molecule to form dimers, which then extend as long-chain polymers, as shown in (c). The 1-AT molecules in white, black, and gray then tangle in the liver to form hepatic inclusions (from the article by Elliott and colleagues [11], with permission from Nature Structural Biology).

spectively (6). This conformational transition also underlies the mild plasma deficiency of the common S (264Gu Val) variant of 1-AT (16). We have developed a novel method of characterizing the conformation of 1-AT in bronchoalveolar lavage fluid (BALF) and we show here that 1-AT can also form inactive loopsheet polymers within the lungs of Z homozygotes. Spontaneous polymerization in vivo will further reduce the antiproteinase screen and exacerbate lung damage.

Materials and Methods

The 1-AT antibodies, bovine -chymotrypsin, and SucAla-Ala-Pro-Phe-pNA substrate used in the study were from Sigma Chemical Co. (Poole, UK), the ECL chemiluminescence assay was from Amersham International PLC (UK), and all other reagents were of analytical grade and from BDH Ltd (Leicester, UK).

Preparation of Control 1-AT Conformations M and Z 1-antitrypsin were purified from the plasma of known homozygotes by 50% and 75% ammonium sulfate fractionation followed by thiol exchange and Q-Sepharose chromatography (Pharmacia, St. Albans, UK) (8). The purified proteins migrated as a single band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE), had normal unfolding transitions on transverseurea-gradient (TUG) gel electrophoresis (17), and were 75% (M) and 50% (Z) active as inhibitors against bovine -chymotrypsin. M 1-AT polymers were prepared by heating plasma M 1-AT (0.25 mg/ml) at 60 C for 3 h as described previously (8), and were confirmed with nondenaturing PAGE and a complete loss of inhibitory activity against bovine -chymotrypsin. Antitrypsinbovine -chymotrypsin complexes were formed by incubating these two agents at a 1:0.8 active-site molar ratio; the pres-

672

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 18 1998

ence of complexes was confirmed by a characteristic band shift on SDS-PAGE, and in all cases there was 1% residual enzyme activity as detected by Suc-Ala-Ala-Pro-PhepNA substrate turnover. Cleaved 1-AT was prepared by incubation with Staphylococcus aureus V8 proteinase (8), which cleaves 1-AT at the P4P5 bond of the reactive loop; full cleavage was confirmed by a 4-kD band shift on SDS PAGE. Samples of cleaved 1-AT incubated with complexes or lavage fluid were treated with the inhibitor 3,4dichloroisocoumarin to a final concentration of 1 mM (17) to prevent V8 proteinase from cleaving the native protein. Detection of 1-AT Conformations by TUG Gel Electrophoresis For TUG gel electrophoresis, 7.5% (wt/vol) polyacrylamide gels were cast with a double lumen tube and a peristaltic pump to give a linear gradient of 0 to 8 M urea with the nondenaturing-PAGE buffer system (18, 19). The gels were rotated through 90 , the stacking gel was poured, and the gels were run in a locally constructed tank 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 (17, 18). 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 in the control preparations were visualized by staining with Coomassie blue. 1-AT was detected in lavage fluids through Western blot analysis. Samples of unconcentrated lavage (300 to 400 l) fluid were mixed with loading buffer and separated as described earlier. Proteins were electroblotted onto nitrocellulose paper in a Biorad mini-PROTEAN II electrophoresis system (Hemels Hempstead, UK) at 80V for 1 h in 0.0125 M Tris, 0.48 M glycine, and 20% (vol/ vol) methanol. The nitrocellulose paper was blocked by shaking for 30 min with 0.05 M Tris, 0.002 M CaCl2, 0.05 M NaCl, pH 8.0, with 5% (wt/vol) skim-milk powder, and 0.02% NP-40. 1-AT was visualized by shaking with 0.1% (vol/vol) polyclonal rabbit anti- 1-AT antibody in blocking buffer for 1 h and, after washing, shaking with 0.1% (vol/vol) horseradish peroxidase-labeled swine antirabbit antibody. The bands were visualized by development with an ECL chemiluminescence kit. Analysis of BALF from Patients with Emphysema Bronchoalveolar lavage (BAL) was obtained from 13 control patients who were undergoing bronchoscopy for the investigation of bronchogenic carcinoma, chronic cough, and hemoptysis. Five 20-ml aliquots of normal saline were instilled into the lower lobe, right middle lobe, or lingula on the side opposite that of the lesion for which the bronchoscopy was being performed. Plasma 1-AT levels were measured and the phenotype was determined for each patient. All of the Z 1-AT homozygotes (phenotype ZZ or Z/null) were ex-smokers, had no history of a recent chest infection, and had radiologic and physiologic evidence of airflow obstruction and gas trapping (all patients had an FEV1 of 1.6 liters and FEV1/FVC ratio 50%, and four of five had an RV/TLC ratio 40%; one patient was unable to tolerate whole-body plethysmography). Four of the five Z 1-AT homozygotes had a significant reduction

