From the Program in Lung and Cell Biology, Hospital for Sick Children, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1X8, Canada
Received for publication, October 9, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Deletion of phenylalanine at position 508 ( Cystic fibrosis (CF)1 is
one of the most prevalent lethal genetic disorders among Caucasian
populations (1). The CF gene encodes the cystic fibrosis transmembrane
conductance regulator (CFTR), a cAMP-regulated Cl The hallmark of CF is the loss of cAMP-activated chloride conductance
in the epithelial plasma membrane of airways, intestine, and exocrine
glands (6-8). More than 900 mutations have been identified in the CF
gene, leading to impaired biosynthesis, processing, activation, and/or
stability of CFTR (7, 8). The most frequent mutation, deletion of
phenylalanine at position 508 ( The recognition that the Here we provide direct biochemical and functional evidence for the
biological instability of the complex-glycosylated Cell Lines--
A mixture of stably transfected baby hamster
kidney (BHK) cells, expressing human wt and Isolation of Microsomes--
Isolation of ER, Golgi, and plasma
membrane-enriched microsomes from BHK cells was performed using
nitrogen cavitation and differential centrifugation as described (11).
Where specified, the core-glycosylated wt or mutant CFTR was eliminated
from the cells during a 3-h incubation in the presence of cycloheximide (CHX, 100 µg/ml). The microsomal pellet was resuspended in HSE medium
(10 mM sodium HEPES, 0.25 M sucrose, pH 7.6)
and used either immediately or after being snap-frozen in liquid nitrogen.
Limited Proteolysis and Glycosidase Digestion--
Microsomes
were isolated from
To distinguish between high mannose and complex-type
N-linked oligosaccharide modification of CFTR, cell lysates
were incubated with endoglycosidase H (7 µg/ml) and peptide
N-glycosidase F (31 µg/ml) at 37 °C for 3 h. The
mobility shift of deglycosylated CFTR was visualized with
immunoblotting using anti-HA mAb.
Electrophoresis and Immunoblotting--
CFTR immunoblotting was
performed with anti-HA mAb (Babco). Proteolytic digestion patterns were
visualized with the mouse monoclonal M3A7 and L12B4 anti-CFTR Abs.
L12B4 localizes to the region of the cytoplasmic NBD1 of CFTR (epitope
within the range of amino acid positions 386 and 412), and M3A7
localizes to the region of the cytoplasmic NBD2 of CFTR (epitope within
the range of amino acid positions 1365 and 1395) (11).
Immunoblotting of ERP72 and GRP78 was performed with the rabbit
polyclonal (SPA720) and mouse monoclonal (SPA826, Stressgen)
antibodies, respectively. Immunoblots, with multiple exposures, were
quantified using DuoScan transparency scanner and N.I.H. Image 6.1 software (developed by the National Institutes of Health and available
at their web site).
Metabolic Labeling and Immunoprecipitation--
Metabolic
labeling and immunoprecipitation were performed essentially as
described (4). Monolayer cells were pulse-labeled under defined
conditions and chased at the indicated temperature for the time
intervals specified for each experiment. Membrane proteins were
solubilized in 1 ml of RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, and 0.5% sodium
deoxycholate, pH 8.0) supplemented with protease inhibitors (10 µg/ml
leupeptin and pepstatin, 0.5 mM phenylmethylsulfonyl
fluoride, and 10 mM iodoacetamide). Immunoprecipitates,
obtained with anti-HA or M3A7 and L12B4 anti-CFTR mAbs, were analyzed
by SDS-polyacrylamide gel electrophoresis and fluorography. The
radioactivity incorporated was quantified using a PhosphorImager
(Molecular Dynamics) with ImageQuant software (Molecular Dynamics)
(4).
Biotinylation of Cell Surface CFTR--
Cells were rinsed
(H-buffer: 154 mM NaCl, 10 mM HEPES, 3 mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM glucose, pH 7.6)
and biotinylated with 1 mg/ml
sulfosuccinimidyl-2-(biotinamido)ethyl-1,2-dithiopropionate (EZ-Link
sulfo-NHS-SS-biotin, Pierce) three times for 15 min at 37 °C.
Following the solubilization of the cells in RIPA buffer, biotinylated
CFTRs were affinity-isolated on streptavidin-Sepharose (Sigma),
separated with SDS-polyacrylamide gel electrophoresis, and visualized
with anti-HA mAb and ECL.
