The Discovery of 1-Antitrypsin

Prof Dr Sabina M. Janciauskiene

Alpha1-Antitrypsin (AAT), an acute-phase , is the prototypic member of the superfamily and a major inhibitor of such as neutrophil and 3. As an acute-phase protein, AAT is thought to play an important role in limiting host tissue injury by proteases at sites of . The clinical importance of AAT is highlighted in individuals with inherited AAT deficiency who exhibit an increased susceptibility to develop chronic inflammatory conditions including chronic obstructive lung , and, sometimes, systemic vasculitis and necrotising panniculitis. Recent findings highlight the potential involvement of AAT in several proteolytic pathways and also indicate that it has broader antiprotease activities than previously anticipated. AAT may also exhibit anti- inflammatory activities independent of its protease inhibitor function, which suggests the existence of receptor-mediated AAT intracellular internalisation, and provides further evidence of a broad protective role of AAT against not only lung, but also other organ injury.

Early discoveries and historic perspectives Discovery of Alpha1-antitrypsin (AAT) The regulation of proteolytic in tissues by endogenous inhibitors is a prerequisite for the maintenance of homeostasis. The inhibitor superfamily () is evolutionarily conserved and present in higher , and (1, 2). The serpins now number in several hundred and includes involved in the regulation of many critical biological processes, including , , complement activation, , , inflammation, neoplasia and viral pathogenesis. The protease inhibitor activity of human plasma was first discovered by Fermi and Pernossi in 1894 (3). In 1897 studies by Hahn (4) and by Pugliese and Coggi (5) clearly noted that normal serum possesses the property of hindering or preventing the action of . However, at that time it was impossible to identify the specific protein which possessed the trypsin inhibitory activity. Only several years later did the development and improvement of a number of methods in , such as electrophoresis, chromatography, gel filtration, etc., open the possibility for the isolation and characterisation of individual serum proteins. During the 1930s the Swedish chemist Arne Tiselius at Uppsala University introduced the use of electrophoresis as a technique for protein separation. Tiselius received the Nobel Prize in 1948 for his research on electrophoresis and adsorption analysis and, in particular, for his discoveries concerning the complex nature of the serum proteins. Thus, the availability of new techniques encouraged many investigators to study the serum protein and, among others, trypsin inhibitors. Ultimately, the main inhibitor responsible for antiprotease activity was first isolated in 1955 by Herman Schultze in Germany and named alpha1-antitrypsin (AAT) because of its location in the α1- fraction and its ability to inhibit trypsin (6).

© 2010 Alpha1 Awareness UK page 1 Discovery of Alpha1-antitrypsin deficiency (AATD) In 1952, C-B Laurell at Malmö University Hospital made outstanding contributions to protein research by introducing plasma protein electrophoresis as a tool for clinical investigations. This originated with the use of paper electrophoresis following his visit to the laboratory of A. Tiselius in Uppsala in 1952 and then, in 1961, in the introduction of the much more selective agarose gel electrophoresis (7). Other medical investigators in Malmö began to use this technique, and in 1961 a newly appointed a specialist in respiratory medicine introduced the use of plasma electrophoresis for all his patients. C-B Laurell personally checked every electrophoretic result and, unexpectedly, noted the absence of the alpha1 band in two samples, both of which were from a hospital specialised in respiratory medicine (8). In conjunction with receiving the award, C-B Laurell gave a speech entitled “Bench Side Medicine” at the Oak Ridge Conference, 2001(9) in which he described his discovery of AATD. According to C-B Laurell, in 1963 he observed two patient samples with similar paper electrophoretic patterns both lacking the normal 1-band. Both sera derived from a hospital specialised in pulmonary diseases. Laurell thought that the electrophoretic 1-band was caused by – a well characterised by Winzler already in 1955 in USA. C-B Laurell had a specific antiserum against orosomucoid and tested it on these two patient sera. To his great surprise the antiserum disclosed a normal content of orosomucoid in both sera despite the missing the 1-band! However, at the same time, while C-B Laurell was working on these interesting serum samples, a report was published from Behringwerke stating that Herman Schultze`s group had purified the major 1-protein and identified it as the major plasma inhibitor of trypsin. At this point C-B Laurell and his co-workers verified that the two sera had a very low AAT activity and, therefore the two patients had an apparent deficiency of AAT. Both suffered from severe respiratory insufficiency caused by emphysema. This finding led to a retrospective analysis of hundreds of stored paper electrophoretic patterns in the laboratory archives of the Malmö and Lund hospitals. Investigators found several more AATD patterns, which had earlier been overlooked. C-B Laurell asked Sten Eriksson, a young physician working at the internal medicine unit, to recall these patients for examination by spirometry, pulmonary X-ray and to draw new plasma samples. It then became evident from family studies that an association existed between degenerative pulmonary diseases and AAT deficiency. The critical finding published in 1963 by C-B. Laurell and S. Eriksson (10) was that AAT deficiency is inherited and relates to emphysema. Early during the clinical evaluation of AAT deficiency, S. Eriksson had the good fortune to come across a family with three siblings suffering from a severe form of emphysema. This family was the basis of an article published in Acta Medica Scandinavica in 1964(11). Later, in 1965 S. Eriksson collected a larger number of AAT deficiency cases, including their families. All findings were presented in his thesis as a supplement to Acta Medica Scandinavica (12) which provided the first comprehensive evidence for the relationship between AAT deficiency and emphysema. At that time it was suggested that the primary reason for AAT deficiency is an inborn error of AAT metabolism. In 1958, the concept of a proteolytic destruction in emphysema was presented and accepted at the Ciba Guest Symposium in London and was further developed in 1961 by a committee of the WHO(13) albeit not on experimental evidence. Therefore, the discovery of AAT deficiency and its association with emphysema appeared at the right time. Moreover, in 1964, Gross and collaborators published an important work showing that intra-tracheal administration of , a protease, induces emphysema in rats(14). This study, together with the Laurell`s and Eriksson`s discoveries, supported the concept of a proteolytic mechanism in emphysema and opened a new chapter in experimental and clinical emphysema research. In the following years, several research groups in Europe and USA demonstrated that AAT is an effective inhibitor of and emphasised a possible role of elastase in the pathogenesis

