Sep 29, 1988 - Crystal, R. G. (1987) Biochemistry 26:5259. 21. Brantly, M., Nukiwa, T., Ogushi, F., Fells, G. A., Stier, L. and Crystal, R. G. (1988) Am. Rev. Resp.
Bioscience Reports, Vol. 9, No. 2, 1989
REVIEW
Alpha,-Antitrypsin: Structure, Function and Molecular Biology of the Gene N. Kalsheker Received September 29, 1988
Alphal-antitrypsin (AAT) deficiencyis one of the commonest inherited disorders in white Caucasians. This association has provided major insights into the pathogenesis of chronic lung disease. The three dimensional structure of the protein and the structure of the gene have been determined. Some of the signals required for regulation of expression and tissue-specificity have been defined. Genetic manipulation of active site residues may provide a new generation of biological compounds with potential therapeutic applications. KEY WORDS: alphal-antitrypsin; protein structure; function; variants; genetically engineered mutants; gene structure; macrophages; alternative transcripts; hepatocyte cis and trans regulators; serpin gene family.
INTRODUCTION Alphal-antitrypsin ( A A T ) , a serine proteinase inhibitor, is a major contributor to protection against proteolytic digestion by human neutrophil elastase ( H N E ) in the lower respiratory tract (1). Laurell and Eriksson (2) first demonstrated the absence of the 0~l-globulin fraction in a number of patients' sera by paper electrophoresis and observed an association with pulmonary e m p h y s e m a - - a chronic disabling disorder of the lung resulting from destruction of lung connective tissue and a reduction in the available surface area for gaseous exchange. The major contributor to the serum aq-band seen on paper electrophoresis was subsequently shown to be A A T . The progress of pulmonary emphysema in individuals deficient in A A T is accelerated markedly by cigarette smoking. This can be explained in part by the recruitment of neutrophils to the lung with a consequent increase in the amount of H N E (3) in the lung and this is compounded by the potential of activated neutrophils to inactivate A A T by oxidation of an active site residue (4). Study of the structure and function of A A T has provided us with major insights into the pathophysiology of chronic respiratory disease and the characterisation of the gene has revealed a large group of genes belonging to the serpin (serine proteinase inhibitor) family that probably arose by gene duplication of an ancestral gene. STRUCTURE
AND FUNCTION
Nascent A A T consists of 418 amino acids with a 24 amino acid signal peptide (1). The mature protein is a single polypeptide chain containing 394 amino acids Department of Medical Biochemistry, University of Wales College of Medicine, Royal Infirmary, Newport Road, Cardiff CF2 1SZ. 129 0144-8463/89/1M00-0129506.00/0 (~) 1989 Plenum Publishing Corporation
130
Kalsheker
with a molecular weight of about 52 kDa of which about 15% is made up of carbohydrate (1). The carbohydrate side chains are linked to the protein by asparagine residues at positions 46, 83 and 247 and exist as bi-antennary or tri-antennary forms (5). The side chains at positions 46 and 247 are predominantly bi-antennary and at position 83 can exist as either bi-antennary or tri-antennary forms with a variable number of sialic acid residues. This heterogeneity contributes to the multiple banding patterns seen on isoelectric focusing and acid starch gel electrophoresis. The tertiary structure of cleaved A.A.T has been determined by crystallography (6). A A T is a globular, highly ordered molecule. The carbohydrate side chains occur in bends of the polypeptide chain protruding from the surface of the molecule. The amino-terminal sequence of about 20 amino acid residues is exposed and susceptible to cleavage. A pentapeptide is released and the physiological significance of this event, if any, is not known. About 30% of its structure is helical and 40% consists of fl-pleated sheets. There are nine tr-helices and 3 fl-pleated sheets. At the N terminus the first five tr-helices are buried under the first fl-pleated sheet. The reactive centre is thought to be on an exposed loop of the molecule which is under tension and methionine at position 358 is a critical residue. A mechanism for inhibition