(Received for publication, November 22, 1996, and in revised form, January 13, 1997)
From the Department of Pathology, Protection of lung elastin fibers from
proteolytic destruction is compromised by inefficient secretion of
incompletely folded allelic variants of human
Conformational maturation of nascent polypeptides is facilitated
by transient physical interaction with molecular chaperones. The
general consensus is that rounds of binding prevent misfolding and
subsequent entrance of partially folded intermediates into nonproductive folding pathways (1). In eukaryotic cells, nascent polypeptides destined to traverse compartments of the secretory pathway
are translocated into the lumen of the endoplasmic reticulum (ER)1 during biosynthesis (2). Incompletely
folded proteins often exhibit a persistent physical association with
one or more molecular chaperones and are retained in the ER prior to
intracellular disposal (for a review, see Ref. 3). This mechanism has
been termed "quality control" (4) and apparently functions to
ensure transport of only correctly folded proteins beyond the ER.
Calnexin (also designated p88 or IP90), a calcium-binding molecular
chaperone of the ER membrane (5), forms a transient noncovalent
association with several newly synthesized proteins in the ER (6-14).
An important feature of calnexin is that it exhibits an affinity for
monoglucosylated oligosaccharides (15), which are intermediates formed
during cotranslational trimming by ER Protein folding and quality control machinery may participate in the
molecular pathogenesis of plasma Intracellular retention of most "null" AAT variants results from
mutations that cause premature truncation of the polypeptide at its
carboxyl terminus (34), a phenomenon predicted to prevent formation of
specific secondary structural features (32, 35). In this study,
conformation-based quality control of human AAT secretion was
investigated in mouse hepatoma cells stably expressing the
nonfunctional allelic variant QO Hong Kong (null(Hong Kong)), which is
incapable of folding into the appropriate native structure. Null(Hong
Kong) exhibits a Ca2+-sensitive physical interaction
with the molecular chaperone calnexin during intracellular retention
(36) and requires release from the molecular chaperone as well as
post-translational trimming of its asparagine-linked oligosaccharides
for normal disposal. Predicted roles for each of these events in the
quality control of human AAT secretion are discussed.
Protein G-agarose, castanospermine,
and 1-deoxymannojirimycin were purchased from Calbiochem. Importantly,
1-deoxymannojirimycin purchased from this source did not inhibit
protein synthesis. Tunicamycin (homolog A1), cycloheximide, and all
salts and buffers were purchased from Sigma. Easy Tag
[35S]methionine was purchased from DuPont NEN. All media
used in tissue culture were purchased from Life Technologies, Inc.
Fetal bovine serum was procured from Summit Biotechnology.
An immunoglobulin fraction of goat anti-human AAT
was purchased from Organon Teknika-Cappel. Rabbit polyclonal antisera
against a synthetic polypeptide homologous to amino acids 487-505 in
the cytoplasmic tail of canine calnexin was a gift from Drs. John J. M. Bergeron (McGill University) and Ari Helenius (Yale University).
The cell line H1A/N13 was previously
generated by stable transfection of the mouse hepatoma cell line Hepa
1a with subcloned DNA encoding the human null(Hong Kong) AAT variant
(37). Cells were maintained as monolayers in Dulbecco's modified
Eagle's/Waymouth medium (3:1) containing 15% fetal bovine serum,
1 × glutathione, and 1 × penicillin/streptomycin. The cell
line remained under selective pressure with the antibiotic G418
(37).
H1A/N13 cells were
maintained in 100-mm diameter dishes. For metabolic radiolabeling of
proteins, confluent cell monolayers were washed with phosphate-buffered
saline and then incubated for the specified period in methionine-free
Dulbecco's modified Eagle's medium containing
[35S]methionine (150-300 µCi). A 30-min incubation in
methionine-free Dulbecco's modified Eagle's medium preceded addition
of radiolabel in all experiments in which the pulse was for 15 min.