© 2010 Alpha1 Awareness UK page 2 of emphysema. Based on these developments was generally accepted that the protease- antiprotease balance as an essential requirement for respiratory health, and that perturbations of this balance may result in the loss of lung tissue and the development of emphysema. Furthermore, this concept opened new insights into why in emphysema and proteolytic overload are more common in cigarette smokers. For instance, in a classical paper from 1978, by L. Larsson and collaborators, it was very convincingly demonstrated that the age of onset of emphysema in Z homozygous who were also current or ex-smokers was very rapidly reduced(15). Since AAT is an acute phase protein produced mainly in the liver, investigators in Malmö expected to find abnormal liver function in AATD patients. However, they were disappointed to find normal results for liver function tests in a number of AATD cases. S Eriksson wrote that in 1962 they had a patient with cryptogenic, decompensated who also lacked AAT band in the electrophoresis strip. Unfortunately, the finding was misinterpreted in the belief that the patient had developed an acquired AAT deficiency secondary to a severely impaired liver function(16). The association between liver diseases and inherited AATD, therefore, was documented for the first time not in Malmö, but in USA by Sharp and colleagues, in 1969 (17). The biochemistry of AAT The detection and biochemistry of AAT variants became the greatest challenge from 1963 to 1978. The molecule AAT was found to occur in many genetic variants, and C-B Laurell together with Magne Fagerhol in Oslo assembled them as the protease inhibitor Pi-system. Jan-Olof Jeppsson, who joined the Malmö group in 1970, took the lead in tracking the structural differences of the numerous molecular Pi-variants by isoelectric focusing. The Pi- system was developed, based on the migration of the AAT variants in an electric field. The position of the migrated proteins is identified by a letter, where M is normal, while the positions of the slower-moving proteins are marked by letters before M in the alphabet and those of the faster-moving proteins are denoted by letters after M. Many variants of AAT have been extensively studied. Initially it was believed that the intracellular accumulation of Z AAT resulted from aberrant . However, it soon became apparent that the underlying abnormalities of the AAT variants were due to single substitutions. During the 70s Robin Carrell analysed the S variant of AAT while the Malmö group analyzed the Z variant of AAT by mapping its tryptic peptides. By 1975, R Carrell and co-workers were able to show that S variant abnormality is due to the substitution of a glutamic acid by a valine (18). Soon after this, the abnormality of Z AAT (glutamic acid substitution by lysine) was solved by Jan-Olof Jeppson in Malmö(19). At this time, C-B Laurell started a collaboration with R. Carrell in Cambridge and presented the 3-dimensional structure of the dominating molecular variants of AAT. It became apparent that the active AAT protease inhibitory centre contained a crucial serine. A series of publications in the late 70s demonstrated a rate of association 1000-fold more rapid between and AAT than between trypsin and AAT (20). Therefore, the most important function of AAT was suggested to be the control of activity of neutrophil elastase but not of the trypsin. Structure and function of AAT: The Serpin Superfamily The AAT sequence was completed in 1982 and it was shown where the reactive site of AAT, where the S and Z occur, and where the points of attachment of three oligosaccharide side-chains of the molecule are located(21). The multiplicity of elecrophoretic bands of AAT was explained to be due to the presence of different glycoforms of AAT, each with variations in the antennary structure of their oligosaccharides. Moreover, by using mRNA isolated from normal (MM) and variant (ZZ) it was possible to show that both proteins

