48
α 1-ANTITRYPSIN DEFICIENCY
DAVID H. PERLMUTTER
CLINICAL MANIFESTATIONS OF LIVER DISEASE 700
PATHOGENESIS OF LIVER DISEASE 705
STRUCTURE, FUNCTION, AND PHYSIOLOGY 701
FATE OF THE MUTANT α1-ATZ IN THE
ENDOPLASMIC RETICULUM 707
VARIANTS OF α1-ANTITRYPSIN 703
Normal Allelic Variants 704
Null Allelic Variants 704
Dysfunctional Variants 704
Deficiency Variants 704
MECHANISM OF LIVER CELL INJURY 711
OTHER DISORDERS WITH ENDOPLASMIC
RETICULUM RETENTION OF MUTANT OR
UNASSEMBLED PROTEINS 712
MECHANISM OF DEFICIENCY 704
TREATMENT 713
The classical form of α1-antitrypsin (α1-AT) deficiency,
homozygous for the mutant α1-ATZ allele, is a relatively
common disease. It affects approximately 1 in 1,600 to 1 in
2,000 live births in most populations of Northern European ancestry (1,2). Although only a subgroup of deficient
individuals develop liver disease, it represents the most
common metabolic cause of liver disease in children (3) and
can be associated with chronic liver disease and hepatocellular carcinoma in adults (4). This deficiency also causes
premature development of pulmonary emphysema in
adults.
The α1-AT molecule is a single-chain secretory glycoprotein that inhibits destructive neutrophil proteases
including elastase, cathepsin G and proteinase 3. It is
often referred to as a hepatic acute phase reactant in that
plasma α1-AT is predominantly derived from the liver
and plasma levels increase 3- to 5-fold during the host
response to tissue injury/inflammation. It is the archetype
of a family of structurally related circulating serine protease inhibitors termed SERPINS. In the deficient state,
there is an approximately 85% to 90% reduction in serum
concentrations of α1-AT. A single amino acid substitution
results in a mutant protein that is unable to traverse the
secretory pathway. This α1-ATZ protein is retained in the
D. H. Perlmutter: Departments of Pediatrics, Cell Biology, and Physiology, Washington University School of Medicine; Division of Gastroenterology
and Nutrition, St. Louis Children’s Hospital, St. Louis, Missouri 63110.
endoplasmic reticulum (ER) rather than secreted into the
blood and body fluids.
The classical deficient state is also unique as a genetic
disease in that it causes injury to one target organ, lung, by
a loss-of-function mechanism and injury to another target
organ, liver, by what appears to be a gain-of-function mechanism. Most of the data in the literature indicate that
emphysema results from a decreased number of α1-AT
molecules within the lower respiratory tract, allowing
unregulated elastolytic attack on the connective tissue
matrix of the lung (5,6). Oxidative inactivation of residual
α1-AT as a result of cigarette smoking accelerates lung
injury (7). Moreover, the elastase–antielastase theory for the
pathogenesis of emphysema is based on the concept that
oxidative inactivation of α1-AT as a result of cigarette
smoking plays a key role in the emphysema of α1-AT-sufficient individuals, the vast majority of cases of emphysema
(5,8). It has been more difficult to explain the pathogenesis
of liver injury in this deficiency. Results of transgenic animal experiments have provided further evidence that the
liver disease does not result from a deficiency in antielastase
activity (9,10). Most of the data in the literature corroborate the concept that liver injury in α1-AT deficiency results
from the hepatotoxic effects of retention of the mutant α1ATZ molecule in the ER of liver cells.
Although it is a single gene defect, there is extraordinary
variation in the phenotypic expression of disease in the classical form of α1-AT deficiency. For instance, nationwide
prospective screening studies done by Sveger in Sweden
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Chapter 48
have shown that only 10% to 15% of the PIZZ1 population
develop clinically significant liver disease over the first 20
years of life (1,11). These data indicate that other genetic
traits and/or environmental factors predispose a subgroup
of PIZZ individuals to liver injury. There is also variation in
incidence and severity of lung injury among α1-AT-deficient individuals. Environmental factors, such as cigarette
smoking, obviously play an important role in the phenotypic expression of lung disease (12,13). However, there are
well documented examples of siblings and other relatives of
deficient individuals with severe emphysema who have the
same genotype, a history of heavy cigarette smoking and
only mild, subclinical pulmonary function abnormalities
even at advanced ages (14). This suggests that there are
other genetic traits that play a role in determining the phenotypic expression of lung disease as well as liver disease in
this genetic disorder.
The diagnosis of α1-AT deficiency is based on the altered
migration of the abnormal α1-ATZ molecule in serum specimens subjected to isoelectric focussing gel analysis. Treatment of α1-AT deficiency-associated liver disease is mostly
supportive. Liver replacement therapy has been used successfully for severe liver injury. Although the clinical efficacy has
not been demonstrated, many patients with emphysema due
to α1-AT deficiency are currently being treated by intravenous and intratracheal aerosol administration of purified
plasma α1-AT. An increasing number of patients with severe
emphysema have undergone lung transplantation.
Recent studies of other genetic diseases have shown that
α1-AT deficiency is a prototype of diseases in which mutant
proteins accumulate in an inappropriate subcellular compartment. Studies of the basic cell biology of protein folding and
trafficking have also shown ancient pathways within cells that
constitute protective mechanisms for responding to the presence of mutant proteins in specific organelles. These “quality
control” pathways are mediated by molecular chaperones,
folding catalysts and translocation processes by which mutant
proteins may be delivered to degradative systems within the
cell. In some cases, it appears that cells attempt to protect
themselves from mutant proteins by facilitating aggregation
and then sequestering protein aggregates within specific locations to limit potential damage. There are also now well
described protective signal transduction pathways such as the
unfolded protein response in which the cell can produce
more chaperones, folding catalysts and components of membrane structure to prevent damage from mutant or unassembled proteins. An understanding of these “quality control”
mechanisms and cellular response pathways has already
resulted in several new genetic and pharmacologic strategies
for the prophylaxis of both liver and lung disease in α1-AT
deficiency, currently under development for clinical application. However, further studies of these pathways will be
1
Nomenclature for the Protease Inhibitor genotype, homozygous for the
Z allele.
needed to more clearly understand the variation in disease
among affected patients and to develop the most effective
strategies for prophylaxis and treatment.
CLINICAL MANIFESTATIONS OF LIVER
DISEASE
Liver involvement is often first noticed at 1 to 2 months of
age, because of persistent jaundice. Conjugated bilirubin
levels in the blood and serum transaminase levels are mildly
to moderately elevated (15,16). A few infants are recognized
initially because of a cholestatic clinical syndrome characterized by pruritus and hypercholesterolemia. The clinical
picture in these infants resembles extrahepatic biliary atresia, but histologic examination shows paucity of intrahepatic bile ducts (17).
Liver disease may be first discovered in late childhood or
early adolescence when the affected individual is seen with
abdominal distention from hepatosplenomegaly and/or
ascites or has upper intestinal bleeding caused by esophageal
variceal hemorrhage. In some of these cases, there is a history of unexplained prolonged obstructive jaundice during
the neonatal period. In others, there is no evidence of any
previous liver injury, even when the neonatal history is carefully reviewed.
The incidence and natural history of liver disease in α1AT deficiency has been determined by Sveger, who carried
out a nationwide screening study of newborn infants in Sweden (1). From 200,000 infants, 127 PIZZ newborns were
identified and have been followed prospectively until 18 years
of age at the time of the last report (11). The results show that
more than 85% of these children have persistently normal
serum transaminases with no evidence of liver dysfunction.
One issue not addressed by the Sveger study is whether 18year-olds with α1-AT deficiency have persistent subclinical
histologic abnormalities, despite a lack of clinical or biochemical evidence of liver injury, and whether liver disease
will eventually become clinically evident during adulthood.
α1-AT deficiency should be considered in the differential diagnosis of any adult who presents with chronic
hepatitis, cirrhosis, portal hypertension, or hepatocellular
carcinoma of unknown origin. An autopsy study in Sweden
shows a higher risk of cirrhosis and primary liver cancer in
adults with α1-AT deficiency than was previously suspected
(4). In many of these cases, cirrhosis or hepatocellular carcinoma was found incidentally without any evidence of
clinical liver disease during an entire lifetime. In many
cases, however, there was no evidence of significant liver
injury in α1-AT-deficient individuals at autopsy. Cases in
which cirrhosis and/or hepatocellular carcinoma are found
incidentally at autopsy also represent examples of the
hepatic effects of α1-AT deficiency that are in striking contrast to other cases in which liver disease is severe enough to
require liver transplantation by 6 to 24 months of age for
α1-Antitrypsin Deficiency
survival. Thus, the overwhelming clinical experience with
this disease indicates that there is wide variation in liver disease phenotype among PIZZ α1-AT-deficient individuals,
with many ”protected” from liver disease, or having very
slowly progressing liver disease.