When appropriate, cells were "chased" by washing monolayers with
warmed phosphate-buffered saline and then incubated in regular growth
medium containing a 4-fold excess of unlabeled methionine. Inhibitors
of oligosaccharide processing activities were added to cells 60 min
prior to methionine starvation and were present in pulse-radiolabeling
and chase media. Cell lysis was performed by first washing monolayers
with cold phosphate-buffered saline, followed by scraping with a rubber spatula in 0.1 M Tris-HCl, pH 7.4, containing 0.5% Nonidet
P-40 and a mixture of protease inhibitors (36). Insoluble material was
removed by centrifugation for 10 min at 10,000 × g.
When appropriate, lysates were frozen on dry ice. A previously
described technique (38) was used for immunological detection of
radiolabeled human AAT in the insoluble fraction of the cell lysate,
but in no case was any detected.
After metabolic
radiolabeling, cells were lysed as described above, and intracellular
complexes containing variant null(Hong Kong) were separated by
sedimentation in linear 5-20% sucrose gradients as described
previously (39). Normally, the lysate generated from a single 100-mm
diameter dish of cells was applied to a single gradient. Following
centrifugation, gradients were fractionated, from the top, with a
Buchler Densi-Flow II apparatus (39) prior to immunoprecipitation of
specific proteins.
Coprecipitation of proteins was
performed by incubating soluble cell lysates for 2 h at 4 °C in
the presence of protein G-agarose containing specific immobilized
antiserum against human AAT or canine calnexin (36). Immunocomplexes
were washed by agitation at 4 °C with cell lysis buffer containing
0.5 M NaCl. For detection of newly synthesized null(Hong
Kong) bound to calnexin, double immunoprecipitations were performed as
described previously (6). For this, calnexin was quantitatively
immunoprecipitated from a selected sucrose gradient fraction during a
1-h incubation with a vast excess of anti-calnexin preimmobilized to
protein G-agarose. Immunoprecipitates were washed at 4 °C with cell
lysis buffer containing 0.5 M NaCl. Coprecipitated
null(Hong Kong) was dissociated from the calnexin immunoprecipitate by
incubation for 10 min at 37 °C in 0.05 M Tris-HCl, pH
7.4, containing 0.5% Nonidet P-40 and 2 mM EGTA (36), and
the immunocomplex was washed twice with 0.5 ml of cell lysis buffer.
The supernatant and washes were combined and incubated at 4 °C for
16 h with an excess of anti-human AAT preimmobilized to protein
G-agarose for quantitative immunoabsorption of released null(Hong
Kong). For fractionation by SDS-PAGE, immunocomplexes were heated for
10 min at 75 °C in disruption buffer containing 2% sodium dodecyl
sulfate. Radiolabeled proteins were detected by fluorographic
enhancement of the vacuum-dried gel and quantitated by scintillation
spectrophotometry of excised gel pieces.
Proteins were electrophoretically
transferred to Hybond nitrocellulose (Amersham Corp.) following
fractionation by SDS-PAGE. Blots were incubated with a 1:1000 dilution
of rabbit polyclonal antisera raised against a synthetic polypeptide
homologous to amino acids 487-505 in the cytoplasmic tail of canine
calnexin (see above). Immunoreactivity was detected by enhanced
chemiluminescent Western blotting (ECL, Amersham Corp.) according to
the manufacturer's instructions, except that SuperSignal reagent
(Pierce) was used as the detection reagent.
Variant null(Hong Kong) is not secreted from stably
transfected mouse hepatoma cells (37), and its interaction with the molecular chaperone calnexin has been reported (36). Intracellular populations of null(Hong Kong) were separated by sucrose gradient sedimentation and detected by immunoprecipitation following incubation of H1A/N13 cells with [35S]methionine for 5 h (Fig.
1). Approximately 70% of the radiolabeled molecules
exhibited a sedimentation coefficient of ~6.8 S (Fig. 1A)
and were physically associated with a coprecipitating 90-kDa protein
(p90) (Fig. 1B, lane 4) recognized by antiserum
against calnexin (Fig. 1C, lane 4). The absence
of p90, as well as no detectable immunoreactivity with
anti-calnexin in mock-immunoprecipitated material (Fig. 1,
B and C, lane 3), was consistent with the
previous finding (36) that calnexin is physically associated with
variant null(Hong Kong). A smaller population of molecules (~30% of
the total) was detected by this method and exhibited a sedimentation coefficient of 4.5 S (Fig. 1A). Coprecipitation of calnexin
was not detected (Fig. 1, B and C, compare
lanes 1 and 2), consistent with a previous report
(39) in which the 4.5 S species was identified as the human AAT
monomer.