( 50% predicted) in carbon monoxide gas-transfer factor, in keeping with emphysema, and one had a gas-transfer factor that was only modestly reduced. Three of the five Z 1-AT homozygotes had evidence of reversible airflow obstruction and were taking inhaled bronchodilators and inhaled corticosteroids. Z 1-AT homozygotes were lavaged from the lower lobes with 20-ml aliquots of normal saline, and the samples were stored on ice prior to assay. Sham bronchoscopy and lavage of purified monomeric Z 1-AT did not induce conformational transitions or polymerization when specimens were assayed with TUG gel analysis. The samples of unconcentrated lavage fluid were assayed with 0 to 8 M TUG gels, with Western blot analysis for 1-AT, and the results were compared with the profiles of 1-AT controls. The study was approved by the local research ethics committee, and all patients gave informed consent for their participation.

Results
The characterization of 1-AT in lung lavage fluid was technically difficult, since the protein was dilute and concentrating the sample was avoided so as not to induce conformational transitions and artifactual polymerization. Polymers could not be confidently detected in lavage fluids with nondenaturing PAGE followed by Western blot analysis for 1-AT, but could be detected by TUG gel electrophoresis, which allowed the loading of much larger volumes of lavage fluids. This biochemical assay technique measures the unfolding and retardation of proteins by urea, and each 1-AT conformation had a characteristic signature, as shown in Figure 2. Native M or Z 1-AT unfolded at approximately 1 M urea, as detailed previously (7, 17, 19), but 1-AT cleaved in the reactive loop (Figure 1) and complexed with enzyme was resistant to unfolding in up to 8 M urea, suggesting that the protein is stabilized by insertion of the reactive loop into the A -sheet. Similarly, 1-AT polymers do not unfold, since the A -sheet is filled by the reactive loop of a second 1-AT molecule. We examined BALF from 13 consecutive control patients with the MM (11 patients), MZ (1 patient), or MS (1 patient) 1-AT phenotype. This group included smokers, ex-smokers, and nonsmokers, and patients with and without chronic bronchitis and emphysema, who were undergoing bronchoscopy for the investigation of chronic cough, hemoptysis, and suspected bronchogenic carcinoma. BALF from these patients contained native (Figure 2e), reactiveloop-cleaved and proteinase-complexed 1-AT as detected with SDSpolyacrylamide and TUG-gel electrophoresis followed by Western blot analysis and chemiluminescence. None of the lavage specimens from these controls contained a prominent ladder of high-molecular-mass 1-AT polymers (Figure 2d), although there were faint bands that might have represented loopsheet dimers. 1-AT loopsheet polymers represented the major conformation of 1-AT in BALF in two of five patients with Z 1-AT deficiency-related emphysema (Figure 2f). The polymers obtained by lung lavage were composed of approximately two to seven 1-AT molecules, and migrated further into the gel than did the M 1-AT control heated at 60 C for 3 h (Figure 2d), which generated polymers of 15