Determination of cAMP-stimulated Iodide Conductance--
The
plasma membrane cAMP-dependent halide conductance of
transfected BHK cells was determined with iodide efflux as described (35). In brief, the chloride content was replaced with iodide by
incubating the cells in loading buffer (136 mM NaI, 3 mM KNO3, 2 mM
Ca(NO3)2, 11 mM glucose, 20 mM HEPES, pH 7.4) for 60 min at room temperature. Iodide
efflux was initiated by replacing the loading buffer with efflux medium
(composed of 136 mM nitrate in place of iodide). The
extracellular medium was replaced every minute with efflux buffer (1 ml). After a steady-state was reached, the intracellular cAMP level was
raised by agonists (10 µM forskolin, 0.2 mM
CTP-cAMP, and 0.2 mM isobutylmethylxanthine) to achieve maximal phosphorylation of Thermoaggregation Assay--
Following the solubilization of BHK
cells in Laemmli sample buffer, the aggregation tendency of wt and
mutant CFTR was compared by exposing the lysate to temperatures ranging
from 37 to 100 °C for 5 min. SDS-resistant macromolecular aggregates
were sedimented with centrifugation (17,000 × g for 15 min). Monomeric mutant and wt CFTR, ERP72, GRP78, and
Na+/K+-ATPase remaining in the supernatant were
measured with quantitative immunoblotting using the appropriate Ab and
ECL.
Deletion of Phe-508 Compromises the Stability of CFTR in the
Post-ER Compartments--
To attain high sensitivity immunodetection,
the influenza HA epitope-tagged
The biological stability of the complex-glycosylated wt and rescued
To verify that the deletion of Phe-508 destabilizes the
complex-glycosylated CFTR, metabolic pulse-chase experiments were performed on transfectants expressing wt or rescued Biochemical and Functional Stability of
The plasma membrane proteins of BHK cells, expressing
The lack of endogenous cAMP-dependent anion conductance of
BHK cells permitted us to monitor the arrival of functional
Taken together, the immunoblot, metabolic pulse-chase, biotinylation,
and iodide measurements provide the first direct evidence that the
functional and biochemical half-life of the complex-glycosylated The Thermostability of Rescued The Impact of
The Ta of rescued
Limited proteolysis in conjunction with immunoblot analysis was used as
an alternative and more direct method to demonstrate that the rescued
The in situ protease resistance of rescued While "saturation" of the ER quality control cannot be
precluded in heterologous systems overexpressing A number of mechanisms, or the combination thereof, could explain the
biological instability of the complex-glycosylated The indistinguishable in vivo and in vitro
protease susceptibility profile of the core-glycosylated In summary, the in vivo turnover measurements together with
the protease susceptibility and thermoaggregation results indicate that
whereas the F508) is the most common cystic fibrosis (CF)-associated mutation
in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated
chloride channel. The consensus notion is that
F508 imposes a
temperature-sensitive folding defect and targets newly synthesized CFTR
for degradation at endoplasmic reticulum (ER). A limited amount of CFTR
activity, however, appears at the cell surface in the epithelia of
homozygous
F508 CFTR mice and patients, suggesting that the ER
retention is not absolute in native tissues. To further elucidate the
reasons behind the inability of
F508 CFTR to accumulate at the
plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and
functional measurements show that rescued
F508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4-20-fold accelerated degradation rate between 37 and 40 °C is, most likely, due to decreased conformational stability of the rescued
F508 CFTR, demonstrated by in situ protease susceptibility and
SDS-resistant thermoaggregation assays. We propose that the decreased
stability of the spontaneously or pharmacologically rescued mutant may
contribute to its inability to accumulate at the cell surface. Thus,
therapeutic efforts to correct the folding defect should be combined
with stabilization of the native
F508 CFTR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channel
and conductance regulator, expressed at the apical membrane of
secretory epithelia (2, 3). CFTR, a member of the ABC transporter
family, consists of two structurally homologous halves, each comprised
of six transmembrane (TM) helices and a nucleotide binding domain (NBD1
and NBD2), which are connected by the regulatory (R) domain (3). This
complex, multidomain structure conceivably renders the
posttranslational folding of wild type (wt) CFTR inefficient. More than
50% of the newly synthesized wt CFTR remains incompletely folded and
is degraded at the endoplasmic reticulum (ER), whereas the remaining
25-50% undergoes an ATP-dependent conformational maturation and is exported to the cis/medial-Golgi, where its complex
glycosylation can occur (4, 5).