has been deduced from the structure of the cleaved molecule. Adjacent to the Meth 358 is a serine residue at position 359 and strain is relieved by cleavage of the bond between these two residues and a C-terminal peptide corresponding to residues Ser359-Lys394 is released by HNE (7). In the nicked molecule the two amino acids are 69 A apart. The separated methionine and serine residues may form a pocket which precisely fits the active site of the serine proteinases (Fig. 1). The interaction between the proteinase and anti-proteinase is noncovalent. The bound complex renders the proteinase inactive and the complex is then removed from the circulation. The susceptibility of residues in proximity to the active site to cleavage by a variety of bacterial and snake venom proteinases (8) provides good evidence in support of the concept of a strained loop at the reactive centre. Monocytes in culture secrete AA.T in 3 different forms (9), the native form (52,000mol. wt.), and in forms complexed with serine proteinase (66,000 and 75,000 tool. wt.). During complex dissociation the molecular weight of A A T is reduced to less than 50,000 due to the release of the small C-terminal peptide Ser359-Lys 394. The small peptide fragment shows strong hydrophobic adhesion to the larger A A T molecule. It has recently been demonstrated that the HNE-A.AT complex is a neutrophil chemoattractant (10) underlining the importance of the molecule in inflammation. The predominant proteinase inhibited by A A T is HNE. The association rate constant (Ka) for HNE has been estimated to be 107 M -1 S -1 (11). A comparison of the Kas for other serine proteinases demonstrates a decreasing order with HNE > chymotrypsin > cathepsin G > anionic trypsin > cationic trypsin > plasmin > thrombin (11). The methionine residue at the active site is susceptible to oxidation to methionine sulphoxide which reduces the binding of lINE to A A T and renders it a poor inhibitor of H N E (12). Oxidative inactivation may have
131
Alphal-Antitlypsinand Molecular Biology
Elastase
S
AAT-Elastase Complex Fig. 1. Schematic representation of the interaction between AAT and neutrophil elastase.
a physiological role. At the site of injury, inactivation of AAT permits localised tissue destruction by uninhibited HNE. This process may facilitate removal of damaged tissue which is subsequently replaced by new tissue. It is suggested that oxidative inactivation can only occur in the immediate vicinity of neutrophils as oxygen radicals have a short half-life and do not diffuse much beyond the neutrophils. This restricts the sphere of activity of HNE as it will.be inhibited by native AAT beyond a certain radius. A naturally occurring variant of AAT (AATpittsburgh)in which the M e t 358 is replaced by Arg (13), alters the specificity of the protein converting it from an inhibitor of HNE to an inhibitor of thrombin. The replacement of the methionine residue at the active site with arginine by site-directed mutagenesis has also resulted in a molecule with antithrombin specificity (14). Because the methionine residue is susceptible to oxidative inactivation, oxidant resistant amino acid residues have been introduced into the molecule, with a view to potential therapeutic applications. For example, the introduction of Va1358 renders the molecule functionally effective as an inhibitor of HNE and the residue is not oxidised. The introduction of leucine at this position results in a molecule that is more potent as an inhibitor of elastase than the native molecule and is also a very effective inhibitor of cathepsin G. A number of other mutations have been introduced (15) and these are summarised in Table 1.
K~sheker
132 Table 1. P5 Glu 354
P4 Ala 355
Site-directed mutagenesis of A A T around the active centre P3 Ile 356
P2 Pro 357
P1 Met 358
P'I Set 359
P'2 Ile 360
P'3 Pro 361
P'4 Pro 362
P'5 Glu 363
Met 358---~Val, Ile, Ala, Leu, Phe, Arg Ile356--* Ala mutations introduced.
SITES OF SYNTHESIS
A A T is synthesised predominantly in the liver and to a much lesser extent by monocytes and alveolar macrophages (16). Using transgenic mice it has been demonstrated that other tissues also produce AAT, most notably Paneth cells of the gastrointestinal tract, the kidneys and lung (17, 18).