Elliott, Bilton, and Lomas: Antitrypsin Polymers and Emphysema

673

Discussion
The flexibility of the reactive center loop of 1-AT allows it to adopt a range of conformations. In the native protein, the loop is an extended -pleated canonical structure that forms a sterically acceptable complex with the cognate proteinase (11). Following docking with the target proteinase, the loop is believed to insert into the A -sheet to form an irreversibly locked enzyme-inhibitor complex (20, 21). Reactive-loop cleavage by nontarget proteinases results in a similar transition, with the loop inserting into the gap between strands 3 and 5 of the A -sheet to form a sixmembered A -sheet (Figure 1a). The flexibility of the reactive loop allows it to fully insert into the A -sheet in the absence of cleavage, to form the latent conformation when heated in stabilizing concentrations of sodium citrate (17, 22, 23). Moreover, the flexibility of the reactive loop also allows insertion of the reactive center loop of a second 1-AT molecule (Figure 1c) into the A -sheet to form a loopsheet dimer, which then extends to form chains of polymers (8, 19, 24). It is this polymerization that occurs spontaneously in Z 1-AT and underlies the formation of hepatic inclusions and the associated plasma deficiency of 1-AT. The lack of 1-AT within the lungs of patients with genetic 1-AT deficiency results in uncontrolled digestion of elastin and the development of emphysema (1). There has been little detailed examination of the conformation of 1AT from BALF in PiM or PiZ patients because this is technically difficult. The observation that Z, Siiyama (14), and Mmalton (15) 1-AT polymers can exist in the plasma raised the possibility that they may also form in other tissues of the body. Moreover, because loopsheet polymers are inactive as proteinase inhibitors (8), they will be unable to contribute to the antiproteinase screen in the lung. We have shown in the present study that TUG gels and Western blot analysis with chemiluminescence constitute a sensitive method for detecting conformations of 1-AT in vivo. This method detected native, reactive-loop-cleaved and complexed 1-AT in dilute samples of BALF from patients with M, MZ, and MS 1-AT phenotypes and a variety of lung pathologies. Loopsheet polymers were detected in the lavage fluid from two of the five Z 1-AT-deficient patients with emphysema. The formation of polymers was not related to inhaled medication, since one of the patients was taking no medication at the time of bronchoscopy. Moreover, sham bronchoscopy with purified Z 1-AT showed that the bronchoscopy itself did not induce conformational transitions in the 1-AT. It is unclear why only two of the five Z 1-AT homozygotes in our study had polymers in their BALF. Polymerization is known to be concentration and temperature dependent (8, 9), but there was no evidence of recent infection in either of the affected individuals, and they did not appear to have a more rapid rate of decline of lung function. It is likely that the quantity of 1-AT polymers will vary with time, and their relationship to infection, smoking, and decline in lung function will need to be determined by sequential lavage in many patients. Moreover, although chemiluminescence is a sensitive assay technique, it may not detect small amounts of polymers mixed with native lung

Figure 2. TUG-gel electrophoresis followed by Western blot analysis and chemiluminescence to detect 1-AT. The left of each gel represents 0 M urea and the right 8 M urea. TUGs a through d contain 40 to 50 g protein, and TUGs e through f were loaded with 300 to 400 l unconcentrated BALF with subsequent Western blot analysis and chemiluminescence to detect antitrypsin. (a) Purified M 1-AT (a similar profile was obtained for Z 1-AT (15); (b) reactive-loop-cleaved 1-AT; (c) 1-ATbovine -chymotrypsin complexes and reactive-loop-cleaved 1-AT mixed and run on the same gel (40 g each sample) (the upper band represents complexed 1-AT, and the lower band represents reactive-loop-cleaved 1-antitrypsin); (d) M 1-AT polymer control generated by heating M 1-AT at 60 C for 3 h; (e) a normal antitrypsin unfolding transition in a BALF specimen from a control M 1-AT homozygote investigated for chronic cough; (f) characteristic profile of loopsheet polymers in a BALF specimen from a Z 1-AT homozygote with emphysema.

