Antitrypsin Deficiency, the Serpinopathies, and Chronic Obstructive Pulmonary Disease

Abstract

α₁-Antitrypsin deficiency is the only genetic factor that is widely recognized to predispose smokers to chronic obstructive pulmonary disease. We have shown that the plasma deficiency results from point mutations perturbing the structure of the protein to favor sequential linkage between the reactive center loop of one molecule and β-sheet A of another. These polymers are retained within the liver to form the periodic acid-Schiff–positive inclusions that are characteristic of the disease. Intracellular polymerization also explains the retention of mutants of other members of the serine proteinase inhibitor (or serpin) superfamily to cause diseases as diverse as thrombosis, angio-edema, and dementia. In view of the common mechanism, we have grouped these conditions together as the serpinopathies. Intrapulmonary Z α₁-antitrypsin similarly forms polymers within the alveolar space. These polymers are inactive as a proteinase inhibitor and act as a chemoattractant for neutrophils. This conformational transition may explain the excessive inflammation that underlies the progressive emphysema associated with Z α₁-antitrypsin deficiency.

α₁-ANTITRYPSIN DEFICIENCY RESULTS FROM THE POLYMERIZATION OF MUTANT α₁-ANTITRYPSIN WITHIN HEPATOCYTES

α₁-Antitrypsin is an acute phase glycoprotein that is synthesized and secreted by the liver. It bathes all the tissues of the body, with its primary role being to inhibit the enzyme neutrophil elastase. The most important deficiency mutation of α₁-antitrypsin is the Z allele (Glu342Lys). Approximately 4% of northern Europeans are heterozygous for the Z allele (PI*MZ), with approximately 1 in 2,000 being homozygotes (PI*Z). The Z allele results in the retention of synthesized α₁-antitrypsin within the endoplasmic reticulum of hepatocytes. The accumulation of abnormal protein starts in utero and is characterized by diastase-resistant, periodic acid-Schiff (PAS)–positive inclusions of α₁-antitrypsin within the periportal cells. This intrahepatic accumulation of Z α₁-antitrypsin predisposes the homozygote to neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The lack of circulating plasma α₁-antitrypsin leaves the lungs exposed to enzymatic damage that is believed to underlie the adult-onset emphysema.

We have shown that the Z variant of α₁-antitrypsin is retained within hepatocytes as it causes a unique conformational transition and protein–protein interaction (1). The mutation distorts the relationship between the reactive center loop and β-sheet A (Figure 1A). The consequent perturbation in structure allows the formation an unstable intermediate (M*) and the formation of polymers in which the reactive center loop of one α₁-antitrypsin molecule sequentially inserts into β-sheet A of another (1–6). Spectroscopic analysis has demonstrated that oligomers of α₁-antitrypsin form during an initial lag phase and that these then condense to form a heterogenous mixture of longer polymers (2, 7). It is these polymers (Figure 1B) that accumulate within the endoplasmic reticulum of hepatocytes to form the PAS-positive inclusions that are the hallmark of Z α₁-antitrypsin liver disease (1, 6, 8). The process of intrahepatic polymerization also underlies the severe plasma deficiency of the Siiyama (Ser53Phe) and Mmalton (deletion of phenylalanine at position 52) alleles that are the commonest cause of α₁-antitrypsin deficiency in Japan and Sardinia, respectively. Moreover, this process explains the mild plasma deficiency of the common S (Glu264Val) and rare I (Arg39Cys) alleles of α₁-antitrypsin (see References 9 and 10 for reviews). In each case, there is strong genotype–phenotype correlation that can be explained by the molecular instability caused by the mutation and, in particular, the rate at which the mutant forms polymers. Those mutants that cause the most rapid polymerization cause the most retention of α₁-antitrypsin within the liver. This, in turn, correlates with the greatest risk of liver damage and cirrhosis, and the most severe plasma deficiency.

Figure 1.

Mutant Z α₁-antitrypsin is retained within hepatocytes as polymers. (A) The structure of α₁-antitrypsin is centered on β-sheet A (green) and the mobile reactive center loop (red). Polymer formation results from the Z variant of α₁-antitrypsin (Glu342Lys at P₁₇; arrowed) or mutations in the shutter domain (blue circle) that open β-sheet A to favor partial loop insertion and the formation of an unstable intermediate (M*). The patent β-sheet A can then accept the loop of another molecule to form a dimer (D), which then extends into polymers (P). The individual molecules of α₁-antitrypsin within the polymer are colored red, yellow, and blue. Support for this came from the finding of polymers within hepatocytes and in plasma of individuals with α₁-antitrypsin deficiency (1, 6, 14). (B) The polymers have a “beads on a string appearance” on electron microscopy (Figure 1A modified by permission from Reference 5 and Figure 1B reproduced by permission from Reference 14).