Sucrose gradient
sedimentation and coprecipitation techniques were used to characterize
the time course of assembly between calnexin and newly synthesized
null(Hong Kong). The entire population of newly synthesized molecules
radiolabeled during a 15-min incubation with
[35S]methionine sedimented at 6.8 S (Fig.
2A). In a double immunoprecipitation analysis, radiolabeled null(Hong Kong) was detected in a calnexin immunoprecipitate generated from the 6.8 S species (Fig.
3A, lane 2). Because less than
quantitative coprecipitation may occur by this methodology, an
experiment was performed in which the null(Hong Kong)-calnexin
interaction was disrupted by chelation of calcium ions (36) prior to
sucrose gradient sedimentation. This shifted sedimentation of the
entire population of radiolabeled molecules to 4.5 S (Fig.
2A) and coincided with the disappearance of radiolabeled null(Hong Kong) in the calnexin immunoprecipitate (Fig. 3A,
lane 4). Importantly, sedimentation of the 6.8 S complex was
unaffected in response to an identical incubation without EGTA (data
not shown), suggesting that dissociation was specific.
Ware
et al. (15) have reported that calnexin exhibits an affinity
for monoglucosylated oligosaccharides, and for many glycoproteins, interaction with calnexin is prevented by compounds that inhibit asparagine-linked glycosylation or that arrest cotranslational trimming
of attached glucose residues (6, 7, 20-22). Since covalent addition of
the oligosaccharide moiety
Glc3-Man9-GlcNAc2 occurs
cotranslationally (16), the necessity of oligosaccharides in generating
the null(Hong Kong)-calnexin association was tested by preincubating
H1A/N13 cells with tunicamycin prior to pulse radiolabeling (Fig.
4). Inhibition of asparagine-linked glycosylation was
confirmed by increased electrophoretic mobility of pulse-radiolabeled molecules (Fig. 4A, panel TUN, lane
1). Assembly with calnexin was not detected (Table
I). Since the three terminal glucose residues are
subjected to cotranslational hydrolysis (16), cells were preincubated
with castanospermine, an inhibitor of ER
Asparagine-linked glycosylation and trimming of oligosaccharides effect
assembly of nascent null(Hong Kong) with calnexin
1-antitrypsin from hepatocytes. Pulse-chase radiolabeling with [35S]methionine and sucrose gradient
sedimentation and coimmunoprecipitation techniques were employed to
investigate quality control of human
1-antitrypsin
secretion from stably transfected mouse hepatoma cells. The
secretion-incompetent variant null(Hong Kong) (Sifers, R. N.,
Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335) cannot fold
into a functional conformation and was quantitatively associated with the molecular chaperone calnexin following biosynthesis. Assembly with
calnexin required cotranslational trimming of glucose from asparagine-linked oligosaccharides. Intracellular disposal of pulse-radiolabeled molecules coincided with their release from calnexin. Released monomers and intracellular disposal were nonexistent in cells chased with cycloheximide, an inhibitor of protein synthesis. Post-translational trimming of asparagine-linked oligosaccharides and intracellular disposal were abrogated by 1-deoxymannojirimycin, an
inhibitor of
-mannosidase activity, without affecting the monomer
population. The data are consistent with a recently proposed quality
control model (Hammond, C., Braakman, I., and Helenius, A. (1994)
Proc. Natl. Acad. Sci. U. S. A. 91, 913-917) in which intracellular disposal requires dissociation from calnexin and post-translational trimming of mannose from asparagine-linked oligosaccharides.
-glucosidases (16).
Importantly, it can also be generated by post-translational
reglucosylation of glycans, an event catalyzed by the ER resident
protein UDP-glucose:glycoprotein glucosyltransferase (17-19).
Interaction with calnexin can be prevented by drugs that either inhibit
asparagine-linked glycosylation or arrest cotranslational trimming of
attached glucose residues (6, 7, 20-22) and is virtually nonexistent
in mutant cell lines deficient in ER
-glucosidase I or II (23).
