From the Departments of Pathology and
§ Cell Biology, ¶ Cell and Molecular Biology Program,
Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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The role of conformation-based quality control in
the early secretory pathway is to eliminate misfolded polypeptides and
unassembled multimeric protein complexes from the endoplasmic
reticulum, ensuring the deployment of only functional molecules to
distal sites. The intracellular fate of terminally misfolded human
The endoplasmic reticulum
(ER)1 functions as the
intracellular site where nascent polypeptides enter the central
vacuolar system (1) and fold into their correct functional conformation
(2), which is dictated by the primary amino acid sequence (3). Quality control machinery resident to that compartment facilitates the selective elimination of incompletely folded proteins to ensure that
only functional molecules are deployed to distal sites (4, 5). As such,
the role of conformation-based quality control is fundamental to normal
cell physiology.
Although initially unexpected, it is now recognized that the 26 S
proteasome, a constituent of the cytosol (6), is responsible for the
degradation of many ER-situated proteins (7-10). Indeed, the
cytoplasmic delivery of proteasomal substrates has been reported (11-14). As yet, however, the molecular basis by which proteins of the
ER are selected for proteasomal degradation remains unclear, although
the molecular chaperone calnexin, which binds monoglucosylated oligosaccharides (15), has been implicated as a possible participant in
the sorting process (16, 17).
The molecular pathogenesis of several human diseases, including cystic
fibrosis, familial hypercholesterolemia, and a heritable form of
pulmonary emphysema, are caused, in part, by the participation of
conformation-based quality control factors (for reviews, see Refs. 18
and 19). The latter disorder is caused by the impaired secretion of
misfolded genetic variants of human Defective secretion of AAT has been investigated by us (21-26) and
others (10, 17, 27, 28) as a model to dissect the involvement of
quality control machinery in the molecular pathogenesis of human
disease. As observed for other hepatic secretory glycoproteins (29),
rounds of binding to calnexin are predicted to facilitate the folding
of the newly synthesized AAT into its functional conformation (25). Our
analyses have shown that prolonged physical association with calnexin
accompanies intracellular retention of the terminally misfolded
glycoprotein (25) and that the removal of mannose from
asparagine-linked oligosaccharides is essential for subsequent degradation (25).
The focus of the present study was to investigate the intracellular
degradation of misfolded AAT as a model to elucidate the role of
intracellular mannosidase activity in glycoprotein quality control, a
phenomenon recently observed by us (25) and others (30-33). Results
from the present investigation are consistent with a model in which
modification by ER mannosidase I mediates the proteasomal degradation
of terminally misfolded AAT by abrogating its dissociation from
calnexin in response to the attenuated deglucosylation of
asparagine-linked oligosaccharides. This finding provides evidence that
ER-situated mannosidase activity occupies a previously unrecognized elevated hierarchical position among known members of the glycoprotein folding and quality control machinery and extends the role of calnexin
beyond that of a simple molecular chaperone.
Reagents and Antisera--
All salts, buffers, and protease
inhibitors were purchased from Sigma, except for lactacystin, which was
synthesized by Dr. E. J. Corey (Harvard University).
Endoglycosidase H, jack bean mannosidase, and all oligosaccharide
processing inhibitors were purchased from Boehringer Mannheim, with the
exception of castanospermine (CalBiochem) and kifunensine (Toronto
Research Chemicals, Inc). All tissue culture media were purchased from
ICN Biochemicals and Life Technologies, Inc. Fetal bovine serum was
procured from Summit Biotechnology. Polyclonal rabbit antiserum against
a synthetic peptide homologous to the cytoplasmic tail of canine
calnexin was purchased from StressGen, and an IgG fraction of
polyclonal goat anti-human AAT was procured from Organon
Teknika-Cappel.
Metabolic Radiolabeling and Immunoprecipitation--
Mouse
hepatoma cells were stably transfected with DNA encoding human AAT in
which 33 amino acids are absent from the carboxyl terminus of the
394-amino acid polypeptide (cell line H1A/N13) (21). Unless stated
otherwise, incubation of cells with inhibitors was performed for 60 min
in regular growth medium (25) prior to a 15-min pulse in
methionine-free medium containing [35S]methionine (NEN
Life Science Products) (23). Cell lysis was performed with buffered
Nonidet P-40 (25), and protein immunoprecipitation was accomplished by
a 2-h incubation of the soluble cell lysate with an excess of specific
antiserum immobilized to protein G-agarose, as described (25).