It is still not clear whether liver injury results from the
heterozygous α1-AT MZ state by itself. Studies of liver
biopsy collections (18) and liver transplant databases (19)
have identified heterozygous patients with severe liver disease and no other explanation. However, these studies are
biased in ascertainment and one is never assured about the
exclusion of environmental causes of liver disease. A crosssectional study of patients with α1-AT deficiency in a referral-based Austrian university hospital who were reexamined
with the most sophisticated and sensitive assays available
suggests that liver disease in heterozygotes can be accounted
for, to a great extent, by infections with hepatitis B or C
virus or autoimmune disease (20).
Liver disease has been described for several other allelic
variants of α1-AT. Children with compound heterozygosity
type PISZ are affected by liver injury in a manner similar to
PIZZ children (1). There are several reports of liver disease in
α1-AT deficiency type PIMmalton(21,22). These are particularly interesting associations because the abnormal PIMmalton
α1-AT molecule has been shown to undergo polymerization
and retention within the ER (23), and the α1-ATS molecule
has been shown to form heteropolymers with the α1-ATZ
molecule (24). Liver disease has been detected in single
patients with several other α1-AT allelic variants (3), but it is
not clear whether other causes of liver injury for which we
now have more sophisticated diagnostic assays, such as infection with hepatitis C and autoimmune hepatitis, have been
completely excluded in these cases.
Diagnosis is established by a serum α1-AT phenotype
determination in isoelectric focusing or by agarose electrophoresis at acid pH. Liver histology is characterized by
periodic acid–Schiff-positive, diastase-resistant globules in
the ER of hepatocytes. These globules are most prominent
in periportal hepatocytes, but may also be seen in Kupffer
cells and cells of biliary ductular lineage (25). There may be
evidence of variable degrees of hepatocellular necrosis,
inflammatory cell infiltration, periportal fibrosis, and/or
cirrhosis. There is often evidence of bile duct epithelial cell
destruction, and occasionally there is a paucity of intrahepatic bile ducts. Our recent study has shown that there may
also be an intense autophagic reaction detected by electron
microscopic examination of liver biopsies with a full array
of nascent and degradative-type autophagic vacuoles (26).
STRUCTURE, FUNCTION, AND PHYSIOLOGY
α1-AT is encoded by a single approximately 12.2-kb gene
on human chromosome 14q31-32.3 (27,28). The α1-AT
gene is organized in 7 exons and 6 introns. The first three
701
exons and a short 5⬘ segment of the fourth exon code for 5⬘
untranslated regions of the α1-AT mRNA. The first two
exons and a short 5⬘ segment of the third exon are included
in the primary transcript in macrophages but not in hepatocytes, accounting for a slightly longer mRNA. There are,
in fact, two mRNA species in macrophages, depending on
alternative posttranscriptional splicing pathways involving
one of the two most 5⬘ exons (29,30). Most of the fourth
exon and the remaining three exons encode the protein
sequence of α1-AT.
The α1-AT protein is a single-chain, approximately 55kd polypeptide with 394 amino acids and 3 asparaginelinked complex carbohydrate side chains. There are two
major isoforms in serum, depending on the presence of a
biantennary or triantennary configuration for the carbohydrate side chains. X-ray crystallography studies have shown
that α1-AT has a globular shape and a highly ordered internal domain composed of two central β sheets surrounded
by a small β sheet and nine α helices (31,32). The dominant structure is the 5-stranded β-pleated sheet termed the
A sheet (Fig. 48.1).
α1-AT is the archetype of a family of structurally related
proteins called SERPINS, including antithrombin III, αantichymotrypsin, C1 inhibitor, α2-antiplasmin, protein C
inhibitor, heparin cofactor II, plasminogen activator
inhibitors, protease nexin I, ovalbumin, angiotensinogen,
corticosteroid-binding globulin, and thyroid-binding globulin (3,32). These proteins share about 25% overall homology, with higher degrees of regional homology in functional
domains. Most SERPINS function as suicide inhibitors by
forming complexes with a specific target protease. Other
SERPINS are not inhibitory. For instance, corticosteroid
and thyroid hormone-binding globulins, which are thought
to represent carriers for corticosteroid and thyroid hormones, respectively, form complexes but do not inactivate
their hormone ligands.
A comparison of α1-AT with other members of the
SERPIN supergene family has generated several important
concepts about the structure and function of α1-AT. For
instance, the reactive site, P1 residue, of α1-AT is localized
to a mobile loop which rises above the gap in the center of
the A sheet (33,34). The P1 residue itself is the most important determinant of functional specificity for each SERPIN
molecule. This concept was dramatically confirmed by the
discovery of α1-AT Pittsburgh, a variant in which the P1
residue of α1-AT, Met 358, is replaced by Arg 358. In this
variant, α1-AT functions as a thrombin inhibitor, and
severe bleeding diathesis results (35).
α1-AT is an inhibitor of serine proteases in general, but
under physiologic conditions its targets are probably only
neutrophil elastase, cathepsin G and proteinase 3, proteases
released by activated neutrophils. The kinetics of association of α1-AT and these enzymes are more favorable, by
several orders of magnitude, than those for α1-AT and any
other serine protease (36).
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Chapter 48
A–D
FIGURE 48.1. Ribbon diagrams of the A-sheet and reactive-site loop of α1-AT in several different
states. The positions of the residue P1 in the reactive site loop and of glutamate 342 are indicated.
A: Presumed native α1-AT. This state is presumed because it has not been crystallized. However, it
is generated by computer models based on the crystal structures of cleaved α1-AT and native ovalbumin. The reactive-site loop is shown in dark gray with residues P10, P14 numbered from the reactive-site methionine P1. The carboxyl-terminal fragment is shown as an open ribbon. α-helices of
the A-sheet are shown in light gray and referred to as S1, S2, S3, S5 and S6. The position of glutamate
342 is indicated. There is a gap in the A-sheet between S3 and S5. B: Cleaved α1-AT. The reactivesite loop in dark gray is cleaved and inserts into the A-sheet. It is referred to as S4 in between α
helices S1–S3 and S5–S6 of the A sheet shown in light gray. The positions of the P3 residue of the reactive site loop and of glutamate 342 are indicated. C: Presumed native α1-AT (Z). The reactive-site
loop simultaneously collapses into the gap in the A-sheet but because of the substitution of lysine
at residue 342, it cannot fully insert. D: Presumed native α1-AT (Z) with peptide. A synthetic peptide that mimics the insertion of a reactive site loop from an adjacent α1-AT is shown in black. The
insertion of peptide would prevent insertion from an adjacent α1-AT molecule and therein prevent
polymerization. (From Carrell RW, Evans DL, Stein DE. Mobile reactive centre of serpins and the control of thrombosis. Nature 1991;353:576–578.)
α1-AT acts competitively by allowing target enzymes to
bind directly to its reactive loop, a substrate-like region
within the carboxyl-terminal region of the inhibitor molecule. This reaction between enzyme and inhibitor is essentially second-order, and the resulting complex contains one
molecule of each of the reactants. A peptide bond in the
inhibitor is hydrolyzed during formation of the
enzyme–inhibitor complex. However, hydrolysis of this reactive-site peptide bond does not proceed to completion. An
equilibrium, near unity, is established between complexes in
which the reactive-site peptide bond of α1-AT is intact
(native inhibitor) and those in which this peptide bond is
cleaved (modified inhibitor). The complex of α1-AT and ser-
ine protease is a covalently stabilized structure, resistant to
dissociation by denaturing compounds including sodium
dodecyl sulfate and urea. The interaction between α1-AT
and serine protease is suicidal in that the modified inhibitor
is no longer able to bind and/or inactivate enzyme. Studies
have now shown that the irreversible trapping of target
enzyme is mediated by a profound conformational change in
α1-AT, such that the cleaved reactive loop, binding enzyme,
inserts into the gap in A sheet (37). Carrell and Lomas have
likened the inhibitory mechanism to a “mousetrap, with the
active inhibitor circulating in the metastable stressed-form
and then springing into the stable, relaxed form to lock the
complex with its target protease” (37).
α1-Antitrypsin Deficiency
The functional activity of α1-AT in vivo may be regulated by several factors. For one, it may be rendered inactive
as an elastase inhibitor by active oxygen products, intermediates of activated neutrophils and macrophages that can
oxidize the reactive-site methionine of α1-AT (7). This
effect is thought to constitute the basis for increased susceptibility to emphysema in smokers, whether deficient in
α1-AT or not. The α1-AT molecule may also be inactivated
in vivo by the proteolytic action of thiol proteases as well as
metalloproteases, such as collagenase and pseudomonas
elastase (38).
Several studies have indicated that α1-AT protects
experimental animals from the lethal effects of tumor
necrosis factor (TNF) (39,40). Most of the evidence from
these studies indicates that this protective effect is due to
inhibition of the synthesis and release of platelet-activating
factor from neutrophils (40,41), presumably through the
inhibition of neutrophil-derived proteases.