1-antitrypsin (AAT) deficiency (24, 25). Human AAT is a monomeric glycoprotein of 394 amino
acids (26, 27) and is secreted from liver hepatocytes (28). It is a
member of the serine proteinase inhibitor superfamily (29) and protects
lung elastin fibers from proteolytic destruction by inhibiting the
activity of elastase released from activated neutrophils (30). Several
allelic variants of the inhibitor exist (31), and many exhibit a
distinct mutation predicted to preclude conformational maturation of
the encoded polypeptide following biosynthesis (32). Defective
intracellular transport of the aberrantly folded protein through
compartments of the secretory pathway can diminish circulating levels
of the inhibitor (25). Proteolytic destruction of lung elastin is
associated with severe plasma AAT deficiency and is implicated in
the pathogenesis of chronic obstructive lung disease (33).
Chemicals and Reagents
Differential Sedimentation of Intracellular Null(Hong Kong)
Populations
Fig. 1.
Detection of monomeric and
calnexin-associated variant null(Hong Kong). H1A/N13 cells were
incubated for 5 h with medium containing
[35S]methionine, and the soluble cell lysate was
subjected to sucrose gradient sedimentation prior to
immunoprecipitation of human AAT from gradient fractions. A,
the sedimentation profile of immunoprecipitated proteins is expressed
as percent of total immunoprecipitating radioactivity associated with
variant null(Hong Kong) (NHK) (closed circles)
and coprecipitating p90 (open circles). Calculated Svedberg units (S) are shown for each peak. B, shown is a fluorogram
after SDS-PAGE of proteins detected in the 4.5 S peak (fraction 12) and
6.8 S peak (fraction 22) following incubation with (lanes 2 and 4) or without (lanes 1 and 3)
human AAT antiserum. C, proteins in a gel identical to that
in B were electrophoretically transferred to nitrocellulose
and incubated with a 1:1000 dilution of antiserum against canine
calnexin to detect immunoreactivity by ECL Western blotting. The lanes
are identical to those shown in B. Lane 5 is a blank, and
lane 6 is a crude cell extract used for detection of
endogenous calnexin (Cxn) in H1A/N13 cells.
[View Larger Version of this Image (41K GIF file)]
Fig. 2.
Cotranslational association with calnexin and
evidence for release of monomers. H1A/N13 cells were
pulse-radiolabeled for 15 min with medium containing
[35S]methionine, and soluble cell lysates were subjected
to sucrose gradient sedimentation following the pulse (A) or
after a 2-h chase (B) prior to immunoprecipitation of human
AAT from selected gradient fractions (closed circles). Also
shown are cell lysates incubated for 10 min at 37 °C in the presence
of 2 mM EGTA prior to sucrose gradient sedimentation
(open circles). Data are expressed as percent of total
immunoprecipitating radioactivity. Calculated Svedberg units (S) are
shown for individual peaks.
[View Larger Version of this Image (25K GIF file)]
Fig. 3.
Detection of pulse-radiolabeled variant
null(Hong Kong) bound to calnexin. Double immunoprecipitation
analysis (see "Materials and Methods") was carried out on
radiolabeled null(Hong Kong) as shown in Fig. 2. A, pulse;
B, 2-h chase. Lanes 1, 3, and
5, direct immunoprecipitation of null(Hong Kong)
(NHK) with human AAT antiserum (A); lanes
2, 4, and 6, immunoprecipitated null(Hong
Kong) following release from a calnexin immunoprecipitate (Cxn
A). In each case, fraction 12 was used for the 4.5 S peak, and
fraction 22 was used for the 6.8 S peak.
[View Larger Version of this Image (35K GIF file)]
-glucosidases I and II
(16), to maintain the oligosaccharide in the original Glc3-Man9-GlcNAc2 structure.
Inhibition of glucose was confirmed by retarded electrophoretic
mobility of pulse-radiolabeled null(Hong Kong) (Fig. 4A,
panel CST, lane 1). Again, assembly with calnexin was prevented (Table I).
Fig. 4.
Asparagine-linked glycosylation and trimming
of oligosaccharides effect disposal of nascent null(Hong Kong).