Following immunoprecipitation, radiolabeled proteins were resolved by
SDS-PAGE and detected by fluorographic enhancement of vacuum-dried
gels. Increased electrophoretic mobility of radiolabeled AAT, resulting
from oligosaccharide modification, was resolved in 20-cm gels (25).
Velocity Sedimentation--
Calnexin-associated AAT was
separated from released monomers by velocity sedimentation of Nonidet
P-40 cell lysates in linear 5-20% sucrose gradients as described
previously (24, 25). Physical interaction with calnexin was confirmed
by sequential coimmunoprecipitation of radiolabeled molecules with
antiserum against the molecular chaperone as described previously (25). The relative amount of AAT in each sedimenting species was assessed by
scintillation spectrophotometry of immunoprecipitated protein excised
from the gel.
Detection of Asparagine-linked Glucosylated
Oligosaccharides--
Prior to treatment, calnexin was dissociated
from immunoprecipitated AAT by incubation with 5 mM EDTA at
37 °C (24). Digestions were performed for 16 h at 37 °C with
0.03 µg of jack bean mannosidase in 50 µl of 10 mM
citrate, pH 5.0, followed by an additional 8-h incubation upon the
addition of another aliquot of enzyme. Resolution of AAT glycoforms was
accomplished by SDS-PAGE and fluorography. Electrophoretic AAT
standards in which all three attached oligosaccharides contain
glucose was generated by preincubating cells with 0.2 µg/ml
castanospermine prior to pulse-radiolabeling and immunoprecipitation. The standard in which one of the attached oligosaccharides contain glucose was generated by chasing pulse-radiolabeled cells in medium containing metabolic poisons to disrupt the physical interaction with
calnexin (26), resulting in the hydrolysis of glucose residues prior to immunoprecipitation.
Calnexin Is an Active Participant in the Proteasome-mediated
Disposal of Misfolded AAT--
In the absence of COOH-terminal amino
acids, human AAT is unable to progress to the correctly folded
conformation (34). In pulse-chase studies using
[35S]methionine, the terminally misfolded molecules were
completely eliminated from stably transfected mouse hepatoma cells
within 4 h of chase (t1/2 = 2 h) (Fig.
1a). Although unaffected by
the lysosomotrophic amine chloroquine, intracellular disposal was
inhibited >85% upon incubation of cells with the membrane-permeable
peptide aldehydes N-acetyl-leu-leu-norleucinal and
N-acetyl-leu-leu-methioninal (Fig. 1a), both of
which are nonspecific inhibitors of proteasomal degradation (35).
Nearly complete inhibition occurred upon incubation with lactacystin, a
specific irreversible covalent inhibitor of the proteasome (36), confirming the participation of the multicatalytic proteolytic complex
in the disposal process.
Incubation of cells with the glucosidase inhibitor castanospermine
prior to pulse-radiolabeling with [35S]methionine was
performed to maintain asparagine-linked oligosaccharides in the
original Glc3Man9GlcNAc2 structure,
preventing posttranslational assembly between the newly synthesized
glycoprotein and calnexin (25). Under these conditions, intracellular
turnover was unaffected by each of the three proteasome inhibitors
(Fig. 1a). Because no insoluble radioactive AAT was detected
under the latter conditions (not shown), these findings indicate that
misfolded molecules are substrates for degradation by an alternative
proteolytic system under conditions that prevent physical interaction
with calnexin.
Endogenous Mannosidase Activity Abrogates the Dissociation of
Misfolded AAT from Calnexin Prior to Proteolysis--
Simultaneous
detection of calnexin-associated (6.8 S) and released AAT monomers (4.5 S) by velocity sedimentation (25) has provided evidence for the
partitioning of the unfolded glycoprotein between the
chaperone-associated and unbound state. A sedimentation coefficient of
6.8 S was exhibited by the entire population of misfolded AAT molecules
following a 4-h chase with lactacystin (Fig.
2a, LCT), and the observation
was duplicated in two additional experiments. This anomaly coincided
with the quantitative coimmunoprecipitation of radiolabeled AAT with
antiserum against calnexin (Fig. 2b). An identical
manipulation did not hinder the secretion of wild-type AAT (not shown),
indicating that the ability of lactacystin treatment to prevent the
release of the glycoprotein from calnexin was restricted to the
terminally misfolded molecule. Because the detection of 4.5 S AAT
monomers requires posttranslational release from calnexin (25), it
became apparent that the abrogated dissociation of the 6.8 S complex
precedes proteasomal degradation, possibly as a normal step in the
disposal process.