GENETIC VARIANTS The protein is encoded by two independent alleles in an autosomal codominant fashion. Isoelectric focusing in polyacrylamide or agarose gels is currently the method of choice for the characterisation of protein variants. There are several bands observed by isoelectric focusing reflecting the microheterogeneity arising from the differences in carbohydrate side chains and the length of the polypeptide chain (5, 19). The A A T locus is highly polymorphic. Over 40 proteinase-inhibitor (Pi) protein variants have been detected, many presumably arising from uncharacterised amino acid substitutions. The predominant allele in the general population is referred to as M and about 6 sub-types have been defined. The M sub-types are designated with the numbers 1-6. The molecular basis for some of the charge substitutions for a number of the normal and deficient variants have been determined (Table 2). A Glu-Lys 342 substitution results in a slow moving variant designated Z, associated with deficiency of AAT. 24 The Z protein is synthesised at a normal rate but accumulates in the rough endoplasmic reticulum of hepatocytes. The protein is thought to be partially glycosylated (1), with a high mannose content and the substitution is thought to mainly affect folding of the protein, preventing its passage to the Golgi apparatus and the accumulation of protein leads to liver cell damage. It has also been proposed that the G I u 342 position forms a salt bridge with Lys~9~ However, this has been disputed (31). The Z protein isolated from hepatocytes is markedly insoluble and may be complexed with other proteins. The clinical consequence of accumulation is that approximately 10% of patients develop severe liver cirrhosis and liver failure. The frequency of the Z allele in the United Kingdom is about 3% (1). Another common cause of deficiency is a single amino acid substitution at 264 where valine replaces glutamic acid (23). This is referred to as the S allele and occurs in about 7% of the population of the United Kingdom.
Alphaa-Antitrypsin and Molecular Biology Table 2.
Mutations in normal and deficient AAT variants
Pi type
Mutation
(a) Normal alleles M1 M2 M3 M3 (b) Deficiency alleles S Z X
Mheerlen Mprocida Null forms bellingham granite falls mattawa hong-kong
cardiff
133
Ref.
Ala 213 (20%) Va1213 (80%) Arg--~ His 1~ Glu ~ Asp 376 GIn---) Asp 376
20 20 21 21 22
Glu---) Va1264 GIn ~ Lys342 Glu ~ LysTM P r o ~ Leu 369 Leu--* Pro 41
23 24 22 25 26
Lys---~stop 217 Tyr---~stop 160 insertion---~ stop 376 Leu 318---)TC dinucleotide deletion 5'--)stop codon at 334 Asp---~Val 256
27 28 29 30
*
* Unpublished observations.
Deficiency in the S form may arise because the G l u 264 residue is thought to form a salt bridge with t y s 387. Consequently, an unstable form of A A T may form intracellularly prior to glycosylation (1). An alternative explanation has been offered on the basis of DNA sequence information where it has been suggested a new splice site is generated (32). However, there is no direct evidence to support the latter hypothesis. In addition to the mutations described in Table 2 several polymorphisms have been described in the gene in non-coding sequences (33, 34) and some of these may prove to be useful in genetic studies of familial chronic obstructive airways disease (35, 36).
POPULATION GENETICS
Deficiency alleles occur at high frequency in Northern Europeans and white Caucasians. PiS is found in about 11-20% of the population in the Iberian peninsula (37). The most common phenotypes in the population are MM, MZ and MS. The presence of deficiency alleles at high frequency in a population suggests some selective advantage that allows them to remain as a balanced polymorphism. It has been proposed (38) that deficiency states may promote increased sperm migration because of the effects of proteinase activity in reducing cervical mucus viscosity and enhance fertility (39). Despite these reports the selective forces responsible for maintaining the Z allele have not yet been adequately explained (40).