A, H1A/N13 cells were preincubated for 1 h with medium
containing no additions (CO), tunicamycin (TUN),
castanospermine (CST), or 1deoxymannojirimycin (DM) prior to a 15-min pulse with
[35S]methionine. Concentrations of each compound are
shown in Table I. SDS-PAGE was carried out on cells lysed after the
pulse (lane 1) or following 180 min of chase (lane
2) prior to immunoprecipitation and detection of radiolabeled
variant null(Hong Kong) (NHK). The mobility of two protein
molecular mass markers is shown in kilodaltons, and the
asterisk shows the increased mobility of null(Hong Kong) that accompanies its intracellular retention. B, percent of
pulse-radiolabeled null(Hong Kong) molecules immunoprecipitated from
cells after the 180-min chase is shown in the form of a bar graph. The
percent of calnexin-associated molecules (6.8 S peak) is shown in
black, and the percent of monomers (4.5 S peak) is shown in
white.
[View Larger Version of this Image (31K GIF file)]
Preincubationa
Inhibits
Predicted oligosaccharide
structure
NHKb associated with calnexinc
%
Control
None
Man8-9-GlcNAc2
100
Tunicamycin (10 µg/ml)
N-Linked
glycosylation
None present
0
Castanospermine (0.1 mg/ml)
-Glucosidases I and
II
Glc3-Man9-GlcNAc2
0
Deoxymannojirimycin (1 mM)
-Mannosidase
Man9-GlcNAc2
100
a
Monolayers were incubated with various inhibitors for
60 min prior to pulse radiolabeling.
b
NHK, null(Hong Kong).
c
Values were determined by sucrose gradient sedimentation and
double immunoprecipitation.
Variant null(Hong Kong) undergoes intracellular disposal with a half-life of ~2 h (40). However, enhanced instability was observed in cells preincubated with either tunicamycin or castanospermine as compared with control cells (Fig. 4A), suggesting that interaction with calnexin protects from intracellular proteolysis. Since the entire population of pulse-radiolabeled molecules of variant null(Hong Kong) was bound to calnexin, we asked whether appearance of the 4.5 S monomer population, representing release from calnexin, coincided with the onset of intracellular disposal. Approximately 70% of the radiolabeled molecules continued to sediment at 6.8 S after 2 h of chase (Fig. 2B). Double immunoprecipitation analysis demonstrated their physical interaction with calnexin (Fig. 3B, lane 4). Furthermore, the 6.8 S species was quantitatively shifted to 4.5 S following incubation of the cell lysate with 2 mM EGTA (Fig. 2B), which coincided with loss of interaction with calnexin (Fig. 3B, lane 6). Importantly, ~30% of the radiolabeled molecules sedimented at 4.5 S after the 2-h chase (Fig. 2B), and no physical interaction with calnexin was detected by double immunoprecipitation (Fig. 3B, lane 2). Since the entire population of pulse-radiolabeled molecules was associated with calnexin immediately following biosynthesis (Fig. 2A), detection of the 4.5 S species was consistent with the idea that this population had been released during the chase.
Intracellular Disposal Coincides with Appearance of the 4.5 S SpeciesSince intracellular disposal of pulse-radiolabeled
null(Hong Kong) is preceded by a 45-min lag (40), we asked whether
appearance of released monomers coincided with the onset of disposal.
The radiolabeled 4.5 S species was not detected until 60 min of chase (Fig. 5). At this time point, 20% of the radiolabeled
molecules sedimented at 4.5 S (Fig. 5). Approximately 80% sedimented
at 6.8 S, and only these exhibited a physical association with calnexin as judged by double immunoprecipitation analysis and altered
sedimentation in response to EGTA (data not shown). Significantly,
appearance of the 4.5 S species coincided with the first detectable
loss of null(Hong Kong). The ratio of calnexin-associated (6.8 S) and monomeric (4.5 S) molecules remained relatively constant during the
next 2 h, during which the majority of molecules were removed from
the cell. It should be noted that radiolabeled null(Hong Kong) was not
detected in the insoluble portion of the cell lysate (data not shown),
which would be expected if loss of immunopreciptable molecules had
resulted from their insolubility.