to 20 1-AT molecules (15). The length of the polymers identified in BALF was comparable to that of polymers obtained previously upon incubating isolated plasma Z 1AT under physiologic conditions (9). Both of the patients whose lavage fluid contained polymers had pulmonary physiology consistent with emphysema, and one was taking no medication. The second patient had partly reversible airflow obstruction for which he was receiving inhaled salbutamol, oxitropium bromide, salmeterol, and inhaled corticosteroids. Lavage fluid from the other three Z 1-AT homozygotes with emphysema revealed -ATproteinase complexes in one and a normal 1-AT unfolding transition in the other two. In order to demonstrate that Z 1-AT polymers formed within the lungs, we purified 1-AT from the plasma of the Z homozygote shown in Figure 2f. The protein was predominantly monomer, with less than 5% being present as loopsheet polymers.

674

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 18 1998

1-AT in either M or Z 1-AT homozygotes, and there is always concern that the conformations of 1-AT in lavage fluid may not represent the conformations at the alveolar surface. Nevertheless 1-AT loopsheet polymerization must be added to reactiveloop cleavage, enzymeinhibitor complex formation, and oxidation of the P1 methionine (25) as a mechanism of inactivating the most important proteinase inhibitor in the lung. TUG-gel electrophoresis and Western blot analysis may provide a useful means for assessing the conformation of 1-AT in longitudinal studies and correlating this with the development of chronic bronchitis and emphysema. In summary, this study provides the first demonstration of Z 1-AT polymers in the lungs of patients with emphysema. The inactivated 1-AT is unable to play any role in the antiproteinase screen, and this will serve to exacerbate the lung disease associated with plasma deficiency of the Z mutation of 1-AT.

Acknowledgments: The authors are grateful to all the patients, particularly the Z 1-AT homozygotes, who took part in this study. This work was supported by the Medical Research Council of the United Kingdom and the Wellcome Trust.

References
1. Brantly, M., T. Nukiwa, and R. G. Crystal. 1988. Molecular basis of alpha-1antitrypsin deficiency. Am. J. Med. 84(Suppl. 6A):1331. 2. Sharp, H. L., R. A. Bridges, W. Krivit, and E. F. Freier. 1969. Cirrhosis associated with alpha-1-antitrypsin deficiency: a previously unrecognised inherited disorder. J. Lab. Clin. Med. 73:934939. 3. Sveger, T. 1976. Liver disease in alpha1-antitrypsin deficiency detected by screening of 200,000 infants. N. Engl. J. Med. 294:13161321. 4. Eriksson, S., J. Carlson, and R. Velez. 1986. Risk of cirrhosis and primary liver cancer in alpha1-antitrypsin deficiency. N. Engl. J. Med. 314:736739. 5. Huber, R., and R. W. Carrell. 1989. Implications of the three-dimensional structure of 1-antitrypsin for structure and function of serpins. Biochemistry 28:89518966. 6. Stein, P. E., and R. W. Carrell. 1995. What do dysfunctional serpins tell us about molecular mobility and disease? Nature Structural Biol. 2:96113. 7. Yu, M.-H., K. N. Lee, and J. Kim. 1995. The Z type variation of human 1-antitrypsin causes a protein folding defect. Nature Structural Biol. 2: 363367. 8. Lomas, D. A., D. L. Evans, S. R. Stone, W.-S. W. Chang, and R. W. Carrell. 1993. Effect of the Z mutation on the physical and inhibitory properties of 1-antitrypsin. Biochemistry 32:500508. 9. Lomas, D. A., D. L. Evans, J. T. Finch, and R. W. Carrell. 1992. The mecha-