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Kalsheker
C H R O M O S O M A L LOCALISATION AND GENE S T R U C T U R E OF A A T The A A T gene has been localised to the long arm of chromosome 14 at position 14q31-32 (41). The DNA sequence of a full length A A T cDNA clone prepared from liver messenger R N A has been determined as well as the sequence of a genomic clone containing 10266 bp (42). The full length cDNA insert was 1434 bp comprising a 5' non-coding region of 49 bp, a coding region of 1254 bp, a stop codon, a 3' non-coding region of 79 bp and a poly A tail. Initially the gene was thought to consist of five exons and four introns (43). The first coding sequence for methionine was in the second exon and the transcription start site was shown to be at the beginning of the first exon in liver messenger R N A with a canonical T A T A box at position - 2 5 to - 2 0 , whereas no homology for the upstream CAAT consensus sequence was found (44). It was subsequently demonstrated that there are in fact two promoters, one in macrophages and one in hepatocytes (45). The macrophage-specific promoter is located approximately 2000 bp upstream to the hepatocyte-specific promoter. Transcription from the two A A T promoters is specific; the macrophage promoter is inactive in hepatocytes and the hepatocyte promoter is inactive in macrophages. There are two distinct macrophage-specific transcription initiation points 37 bases apart. Furthermore, there are two distinct mRNAs in macrophages generated by alternative splicing arising from two additional exons designated A and B. Exon A contains 207 bases and exon B contains 209 bases which may not be present in all macrophage transcripts (Fig. 2). In macrophages the third exon potentially contains an additional 56 bases and a 44 base sequence common to both liver and macrophages (45). The reason for two transcripts in macrophages is not known. There are two short open reading frames, one within exon B and the other is present in the 56 additional bases of the third exon before it joins the first liver specific exon. An analogous situation occurs in other genes including the human and chicken oestrogen receptors (46, 47), the human transferrin receptor (48) and the yeast regulatory protein GcN4 (49). In the case of the latter there is convincing evidence suggesting that the short upstream open reading frames are essential for translational repression (49). Although AAT is produced predominantly in the liver, in certain situations localised production of A A T by macrophages may be important and consequently the regulation of this expression may turn out to have physiological and pathological effects. It has been demonstrated that the 5' flanking region of the human A A T gene contains cis-acting signals for liver-specific expression. In the liver a tissue-specific element has been identified between the nucleotides -137 to - 3 7 from the transcriptional start site (50). Two domains - 1 2 5 / - 1 0 0 and (-84/70) are essential for transcription. There are at least two further domains located between -261 to -210 from the 5' cap site which are capable of activating heterologous promoters (50). The fragment - 1 3 7 / + 4 4 is an autonomous regulatory element able to act as a cell-specific activator (50). Furthermore it has been demonstrated that proteins from rat liver nuclear extracts specifically bind to the domains essential for transcription (51, 52). To
Alphal-Antitrypsin and Molecular Biology
135
b/
I o IEI
Fpo,y A Macrophage transcripts
ol D IEI FIGI "P~ A
Icl
D
IEI
FIGI-Poly A
Liver
transcript
Fig. 2. Processing of A A T transcripts in the macrophage and the hepatocyte. PM corresponds to the macrophage-specific promoter and PH is the hepatocyte-specific promoter. A to G represent the exons and the lines represent introns. Exon C is common to macrophages and hepatocytes but an additional 56 bp sequence is present in macrophages. Macrophage transcripts may include exon B.
date, two proteins which bind to the promoter region have been identified and shown to be common positive trans-acting factors required for the expression of several other genes in hepatocytes apart from A A T (50). The tissue-specific elements in macrophages and the precise sequences important in regulation have not been mapped to date.
R E G U L A T I O N OF A A T EXPRESSION
A A T is an acute-phase reactant and its serum concentration can increase three to fourfold during inflammation (53). The mechanisms for the increase are poorly understood and it seems likely that cytokines may regulate its production. It has been demonstrated that interleukin 6 probably regulates A A T production in hepatocytes (54) and tumour necrosis factor has been shown to stimulate mRNA production in monocytes (55, 56). However the presence of alternative transcripts may be important factors in determining R N A stability and consequently the amount of AAT produced. Although many cytokines can stimulate A A T production these increases may occur by post-translational mechanisms. The ability of H N E to stimulate A A T expression (57) may provide an important clue as to how monocyte A A T may be regulated by a substrate dependent feedback mechanism.