α1-AT also appears to have functional activities that do
not involve the inhibition of neutrophil proteases. The carboxyl-terminal fragment of α1-AT, which can be generated
during the formation of a complex with serine protease or
during proteolytic inactivation by thiol- or metalloproteases, is a potent neutrophil chemoattractant (42,43).
The predominant site of synthesis of plasma α1-AT is
the liver (44).Tissue-specific expression of α1-AT in human
hepatoma cells is directed by structural elements within a
750-nucleotide region upstream of the hepatocyte transcriptional start site in exon Ic. Within this region, there are
structural elements that are recognized by nuclear transcription factors including HNF-1α and HNF-1β, C-EBP,
HNF-4, and HNF-3 (45). HNF-1α and HNF-4 appear to
be particularly important for expression of the human α1AT gene (46,47).
Plasma concentrations of α1-AT increase 3- to 5-fold
during the host response to inflammation and/or tissue
injury (3). The source of this additional α1-AT has always
been considered the liver; thus, α1-AT is known as a positive hepatic acute phase reactant. Synthesis of α1-AT in
human hepatoma cells (HepG2, Hep3B) is upregulated by
interleukin 6 (IL-6) but not by interleukin 1 (IL-1) or TNF
(48). Plasma concentrations also increase during oral contraceptive therapy and pregnancy (49).
α1-AT is also synthesized and secreted in primary cultures of human blood monocytes as well as bronchoalveolar
and breast milk macrophages (50). Expression of α1-AT in
monocytes and macrophages is profoundly influenced by
products generated during inflammation (48,51). Synthesis
of α1-AT in liver cells and mononuclear phagocytes is also
regulated by a feed-forward mechanism. In this regulatory
loop, elastase-α1-AT complexes mediate an increase in the
synthesis of α1-AT through the interaction of a pentapeptide domain in the carboxylterminal tail of α1-AT with a
novel cell surface receptor (52). This class of receptor molecules is now referred to as serpin-enzyme complex (SEC)
703
receptors because they recognize the highly conserved
domains of several other serpin–enzyme complexes (53,54).
α1-AT mRNA has been isolated from multiple tissues in
transgenic mice (55,56), but only in some cases have studies distinguished whether such α1-AT mRNA is in ubiquitous tissue macrophages or other cell types. For instance,
α1-AT is synthesized in enterocytes and intestinal paneth
cells, as determined by studies in intestinal epithelial cell
lines, ribonuclease protection assays of human intestinal
RNA, and in situ hybridization analysis in cryostat sections
of human intestinal mucosa (30,57). Expression of α1-AT
in enterocytes increases markedly as they differentiate from
crypt to villus, in response to IL-6, and during inflammation in vivo. α1-AT is also synthesized by pulmonary
epithelial cells (58,59). Synthesis of α1-AT in pulmonary
epithelial cells is less responsive to IL-6 than to a related
cytokine, oncostatin M (59).
The half-life of α1-antitrypsin in plasma is approximately 5 days (60). It is estimated that the daily production
rate of α1-AT is 34 mg per kilogram of body weight, with
33% of the intravascular pool of α1-AT degraded daily.
Several physiologic factors may affect the rate of α1-AT
catabolism. First, desialylated α1-AT is cleared from the circulation in minutes (3), probably via hepatic asialoglycoprotein receptor-mediated endocytosis. Second, α1-AT in
complex with elastase or proteolytically modified is cleared
more rapidly than native α1-AT (61). Because its ligand
specificity is similar to that required for in vivo clearance of
serpin-enzyme complexes, the SEC receptor may also be
involved in the clearance and catabolism of α1-AT-elastase
and other serpin-enzyme complexes (54,62). The low-density protein receptor-related protein (LRP) can also mediate
clearance and catabolism of α1-AT-elastase complexes
(63,64). α1-AT diffuses into most tissues and is found in
most body fluids (5).
VARIANTS OF α1-ANTITRYPSIN
Variants of α1-AT in humans are classified according to the
protease inhibitor (PI) phenotype system as defined by
agarose electrophoresis or isoelectric focusing of plasma in
polyacrylamide at acid pH (65). The PI classification
assigns a letter to variants, according to migration of the
major isoform, using alphabetic order from anode to cathode, or from low to high isoelectric point. For example, the
most common normal variant migrates to an intermediate
isoelectric point, designated M. Individuals with the most
common severe deficiency have a α1-AT allelic variant that
migrates to a high isoelectric point, designated Z.
In recent years it has become possible to identify greater
polymorphic variation of α1-AT by direct DNA sequence
analysis. Using these techniques in addition to isoelectric
focusing, more than 100 allelic variants have been reported
(66).
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Chapter 48
Normal Allelic Variants
The most common normal variant of α1-AT is termed M1
and is found in 65% to 70% of Caucasians in the United
States (67). The M2 allele, characterized by an additional
base change from the M3 sequence, occurs in 15% to 20%
of the Caucasian population (66). Each of the normal allelic
variants are associated with serum concentrations of, and
functional activity for, α1-AT within the normal range.
Null Allelic Variants
α1-AT variants in which α1-AT is not detectable in
serum are called null allelic variants and, when inherited
with another null variant or deficiency variant, are associated with premature development of emphysema (8). Several types of defects, including insertions and deletions,
appear to be responsible for these variants (3,8,68). A single-base substitution has been discovered in the NullLudwigshafen allele (69). A recent study suggests that this mutant
α1-AT molecule is synthesized and secreted in transfected
heterologous cells, but there is a slight decrease in its rate of
secretion and it completely lacks functional activity (70). It
is not yet known whether instability or accelerated catabolism in vivo are the explanation for the inability to detect
this mutant α1-AT molecule in serum specimens.
Dysfunctional Variants
Dysfunctional variants of α1-AT include α1-AT Pittsburgh
(35). There also is a decrease in serum concentration and
functional activity for α1-AT MMineral Springs (71).
Deficiency Variants
Several variants of α1-AT associated with a reduction in
serum concentrations of α1-AT have been described and
are called deficiency variants. Some of these variants are not
associated with clinical disease, such as the S variant (27,
72). Other deficiency variants are associated with emphysema such as MHeerlen (73), MProcida (74), MMalton (21),
MDuarte (3), MMineral Springs (71), PLowell (75), and WBethesda
(76). In two persons with MMalton and one with MDuarte,
hepatocyte α1-AT inclusions and liver disease have been
reported (3,21,22). In one person with the deficiency variant SIiyama, emphysema and hepatocyte inclusions were
reported but this person did not have liver disease (77).
MECHANISM OF DEFICIENCY
A point mutation results in the substitution of lysine for
glutamate 342 (37) and a mutant α1-ATZ molecule which
is retained in the ER rather than secreted (78). Defective
secretion is observed in liver cells, macrophages and transfected cell lines (3). Site-directed mutagenesis studies have
shown that the single amino acid substitution (E342K) is
sufficient to produce the defect in secretion (79–81). The
mutant α1-ATZ molecule is partially functionally active,
having about 50% to 80% of the elastase inhibitory capacity of wild-type α1-ATM (82–84). There is a modest
increase in the rate of in vivo clearance/catabolism of radiolabeled α1-ATZ compared with wild-type α1-ATM when
infused into normal individuals, but this difference does
not account for the decrease in blood levels of α1-AT in
deficient individuals (60).
A series of studies by Carrell and colleagues have provided a mechanistic explanation for misfolding of α1-ATZ
in the ER (37). Apparently, substitution of Glu 342 by Lys
in the α1-ATZ variant reduces the stability of the molecule
in its monomeric form and increases the likelihood that it
will form polymers by means of a so-called ”loop-sheet”
insertion mechanism (85). In this mechanism, the reactive
center loop of one α1-AT molecule inserts into a gap in the
β-pleated A sheet of another α1-AT molecule. Carrell and
co-workers were the first to notice that the site of the amino
acid substitution in the α1-ATZ variant was at the base of
the reactive center loop, adjacent to the gap in the A sheet
(Fig. 48.1). These investigators predicted that a change in
the charge at this residue, as occurs with the substitution of
Lys for Glu, would prevent the insertion of the reactive-site
loop into the gap in the A sheet during interaction with
enzyme; therefore, the mutant α1-ATZ would be susceptible to the insertion of the reactive center loop of adjacent
molecules into the gap in its A sheet. This would, in turn,
cause the mutant α1-ATZ to be more susceptible to polymerization than the wild-type α1-AT. In fact, their experiments showed that α1-ATZ undergoes this form of polymerization to a certain extent spontaneously and to a
greater extent during relatively minor perturbations, such as
a rise in temperature. Presumably, an increase in body temperature during systemic inflammation would exacerbate
this tendency in vivo. Polymers could also be detected by
electron microscopy in the ER of hepatocytes in a liver
biopsy specimen from a PIZZ individual (85). Similar polymers have been found in the plasma of patients with the
PISIiyama α1-AT variant and the PIMMalton α1-AT variant
(23,86). The mutation in α1-AT PISIiyama (Ser 53 to Phe)
(77), and in α1-ATPIMMalton (Phe 52 deletion) (21) affect
residues that provide a ridge for the sliding movement that
opens the A sheet. Thus, these mutations would be
expected to interfere with the insertion of the reactive center loop into the gap in the A sheet, and therefore leave the
gap in the A sheet available for spontaneous loop-sheet
polymerization. It is indeed interesting that the hepatocytic
α1-AT globules have been observed in a few patients with
these two variants. Recent observations suggest that the α1ATS variant also undergoes loop-sheet polymerization (24)
and that this may account for its retention in the ER, albeit
a milder degree of retention than that for α1-ATZ (72).