Enhanced electrophoretic mobility of radiolabeled molecules in SDS-PAGE
(Fig. 1b, CO), which results from the removal of mannose from attached oligosaccharides (24, 25), was arrested, as was
intracellular disposal, by incubating cells with 1-deoxymannojirimycin (Fig. 1b, DMJ), an inhibitor of ER mannosidases (37).
However, of the two events, only intracellular disposal was arrested
with lactacystin (Fig. 1b, LCT), confirming that the removal
of mannose from asparagine-linked oligosaccharides precedes
proteolysis. The importance of this finding was revealed in the next
set of experiments, in which a population of released misfolded
monomers, in addition to the 6.8 S complex, was detected following
coincubation of cells with both lactacystin and the mannosidase
inhibitor (Fig. 2a, LCT+DMJ). Under these conditions, the
relative ratio of the 6.8 and 4.5 S species was identical to that
observed when disposal had been arrested with 1-deoxymannojirimycin
alone (Fig. 2a, DMJ). These findings demonstrate that
intracellular mannosidase activity is responsible for abrogating the
dissociation of misfolded AAT from calnexin prior to proteolysis.
The Activity of ER Mannosidase I Is Required for Disposal--
In
the absence of sufficient material to characterize the mannose content
of attached oligosaccharides, we examined the effect of processing
inhibitors on AAT disposal as a means to identify the mannosidase
activity essential for degradation. Because the oligosaccharide-dependent size shift has been localized to
the ER (24), we examined the involvement of two processing
Attenuated Deglucosylation of Attached Oligosaccharides Is a
Rate-limiting Step in AAT Disposal--
Reversible glucosylation of
asparagine-linked oligosaccharides mediates the partitioning of
glycoproteins between the calnexin-associated and unbound state (41,
42). Results from separate in vitro studies have
demonstrated that whereas glucose transfer by UDP-glucose:glycoprotein glucosyltransferase (UGTR) to asparagine-linked
Man8GlcNAc2 remains 70% as efficient as for
the Man9GlcNAc2 precursor (43), subsequent deglucosylation of Glc1Man8GlcNAc2
by glucosidase II is diminished to only 21% of that observed for
Glc1Man9GlcNAc2 substrate (44). Because the deglucosylation of
Glc1Man8GlcNAc2 is very
inefficient, at least for the protein-free oligosaccharide (44), in the
next set of experiments we tested the hypothesis that the removal of a
single mannose unit from multiple oligosaccharides attached to the
unbound monomer prevents complete dissociation of the complex following
assembly with calnexin, as depicted in Fig.
4. The accuracy of this prediction was
first addressed by asking whether any of the oligosaccharides of
calnexin-associated AAT contained glucose following inhibition of
proteolysis with lactacystin. Because the removal of mannose by jack
bean mannosidase is restricted by terminal glucose units (Fig.
5a), differential migration of mannosidase-digested glycoproteins in SDS-PAGE was used as a method to
identify the number of oligosaccharides attached to
[35S]methionine-radiolabeled AAT that are glucosylated
(45). For AAT, a total of four glycoforms can be generated by this
methodology in which either 0, 1, 2, or all 3 attached oligosaccharides
contain glucose. However, two predominant bands were resolved by
SDS-PAGE following lactacystin treatment (Fig. 5b, LCT).
When compared with the migration of known electrophoretic mobility
standards (Fig. 5b, Stds), it was concluded that these
species represented populations of calnexin-associated AAT in which
either two or all three asparagine-linked oligosaccharides contain
glucose (Fig. 5b, LCT). A slight variation in this pattern
has been observed in four additional experiments, including the
infrequent detection of a faster migrating band in which only a single
glycan contains glucose (not shown). Importantly, the latter species
has routinely represented <10% of the total glycoform population,
suggesting that it represents a minor species. Finally, radiolabel from
[14C]galactose (46) was incorporated as glucose into
asparagine-linked oligosaccharides of the misfolded glycoprotein during
cycloheximide treatment (not shown), indicating that the sugar units
had been added posttranslationally, as would be expected for substrates of UGTR.