F508) in the NBD1, is found in >90%
of the patients and detected in ~70% of CF chromosomes (1, 7). It is
believed that deletion of Phe-508 interrupts the posttranslational
folding of CFTR (4, 5, 9-11) and targets the core-glycosylated folding
intermediate for degradation, predominantly via the
ubiquitin-proteasome pathway at the ER (12, 13). Exposure of
ER-retention signals may contribute to the inability of folding
intermediate(s) to exit the ER (14). Accordingly, negligible expression
of
F508 CFTR could be detected at the cell surface by immunochemical
techniques in recombinant cells, CF primary airway cells, and CF
tissues (9, 15, 16).
F508 CFTR channel is functional both
in vivo (17-19) and after its reconstitution into the
phospholipid bilayer (20) suggested that the CF phenotype could be
alleviated by relocating the mutant CFTR from the ER to the plasma
membrane. Reduced temperature (10, 21, 22), chemical chaperones
(23-25), and down-regulation of Hsp70 (26, 27) activity are thought to
partially revert the folding defect of
F508 CFTR and promote the
accumulation of the functional channel at the cell surface. Importantly, using more sensitive electrophysiological techniques, constitutive accumulation of
F508 CFTR was documented in the plasma
membrane of primary epithelia from
F508 homozygous mice (21, 28) and
in the intestinal and gallbladder epithelia of homozygous
F508
patients (29, 30). These studies parallel the results of recent
immunolocation reports to some extent and suggest that the processing
defect of the
F508 CFTR is tissue-specific (30-33). If the ER
retention of the mutant is not complete, accelerated disposal from the
post-ER compartments could contribute to its inability to express at
the physiological level in certain tissues. Indeed, based on indirect
evidence, we proposed that the rescued
F508 CFTR has a short
residence time at the cell surface of Chinese hamster ovary cells
(CHO), expressing
F508 CFTR heterologously (34). However, neither
this nor any other study has provided direct biochemical or structural
information regarding the behavior of
F508 CFTR following its escape
from ER.
F508 CFTR.
Furthermore, we show that the stability defect is
temperature-sensitive, which is likely due to an attenuated
conformational stability of the native state, demonstrated by increased
protease susceptibility and the thermoaggregation tendency of the
rescued mutant relative to its wt counterpart. The implications of
these observations are fundamental with respect to understanding the
cellular phenotype and designing more efficient therapeutic strategies
in CF.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F508 CFTR with a
carboxyl-terminal hemagglutinin (HA) epitope, was generated and
maintained as described (4). Characterization of the HA-tagged CFTR
variants will be described elsewhere.
F508 and wt CFTR expressor BHK cells and
incubated at a protein concentration of 1.3-1.5 and 0.8-1.0 mg/ml,
respectively, in the presence of trypsin or proteinase K for 15 min at
4 °C in digestion buffer (phosphate-buffered saline) as
described. Proteolysis was terminated by the addition of
phenylmethylsulfonyl fluoride to 1 mM, and samples were
immediately denatured in 2× Laemmli sample buffer at 37 °C for 20 min.
F508 CFTR, and collection of the efflux medium resumed for an additional 6-9 min. The amount of iodide in each
sample was determined with an iodide-selective electrode (Orion).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F508 and wt CFTR were expressed
stably in BHK cells. Immunoblot analysis showed that the HA-tagged
F508 CFTR appears as an endoglycosidase H (endo-H)- and peptide
N-glycosidase F (PNGase-F)-sensitive, core-glycosylated
polypeptide with an apparent molecular mass
140-150 kDa (Fig.
1a, empty
arrowhead) at 37 °C (9).
The processing defect of the
F508 CFTR could be partially overcome
by the combination of glycerol and low temperature treatment, similarly
to its nontagged counterpart, as reported in a number of cultured cells
(10, 23, 24). Following the optimization of the rescue conditions,
accumulation of the complex-glycosylated
F508 CFTR was indicated by
the appearance of endo-H-resistant immunoreactive polypeptide with an
apparent molecular mass
170 kDa (Fig.
1a, black
arrowhead) (9). The N-linked oligosaccharide modification and the apparent molecular mass of the rescued
F508 CFTR are virtually identical to that of the HA-tagged
complex-glycosylated wt CFTR (Fig. 1).
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Fig. 1.