Moreover, α1-ATS can apparently form heteropolymers
α1-Antitrypsin Deficiency
with α1-ATZ (24), providing a potential explanation for
liver disease in patients with the SZ phenotype. The precise
mechanism for loop-sheet insertion for each of these
mutant proteins is currently under further investigation
(86,87).
A complementary study by Yu et al. comparing the folding kinetics of mutant α1-ATZ to wild-type α1-ATM in
transverse urea gradient gels (88) has provided further
understanding of the mechanism for misfolding in the ER.
This study shows that α1-ATZ folds at an extremely slow
rate, unlike the wild-type α1-ATM which folds in minutes.
The delay in folding leads to an accumulation of an intermediate which has a high tendency to polymerize, presumably by the loop-sheet insertion mechanism.
By themselves, however, these data do not prove that the
polymerization of α1-ATZ results in retention in the ER.
In fact, many polypeptides must assemble into oligomeric
or polymeric complexes to traverse the ER and reach their
destination within the cell, at the surface of the plasma
membrane, or into the extracellular fluid (89). However,
evidence that polymerization results in the retention of α1ATZ in the ER has been provided by studies in which the
fate of α1-ATZ is examined after the introduction of additional mutations into the molecule. For instance, Kim et al.
(90) introduced a mutation into the α1-AT molecule at
amino acid 51, F51L. This mutation is remote from the Z
mutation, E342K, but apparently impedes loop-sheet polymerization and prevents insertion of synthetic peptide into
the gap in the A sheet, implying that the mutation leads to
closing of this gap. The double-mutated F51L α1-ATZ
molecule was less prone to polymerization and folded more
efficiently in vitro than α1-ATZ. Moreover, the introduction of the F51L mutation partially corrected the intracellular retention properties of α1-ATZ in microinjected
Xenopus oocytes (91) and in yeast (92).
Further evidence has recently come from studies of two
families with autosomal dominantly inherited dementia
(93). This dementia was associated with a histological picture of unique neuronal inclusion bodies and characterized
biochemically by polymers of a neuron-specific member of
the SERPIN family, neuroserpin. Moreover, the mutation
in neuroserpin in one family is homologous to the mutation
in the α1-AT SIiyama allele that is associated with polymerization and inclusions in the ER of liver cells. Taken
together, these studies provide relatively strong evidence
that the polymerization of α1-ATZ results in its retention
with the ER.
However, it is still unclear what proportion of the newly
synthesized mutant α1-ATZ molecules is converted to the
polymeric state in the ER. It is also not known whether
polymeric molecules are degraded in the ER less rapidly
than their monomeric counterpart or whether polymeric
molecules, when retained in the ER, are more hepatotoxic
than their monomeric counterparts. Indeed, recent studies
on the effect of temperature on the fate of α1-ATZ have
705
indicated the high degree of complexity involved in these
issues. Although Lomas et al. showed that a rise in temperature to 42ºC increases the polymerization of purified α1ATZ in vitro (85), Burrows et al. found that a rise in temperature to 42ºC resulted in increased secretion of α1-ATZ
as well as decreased intracellular degradation of α1-ATZ in
a model cell culture system (94). In contrast, lowering the
temperature to 27ºC resulted in diminished intracellular
degradation of α1-ATZ without any change in the small
amount of α1-ATZ secreted (94).
PATHOGENESIS OF LIVER DISEASE
There are several theories for the pathogenesis of liver injury
in α1-AT deficiency. According to the immune theory, liver
damage results from an abnormal immune response to liver
antigens (95). This theory is based on an increase in the
HLA DR3-DW25 haplotype observed in α1-AT-deficient
individuals with liver disease (96). However, there is no difference in the expression of class II major histocompatibility complex (MHC) antigen in the livers of these individuals compared with normal controls (97). Moreover, an
increase in the prevalence of a particular HLA DR haplotype in the affected population does not by itself imply
altered immune function. In fact, because of the linkage
disequilibrium displayed by genes within the MHC, it is
possible that increased susceptibility is caused by the products of unrelated but linked genes. For instance, the MHC
contains genes for several heat shock/stress proteins (98)
which play an important role in the folding/translocation of
polypeptides and could therefore theoretically affect the fate
and hepatotoxicity of α1-ATZ in the ER (see Fate of
Mutant α1-ATZ in the Endoplasmic Reticulum, below).
The accumulation theory, in which liver damage is
thought to be caused by an accumulation of mutant α1-AT
molecules in the ER of liver cells, is the most widely
accepted. Experimental results in transgenic mice are most
consistent with this theory and completely exclude the possibility that liver damage is caused by “proteolytic attack” as
a consequence of diminished serum α1-AT concentrations.
Transgenic mice carrying the mutant human α1-ATZ allele
develop periodic acid–Schiff-positive, diastase-resistant
intrahepatic globules and liver injury (9,10). Because there
are normal levels of antielastases in these animals, as
directed by endogenous genes, the liver injury cannot be
attributed to “proteolytic attack.”
Some have argued that the histologic characteristics of
the liver in the transgenic mouse model are not identical to
those in humans. Detailed histologic characterization of the
liver in one transgenic mouse model by Geller and colleagues has shown that there are focal areas of liver cell
necrosis, microabscesses with an accumulation of neutrophils and regenerative activity in the form of multicellular liver plates, and focal nodule formation during the
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Chapter 48
neonatal period (99). Nodular clusters of altered hepatocytes that lack α1-AT-immunoreactivity are also seen during the neonatal period. With aging, there is a decrease in
the number of hepatocytes containing α1-ATZ globules;
there is also an increase in the number of nodular aggregates
of α1-AT-negative hepatocytes and development of periosinusoidal fibrosis. Within 6 weeks, there are dysplastic
changes in these aggregates. Adenomas occur within 1 year,
and invasive hepatocellular carcinoma is seen between 1
and 2 years of age (99). The relationship between the α1ATZ globules and inflammation or dysplasia is, however,
not yet apparent from these animal studies. The
histopathology of the α1-ATZ transgenic mice is remarkably similar to that of hepatitis B virus surface antigen in
transgenic mice, and is particularly interesting because
hepatitis B virus is retained in the ER, or in the ER-Golgi
intermediate compartment of hepatocytes, often called
“ground-glass hepatocytes” (100). It is still unclear why the
liver injury in this transgenic mouse model is somewhat
milder and less fibrogenic than that seen in children with
α1-AT-deficiency-associated liver disease. It is possible that
there are strain-specific factors that condition the response
to injury in the mouse. There are certainly host-specific factors that determine the amount of liver injury in α1-AT
deficiency (see below), and we find that the amount of
inflammation and fibrosis varies widely among our patients
with liver disease from α1-AT deficiency.
Data from individuals who have null alleles of α1-AT
have also been used as evidence against the “proteolytic
attack” theory. These individuals do not develop liver injury
—at least not enough liver injury to result in clinical detection. However, only a few individuals with null alleles have
been reported, each has a different allele, and based on data
in PIZZ individuals in which about 10% to 15% of the
population develops clinically significant liver injury, it
might be necessary to evaluate seven to eight individuals
with each null allele before detecting one with liver injury.
The recognition that several other naturally occurring
variant alleles of α1-AT associated with deficiency can
undergo polymerization has provided some support for the
accumulation theory. The most important of these is the
compound heterozygous α1-ATSZ phenotype. Recent
work by Lomas and colleagues has shown that α1-ATS and
α1-ATZ may form heteropolymers (24). We know from
the nationwide study of α1-AT deficiency in Sweden that
the incidence of liver disease among individuals with the
PISZ phenotype is similar to that of individuals with the
PIZZ phenotype (1,11). We also now know that the PIMMalton allele undergoes polymerization, and liver injury has
been reported in several patients with this allele (21,22).
However, there is a report of an individual with PISIiyama
allele having hepatocyte α1-AT globules but no liver injury
(77). Moreover, a recent report by Ray and Brown has indicated that PIMHeerlen and PIMProcidia undergo aggregation
and that the PIMMineral Springs and PINullLudwigshafen may
undergo aggregation, but there are no reports of liver disease in individuals carrying these alleles (70). However,
there are only a few patients with the MMalton, SIiyama,
MHeerlen, MProcida, MMineral springs and NullLudwigshafen that have
been identified. It is also not clear how many of these
patients have been thoroughly examined for liver disease.