Next, we attempted to establish a causal relationship between the
attenuation of oligosaccharide deglucosylation and proteasomal degradation of misfolded AAT. Because a dynamic interaction with calnexin allows for the liberation of bound glycoprotein substrates in
response to the deglucosylation of asparagine-linked oligosaccharides (41), we took advantage of the 2-h half-life of the misfolded protein
to test the effect of a posttranslational glucosidase blockade as a
means to bypass the predicted role of mannose removal to attenuate
oligosaccharide deglucosylation. For this, pulse-radiolabeled cells
were chased with medium containing the glucosidase inhibitor castanospermine to directly inhibit the activity of glucosidase II
following cotranslational assembly of molecules with calnexin. Disposal
of misfolded AAT was accelerated >2-fold under these conditions (Fig.
6a, lane 4) as compared with
control cells (lane 2). Accelerated intracellular turnover
was also observed when pulse-radiolabeled cells were chased with
deoxynojirimycin (Fig. 6b, DNJ), an alternative glucosidase
inhibitor and glucose analog (37). In contrast, incubation with the
galactose analog, deoxygalactojirimycin, had no influence on the rate
of degradation (Fig. 6b, DgalJ). A ~1.5-h lag preceded the
acceleration of degradation (not shown), possibly reflecting the time
required for either inhibitor to attain an appropriate concentration in
the ER. It is unlikely that accelerated degradation resulted from the
displacement of misfolded AAT by castanospermine because the compound
is a transition state analog of the glucosidase reaction, rather than a
true structural analog of glucose (37). Coincubation of cells with
1-deoxymannojirimycin during the posttranslational glucosidase blockade
had no inhibitory effect on the accelerated rate of disposal (Fig.
6a, lane 5), as compared with when the mannosidase inhibitor
was used alone (lane 3). These results were consistent with
the notion that the slower means of attenuating oligosaccharide
deglucosylation, which normally occurs through the removal of mannose,
had been successfully bypassed via the direct inhibition of glucosidase
II. Finally, degradation was greatly retarded by incubating cells with
the proteasomal inhibitor lactacystin during the posttranslational glucosidase blockade (Fig. 6b, LCT+CST),
indicating that the rapid rate of degradation had reflected an
accelerated rate of entrance into the proteasome-mediated disposal
pathway. Under none of these conditions was misfolded AAT secreted into
the medium or was the secretion of wild-type AAT impaired (not shown).
Together, these findings are consistent with the notion that the
attenuated removal of glucose from asparagine-linked oligosaccharides
functions as the underlying rate-limiting step in the
proteasome-mediated degradation of the misfolded glycoprotein.
The complete inhibitory effect of lactacystin on misfolded AAT
disposal suggests that its elimination is accomplished almost exclusively by the proteasome, as has been reported for an additional misfolded human AAT variant expressed in transfected yeast (16), transduced human fibroblasts (17), and a cell-free rabbit reticulocyte lysate system supplemented with canine pancreatic microsomal membranes (17). Although the nonproteasomal disposal pathway, which may be of the
same type observed for some other mutant ER-situated proteins (27, 32,
35), was not detected in any of these heterologous expression systems
(17), our data confirm that calnexin is an active participant in the
proteasomal degradation of misfolded AAT, as first reported by Qu
et al. (17).
Although it is well documented that the reversible glucosylation of
asparagine-linked oligosaccharides can facilitate glycoprotein folding
(41, 42), the role of ER-situated mannosidase activity in regulating
the disposal of unfolded glycoproteins has been appreciated only
recently (25, 30-32). A molecular explanation for the role of
ER-situated mannose trimming in AAT disposal is depicted in Fig.
7, and outlined below, extending the
glycoprotein folding model originally proposed by Hammond et
al. (47). In the absence of conformational maturation, persistent
rounds of binding to calnexin, as reflected by the simultaneous
detection of the 6.8 and 4.5 S species, functions as a "retention
phase," preventing movement to the Golgi complex (Fig. 7). Because
glucose transfer by UGTR is restricted to only nonnative glycoproteins (48), it is predicted that conformational maturation results in the
permanent release of AAT from the calnexin binding cycle, allowing
transport of only functional molecules to the Golgi complex, where
further modification of attached glycans can occur (38) prior to
secretion (Fig. 7).