© 2010 Alpha1 Awareness UK page 3 have equivalent rates of synthesis. However, while the M protein was secreted, the Z protein was mainly retained intra-cellularly(22). The conclusions drawn in 1986 from these studies was that the in Z AAT does not effect protein synthesis, but most likely causes a perturbation of structure that results in defective secretion of the protein(23). It was clear that further understanding of the biological functions of AAT required a detailed knowledge of the three-dimensional structure of AAT. Identification of the serpin family occurred with the recognition in the 1980s by Hunt and Dayhoff that , AAT and III share a 30 to 50% sequence (24). Genetic characterisation has revealed a large group of belonging to the serpin family that probably arose by duplication of an ancestral gene(25). The term Serpin (an acronym for serine proteinase inhibitor) was later introduced as a general denominator by R Carrell and J Travis in 1985 to describe a superfamily of serine protease inhibitors of mammalian plasma (26). The individual serpins manifest about 30% common , and this increases to 70% when the comparison is limited to the hydrophobic sequences. Thus, there has been greater conservation of the internal residues of the molecule, indicating conservation of the overall, tertiary structure of the serpins(27). Huber and colleagues in Munich in 1984 resolved the crystal structure of the cleaved AAT for the first time. The cleaved form, with its change from a five-stranded to a six-stranded A-sheet provided an enigma and a challenge to and biochemistry as a whole because no protein had been known to undergo such a remarkable change in its folding(28). Later it was shown in the native form of AAT that the reactive centre loop lies outside the tertiary core of the protein to allow binding of the target protease. A large is triggered upon cleavage of the reactive centre loop by the protease. While the protease is covalently bonded to the AAT during the formation of the acyl- intermediate, the RCL is inserted into the centre of β sheet A, becoming a sixth strand. In the process, the bound protease is inactivated through the distortion of its (29). The inhibited (loop-inserted) form of serpins is known to be considerably more stable than the active (non-loop-inserted) form(30). AAT, similar to other inhibitory serpins, is thus unusual because it folds into a metastable conformation and is converted into a stable conformation during inhibition. In recent years the structures of numerous serpins in a variety of different conformations have been solved by X-ray crystallography (31). Although already in 1969 Sharp and co-workers had described accumulation in the liver of the Z variant of AAT, the understanding of this problem was initiated only in the 1990s when D. Lomas and his group in Cambridge, showed that Z AAT forms due to the insertion of the reactive center loop of one molecule into the opened B-sheet of the next(31). This gives the molecular mechanism that underlies AAT deficiency and showed how an understanding of this mechanism can help us to explain the deficiency of other members of the serpin superfamily. A number of mutations and post-translational modifications have been reported in serpins which result in conformational instability, molecular polymerisation and loss of serpin activity. In many cases these mutations have been associated with enhanced susceptibility to inflammatory, neurodegenerative, cardiovascular diseases and cancer. These include the deficiency of antithrombin, C1-inhibitor and alpha1-antichymotrypsin in association with , angio-oedema and emphysema, respectively. Moreover, the accumulation of mutant within neurones causes the novel , familial encephalopathy with neuroserpin . These conditions were grouped together as the serpinopathies(33- 35). To date much has been learned about AAT; the structure of the protein, the mechanism of its binding to target enzymes, the mechanism of intra-cellular accumulation and the main clinical manifestations.