Inhibition of Protein Synthesis Prevents Appearance of the 4.5 S Species and Arrests Intracellular Disposal
Cycloheximide is an
inhibitor of protein synthesis and somehow blocks intracellular
disposal of several transport-impaired proteins in the secretory
pathway (40, 41). Disposal of pulse-radiolabeled null(Hong Kong) was
nonexistent during a 3-h chase in medium containing 20 µg/ml
cycloheximide (Fig. 6A). Furthermore, the
gradual increase in the electrophoretic mobility of variant null(Hong
Kong) that normally accompanies its intracellular retention (40) was
absent (Fig. 6A, asterisk). Puromycin, another
inhibitor of protein biosynthesis, had these same effects (data not
shown). Significantly, arrested disposal coincided with our inability
to detect the radiolabeled 4.5 S species by sucrose gradient
sedimentation (Fig. 6B). Cycloheximide did not reverse the
intracellular stability of null(Hong Kong) in cells preincubated with
either tunicamycin or castanospermine (Fig. 6B), consistent
with the idea that the mechanism of inhibiting disposal required
association with calnexin.
1-Deoxymannojirimycin Arrests Disposal without Affecting Appearance of the 4.5 S Species
Deglycosylation with endoglycosidase H and
subsequent fractionation by SDS-PAGE indicated that the gradual
increase in the electrophoretic mobility of variant null(Hong Kong)
during intracellular retention had resulted from post-translational
modification of asparagine-linked oligosaccharides (data not shown).
This conclusion was supported by the ability of 1-deoxymannojirimycin,
a mannose analog capable of inhibiting the activity of several ER and
Golgi -mannosidases (42-45), to prevent this post-translational
anomaly (Fig. 4A, panel DM). Preincubation with
this mannose analog did not prevent nascent null(Hong Kong) from
interacting with calnexin (Table I). However, intracellular disposal of
pulse-radiolabeled molecules was completely arrested by this treatment
(panel DM). Unlike inhibition during cycloheximide
treatment, both the 4.5 S and 6.5 S species were detected in cells
after 3 h of chase (Fig. 6B), and their ratio was
almost identical to that detected in untreated cells (Fig.
4B). This was no different at the 60-, 120-, and 180-min
time points (not shown). Assembly of newly synthesized null(Hong Kong)
with calnexin was not affected by preincubation with
1-deoxymannojirimycin (Table I). Furthermore, 1-deoxymannojirimycin did
not prevent disposal of null(Hong Kong) in cells coincubated with
either tunicamycin or castanospermine (Fig. 4B). This
indicated that inhibition of disposal by 1-deoxymannojirimycin required that null(Hong Kong) interact with calnexin. The data suggested that
inhibition of
-mannosidase activity was responsible for arresting
intracellular disposal of variant null(Hong Kong).
Since hepatocytes are the major site for biosynthesis and
secretion of AAT (28, 46), stably transfected mouse hepatoma cells were
used to study conformation-based quality control of human AAT
secretion. Variant null(Hong Kong) was employed for this analysis
because truncation of amino acids at its carboxyl terminus prevents
formation of all three large -sheet structures common to members of
the serine proteinase inhibitor superfamily (32, 35). The correctly
folded variant PI M1(Val-213) is rapidly secreted from these cells,
whereas null(Hong Kong) is subjected to intracellular retention and
disposal (37, 40). In this study, cosedimentation and
coimmunoprecipitation analyses indicated that newly synthesized
molecules sedimented at 6.8 S, which coincided with their quantitative
association with the molecular chaperone calnexin. This indicates that
assembly of the complex occurred cotranslationally or immediately
following biosynthesis.