Stability of the complex-glycosylated
F508 and wt CFTR cellular pool. The expression
of HA-tagged wt and
F508 CFTR in stably transfected BHK cells was
monitored with immunoblotting, using anti-HA mAb and ECL. Black
arrowhead, complex-glycosylated (band C); empty
arrowhead, core-glycosylated (band B); gray
arrowhead, unglycosylated CFTR (band A). a,
the processing defect of the
F508 CFTR was partially reverted by
incubating the cells in medium supplemented with 10% glycerol at
26 °C for overnight. Endo-H and PNGase F digestions were performed
as described under "experimental Procedures." To facilitate
immunodetection of the rescued mutant, cells were incubated for 1 h with cycloheximide. 50, 100, and 20 µg of protein were separated
from
F508, rescued
F508, and wt CFTR expressors, respectively.
b, the disappearance of complex-glycosylated wt CFTR was
monitored in the presence of BFA (5 µg/ml) or CHX (100 µg/ml).
Subsequent to the indicated chase, cells were solubilized and equal
amounts of protein (20 µg) were immunoblotted with anti-HA mAb.
c, a similar approach was used to determine the in
vivo stability of the complex-glycosylated
F508 CFTR, after a
24-h rescue treatment (see a). Expression of
Na+/K+-ATPase was monitored with
anti-Na+/K+-ATPase mAb. For reference, total
protein from untreated wt (20 µg) and
F508 CFTR expressors (100 µg) were loaded (two lanes on the far right).
d, half-life determination of the wt and mutant CFTR. The
complex-glycosylated wt and
F508 CFTR persisting in the cell was
calculated from densitometry of immunoblots shown on b and
c. The chase was performed in the absence or presence of CHX
or BFA, as indicated at 37 °C. Data are expressed as percentage of
the initial complex-glycosylated wt or
F508 CFTR. Mean ± S.E.
(n = 3-8).
F508 CFTR was determined upon inhibition of protein biosynthesis with CHX or vesicular transport from ER to Golgi with brefeldin A
(BFA). After the accumulation of the complex-glycosylated
F508 CFTR
at 26 °C, cells were incubated in the presence of CHX or BFA at
37 °C, and the remaining CFTR was measured with quantitative immunoblotting, using anti-HA mAb as a function of incubation time
(Fig. 1, b and c). Densitometry revealed that the
half-life of the complex-glycosylated
F508 CFTR
(t1/2
5 h) is at least four times shorter
than that of the wt CFTR (t1/2
22 h) at
37 °C, regardless of the inhibitor (Fig. 1d). Similarly
fast disposal of the
F508 CFTR (t1/2
5 h) could be observed in the absence of CHX or BFA, after shifting the
temperature from 26 to 37 °C during the chase (Fig.
1c).
F508 CFTR (Fig.
2a). Phosphorimage analysis
confirmed that the complex-glycosylated
F508 was eliminated four
times faster (t1/2
4.5 h) than wt CFTR
(t1/2
18 h) at 37 °C (Fig.
2b). Similar turnover rates were obtained upon rescuing the
mutant with either glycerol or reduced temperature alone, and on
mock-treated wt CFTR, ruling out that osmotic or cold stress can
account for the difference (data not shown). The accelerated
disappearance of
F508 CFTR also cannot be attributed to the loss of
the carboxyl-terminal epitope, because similar half-lives were measured
with antibodies to epitopes located in NBD1, NBD2, or at the
amino-terminal tail (data not shown). Finally, preliminary results
obtained on pancreatic ductal epithelia expressing the
F508 CFTR
constitutively showed that the mutation impairs the stability of CFTR
in polarized epithelia to a degree comparable with that found in
nonpolarized cells (data not shown). These results collectively
support the notion that the instability is an intrinsic property of the
rescued
F508 CFTR rather than epitope- or cell-specific.
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Fig. 2.
Turnover of metabolically labeled
complex-glycosylated F508 and wt CFTR.
A, wt and
F508 CFTR expressors were metabolically labeled
for 20 min and 3 h, respectively, under the conditions indicated.
Conversion of the core-glycosylated into the complex-glycosylated form
was ensured during a 2-h chase, prior to the stability of the
complex-glycosylated form being assessed. After the chase at
37 °C for the indicated time, CFTR was immunoprecipitated and
visualized with fluorography. b, radioactivity associated
with the complex-glycosylated wt and
F508 CFTR was measured with
phosphorimage analysis and expressed as a percentage of the initial
incorporation at time 0 (means ± S.E., n = 3-4).
F508 CFTR at the Cell
Surface--
To examine whether the turnover of the plasma
membrane-associated mutant CFTR is similar to that of the
complex-glycosylated
F508 CFTR pool, which comprises the
trans-Golgi network, secretory vesicles, and endosomes as
well, the fate of the rescued
F508 CFTR was followed with cell
surface biotinylation and iodide efflux measurements.