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Kalsheker
SERPIN GENE FAMILY
AAT belongs to the serpin gene family. Serine proteinases are important in several inflammatory processes including blood coagulation, fibrinolysis and complement activation. Within each group there are several key serine proteinase inhibitors including antithrombin, antiplasmin and C1 esterase inhibitor which belong to the same gene family. From amino acid and nucleotide sequence comparisons it has become apparent that the serpin family also include genes coding for proteins that do not have antiproteinase activity, e.g. chicken ovalbumin and angiotensinogen (58). New members of the family continue to be discovered and it is probable that the family will turn out to be large. Of potential importance is a highly homologous sequence that occurs approximately 10 kb downstream to AAT and preliminary data suggest that this sequence codes for a new member of the serpin f~imily (59). The cloning of the gene for AAT has provided us with important information into the biology of A A T both in terms of structure and function. The sequences required for tissue specific expression and the precise protein DNA interactions that regulate expression are being unravelled. It is likely that there are as yet uncharacterised members of the serpin gene family and much may be learnt about other members of the family, in terms of specificity and structure, based on our knowledge of AAT. From a practical point of view, our understanding of AAT deficiency and disease has made it possible to contemplate therapy for AAT deficiency. Currently trials of replacement therapy for AAT deficiency are being undertaken and there may also be potential applications for acute shock syndromes where the inappropriate activation of inflammatory cascades can be life threatening. REFERENCES 1. Carrell, R. W., Jeppsson, J.-O., Laurell, C.-B., Brennan, S. O., Owen, M. C., Vaughan, L. and Boswell, D. R. (1983) Nature (Lond.) 298:329. 2. Laurell, C.-B. and Eriksson, S. (1963) Scand. J. Lab. Invest. 15:132. 3. Kilburn, K. H. and McKenzie, W. (1975) Science 189:634. 4. Carp, H., Miller, F. and Hoidal, J. R. (1982) Proc. Natl. Acad. Sci. USA 79:2041. 5. Carrell, R. W., Jeppsson, J.-O., Vaughan, L., Brennan, S., Owen, M. and Boswell, D. R. (1981) FEBS Lett. 135: 301. 6. Loebermann, H., Tokuoka, R., Deisenhofer, J. and Huber, R. (1987) J. Mol. Biol. 177:531. 7. Johnson, D. A. and Travis, J. (1976) Biochem. Biophys. Res. Commun. 72:33. 8. Carrell, R. W. and Owen, M. C. (1985) Nature (Lond.) 317:730. 9. Takemura, S., Rossing, T. H. and Perlmutter, D. H. (1986) J. Clin. Invest. 77:1207. 10. Banda, M. J., Rice, A. G., Griffin, G. L. and Senior R. M. (1988) J. Exp. Med. 167:1608. 11. Beatty, K., Bieth, J. and Travis, J. (1980) J. Biol. Chem. 2,55:3931. 12. Carrell, R. W. (1986)J. Clin. Invest. 78"-1427. 13. Owen, M. C., Brennan, S. O., Lewis, J. H. and Carrell, R. W. (1983) N. Engl. J. Med. 3119:694. 14. Courtney, M., Jallat, S., Tessier, L. H., Benavente, A., Crystal, R. G. and Le Cocq, J. P. (1985) Nature (Lond.) 313:149. 15. Jallat, S., Carvallo, D., Tessier, L. H., Roecklin, D., Roitsch, C., Ogushi, F., Crystal, R. G. and Courtney M. (1986)Prot. Engin. 1-29. 16. Mornex, J.-F., Chytil-Weir, A., Martinet, Y., Courteney, M., Le Cocq, J.-P. and Crystal, R. G. (1986) J. Clin. Invest. 77:1952.
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