Interaction with calnexin was prevented by inhibiting asparagine-linked glycosylation with tunicamycin or by arresting cotranslational trimming of attached glucose residues with castanospermine. This indicates that like many other glycoproteins (11, 47), oligosaccharides may somehow function to facilitate assembly between null(Hong Kong) and calnexin. Intracellular disposal of nascent null(Hong Kong) was accelerated in cells preincubated with either tunicamycin or castanospermine, suggesting that disposal occurs in the absence of the bound molecular chaperone. This idea was consistent with appearance of the 4.5 S species at a time point that coincided with the onset of intracellular disposal. The absence of associated calnexin and sedimentation at 4.5 S suggests that this population of molecules represents the released monomer (36, 39). No changes in the monomer population were observed when cell lysis was performed under less stringent conditions (data not shown), suggesting that the 4.5 S population does not merely reflect weakening of the null(Hong Kong)-calnexin interaction during retention, but represents unassociated molecules. It should be pointed out that in an earlier report (36), an error in our fractionation method, overlap between the 4.5 S and 6.8 S species, and incomplete dissociation of complexes in response to incubation with EGTA led to the incorrect conclusion that an additional sedimenting species was detected under steady-state conditions. It is now apparent that the most abundant forms of null(Hong Kong) are the 4.5 S and 6.8 S species. However, we cannot rule out the possibility that additional, more transient species exist.
Intracellular disposal of null(Hong Kong) was arrested during incubation of pulse-radiolabeled cells with cycloheximide, and this coincided with complete absence of the 4.5 S monomer population. Cycloheximide had no demonstrable effect on the intracellular stability of null(Hong Kong) in cells preincubated with either tunicamycin or castanospermine, which supported the idea that dissociation from calnexin required protein synthesis. Although our experiments have not addressed the mechanism by which dissociation was inhibited, one possibility is that a short-lived protein is required for normal disruption of the null(Hong Kong)-calnexin complex. Cycloheximide treatment had the added effect of preventing post-translational trimming of oligosaccharides attached to null(Hong Kong). At present, we do not know whether this reflects inaccessibility of glycans for trimming while null(Hong Kong) is bound to calnexin. However, previous experiments have suggested that the null(Hong Kong)-calnexin interaction is stabilized by a peptide-peptide interaction (36). Furthermore, incubation with several combinations of monosaccharides has failed to dissociate the complex after coprecipitation,2 suggesting that the glycan moiety is not involved in stabilizing the interaction after assembly. These data are in agreement with a two-step binding model proposed by Tector and Salter (12) in which the function of the monoglucosylated oligosaccharide is to initiate assembly of glycoproteins with calnexin. However, we cannot disregard the possibility that a lectin-like interaction does exist, but is disrupted during immunoprecipitation.
Intracellular disposal of null(Hong Kong) was also arrested in cells
treated with 1-deoxymannojirimycin, which also prevented demonstrable
post-translational trimming of asparagine-linked oligosaccharides.
Originally, only one -mannosidase activity had been assigned to the
ER (16). However, in recent years, several
-mannosidases have been
identified in the ER, many of which are inhibited by
1-deoxymannojirimycin (42-45). Unlike cycloheximide treatment, in
which inhibition of disposal coincided with a complete absence of the
4.5 S peak, inhibition with 1-deoxymannojirimycin did not alter the
monomer population. Taken together, data from this study suggest that
variant null(Hong Kong) is subjected to a quality control pathway
identical to that recently proposed by Hammond and Helenius (11). Their
model proposes that persistent association between unfolded
glycoproteins and calnexin reflects a continuous cycle of binding in
which assembly of the complex is facilitated by reglucosylation of
asparagine-linked oligosaccharides by the ER resident enzyme
UDP-glucose:glycoprotein glucosyltransferase (17-19, 48). Importantly,
the model predicts that permanent dissociation from the binding cycle
will occur when hydrolysis of mannose residues generates an
oligosaccharide unable to participate as a glucose acceptor, thereby
leading to disposal of the unfolded glycoprotein. Consistent with this
model, inhibition of oligosaccharide trimming by 1-deoxymannojirimycin
abrogated intracellular disposal of variant null(Hong Kong) without
affecting the percent population of 4.5 S monomers present in the cell.
This latter finding would be expected if the protein is degraded only
when it can no longer participate in the cycle of binding to calnexin,
which would result after extensive post-translational trimming of
glycans by
-mannosidase. Further dissection of this quality
control pathway will be the subject of future investigations and will
enhance our current understanding of how incompletely folded human AAT
variants are retained and degraded in hepatocytes.
We thank Drs. John J. M. Bergeron and Ari Helenius for gifts of specific antisera to calnexin.