Again, on the basis of what we know about the PIZZ and
PISZ phenotype, at least seven to eight individuals with
each of these alleles would need to be examined to detect
one with liver injury.
It has been difficult to reconcile the accumulation theory
with the observations of Sveger, which show that only a subset of PIZZ α1-AT-deficient individuals develop significant
liver damage. We have made the prediction that a subset of
the PIZZ population is more susceptible to liver injury by
virtue of one or more additional inherited traits or environmental factors that exaggerate the intracellular accumulation
of the mutant α1-AT Z protein or exaggerate the cellular
pathophysiological consequence of mutant α1-AT accumulation. To address this prediction experimentally, we transduced skin fibroblasts from PIZZ individuals, with or without liver disease, with amphotropic recombinant retroviral
particles designed for constitutive expression of the mutant
α1-ATZ gene (101). The PIZZ individuals were carefully
selected to ensure appropriate representation. Susceptible
hosts were defined as having severe liver disease by clinical
criteria. Protected hosts were discovered incidentally and
never had clinical or biochemical evidence of liver disease.
Human skin fibroblasts do not express the endogenous α1AT gene but, presumably, express other genes involved in the
postsynthetic processing of secretory proteins. The results
show that expression of the human α1-AT gene was conferred on each fibroblast cell line. Compared with the same
cell line transduced with the wild-type α1-ATM gene, there
was selective intracellular retention of the mutant α1-ATZ
protein in each case. However, there was a marked delay in
degradation of the mutant α1-ATZ protein after it accumulated in the fibroblasts from PIZZ individuals with liver disease (susceptible hosts) as compared with those without liver
disease (protected hosts) (Fig. 48.2). Thus, these data provide evidence that other factors that affect the fate of the
mutant α1-ATZ molecule, such as a lag in ER degradation,
at least in part determine susceptibility to liver disease.
Data from our most recent studies of different susceptible hosts have suggested that there are several mechanisms
by which ER degradation may be delayed, each affecting a
separate step in the pathway (see Fate of Mutant α1-ATZ in
the Endoplasmic Reticulum, below). Carrell and Lomas
have suggested that differences in the incidence or severity
of febrile illnesses which could affect the relative degree of
polymerization of α1-ATZ provide an explanation for differences in the development of liver disease (85). There are,
however, no data to substantiate this hypothesis. We have
α1-Antitrypsin Deficiency
707
FIGURE 48.2. Fate of secretory proteins in the endoplasmic reticulum (ER). Secretory proteins
are cotranslationally translocated into the lumen of the ER through the import channel (1). These
polypeptides transiently interact with several different chaperones (2) to facilitate folding (3).
Once folding is completed (4), there is dissociation of chaperones (5) and vesicular transport out
of the ER (6). In the case of mutant proteins (7) which remain mutant even after interaction with
chaperones (8), there is an accumulation of mutant proteins bound to their chaperones. The
quality control apparatus of the ER mediates transport of these mutant proteins, free or bound
to chaperones, to the ER membrane (9) or through a channel, perhaps even through the import
channel (10), into the cytoplasm (11) for degradation. The accumulation of mutant proteins also
induces the synthesis of new chaperones (12) and new ER membrane to accommodate the
increased load of mutant proteins and thereby protect the cells. (From Kuznetsov G, Nigam SK.
Folding of secretory and membrane proteins. N Engl J Med 1998;339:1688–1695, with permission.)
not seen any differences in the incidence or severity of
febrile illnesses between our susceptible and protected
hosts. Furthermore, recent studies have suggested that in
addition to its effect on polymerization of α1-ATZ,
enhanced temperature may have independent effects on the
fate of mutant α1-ATZ which are potentially protective
(94).
FATE OF THE MUTANT α1-ATZ IN THE
ENDOPLASMIC RETICULUM
It now appears that the key processes for determining the
folding of secretory proteins during biogenesis and the fate
of mutant secretory proteins occur in the ER. In fact, these
processes have been referred to as the “quality control
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Chapter 48
mechanism” of the cell. After translocation into the lumen
of the ER, nascent secretory polypeptide chains undergo a
series of post-translational modifications, including glycosylation formation of disulfide bonds, oligomerization and
folding. These modifications and transport through the ER
to the Golgi is facilitated by transient and sequential interactions with ER proteins, termed molecular chaperones.
Several families of ER chaperones have been identified.
One has been referred to as the polypeptide chain-binding
protein family and includes several heat-shock/stress proteins, GRP78/BiP and GRP94, protein disulfide isomerase,
and ERp72 (102). Several calcium-binding phosphoproteins of the ER, most notably calnexin and calreticulin,
have also been implicated as having molecular chaperone
activity within the ER. Calnexin is an approximately 88-kd
transmembrane ER resident phosphoprotein (103) now
known to facilitate the folding and assembly of many membrane and secretory glycoproteins (104). In addition to its
chaperone activity (105), calnexin uses a lectin-like mechanism to bind the innermost glucose residue of the
asparagine-linked oligosaccharide side chains present on
most glycoproteins (106). The innermost glucose residue
becomes accessible almost immediately after the secretory
glycoprotein has undergone the initial stages of oligosaccharide side chain trimming in the lumen of the ER,
including the removal of the two outermost glucose
residues by the action of glucosidases I and II (Fig. 48.3).
Once bound to calnexin, monoglucosylated glycoproteins
are retained in the ER until properly folded. Once folding
is complete, the glycoprotein can dissociate from calnexin
for vesicular transport out of the ER. Two studies have indicated that a unique reglucosylating enzyme, uridine diphosphate-glucose:glycoprotein glucosyltransferase (UDGGT),
can transfer glucose onto unfolded or denatured, deglucosylated proteins in the ER (107,108). In fact, the binding of
glycoproteins to calnexin during folding in the ER is now
thought to depend on a cycle of glucosidase II activity, producing the deglucosylated form of a protein and reglucosylation by ER luminal UDGGT, leading to regeneration of
the monoglucosylated form. The glucosyltransferase acts
preferentially on unfolded or denatured proteins. Thus, the
repeated cycles of binding to and dissociation from calnexin
are designed to maximize the possibility that a given
unfolded or denatured protein will undergo proper folding
for transport out of the ER.
As a part of its quality control functions, the ER also
possesses machinery whereby it can degrade any mutant or
unassembled polypeptides that is unable to fold properly
even after interaction with the ER chaperones (Fig. 48.4).
This machinery has come to be called the “ER degradation
pathway” (109). Although the ER degradation pathway was
originally thought to involve a distinct proteolytic system,
it now appears to be mediated in large part from the cytoplasmic aspect of the ER by the ubiquitin system (110) and
the proteasome (111). The role of the proteasome was
originally shown for the mutant membrane protein
CFTRΔF508 (112,113) as well as for α1-ATZ (114,115).
Although it is relatively easy to conceptualize how a trans-
FIGURE 48.3. Differences in endoplasmic reticulum (ER) degradation of α1-ATZ in protected and
susceptible hosts. The block in ER degradation in susceptible hosts is represented by a small dark bar.
RER, rough ER. (From Teckman JH and Perlmutter DH. Conceptual advances in pathogenesis and
treatment of childhood metabolic liver disease. Gastroenterology 1995;108:1263, with permission.)
α1-Antitrypsin Deficiency
709
FIGURE 48.4. Endoplasmic reticulum (ER) degradation of α1-ATZ. In one pathway for ER degradation, α1-ATZ in the lumen of the ER binds to the transmembrane chaperone calnexin. Calnexin
molecules bound by α1-ATZ are then polyubiquitinated, presumably on their cytoplasmic tail. The
polyubiquitinated calnexin-α1-ATZ complexes are degraded by the proteasome. There are many
calnexin molecules still present to provide chaperone activity for endogenous wild-type proteins.
This figure does not show the ubiquitin-independent proteasomal and nonproteasomal mechanisms for ER degradation of α1-ATZ.
membrane protein such as CFTRΔF508 might be accessible on the cytoplasmic aspect of the ER membrane for
ubiquitination and degradation by the proteasome, it is
more difficult to conceptualize how this might occur for a
luminal polypeptide such as α1-ATZ. To address this issue,
we used a cell-free microsomal translocation system and
found that α1-ATZ must interact with the transmembrane
molecular chaperone calnexin in order to be degraded. The
results showed that degradation of the α1-ATZ-calnexin
complex by the proteasome involves both ubiquitin-dependent and -independent mechanisms (115,116). The ubiquitin-dependent mechanism requires recruitment of the
ubiquitin conjugating enzyme E2-F1 from the cytoplasm
onto the ER membrane and polyubiquitination of the cytoplasmic tail of calnexin (117). Degradation of the mutant
secretory protein carboxypeptidase Y in yeast also requires
recruitment of Ubc7p (the yeast homologue of E2-F1) from
the cytoplasm by the ER membrane protein Cue1p to generate a ubiquitin conjugation platform at the surface of the
ER (117). Taken together, these studies show that there are
at least two pathways and several steps in each pathway
involved in ER degradation of α1-ATZ: binding to calnexin; induction of calnexin ubiquitination by a ubiquitin
conjugation platform at the surface of the ER that includes
E2-F1; and degradation of α1-ATZ-calnexin and α1-ATZpolyubiquitinated calnexin complexes by the 26S proteasome (Fig. 48.5). Studies of α1-ATZ expressed in yeast
(114) and of the truncated mutant α1-ATHong Kong in transfected mouse hepatoma cells (118) have also shown that
calnexin and the proteasome are involved in the ER degradation of these molecules.