1-antitrypsin was examined in hepatoma cells to
identify the functional role of asparagine-linked oligosaccharide
modification in the selection of glycoproteins for degradation by the
cytosolic proteasome. Proteasomal degradation required physical
interaction with the molecular chaperone calnexin. Altered
sedimentation of intracellular complexes following treatment with the
specific proteasome inhibitor lactacystin, and in combination with
mannosidase inhibition, revealed that the removal of mannose from
attached oligosaccharides abrogates the release of misfolded
1-antitrypsin from calnexin prior to proteasomal
degradation. Intracellular turnover was arrested with kifunensine,
implicating the participation of endoplasmic reticulum mannosidase I in
the disposal process. Accelerated degradation occurred in a
mannosidase-independent manner and was arrested by lactacystin, in
response to the posttranslational inhibition of glucosidase II,
demonstrating that the attenuated removal of glucose from attached
oligosaccharides functions as the underlying rate-limiting step in the
proteasome-mediated pathway. A model is proposed in which the removal
of mannose from multiple attached oligosaccharides directs calnexin in
the selection of misfolded
1-antitrypsin for degradation
by the proteasome.
INTRODUCTION
Top
Abstract
Introduction
References
1-antitrypsin (AAT)
from liver hepatocytes. Human AAT is a monomeric glycoprotein that is a
member of the serine proteinase inhibitor superfamily that
protects lung elastin fibers from elastolytic destruction. Defective
intracellular transport of the aberrantly folded glycoprotein diminishes circulating levels of the inhibitor, resulting in the elastolytic destruction of lung elastin (reviewed in Ref.
20).
MATERIALS AND METHODS
RESULTS
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Fig. 1.
Effect of proteasomal inhibitors on the
disposal of misfolded AAT and modification of asparagine-linked
oligosaccharides. a, the percentage of radiolabeled
misfolded AAT molecules remaining in cells after a 4-h chase in media
containing either no additions (CO), 50 µM
chloroquine (CLQ), 100 µM
N-acetyl-leu-leu-norleucinal (ALLN), 100 µM N-acetyl-leu-leu-methioninal
(ALLM), or 25 µM lactacystin (LCT)
(solid bars). Cells were treated as above, except that
media, including that used for preincubation, were supplemented with
0.2 mg/ml castanospermine (open bars). b,
electrophoretic mobility of immunoprecipitated misfolded AAT in
SDS-PAGE following a 15-min pulse (P) and subsequent 3-h
chase (C) in cells treated with either no additions
(CO), 25 µM lactacytin (LCT), or 1 mM 1-deoxymannojirimycin (DMJ). Enhanced
electrophoretic mobility is shown by an asterisk.
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Fig. 2.
Differential sedimentation of misfolded AAT
in sucrose gradients following inhibition of disposal.
a, relative ratio of radiolabeled molecules sedimenting at
either 6.8 S (closed bars) or 4.5 S (open bars)
following a 4-h chase in cells incubated with medium containing either
25 µM lactacystin (LCT), 1 mM
1-deoxymannojirimycin (DMJ), or a combination of the two
inhibitors (LCT + DMJ). Each bar represents the
average result of three determinations. b, equal aliquots of
the 6.8 S species resulting from lactacystin treatment were subjected
to coimmunoprecipitation analyses. Shown are immunoprecipitation of
misfolded AAT (A), immunoprecipitation of misfolded AAT
following its release from a calnexin immunoprecipitate (C
A), and incubation with protein G-agarose following the
release of misfolded AAT from a calnexin immunoprecipitate
(C
pG), as a negative control.
-mannosidases, each of which can initiate the removal of mannose
from asparagine-linked oligosaccharides in higher eukaryotes (38) and
is inhibited with 1-deoxymannojirimycin (39). Of the two enzymes, ER
mannosidase I catalyzes the removal of the outermost mannose unit from
the middle branch of Man9GlcNAc2 (Fig.
3a), and is inhibited by the plant alkaloid kifunensine (39). A separate mannose unit is excised by
the kifunensine-resistant ER mannosidase II (40), which is sensitive to
both swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol (38).
Incubation of cells with kifunensine arrested the intracellular disposal of misfolded AAT (Fig. 3b, KIF), whereas no
demonstrable effect was exerted by either of the ER mannosidase II
inhibitors (Fig. 3b, SWN, DIM), indicating that trimming of
attached glycans to Man8GlcNAc2, by ER
mannosidase I, is responsible for mediating the onset of misfolded AAT
degradation by the proteasome in these cells.
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Fig. 3.