© 2010 Alpha1 Awareness UK page 4 REFERENCES 1. Irving JA, Pike RN, Lesk AM, Whisstock JC. Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 2000; 10:1845-64. 2. Gettins PG. Serpin structure, mechanism, and function. Chem Rev 2002; 102: 4751-4804. 3. C. Fermi and L. Pernossi, Untersuchungen uber die enzyme, Vergleichende Studie Z. Hyg. Infektionskr 18 (1894), pp. 83–89 4. Hahn. Berl. klin. Woch. xxXIV. p. 499. 1897. 5. Pugliese and Coggi. Bollet. Scienze Med. VIII. 1897. 6. Schutze et al., Zur Kenntnis der alpha-globulin des menschlichen nirmal serums. Z Naturforsch 1955, 10:463, 7. Protein Purification : principals, high resolution methods, and applications, Edited by Jan-Christer Janson, Lars Rydén Wiley –VCH, pp 1- 695, 1998. 8. „Axplock“ från min tid som klinisk kemist Lund 1944-53 * Malmö 1954-83, pp 1-73,. C-B Laurrell; Book printed in Sjukvården Malmö, 1992 9. C-B. Laurell. Bench Side Medicine. Clinical Chemistry 47, p 613- 614, 2001 (citations received from the personal communication with C-B Laurell son, Dr Martin Laurell) 10. Laurell C-B, Eriksson S. The electrophoretic 1–globulin pattern of serum in 1–antitrypsin deficiency. Scand J clin Lab invest 1963; 15:132-140 11. Eriksson S, Laurell C-B. A new abnormal serum globulin alpha-1-antitrypsin. Acta Chem Scand 1963; 17: 150-153 12. Eriksson S. Studies in 1–antitrypsin deficiency. Acta Med Scand 1965, suppl 432:1-85. 13. Snider LJ, Kleinerman J, Thurlbeck WM, Bengali ZH. The definition of emphysema. Report of a national heart, lung and blood institute, Division of Lund diseases Workshop. Am Rev Respir Dis 1985; 132:182-185. 14. Gross PM, Bubyak E, Tolken E and Kashak. Enzymatically produced pulmonary emphysema. A preliminary report. J Occup med 1964; 6: 481-484. 15. Larsson C. Natural history and expectancy in severe alpha1-antitrypsin deficiency, PiZ. Acta Med Scand 1978; 204: 345-351. 16. Eriksson S. A 30-year perspective on alpha 1-antitrypsin deficiency. Review. Chest 1996; 110:237S-242S. 17. Sharp HL, Bridges RA, Krivit W, Freier EF. Cirrhosis associated with alpha1-antitrypsin deficiency: a previously unrecognised inherided disorger. J Lab Clin Med 1969; 73:934-939. 18. Owen MC, Carrell RW. Alpha1-antitrypsin: molecular abnormality of S variant. Br Med J 1976; 1:130-131. 19. Jeppsson J-O. Amino acid substitution Glu Lys in alpha1-antitrypsin PiZ. FEBS Lett 1976; 65:195-197. 20. Travis J. 1989. Alpha1-proteinase inhibitor deficiency. In Lund Biology in health and Disease. Donald Massaro, ed 41: 1227-1246. 21. Mega T., Lujan E, Yoshida A. Studies on the oligosaccharide chains of human alpha1-protease inhibitor. I. Isolation of . J Biol Chem 1980; 255: 4053-4056. 22. Carrell RW., Jeppsson J-O., Laurell C-B., Brennan SO., Owen Mc., Vaughan L., Boswell. Structure and variations of human alpha1-antitrypsin. Nature 1982; 298: 329-334. 23. Carrell RW. Alpha1-Antitrypsin: molecular pathology, leukocytes and tissue damage. J Clin Invest 1986; 78: 1427- 1431. 24. Hunt LT, Dayhoff MO. A surprising new containing ovalbumin, antithrombin-III, and alpha1- protease inhibitor. Biochem Biophys Res Commun 1980; 95: 864-871. 25. Carrell RW., Pemberton PA., Boswell DR. The serpins: and adaptation in a family of protease inhibitors. Cold Spring Harb Symp Quant Biol 1987; 52: 527-535. 26. Carrell R, Travis J. Alpha1-antitrypsin and the serpins: variation and countervariation. Trends Biochem Sci 1985; 10: 20-24. 27. Potempa J., KorzusE., Travis J. The serpin superfamily of proteinase inhibitors:structure, function, and regulation. J Biol Chem 1994; 269: 15957-15960. 28. Loebermann H., Tokuota R., Deisenhofer J., Huber R. Human alpha1-proteinase inhibitor. Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. J Mol Biol 1984; 177: 531-557. 29. Gettins PG. Serpin structure, mechanism, and function. Chem Rev. 2002;102:4751-804. Review. 30. Kaslik G, Kardos J, Szabó E, Szilágyi L, Závodszky P, Westler WM, Markley JL, Gráf L. Effects of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site. Biochemistry. 1997; 36:5455-5464. 31. Carrell RW, Lomas DA. Conformational disease. Lancet 1997; 350:134-38. 32. Sharp HL, Bridges RA, Krivit W, Freier EF. Cirrhosis associated with alpha-1-antitrypin deficiency: a previously unrecognized inherited disorder J Lab Clin Med 1969; 73: 934-939. 33. Janciauskiene S. Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles. Biochim Biophys Acta 2001; 1535:221-35. 34. Wright HT, Scarsdale JN. Structural Basis for serpin inhibitor activity. Proteins 1995; 22: 210-25. 35. Gooptu B, Lomas DA. Conformational Pathology of the Serpins: Themes, Variations, and Therapeutic Strategies. Annual Rev Biochem. 2009 Feb 26. [Epub ahead of print]

© 2010 Alpha1 Awareness UK page 5