We do not yet know exactly how the entire α1-ATZ-calnexin complex, including the luminal domain of calnexin
associated with α1-ATZ, is degraded. The proteasome may
initiate a process that is completed by other enzymes within
the ER membrane or within the ER lumen. Several other
mechanisms by which the ubiquitin system and the proteasome gain access to membrane-bound and lumenal substrates of the ER degradation pathway have been discussed
in the literature. The retrograde translocation mechanism in
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Chapter 48
FIGURE 48.5. Oligosaccharide side chain trimming in the endosplasmic reticulum (ER). There are
two N-acetyl glucosamine (open squares), nine mannose (open circles) and three glucose residues
(filled triangles) on each asparagine-linked oligosaccharide side chain. Trimming involves removal
of the outermost glucose by glucosidase I and the second glucose by glucosidase II. Calnexin binds
glycoproteins by recognizing the innermost glucose. However, this glucose residue can still be
removed by glucosidase II when the glycoprotein is bound to calnexin. The glycoprotein then dissociates from calnexin. If the polypeptide is mutant or unassembled, it can be recognized by the
reglycosylating enzyme, UPD-Glc:glycoprotein glucosyltransferase, to add back a glucose molecule in a manner that leads to re-association with calnexin. Glucosidase I and II activities can be
inhibited by castanospermine (CST), N-methyl deoxynojirimicin (MDNJ), and N-butyl deoxnojirimicin (BDNJ). There are also α mannosidases in the ER. Mannosidase I can remove a single mannose residue from the inner branch. It is inhibited by kifunensine (KIF) and deoxymannojirimicin
(DMJ). Mannosidase II can remove a single mannose from the outer branch. It is inhibited by 1,4
dideoxy-1,4-imino-O-mannitol hydrochloride (DIM and DMJ).
which substrates are transported from the ER lumen or
membrane through the Sec61p translocon has received the
most attention (119–126). Recently, Mayer et al. have
shown that a chimeric ER membrane protein may be dislocated and degraded directly at the ER membrane by a
membrane-bound assembly of the ubiquitin-dependent
proteasomal system (127). This “membrane extraction”
mechanism, or “dislocation” mechanism, may be particularly relevant to degradation of α1-ATZ because ER degradation of this substrate appears to involve polyubiquitination on the cytoplasmic tail of the transmembrane ER
chaperone calnexin only when it has bound α1-ATZ at the
luminal surface of the ER membrane. It is also possible that
the proteasome gains access to α1-ATZ and/or the α1ATZ-polyubiquitinated calnexin during the formation of
autophagic vacuoles. Our recent studies have shown that
retention of α1-ATZ in the ER is associated with the induction of an autophagic response (26). The autophagic
response is thought to be a general mechanism by which
intracellular organelles, or parts of organelles, are degraded.
It is a highly evolutionarily conserved process that occurs in
many cell types, especially during stress states, such as nutrient deprivation, and during the cellular remodeling that
accompanies morphogenesis, differentiation and senescence. Several studies have suggested that autophagic vacuoles are derived in part from subdomains of ER (128).
Autophagosomes initially form as invaginations from ribosome-free areas of the ER membrane. Together with con-
stituents of the ER, autophagosomes engulf cytosolic constituents including components of the ubiquitin system and
the proteasome (129,130). Thus, it is possible that degradation of α1-ATZ is mediated by proteasomal machinery
engulfed during formation of the autophagosome. Indeed,
in our recent studies an intense autophagic response was
demonstrated in cell culture model systems with ER retention of mutant α1-ATZ and in liver biopsy specimens from
patients with α1-AT deficiency (26). Moreover, α1-AT and
calnexin were colocalized within autophagosomes as well as
within the ER. Finally, degradation of α1-ATZ in the cell
culture model system is partially abrogated by inhibitors of
autophagy including wortmannin, 3-methyladenine and
LY294002. However, it is also possible that α1-ATZ molecules taken up into autophagosomes are degraded by a nonproteasomal mechanism when the autophagosomes
merge/fuse with the lysosomal pathway, and that the
autophagic and proteasomal pathways constitute completely independent mechanisms for degradation of α1ATZ.
A study by Van Leyen et al. has suggested the possibility
that a certain, very specialized type of autophagic response,
termed programmed organelle degradation, allows access of
cytoplasmic proteases to both luminal and integral membrane proteins (131). Whole organelles are degraded during
differentiation of specific cell types. In the central fiber cells
of the eye lens, organelles are degraded so that the cell can
become transparent to incoming light. In reticulocytes,
α1-Antitrypsin Deficiency
organelles are degraded to accommodate the sole need for
globin synthesis by a mechanism that leaves the plasma
membrane intact. These processes appear to involve the
highly regulated recruitment of 15-lipoxygenase from the
cytoplasm to the ER membrane where it presumably oxygenates membrane phospholipids, in turn releasing proteins
from the ER lumen and membrane. A mechanism like this
could possibly account for the ER degradation pathway of
some substrates.
Together, these studies indicate that degradation of α1ATZ is a complex process that may involve more than one
pathway and at least several sequential steps in each pathway. Theoretically, each of these pathways or its individual
steps may be affected in an α1-AT-deficient patient who is
“susceptible” to liver disease — that is, there may be heterogeneity among susceptible hosts in the mechanism by
which ER degradation is delayed. Indeed, there is already
some evidence for distinct mechanisms of delayed ER
degradation among susceptible hosts. In one susceptible
host, the retained α1-ATZ interacts poorly with calnexin
(101). In the liver cells of this host, there is likely to be only
a very little polyubiquitinated calnexin-α1-ATZ complex
that can be recognized for proteolysis by the proteasome. In
several other susceptible hosts, the retained α1-ATZ interacts well with calnexin but is degraded slowly (Teckman J,
Qu D, and Perlmutter DH, unpublished observations).
These hosts may have a defect in calnexin that prevents its
ubiquitination or a defect in the ubiquitin system or the
proteasome. Hosts with the latter defect would also be more
likely to respond to a pharmacological agent such as interferon-γ (132) that enhances the activity of the ubiquitindependent proteasomal system. Recent studies involving
yeast which have identified at least 30 putative recessive
mutants and seven complementation groups of strains
defective in ER degradation of α1-ATZ (133), are likely to
lead to the recognition of other mechanisms for excessive
ER retention of α1-ATZ.
MECHANISM OF LIVER CELL INJURY
There is still relatively limited information about the mechanism by which ER retention of α1-ATZ leads to liver cell
injury. In the transgenic mice that express human α1-ATZ,
there is mild inflammation and necrosis, formation of adenomas and ultimately hepatocellular carcinoma (99), but
this animal model has not yet provided any clues for understanding the mechanism of hepatotoxicity. There is also relatively little information about the short-term and longterm effects of ER retention of α1-ATZ in cell culture
systems. In one report a number of years ago (134), the
accumulation of α1-ATZ in Xenopus oocytes was associated
with the release of lysosomal enzymes, but there has not
been any follow-up study of this potential mechanism. Several more recent studies using model systems have provided
711
interesting and perhaps relevant information. Raposo and
colleagues used a novel approach to establish cell lines with
marked retention of MHC class I molecules in the ER
(135). This led to a marked alteration in the structure of the
ER into an expanded network of tubular and fenestrated
membranes. Marker studies suggest that this altered network is derived from the ER and ER-Golgi intermediate
compartment. Electron-dense compartments resembling
lysosomes appear to bud off from this altered network.
Because ubiquitin and ubiquitin-activating enzymes were
associated with the cytosolic aspect of the electron-dense
bodies, the bodies were thought to represent compartments
in which ER degradation takes place. The electron-dense
bodies also resemble the autophagic vacuoles that we
observed in liver biopsies from α1-AT-deficient patients
and in genetically engineered cell culture model systems
(26).
Work in several labs has shown that a novel structure
called the aggresome is formed in cells when the expression
of mutant membrane proteins, such as CFTRΔF508, other
mutant membrane proteins and mutant viral proteins
exceeds the capacity of the proteasome to degrade them
(136,137). The aggresome is a pericentriolar membranefree cytoplasmic inclusion containing mutant, ubiquitinated protein ensheathed in a case of vimentin and perhaps
other intermediate filaments. Formation of these structures
requires an intact microtubular system that presumably
plays a role in moving the aggregate to the pericentriolar
location. Indeed, formation of aggresomes is now thought
to be a mechanism by which the cell can sequester aggregated proteins to prevent them from having toxic effects on
critical structures within the cell. Because autophagosomes
have been seen in the vicinity of the aggresomes (136), it is
possible that the cell uses the autophagic machinery to ultimately degrade aggresomes. Our recent studies indicate that
retention of α1-ATZ induces an expansion of, and alteration in the structure of, the ER and the formation of
autophagic vesicles but does not cause aggresome formation
(26). Thus, there is specificity in the response of the cell to
different types of protein aggregates, but autophagy may
represent, at least in part, a final common pathway.