Effect of ER mannosidase inhibitors on
misfolded AAT disposal. a, the branched
Man9GlcNAc2 structure is shown that contains
nine mannose units (closed circles) and two GlcNAc units
(closed squares). Shown are the sites of hydrolysis by ER
mannosidases (ER Man) I and II and the site at which glucose
is added by UGTR (shaded arrow). b,
electrophoretic mobility of immunoprecipitated misfolded AAT in
SDS-PAGE following a 15-min pulse (P) and subsequent 3-h
chase (C) in cells incubated with medium containing either
no additions (CO), 100 µM kifunensine
(KIF), 100 µM swainsonine (SWN), or
1 mM 1,4-dideoxy-1,4-imino-D-mannitol
(DIM). Enhanced electrophoretic mobility of misfolded AAT is
shown by an asterisk.
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Fig. 4.
Predicted influence of asparagine-linked
oligosaccharides on the partitioning of misfolded AAT between the
calnexin-associated and unbound state. Vertical arrows
represent the order of mannose removal from oligosaccharides attached
to the misfolded AAT (A) monomer by ER-situated
mannosidases. Numbers above and below the
horizontal arrows represent the results of separate in
vitro studies in which the relative percent efficiency of
glucosylation by UGTR (43) and deglucosylation by glucosidase II (44),
respectively, was determined for each of the depicted oligosaccharide
species. Our prediction was that intracellular dissociation from
calnexin (C) would be abrogated following assembly with the
AAT monomer bearing multiple asparagine-linked oligosaccharides with
the Glc1Man8GlcNAc2 structure
(asterisk) because it is a poor substrate for
deglucosylation. The number of glucose (Glc) and mannose
(Man) units in the oligosaccharides attached to AAT is shown
for each step.
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Fig. 5.
Detection and quantitation of glucosylated
asparagine-linked oligosaccharides. a, strategy for
detecting glycoprotein bearing glucosylated oligosaccharides. A
terminal glucose unit (open square) hinders the removal of
mannose residues (closed circles) by jack bean mannosidase
(JBM). b, electrophoretic mobility in SDS-PAGE of
radiolabeled misfolded AAT. The differential electrophoretic mobility
of standards (Stds) is shown in which all three or none of
the attached oligosaccharides contain glucose (see under "Materials
and Methods"). Following a 4-h chase with lactacystin
(LCT), radiolabeled misfolded AAT was immunoprecipitated
from cells and then was either mock-digested (lane 1),
digested with jack bean mannosidase (lane 2), or digested
with endoglycosidase for total deglycosylation (lane 3)
prior to fractionation by SDS-PAGE and detection by fluorography. For
this experiment, all samples were subjected to SDS-PAGE on the same
gel, and the predicted number of asparagine-linked oligosaccharides
that contain glucose is shown to the right of each
panel.
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Fig. 6.
Disposal of misfolded AAT is accelerated in
response to a posttranslational glucosidase blockade.
a, electrophoretic mobility of immunoprecipitated misfolded
AAT in SDS-PAGE following a 15-min pulse (lane 1) and 3-h
chase (lanes 2-5) in cells incubated with medium containing
combinations of 1 mM 1-deoxymannojirimycin (DMJ)
and 0.2 mg/ml castanospermine (CST). The former compound was
included in preincubation and chase medium, whereas the latter was
present only during the chase. Enhanced electrophoretic mobility of
misfolded AAT is shown by an asterisk. b, quantitation of
the percentage of radiolabeled misfolded AAT molecules remaining in
cells following the above treatments and after a 3-h chase in medium
containing 1 mM deoxynojirimycin (DNJ), 1 mM deoxygalactojirimycin (DgalJ), and 25 µM lactacystin plus and 0.2 mg/ml castanospermine
(LCT + CST) in which the former compound was included in
preincubation and chase medium, whereas the latter was present only
during the chase.
DISCUSSION
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Fig. 7.
Proposed pathway for AAT quality
control. Following cotranslational addition of
Glc3Man9GlcNAc2 (closed
vertical arrow) to nascent AAT (A), partial
deglucosylation by glucosidases I and II (GI, GII) generates
asparagine-linked Glc1Man9GlcNAc2,
which promotes cotranslational assembly with calnexin (C).
The correctly folded glycoprotein (square) is no longer
recognized by UGTR (U), ensuring that only the functional
molecule is delivered to the Golgi complex for subsequent secretion.