nism of Z 1-antitrypsin accumulation in the liver. Nature 357:605607. 10. Lomas, D. A. 1996. New insights into the structural basis of 1-antitrypsin deficiency. Q. J. Med. 89:807812. 11. Elliott, P. R., D. A. Lomas, R. W. Carrell, and J. P. Abrahams. 1996. Inhibitory conformation of the reactive loop of 1-antitrypsin. Nature Structural Biol. 3:676681. 12. Ryu, S.-E., H.-J. Choi, K.-S. Kwon, K. N. Lee, and M.-H. Yu. 1996. The native strains in the hydrophobic core and flexible reactive loop of a serine protease inhibitor: crystal structure of an uncleaved 1-antitrypsin at 2.7. Structure 4:11811192. 13. Sveger, T., and S. Eriksson. 1995. The liver in adolescents with 1-antitrypsin deficiency. Hepatology 22:514517. 14. Lomas, D. A., J. T. Finch, K. Seyama, T. Nukiwa, and R. W. Carrell. 1993. 53 1-antitrypsin Siiyama (Ser Phe); further evidence for intracellular loopsheet polymerisation. J. Biol. Chem. 268:1533315335. 15. Lomas, D. A., P. R. Elliott, S. K. Sidhar, R. C. Foreman, J. T. Finch, D. W. Cox, and R. W. Carrell. 1995. Alpha1-antitrypsin Mmalton (52Phe deleted) forms loop-sheet polymers in vivo: evidence for the C sheet mechanism of polymerisation. J. Biol. Chem. 270:1686416870. 16. Elliott, P. R., P. E. Stein, D. Bilton, R. W. Carrell, and D. A. Lomas. 1996. Structural explanation for the dysfunction of S 1-antitrypsin. Nature Structural Biol. 3:910911. 17. Lomas, D. A., P. R. Elliott, W.-S. W. Chang, M. R. Wardell, and R. W. Carrell. 1995. Preparation and characterization of latent 1-antitrypsin. J. Biol. Chem. 270:52825288. 18. Goldenberg, D. P. 1989. Analysis of protein conformation by gel electrophoresis. In Protein Structure: A Practical Approach. T. E. Creighton, editor. IRL Press, Oxford. 225250. 19. Mast, A. E., J. J. Enghild, and G. Salvesen. 1992. Conformation of the reactive site loop of 1-proteinase inhibitor probed by limited proteolysis. Biochemistry 31:27202728. 20. Whisstock, J., A. M. Lesk, and R. W. Carrell. 1996. Modeling of serpin-protease complexes: antithrombin-thrombin, 1-antitrypsin (358MetArg)thrombin, 1-antitrypsin (358MetArg)-trypsin, and antitrypsin-elastase. Proteins: Structure, Function and Genetics 26:288303. 21. Lawrence, D. A. 1997. The serpin-proteinase complex revealed. Nature Structural Biol. 4:339341. 22. Lomas, D. A., P. R. Elliott, and R. W. Carrell. 1997. Commercial plasma 1-antitrypsin contains a conformationally inactive, latent component. Eur. Respir. J. 10:672675. 23. Koloczek, H., A. Banbula, G. S. Salvesen, and J. Potempa. 1996. Serpin 1-proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy. Protein Sci. 5:22262235. 24. Schulze, A. J., U. Baumann, S. Knof, E. Jaeger, R. Huber, and C.-B. Laurell. 1990. Structural transition of 1-antitrypsin by a peptide sequentially similar to -strand s4A. Eur. J. Biochem. 194:5156. 25. Beatty, K., J. Bieth, and J. Travis. 1980. Kinetics of association of serine proteinases with native and oxidized -1-proteinase inhibitor and -1-antichymotrypsin. J. Biol. Chem. 255:39313934. 26. Loebermann, H., R. Tokuoka, J. Deisenhofer, and R. Huber. 1984. Human 1-proteinase inhibitor: crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. J. Mol. Biol. 177:531556.