The unfolded protein response (UPR) is also induced by
the accumulation of unfolded proteins in the ER (Fig. 48.4).
It results in the induction, or upregulation, of a repertoire of
genes encoding chaperones such as BiP, GRP94, and
enzymes that facilitate disulfide bond formation including
protein disulfide isomerase, ERp72 and ERO1 (138,139).
Enzymes in the phospholipid biosynthetic pathway are also
induced, permitting the synthesis of new ER membrane to
accommodate the increased load (138). These target genes
have a common upstream activating sequence in their promoters, the unfolded protein response element (UPRE),
which directs that transcription as a part of the response
pathway. Studies in the laboratory of Peter Walter using yeast
have shown that the UPR involves the oligomerization and
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Chapter 48
transautophosphorylation of a novel ER transmembrane serine/threonine kinase, Ire1p. Once activated, Ire1p has
endonuclease activity and, together with tRNA ligase, mediates a unique posttranscriptional splicing pathway to generate Hac1pi, a transcription factor capable of binding to the
UPRE (139). In mammalian cells, there appears to be a
homologue of yeast Ire1p (140). Moreover, another protein,
ATF6, appears to be involved in the mammalian UPR (141).
ATF6 is a type II transmembrane glycoprotein that resides in
the ER and is expressed constitutively. When mutant or
unassembled proteins accumulate in the ER, the cytoplasmic
tail of ATF6 is cleaved and translocates into the nucleus,
where it functions as a transcription factor capable of binding
to the UPRE.
The presence of mutant proteins in the ER also leads to
reduced translation of endogenous proteins. Harding et al.
have characterized a novel gene called PERK which encodes
a type 1 transmembrane ER resident protein and appears to
initiate a signal transduction pathway for inhibition of protein translation (142). PERK has a luminal domain that
resembles that of Ire1p and a cytoplasmic tail with a protein
kinase domain that resembles the eukaryotic initiation factor-2α (E1F2α) kinases. Accumulation of mutant proteins
in the ER is associated with increased protein kinase activity of PERK, phosphorylation of a key serine residue on
E1F2α, and inhibition of translation.
Two other signal transduction pathways activated by
mutant proteins in the ER have recently been characterized and may be relevant to α1-AT deficiency. The ER
overload pathway is a signaling pathway that appears to
be distinct from the UPR, and involves activation of
NFκB and release of active oxygen intermediates (143).
So far, this pathway has only been described in experimental conditions associated with ER overload of mutant
or unassembled membrane proteins. Recent studies in the
Morimoto lab have shown that the heat shock factor
HSF2 may be activated by the accumulation of ubiquitinated proteins (144). The downstream effect of HSF2
activation by ubiquitinated protein is induction of the
classical heat shock response including cytoplasmic and
nuclear chaperones HSP90, HSP70, HSC70, HSP27, an
ER chaperone GRP78/BiP, and mitochondrial chaperone
HSP60.
It is not yet known whether one or all of these signaling
pathways are induced by ER retention of α1-ATZ or
whether there is any alteration in their activation in the subgroup of α1-AT-deficient patients who are susceptible to
liver disease. Because these are considered response pathways that are designed to protect the cell, it is presumed
that they must be overwhelmed by the concentration or
intrinsic toxic potential of a particular mutant protein
before cell injury occurs. However, the consequences of
prolonged activation of these response pathways are entirely
unknown and could potentially include cytotoxic and/or
oncogenic effects.
OTHER DISORDERS WITH ENDOPLASMIC
RETICULUM RETENTION OF MUTANT OR
UNASSEMBLED PROTEINS
There are many other examples of disorders in which
mutant or unassembled proteins are retained in the ER
(Table 48.1). For example, in most cases of cystic fibrosis, a
partially active but mutant CFTR molecule (CFTRΔF508)
is retained and degraded in the ER rather than transported
to the apical plasma member from which it ordinarily functions as a chloride transporter (145). Recent studies have
indicated that about 37% of Wilson disease (WD) alleles
have the H1069Q mutation that is associated with retention and degradation of the WD ATPase in the ER (146).
Some patients with Fabry’s disease are characterized by a
mutant galactosidase that does not reach its final destination in the lysosome but rather forms aggregates in the ER
(147).
A study of the combined deficiency of coagulation factors V and VIII has provided some extraordinary information about folding in the secretory pathway (148). It is an
autosomal recessive disorder that has been described in 58
families and linked to a gene on chromosome 18q. Nichols
et al. used positional cloning techniques to identify mutations in ERGIC-53, a transmembrane protein localized to
the ER-Golgi intermediate compartment that has mannose-specific lectin properties. The results therefore imply
that ERGIC-53 mediates cargo-selective ER-to-Golgi transport for the two coagulation proteins. Because there is complete loss of ERGIC-53 but residual levels of factors V and
VIII in the blood, it is likely that ERGIC-53 facilitates the
transport of this subset of proteins but is not absolutely
required for their secretion. A recent study with cell lines
expressing genetically engineered dominant-negative forms
of ERGIC-53 have in fact shown that the ERGIC-53
cycling pathway facilitates secretion of the two coagulation
proteins (149). Thus, the interaction between factors V and
VIII and ERGIC-53 constitutes one of the first examples of
selective packaging of secretory proteins as cargo for export
from the ER.
There are also several examples of infectious diseases in
which mutant or unassembled proteins accumulate in the
ER of liver cells. Hepatitis B virus infection is one of the
most well-characterized of these. Ground-glass hepatocytes which are seen in individuals infected with hepatitis
B virus are thought to arise from the accumulation of the
large form of the hepatitis B virus surface antigen and
empty virions in the ER (100). Recent studies have suggested that this process is associated with alterations in
the morphology of the ER with the formation of large
cisternae and budding tubules (150), in some ways similar to what we have seen in liver biopsy specimens from
α1-AT-deficient patients and in cell culture model systems of α1-AT deficiency (26). It is not yet clear whether
this process is cytopathic, whether it involves an apop-
α1-Antitrypsin Deficiency
713
TABLE 48.1. EXAMPLES OF INBORN ERRORS OF METABOLISM IN WHICH ABNORMAL PROTEINS ARE
RETAINED IN ENDOPLASMIC RETICULUM
Proteins
α1-Antitrypsin
α1-Antichymotrypsin
Complement component C2
Complement component factor H
Fibrinogen
α2-plasmin inhibitor
Protein C deficiency
LDL Receptor
CFTR
β-Hexosaminidase
Microsomal triglyceride transfer protein
Palmitoyl protein thioesterase
Sucrase-isomaltase
Thyroglobulin
Type I procollagen
Vasopressin precursor
Proteolipid protein
Vasopressin receptor
Water channel (aquaporin)
Unknown
ERGIC-53
Von Willebrand
Factor VII
Glycoprotein GPIIb/IIIa
Rhodopsin
Fibrillin
Wilson disease ATPase
α-Galactosidase A
Myeloperoxidase
HFE
Type of Protein
Secreted
Secreted
Secreted
Secreted
Secreted
Secreted
Secreted
Membrane
Membrane
Lysosomal
Secreted
Secreted
Membrane
Secreted
Secreted
Secreted
Membrane
Secreted
Secreted
Secreted/Membrane
Membrane
Secreted
Secreted
Membrane
Membrane
Secreted
Membrane
Lysosomal
Granular
Membrane
totic mechanism, or, conversely, whether it induces resistance to apoptosis and a tendency toward malignant
transformation. Transgenic mice which overexpress
hepatitis B virus surface antigen have ground-glass hepatocytes (100) and have a hepatic histopathology that is
remarkably similar to that in mice transgenic for α1-ATZ
(99). Two of the envelope glycoproteins of hepatitis C
virus also accumulate in the ER of infected cells (151),
and it has been speculated that the retention of hepatitis
C virus envelope proteins in the ER results in apoptosis.
In some of the metabolic diseases associated with an
“ER storage” state, a mutant protein is retained in the ER
of liver cells, and yet there is no evidence that these disorders are associated with liver injury — for example, complement C2 and factor H deficiency. When compared to
what we know about α1-AT deficiency, this observation
has important implications. It may indicate that α1-ATZ
has intrinsic hepatotoxic properties. Alternatively, the
hepatotoxic effect may be dependent on the relative concentration of mutant protein that is retained in the ER.
Because α1-AT is one of the most abundant products of
the liver cell, α1-ATZ is likely to reach particularly high
levels in the ER.