Removal of mannose from glycans attached to correctly folded AAT does
occur, but it does not result in degradation because these are not
substrates for UGTR. In the absence of bound calnexin, which occurs
upon preincubation of cells with the glucosidase inhibitor
castanospermine (CST), misfolded molecules are degraded by a
nonproteasomal pathway, confirming that calnexin is an active
participant in proteasomal degradation and identifying the existence of
an alternative disposal mechanism. The terminally misfolded
glycoprotein (circle) enters a retention phase consisting of
rounds of binding to calnexin (represented by the 4.5 S and
6.8 S species), in which assembly and dissociation are
driven by the activities of UGTR and glucosidase II, respectively.
Modification by ER mannosidase I (ER Man I) allows the
glucosylation of oligosaccharides attached to the monomer, but it
attenuates subsequent deglucosylation and abrogates dissociation of the
6.8 S complex because
Glc1Man8GlcNAc2 is a poor substrate
for glucosidase II. Abrogated dissociation from calnexin initiates the
entrance of misfolded AAT into the degradation phase of the proteasomal
pathway in which several steps are arrested by the inhibition of
proteolysis during treatment with lactacystin (LCT).
Inhibition of ER mannosidase I (ERMI) with
1-deoxymannojirimycin (DMJ) or kifunensine (KIF)
uncouples the retention and degradation phases. The predicted number of
glucose (Glc) and mannose (Man) units in the
oligosaccharides attached to AAT is shown for each step.
Predominance of the 6.8 S species during intracellular retention of the misfolded protein (25) suggests that binding to calnexin is kinetically favored over that of release. In a dynamic, rather than static, interaction with calnexin, as previously suggested by Zapun et al. (41), oligosaccharide deglucosylation can precede the complete dissociation and detection of the monomer as the 4.5 S species. In an earlier report (25), we observed that the degradation of misfolded AAT coincided with the appearance of the released 4.5 S monomer. It now seems likely that this observation might reflect a greater level of accessibility of oligosaccharides to ER mannosidases when misfolded AAT exists as the dissociated monomer and can rebind calnexin only after reglucosylation by UGTR. UGTR, which reglucosylates oligosaccharides attached to only nonnative proteins (47), is responsible for selectively recognizing the misfolded AAT monomer and promoting its reassembly with calnexin (Fig. 7). Gradual modification of oligosaccharides attached to the monomer, regardless of the conformational state of the underlying protein, serves as a timer that marks a period during which the polypeptide is provided with multiple attempts to fold properly. Following the removal of a critical number of mannose residues, attenuated deglucosylation by glucosidase II targets the misfolded molecule for degradation by the proteasome (Fig. 7). As such, the removal of mannose functions as a molecular switch to initiate the disposal of misfolded AAT, whereas modification of oligosaccharides attached to correctly folded glycoproteins, even those resident to the ER, should have no effect on their stability because these glycans are not substrates for reglucosylation by UGTR (48). Possibly, the enhanced migration of misfolded AAT in SDS-PAGE may correspond to what Qu et al. (17) had considered to represent an endoproteolytic intermediate.
In the present study, inhibitors of ER mannosidase I arrested proteasomal disposal without disturbing the relative ratio of the 4.5 and 6.8 S species, resulting in a futile cycle of binding to calnexin. Thus, it is likely that trimming of attached oligosaccharides to Man8GlcNAc2, by ER mannosidase I, couples intracellular retention and degradation processes. Although ER mannosidase II apparently is not essential for triggering the proteasome-mediated degradation of misfolded AAT in the mouse hepatoma cell line, we cannot disregard the possibility that it may play a pivotal role in glycoprotein quality control in other cell lines in which the relative level of its activity is elevated (40). Nevertheless, our findings indicate that an oligosaccharide modification that is common to several distinct proteins, rather than a specific amino acid sequence, can function as a signal to "recognize" a misfolded molecule for degradation by the proteasome. By employing this posttranslational modification as the criterion for degradation, it can now be understood how misfolded molecules are preferentially degraded whereas nascent folding intermediates are provided with ample opportunity to progress to the native conformation, if attainable, prior to oligosaccharide modification.