F508, rescued
F508, or wt CFTR, were covalently tagged with NHS-SS-biotin, affinity-isolated on streptavidin beads, and immunoblotted with anti-HA
mAb. Both the rescued
F508 and the wt CFTR are amenable to
biotinylation, in contrast to the ER-resident, core-glycosylated
F508 CFTR (Fig. 3a).
Densitometric analysis revealed that
50% of the biotinylated
F508 CFTR disappeared after 4 h and became undetectable by
10 h of chase at 37 °C (Fig. 3b). In contrast, the
turnover of biotinylated wt CFTR was more than 4-fold slower (t1/2
18 h, data not shown) than the
rescued
F508 CFTR but comparable with the complex-glycosylated wt
CFTR pool (Fig. 1d and 2b).
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Fig. 3.
The turnover of F508
CFTR at the cell surface. a, the turnover of
biotinylated
F508 CFTR. Rescued
F508 CFTR were covalently labeled
with NHS-SS-biotin and chased for 0-10 h at 37 °C. Biotinylated
F508 CFTR was affinity-isolated on streptavidin beads and
immunoblotted with anti-HA mAb. For comparison, biotinylated and
nonbiotinylated wt CFTR are also shown. The core-glycosylated forms of
neither the wt nor the mutant CFTR are susceptible to biotinylation. A
fraction of cell lysate from the corresponding samples was directly
probed with anti-HA mAb (lower panel). b,
disappearance kinetic of biotinylated
F508 CFTR at 37 °C. The
amount of biotinylated
F508 CFTR remaining was measured with
densitometry of immunoblots, obtained in experiments shown on
a, and expressed as the percentage of biotinylated
F508
CFTR before the chase (means ± S.E., n = 3).
F508 CFTR to the plasma membrane by the iodide efflux assay. At permissive temperatures, the cAMP-stimulated iodide release was proportional with
the length of the rescue period up to 8 h (Fig.
4a, inset). After
allowing
F508 CFTR to accumulate at the cell surface for 5 h,
raising the temperature to 37 °C evoked a rapid disappearance of the
mutant. The amount of iodide released by cAMP-dependent protein kinase stimulation decreased by 50% after 4 h and
became undetectable after 10 h of chase at 37 °C (Fig. 4,
a and b) in cells expressing rescued
F508,
whereas no decrease was apparent over 10 h in wt CFTR expressors.
Because the cAMP-activated iodide release could not be detected in
rescued mock-transfected and parental BHK cells (data not shown), the
functional and the biotinylation studies jointly indicate that the
F508 CFTR channels are as unstable at the cell surface as in the
post-ER compartments.
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Fig. 4.
Residence time of functional
F508 CFTR at the plasma membrane.
a, expression level of
F508 CFTR at the cell surface was
inferred based on the amount of cAMP-stimulated iodide release from BHK
cells. Iodide release was proportional to the duration of the rescue
period up to 8 h (inset). To avoid saturation of iodide
release assay, subsequent measurements were performed on cells exposed
to 26 °C and glycerol for 5 h and to 37 °C for 2 h (no
chase). Continuous incubation at 37 °C up to 10 h progressively
eliminated the cAMP-stimulated iodide release (4 h and 10 h
chase). cAMP-dependent protein kinase-agonist mixture was
added at 0 min (arrow). Data points are averages of
triplicate determinations in a representative experiment. b,
the disappearance kinetic of functional
F508 and wt CFTR from the
cell surface. The magnitude of mean cAMP-stimulated iodide release was
determined on
F508 and wt CFTR expressors before and after the
rescue as described in a. CHX (100 µg/ml) was added to the
chase medium for the wt CFTR-expressing cells. The iodide efflux was
measured after 1 and 2 min of cAMP-dependent protein kinase
stimulation for the wt and mutant CFTR, respectively, and expressed as
percentage of that in the absence of chase (mean ± S.E.,
n = 3-4).
F508 CFTR is 4-5-fold shorter than its wt counterpart at 37 °C, suggesting that structural differences may persist between the rescued
F508 and wt CFTR at the plasma membrane.
F508 CFTR in Vivo--
To test
whether the stability defect of the complex-glycosylated
F508 CFTR
at 37 °C is related to its thermolability, pulse-labeled cells were
chased at temperatures ranging from 28 to 40 °C. Although the
t1/2 of wt and rescued
F508 CFTR converged at
28 °C (
50 h), a striking difference became apparent at
temperatures above 30 °C. Rescued
F508 CFTR was at least 20-fold
more unstable (t1/2
0.8 h) than its wt
counterpart (t1/2
18 h) at 40 °C (Fig.