Related Disease
Emphysema, liver disease
α1-antichymotrypsin deficiency
Type II complement C2 deficiency
Factor H deficiency
Familial hypofibrinogenemia
α2-plasmin inhibitor deficiency
Hereditary protein C
Familial hypercholesterolemia
Cystic fibrosis
Tay–Sachs disease
Abetalipoproteinemia, Hypolipoproteinemia
Infantile neuronal ceroid lipofuscinosis
Congenital sucrase-isomaltase deficiency
Congenital hypothyroid
Osteogenesis imperfecta type II
Central diabetes insipidus
Pelizaeus–Merzbacher disease
Congenital nephrogenic diabetes insipidus
Congenital nephrogenic diabetes insipidus
Carbohydrate-deficient glycoprotein syndrome
Combined deficiency of coagulation factors V and VIII
Von Willebrand’s disease
Hereditory factor VII deficiency
Glanzmann’s thrombasthenia
Autosomal dominant retinitis pigmentosa
Marfan’s syndrome
Wilson disease
Fabry disease
Hereditary myeloperoxidase deficiency
Hemochromatosis
TREATMENT
The most important principle in the treatment of α1-AT
deficiency is avoidance of cigarette smoking. Cigarette
smoking markedly accelerates the destructive lung disease
that is associated with α1-AT deficiency, reduces the quality of life, and significantly shortens the longevity of these
individuals (12,13).
There is no specific therapy for α1-AT deficiency-associated liver disease. Therefore, clinical care largely involves
supportive management of symptoms due to liver dysfunction and for the prevention of complications. Progressive
liver dysfunction in α1-AT-deficient patients has been
treated by orthotopic liver transplantation, with survival
rates approaching 90% at 1 year and 80% at 5 years (152).
Several studies have shown that a class of compounds
called chemical chaperones can reverse the cellular mislocalization or misfolding of mutant plasma membrane, lysosomal, nuclear and cytoplasmic proteins including
CFTRΔF508, prion proteins, mutant aquaporin molecules
associated with nephrogenic diabetes insipidus, and mutant
galactosidase A associated with Fabry disease (153–155).
These compounds include glycerol, trimethylamine oxide,
714
Chapter 48
deuterated water and 4-phenylbutyric acid (PBA). We
recently found that glycerol and PBA mediate a marked
increase in the secretion of α1-ATZ in a model cell culture
system (94). Moreover, oral administration of PBA was well
tolerated by PiZ mice (transgenic for the human α1-ATZ
gene) and consistently mediated an increase in blood levels
of human α1-AT, reaching 20% to 50% of the levels present in PiM mice and normal humans. PBA did not affect
the synthesis or intracellular degradation of α1-ATZ. The
α1-ATZ secreted in the presence of PBA was functionally
active, in that it could form an inhibitory complex with
neutrophil elastase. Because PBA has been used safely for
years in children with urea cycle disorders as an ammonia
scavenger and because clinical studies have suggested that
only partial correction of the deficiency state is needed for
the prevention of both liver and lung injury in α1-AT deficiency (3,8,156), PBA constitutes an excellent candidate for
chemoprophylaxis of target organ injury in α1-AT deficiency.
It also now appears that several iminosugar compounds
(Fig. 48.3) may be potentially useful for chemoprophylaxis
of liver and lung disease in α1-AT deficiency. These compounds are designed to interfere with oligosaccharide side
chain trimming of glycoproteins and are now being examined as potential therapeutic agents for viral hepatitis and
other types of infections (157,158). We have examined several of these compounds initially to determine the effect of
inhibiting glucose or mannose trimming from the carbohydrate side chain of mutant α1-ATZ on its fate in the ER,
but found to our surprise that one glucosidase inhibitor,
castanospermine (CST) and 2 α mannosidase I inhibitors,
kifunensine (KIF) and deoxymannojirimicin (DMJ), actually mediate increased secretion of α1-ATZ (159). The α1ATZ that is secreted in the presence of these drugs is partially functionally active. KIF and DMJ are less attractive
candidates for chemoprophylactic trials because they delay
degradation of α1-ATZ in addition to increasing its secretion and therefore have the potential to exacerbate susceptibility to liver disease. However, CST has no effect on the
degradation of α1-ATZ and, therefore, may be targeted for
development as a chemoprophylactic agent. The mechanism of action of CST on α1-ATZ secretion is unknown.
An interesting hypothesis for the mechanism of action of
KIF and DMJ has mutant α1-ATZ interacting with
ERGIC-53 for transport from ER to Golgi when mannose
trimming is inhibited.
Novoradovskaya et al. have suggested that inhibition of
ER degradation of α1-ATZ by proteasome inhibitor lactacystin and by protein synthesis inhibitor cycloheximide is
associated with increased secretion of α1-ATZ (160). We
have been unable to confirm this result (94). Moreover,
there are now several lines of evidence indicating that there
is not a simple relationship between ER degradation of α1ATZ and its secretion such that perturbations that delay
degradation are automatically accompanied by increased
secretion. Some physiologic and pharmacologic perturbations are associated with delayed degradation without any
change in secretion. Other perturbations increase secretion
without any change in degradation. Increased temperature
is associated with both delayed degradation and increased
secretion (94).
Some patients with α1-AT deficiency and emphysema
are currently receiving replacement therapy with purified
and recombinant plasma α1-AT either by intravenous or
intratracheal aerosol administration (8). This therapy is
associated with improvement in serum concentrations of
α1-AT and in α1-AT and neutrophil elastase inhibitory
capacity in bronchoalveolar lavage fluid without significant
side effects. Although initial studies have suggested that
there is a slower decline in forced expiratory volume in
patients on replacement therapy, this only occurred in a
subgroup of patients and the study was not randomized
(161). Protein replacement therapy is designed only for
individuals with established and progressive emphysema. It
is not being considered for individuals with liver disease,
because there is no information to support the notion that
deficient serum levels of α1-AT are mechanistically related
to liver injury.
A number of patients with severe emphysema from α1AT deficiency have undergone lung transplantation in the
past ten years. The latest data from the St. Louis International Lung Transplant Registry shows actuarial survival for
patients in this category who underwent transplantation
between 1987 and 1994 at approximately 50% for 5 years.
Lung function and exercise tolerance are significantly
improved (162).
Replacement of α1-AT by somatic gene therapy has also
been discussed in the literature (163). This strategy is
potentially less expensive than replacement therapy with
purified protein. Again, this therapeutic strategy would
only be useful in ameliorating emphysema. It would be
helpful to know that replacement therapy with purified α1AT, as it is currently applied, is effective in ameliorating
emphysema in this deficiency before embarking on clinical
trials involving gene therapy. Several novel types of gene
therapy, such as repair of mRNA by trans-splicing
ribozymes (164) and chimeric RNA/DNA oligonucleotides
(165), are theoretically attractive alternative strategies for
liver disease in α1-AT deficiency because they would prevent the synthesis of mutant α1-ATZ protein and ER retention. In fact, a chimeric RNA/DNA oligonucleotide based
on the sequence of coagulation factor IX in complex with
lactose so that it could be taken up by asialoglycoprotein
receptor-mediated endocytosis was delivered to hepatocytes
with high efficiency after intravenous administration (165).
Other studies have shown that transplanted hepatocytes
can repopulate the diseased liver in several mouse models
(166,167), including a mouse model of a childhood metabolic liver disease termed hereditary tyrosinemia. Replication of the transplanted hepatocytes occurs only when there
α1-Antitrypsin Deficiency
is injury and/or regeneration in the liver. The results provide evidence that it may be possible to use hepatocyte
transplantation techniques to treat hereditary tyrosinemia
and, perhaps, other metabolic liver diseases in which the
defect is cell-autonomous. For instance, α1-AT deficiency
involves a cell-autonomous defect and would be an excellent candidate for this strategy.
Alternative strategies for at least partial correction of α1AT deficiency may result from a more detailed understanding of the fate of the α1-ATZ molecule in the ER. For
instance, delivery of synthetic peptides to the ER to insert
into the gap in the A-sheet or into a particular hydrophobic
pocket of the α1-AT molecule (34) and prevent polymerization of α1-AT might result in release of the mutant α1ATZ molecules into the extracellular fluid and prevent
accumulation in the ER. Although it is not yet entirely
clear, there is some evidence from studies on the assembly
of MHC class I molecules that synthetic peptides may be
delivered to the ER from the extracellular medium of cultured cells (168). There is also evidence that certain molecules may be transported retrograde to the ER by receptormediated endocytosis (169). Second, elucidation of the
biochemical mechanism by which abnormally folded α1AT undergoes intracellular degradation might allow pharmacological manipulation of this degradative system, such
as enhancing proteasomal activity with interferon γ in the
subpopulation of the PIZZ individuals predisposed to liver
injury.
ACKNOWLEDGMENTS
The author is indebted to Mary Pichler for preparing this
manuscript and to the support from the U.S. National
Institutes of Health (HL37784, DK52526, DK56783).
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