THE SERPINOPATHIES

The phenomenon of loop-sheet polymerization is not restricted to α₁-antitrypsin and has now been reported in mutants of other members of the serpin superfamily to cause disease. Naturally occurring mutations have been described in the shutter (Figure 1A) and other domains of the plasma proteins C1-inhibitor, antithrombin, and α₁-antichymotrypsin. These mutations destabilize the serpin architecture to allow the formation of inactive reactive loop–β-sheet polymers that are also retained within hepatocytes. The associated plasma deficiency results in uncontrolled activation of proteolytic cascades and angio-edema, thrombosis, and chronic obstructive pulmonary disease, respectively (see References 9 and 10 for reviews). More recently, a mutation in heparin cofactor II (Glu428Lys) has been associated with plasma deficiency, but as yet this has not been shown to cause disease (11). The mutation is of particular interest as it is the same as the Z allele that causes polymerization and deficiency of α₁-antitrypsin. We have shown that this same mutation also causes temperature-dependent polymerization and inactivation of the Drosophila serpin necrotic (12).

Perhaps the most striking finding of polymer-associated disease is the inclusion body dementia of familial encephalopathy with neuroserpin inclusion bodies (FENIB) (13). This is an autosomal dominant dementia characterized by eosinophilic neuronal inclusions of neuroserpin (Collins' bodies) in the deeper layers of the cerebral cortex and the substantia nigra. The inclusions are PAS positive and diastase resistant and bear a striking resemblance to those of Z α₁-antitrypsin that form within the liver. The observation that FENIB was associated with a mutation (Ser49Pro) in the neuroserpin gene that was homologous to one in α₁-antitrypsin that causes cirrhosis (Ser53Phe) (14) strongly indicated a common molecular mechanism. This was confirmed by the finding that the neuronal inclusion bodies of FENIB were formed by entangled polymers of neuroserpin with identical morphology to those isolated from hepatocytes from an individual with Z α₁-antitrypsin–related cirrhosis (13). Five families with four different point mutations have now been identified with FENIB (15). These have allowed comparison of the severity of the mutation (as predicted by molecular modeling), the number of inclusions, and the age of onset of dementia. Once again, there is a striking genotype–phenotype correlation that can be explained by the rate at which the mutants form intracellular polymers (Table 1). The more rapid the rate of polymerization, the more the protein is retained as intraneuronal inclusions and the earlier the onset of the clinical phenotype (see Reference 16 for review).

TABLE 1.

CORRELATION BETWEEN THE RATE OF POLYMERIZATION OF MUTANTS OF NEUROSERPIN, THE NUMBER OF INCLUSIONS, AND THE SEVERITY OF THE ASSOCIATED DEMENTIA

Mutation	Rate of Polymerization	Inclusions	Age of Onset (yr)	Clinical Features
Ser49Pro	+	+	45–63	Dementia, seizures
Ser52Arg	++	++	20–40	Dementia, myoclonus
His338Arg	+++	+++	15	Progressive myoclonus epilepsy
Gly392Glu	++++	++++	13	Progressive myoclonus epilepsy, chorea

Reproduced by permission from Reference 16.

In view of the common mechanism (serpin polymerization) that underlies all these conditions, we have grouped them together as the serpinopathies (9, 10). We and others have shown that, at least in vitro, serpin polymerization can be attenuated by competing reactive loop peptides, chemical chaperones, and manipulating an exposed surface cavity (see Reference 17 for review). These approaches now need to be converted into effective drugs if we are to develop novel therapies for the serpinopathies.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Emphysema associated with plasma deficiency of α₁-antitrypsin is widely believed to be due to the reduction in plasma levels of α₁-antitrypsin to 10 to 15% of normal. This makes the lungs vulnerable to uncontrolled digestion by neutrophil elastase. The situation is exacerbated because the Z mutation reduces the association rate between α₁-antitrypsin and neutrophil elastase by approximately fivefold, so the α₁-antitrypsin available within the lung is not as effective as the normal M protein (18, 19). α₁-Antitrypsin is present within the lung by passive diffusion or local secretion by bronchial epithelial cells and macrophages. In each case, the secreted protein contains the Z (or other) mutation and hence the propensity to spontaneously form polymers. We have detected polymers within lung lavage (20) and explanted tissue (21) from patients with emphysema associated with Z α₁-antitrypsin deficiency but not in samples from individuals with emphysema and normal α₁-antitrypsin phenotypes. This conformational transition inactivates α₁-antitrypsin as a proteinase inhibitor, thereby further reducing the already depleted levels of α₁-antitrypsin that are available to protect the alveoli (see Reference 9 for review). Moreover, the conversion of α₁-antitrypsin from a monomer to a polymer changes it to a chemoattractant for human neutrophils (22, 23). The magnitude of the effect is similar to that of the chemoattractant C5a and is present over a range of physiologic concentrations (EC₅₀, 4.5 ± 2 μg/ml). These chemoattractant properties of polymers may explain the excess number of neutrophils in bronchoalveolar lavage (24) and in tissue sections of lung parenchyma (21) from individuals with Z α₁-antitrypsin deficiency. Moreover, polymers may contribute to the excess inflammation that is apparent even in individuals with Z α₁-antitrypsin deficiency with very early lung disease (25) and may drive the progressive inflammation that continues even after cessation of smoking. The inflammatory properties of polymers may also explain other inflammatory conditions that have been associated with Z α₁-antitrypsin deficiency: panniculitis (26), pancreatitis (27), Wegener's granulomatosis (28), glomerulonephritis, and asthma (29).