5, a and b). In
sharp contrast, no difference could be resolved in the relative
turnover rates of the core-glycosylated (ER-resident) wt and
F508
CFTR between 28 and 40 °C (Fig. 5b), consistent with the
notion that the core-glycosylated form represents a common folding
intermediate, which is less susceptible to thermal denaturation (4, 5,
11). Considering that unfolding of soluble and membrane proteins upon
heat shock accelerates their cellular degradation (36), our results
suggest that the thermal resistance of the complex-glycosylated
F508
CFTR toward unfolding is substantially lower than its wt counterpart.
Because large quantities of purified CFTR are not available to test
this hypothesis directly, the protease susceptibility of native and the
thermoaggregation propensity of solubilized CFTR variants were measured
as indirect indicators of their structural stability.
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Fig. 5.
The stability defect of the rescued
F508 CFTR is temperature-sensitive.
a, after the pulse labeling of wt (20 min at 37 °C) and
F508 CFTR (3 h at 26 °C in the presence of 10% glycerol)
expressors, the conversion of core- to complex-glycosylated form was
allowed to occur for 2 h at 37 °C. Subsequent chase was
performed at the indicated temperatures. For comparison, both wt and
rescued
F508 CFTR were loaded. b, the turnover of the
core- and complex-glycosylated
F508 and wt CFTR was determined with
phosphorimage analysis from experiments shown on a
(means ± S.E., n = 3-4, inset and
data not shown). The ratio of complex-glycosylated wt and
F508 CFTR
half-lives (filled circle) is plotted as a function of
temperature. In contrast, the relative turnover rate of the
core-glycosylated wt and mutant CFTR (empty circles) is
insensitive to the same temperature range.
F508 Mutation on the Thermoaggregation and
Protease Susceptibility of CFTR--
SDS-solubilized cell lysates,
obtained from BHK cells expressing
F508, rescued
F508, or wt
CFTR, were heat-denatured at temperatures ranging between 37 and
100 °C. Insoluble aggregates were sedimented by centrifugation, and
monomeric CFTR remaining in the supernatant was quantified by
immunoblotting. The thermostability of solubilized CFTR was
characterized by measuring the aggregation temperature
(Ta), at which 50% of monomeric CFTR is converted into SDS-resistant aggregates (Fig.
6a).
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Fig. 6.
Thermoaggregation of
F508 and wt CFTR. a, wt,
F508,
and rescued
F508 CFTR-expressing cells were solubilized in 2×
Laemmli sample buffer, and samples were incubated at the indicated
temperature for 5 min. Aggregates were sedimented, and wt,
F508, and
rescued
F508 CFTR remaining in the supernatant were visualized with
anti-HA mAb and ECL. To eliminate the core-glycosylated forms in wt and
rescued
F508 CFTR expressors, cells were treated with CHX (100 µg/ml) for 2 h before solubilization. ERP72 and GRP78 were
detected with antibodies described under "Experimental Procedures."
A significant fraction of the SDS-resistant aggregates, containing CFTR
variants, could be recovered in Laemmli sample buffer comprising 8 M urea at room temperature (data not shown). Where
indicated, heat denaturation was performed in 2× Laemmli sample buffer
supplemented with 8 M urea. b and c,
quantitative assessment of the thermoaggregation tendencies.
b, the monomeric complex-glycosylated wt and
F508 CFTR
(rescued
F) and the core-glycosylated
F508
CFTR (
F) were quantified with densitometry on immunoblots
shown on a and expressed as the percentage detected without
heat denaturation (means ± S.E., n = 3-7).
c, the thermoaggregation of ERP72 (solid symbols)
and Na+/K+-ATPase (open symbols) was
determined as described for CFTR in untransfected BHK cells
(triangles) and wt (diamonds), rescued
F508
(squares), or core-glycosylated
F508 CFTR
(circles) expressors (mean ± S.E., n = 3-7).
F508 CFTR was 10 °C lower
(Ta
65 °C) than its wt counterpart
(Ta
75 °C) but 10 °C higher than the
core-glycosylated
F508 CFTR (Ta
55 °C)
(Fig. 6b). The following observations suggest that the progressively decreasing thermostability of the rescued and
core-glycosylated
F508 CFTR is likely the consequence of structural
differences. Firstly, distinct aggregation pattern was also measured on
immunoprecipitated wt and mutant CFTR (data not shown), implying that
their aggregation propensity is independent of other polypeptides.