Although some hydrophobic interaction does exist between misfolded AAT and calnexin (24), several lines of evidence, including (i) the detection of only the 6.8 S complex following lactacystin treatment, (ii) the reversal of this anomaly upon mannosidase inhibition, (iii) the presence of glucose on multiple asparagine-linked glycans of calnexin-associated AAT following lactacystin treatment, and (iv) the accelerated degradation of molecules in response to a simple posttranslational glucosidase blockade, indicate that attenuated oligosaccharide deglucosylation is the molecular event that initiates entrance into the proposed "degradation phase," which sorts misfolded AAT for proteasomal degradation (Fig. 7). Although our study indicates that oligosaccharides of calnexin-associated AAT do contain glucose, these findings do not necessarily reflect an accurate assessment of the number of possible lectin-binding sites present in the molecular chaperone because the persistence of glucose could result from the attenuated deglucosylation of an unbound glycan in response to the removal of mannose. However, it is now apparent that by extending the role of calnexin beyond that of a mere molecular chaperone, ER-situated mannosidase activity occupies a previously unrecognized elevated hierarchical position among the conformation-based glycoprotein quality control machinery.
Abrogated dissociation from calnexin following lactacystin treatment precluded a detailed analysis of the predicted role that calnexin might play in the degradation phase of proteasome-mediated disposal. However, one hypothesis to be considered is that because calnexin is a type I transmembrane protein (49), abrogated dissociation might function to tether misfolded lumenal AAT to the ER membrane, providing a kinetically favorable situation for subsequent physical interaction with retrograde transport machinery, members of which have been implicated as components of the ER translocon (11, 12, 14). Calnexin would serve as a suitable candidate for this purpose because at least one copurifying protein has been shown to cross-link with nascent polypeptides during their translocation into the ER (49). In support of this notion, physical interaction with calnexin has been identified following the inhibition of the degradation of a COOH-terminally truncated form of ribophorin under conditions arresting ubiquitin conjugating activity (14). Furthermore, Qu et al. (17) have reported evidence that the induced ubiquination of the cytoplasmic tail of associated calnexin precedes proteasomal degradation of an additional genetic variant of human AAT expressed in a cell-free rabbit reticulocyte system supplemented with canine pancreatic microsomal membranes (17). In the present study, ubiquitin was associated with <10% of calnexin-associated AAT following lactacystin treatment, and none was associated with the bound molecular chaperone (not shown). Also, indirect immunfluorescence microscopy and protease protection analyses have verified that the delivery of misfolded AAT to the cytoplasm is blocked by lactacystin treatment.2 Because polyubiquination is often required for the cytoplasmic delivery of ER-derived protein substrates (8, 9, 14), removal of the covalent modification by isopeptidases during inhibition of proteolysis might account for the continued ER residence and physical interaction of misfolded AAT with calnexin. Not all proteasomal substrates bear asparagine-linked oligosaccharides, indicating that alternative mechanisms must function in the selection of nonglycosylated ER-situated proteins for degradation by this pathway, none of which apparently recognize terminally misfolded AAT as substrate. Therefore, it is tempting to consider the possibility that a common mechanism for sorting ER-derived proteins to the proteasome is to maintain or promote a constant physical interaction with the translocon.
An essential role for asparagine-linked
Man8GlcNAc2 in initiating the degradation of
mutant carboxypeptidase Y in Saccharomyces cerevisiae was
recently reported by Jakob et al. (32). Although the
predicted role of the attached glycan in the disposal process in yeast
differs from what is proposed to occur in hepatoma cells, data from the
two studies agree that the development of systems to eliminate soluble
secretory glycoproteins in lower and higher eukaryotes has centered
around the conserved Man8GlcNAc2 structure. Elucidation of the participation of calnexin in proteasomal disposal pathway awaits further experimentation, as does an assessment of how
the nonproteasomal pathway might participate in heritable plasma AAT deficiency.
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ACKNOWLEDGEMENTS |
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We express our appreciation to Drs. Kelley W. Moremen and Robert G. Spiro for their expert advice with regard to the differential inhibition of ER mannosidase activities and to Dr. David B. Williams for sharing his knowledge of the optimal oligosaccharide structure necessary for binding calnexin.
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FOOTNOTES |
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* This work was supported in part by an American Lung Association research career investigator award, by an American Heart Association grant-in-aid, by the Moran Foundation (to R. N. S), and by an American Lung Association research training fellowship (to P. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Pathology, Baylor College of Medicine, One Baylor Plaza, Houston,
TX 77030. Tel.: 713-798-3169; Fax: 713-798-5838; E-mail:
rsifers{at}bcm.tmc.edu.
2 P. Choudhury, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
ER, endoplasmic
reticulum;
AAT, 1-antitrypsin;
PAGE, polyacrylamide gel
electrophoresis;
UGTR, UDP-glucose:glycoprotein
glucosyltransferase.
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REFERENCES |
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