Secondly, no difference could be documented in the thermoaggregation of
the polytopic Na+/K+-ATPase, in parental BHK
cells, or in cells expressing wt,
F508, or rescued
F508 CFTR
(Fig. 6c). Conversely, ERP72 and GRP78, soluble ER proteins,
were resistant to aggregation, presumably due to their fully denatured
state in SDS, in contrast to polytopic membrane proteins, which tend to
preserve their aggregation tendency following detergent solubilization
(37) (Fig. 6c). Finally and most importantly, no significant
difference between the thermoaggregation tendency of
F508, rescued
F508, and wt CFTR was detected (Ta
75 °C)
when heat-denaturation was performed in Laemmli sample buffer
supplemented with 8 M urea (Fig. 6, a and
b); suggesting that urea denaturation of residual
structural elements, prevailing in SDS micelles (37), abolishes the
differences in the thermostability of CFTR variants.
F508 CFTR is structurally distinct from its wt counterpart. This
approach was instrumental in revealing the distinct conformation of the
cytosolic domains (representing more than 70% of the CFTR polypeptide)
in the complex-glycosylated wt and the core-glycosylated
F508 CFTR
and to monitor the folding of wt CFTR (11). Microsomes were isolated
with differential centrifugation, and the cleavage patterns of wt,
F508, and rescued mutant CFTR, obtained with limited trypsin and
proteinase K digestions, were visualized with immunoblotting. CHX
treatment of the cells ensured that the core-glycosylated CFTR was
degraded prior to the isolation of microsomes, enriched in the
complex-glycosylated wt or rescued
F508 CFTR (Fig.
7a).
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Fig. 7.
In situ protease susceptibility of
wt, F508, and rescued
F508 CFTR. a, complex-glycosylated
F508 CFTR was accumulated during a 24-h rescue procedure as
described in Fig. 1a. To substantially reduce the
core-glycosylated form in wt and rescued
F508 CFTR expressors, cells
were treated with CHX (100 µg/ml) for 3 h, and the lysates were
probed with anti-HA mAb. CHX treatment decreased the core-glycosylated
form by 80-86% according to densitometric analysis. b and
c, enrichment of complex-glycosylated wt and
F508 CFTR
was achieved in BHK cells as described on a. Microsomes were
isolated with differential centrifugation, and limited proteolysis was
performed at the indicated concentrations of trypsin (b) and
proteinase K (c) for 15 min at 4 °C. Samples (50-75 µg
of protein/lane) were immunoblotted with the L12B4 (NBD1
domain-specific) or M3A7 (NBD2 domain-specific) anti-CFTR mAbs and
visualized with ECL.
F508 CFTR to
trypsin and proteinase K was consistently 2-fold lower than that of the
wt CFTR, regardless of whether the NBD2- or the NBD1-specific mouse
monoclonal M3A7 and L12B4 anti-CFTR antibody was used (Fig. 7,
b and c, and data not shown). Differences in the
banding patterns between the rescued
F508 and wt CFTR could also be
recognized, which was more obvious with the NBD2-specific M3A7 than
with the NBD1-specific L12B4 mAb. This suggests that the altered
accessibility of the proteolytic cleavage sites are not restricted to
the NBD1, but encompasses the NBD2 in the rescued
F508 CFTR (Fig. 7,
b and c). A more substantial difference was
observable in the protease resistance and proteolytic digestion
patterns of the rescued and core-glycosylated
F508 CFTR (Fig. 7,
b and c). Because neither the location,
N-linked glycosylation, nor the association with peripheral
membrane proteins alters the protease susceptibility of the wt CFTR
(11), these results imply that the conformation and/or conformational
stability of the rescued
F508 CFTR lies between that of the
core-glycosylated
F508 CFTR and the fully mature wt CFTR.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F508 CFTR, this is not the case in the homozygous knock-in transgenic mouse, expressing
F508 CFTR under its endogenous promoter (21, 28). Short circuit current measurements have shown that at least 4% of the wt CFTR activity is present in the apical membrane of the intestinal epithelium and approximately 1-2% in cultured gall bladder epithelium of the
homozygous
F508 mouse, which could be augmented by 16-18-fold at
the reduced temperature (21). Extrapolating from our results, stabilization of the
F508 CFTR could be responsible for a 4-fold increase of the plasma membrane chloride conductance, whereas the rest
of the difference could be attributed to increased folding efficiency.
Conversely, at physiological temperature, the short residence time of
the spontaneously or pharmacologically rescued
F508 CFTR compromises
its ability to accumulate at the plasma membrane, thus providing
additional explanation for the difficulties in detecting spontaneously
escaped
F508 CFTR both in native tissues and in cultured cells (9,
15, 16).
F508 CFTR. First,
structural alterations may accelerate the endocytosis and inhibit the
recycling of the
F508 CFTR from the endosomes to the cell surface,
promoting the proteolytic degradation in the endolysosome. Second,
preferential delivery of the mutant from post-ER compartments to the
lysosomes may occur, similarly to the progressive aggregation of furin
in the trans-Golgi network (38). Because repeated attempts
failed to detect aggregated
F508 CFTR in the detergent-insoluble
fractions and the cell surface appearance of the mutant was verified
with both biochemical and functional assays, this scenario is unlikely
to be the case. Finally, deletion of Phe-508 may promote the exposure
of a dispersed degradation signal, comprised of hydrophobic patches and
flexible loops in the rescued mutant. These motifs, similar to those
described in the hydroxymethylglutaryl-CoA reductase (39) and
conceivably exposed in the structurally destabilized G-protein-coupled
receptor (40), can be recognized by the cellular proteolytic mechanisms and are perhaps responsible for the degradation. Whether a global structural destabilization, affecting the cytosolic and the
transmembrane domains, is exclusively responsible for the accelerated
degradation of the complex-glycosylated
F508 CFTR or altered
targeting mechanisms are also involved remains to be established. Based
on the observations that structural destabilization of
carboxyl-terminally truncated CFTR and G-protein-coupled receptors
coincides with their accelerated disposal, and in the case of the
mutant CFTR this process depends upon the activity of the
ubiquitin-proteasome degradation
pathway,2 we favor the third scenario.
F508 and
the early folding intermediate of wt CFTR, with the absence of
aggregated
F508 CFTR in the detergent-insoluble fraction, suggested
that
F508 favors the formation of folding intermediate(s) (11). This
may occur by imposing a kinetic block on the folding reaction of CFTR, by energetically destabilizing the native form, or a combination of
these. Whereas previous data obtained on recombinant NBD1 domain are
compatible with a kinetic block (41, 42), the present results and data
derived using synthetic peptides (43) suggest that deletion of Phe-508
has multiple effects. The mutation not only interferes with the
posttranslational folding in a temperature-dependent manner, as reported by a number of laboratories (4, 5, 10, 11, 21), but
also renders temperature-sensitive stability defect to the
complex-glycosylated (or native)
F508 CFTR. According to the
classical terminology the distinctive feature of the
temperature-sensitive folding mutants is the absence of detectable
defects in the protein formed at the permissive temperature (44).
Therefore,
F508 CFTR appears not to belong to this category of
mutations. Intriguingly, while glycerol could partially rescue the
folding defect of the
F508 CFTR at 37 °C, it was unable to
restore the impaired stability of the rescued form,3
suggesting that distinct structural alterations are responsible for the
folding and the stability defect.
F508-imposed folding defect can be partially overcome,
the biological and structural characteristics of the rescued
F508
CFTR, generated at permissive temperature, remain distinct from its wt counterpart. Development of novel strategies to
stabilize the native
F508 CFTR would complement the present therapeutic approaches aiming to correct the folding defect at the ER,
in tissue culture models (10, 22-25, 45), in CF mice (21), and in
human trials (46, 47).
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. N. Kartner for providing the L12B4 and M3A7 mAbs. We thank Drs. A. Davidson and R. Reithmeier for critical reading of the manuscript, Dr. C. Deber for stimulating discussions, and S. Jandu for secretarial help.
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FOOTNOTES |
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* This work was supported by the Canadian Cystic Fibrosis Foundation, the Medical Research Council of Canada, and the NIDDK, National Institutes of Health. Instrumentation was covered in part by a Block Term Grant of the Ontario Thoracic Society.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.
Supported by a Canadian Cystic Fibrosis Foundation Postdoctoral Fellowship.
§ Scholar of the Medical Research Council of Canada. To whom correspondence should be addressed: Program in Lung and Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5125; Fax: 416-813-5771; E-mail: glukacs@sickkids.on.ca.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M009172200
2 M. Benharouga, M. Haardt, and G. L. Lukacs, unpublished observation.
3 M. Sharma and G. L. Lukacs, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; BFA, brefeldin A; CHX, cycloheximide; endo-H, endoglycosidase H; PNGase F, peptide N-glycanase; HA, hemagglutinin; mAb, monoclonal antibody; TM, transmembrane; NBD, nucleotide binding domain; wt, wild type; ECL, enhanced chemiluminescence; NHS-SS-biotin, sulfo-succinimidyl-2-(biotinamido)ethyl-1,2-dithiopropionate.
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