Department of Pediatrics, Case Western Reserve University at Rainbow Babies and Children's Hospital, Cleveland, Ohio 44106
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reduced terminal sialylation
at the surface of airway epithelial cells from patients with cystic
fibrosis may predispose them to bacterial infection. To determine
whether a lack of chloride transport or misprocessing of mutant cystic
fibrosis transmembrane conductance regulator (CFTR) is critical for the
alterations in glycosylation, we studied a normal human tracheal
epithelial cell line (9/HTEo) transfected with the
regulatory (R) domain of CFTR, which blocks CFTR-mediated chloride
transport;
F508 CFTR, which is misprocessed, wild-type CFTR; or
empty vector. Reduced cAMP-stimulated chloride transport is seen in the
R domain and
F508 transfectants. These two cell lines had
consistent, significantly reduced binding of elderberry bark lectin,
which recognizes terminal sialic acid in the
-2,6 configuration.
Binding of other lectins, including Maakia amurensis lectin,
which recognizes sialic acid in the
-2,3 configuration, was
comparable in all cell lines. Because the cell surface change occurred
in R domain-transfected cells, which continue to express wild-type
CFTR, it cannot be related entirely to misprocessed or overexpressed
CFTR. It is associated most closely with reduced CFTR activity.
cystic fibrosis; cystic fibrosis transmembrane conductance
regulator; F508; regulatory domain; lectins; fluorescent imaging; immunogold labeling; 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CYSTIC FIBROSIS (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cAMP-responsive chloride channel. However, not all the abnormalities associated with the CF genotype can yet be directly related to CFTR. Recent studies show that CFTR itself regulates other ion channels in the plasma membrane, including the epithelial sodium channel (7) and the outward rectifying chloride channel (11, 24). Some consistent abnormalities such as the abnormal glycosylation of mucins and proteins and lipids at the cell surface (18, 19) have not yet found ready explanations, although they may be clinically important. For example, abnormal surface sialylation is associated with increased adherence to CF cells of Pseudomonas aeruginosa (21, 31), an organism with a predilection for the CF airway, acquisition of which is associated with a poorer prognosis.
The abnormal glycosylation patterns in CF may be explained, in part, by
the failure of activity of CFTR at intracellular sites, which could
lead to abnormal acidification of particular intracellular compartments, notably the trans-Golgi network (TGN)
(2, 4, 5). Failure to acidify this compartment normally
might lead to reduced activity of glycosyltransferases such as
-2,6-sialyltransferase with narrow, acidic pH optima and favor
activity of enzymes such as sulfotransferases with broader pH optima.
Barasch et al. (5) presented several lines of evidence in
support of this hypothesis, including electron microscopy data showing
increased accumulation of the weak base
3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), in the TGN of SV40-transformed airway epithelial cells compared
with SV40-transformed CF cell lines. However, a study using
ratio-imaging confocal microscopy on various cell lines that do and do
not express CFTR suggested that the pH in all intracellular compartments was normal in CF cells, even with cAMP stimulation (25). An alternative suggestion is that the abnormal
processing of some mutant forms of CFTR, including the most common
mutant
F508, interferes with processing, trafficking, and routing of other glycoconjugates including sialyltransferases (4,
29). Because most CF cell lines now in use contain at least one
F508 allele, it is difficult to distinguish the effects of
processing mutants from those of reduced CFTR activity.
To investigate the link between the CF phenotype and abnormal
glycosylation, we used 9/HTEo cells (normal human
tracheal epithelial cells known to express CFTR) stably transfected
with an episomal expression vector (pCEP) that contains either the
regulatory (R) domain of CFTR (pCEP-R), wild-type (WT) CFTR
(pCEP-WTCFTR),
F508 CFTR (pCEP-
F508), or no expressed gene except
that for hygromycin resistance (pCEP). These cell lines were isogenic
except for the transfected gene. Overexpression of the R domain of CFTR
(amino acids 588-857) in 9/HTEo
cells inhibits CFTR
function as previously described (17). When the R domain
is overexpressed in these cells, transcription of CFTR as assessed by
RT-PCR continues. Although levels of CFTR protein are too low to be
detected in 9/HTEo
cells either by Western blot or by
immunofluorescence assay, after R domain transfection, antibody to the
R domain shows an increase in specific fluorescence in a cytoplasmic
staining pattern, suggesting that the overexpressed R domain is not
retained in the endoplasmic reticulum and thus probably does not retard
CFTR maturation either. We demonstrate in this paper that in a
heterologous expression system, coexpression of the R domain with CFTR
doesn't interfere with CFTR processing. R domain expression is
associated with a reduction in basal and cAMP-stimulated chloride
efflux as measured by
6- methoxy-N-(3-sulfopropyl)quinolinium (SPQ) fluorescence in a pattern similar to that produced by incubation of
9/HTEo
cells with antisense oligonucleotides to CFTR,
indicating that it is due to CFTR. Calcium-stimulated chloride efflux
is not reduced in R domain-transfected (or antisense-treated) cells,
indicating the specificity of the R domain effect. R domain
transfection does not alter cAMP levels or protein kinase (PK) A
activity so its effect is not at that level. In planar lipid bilayers,
unphosphorylated R domain on the intracellular side of the CFTR channel
results in channel closure (15, 16), and it is likely that
this is the mechanism of reduction of CFTR function in the pCEP-R
cells. Besides the R domain cells, we developed and tested
9/HTEo
cells transfected with
F508 CFTR as well as
with WT CFTR to assess the effect of the misprocessing known to occur
with
F508-CFTR.
We studied the binding of various fluorescent lectins to these cell
lines using computer-assisted quantitative fluorescence microscopy. We
tested these hypotheses: first, that human airway epithelial cells that
express only normal CFTR but have no CFTR activity (pCEP-R cell lines)
have abnormal glycosylation as detected by lectin binding due to the
alkalinized pH of the TGN relative to the that in the control line
(pCEP) and second, that overexpression of F508 CFTR in a human
airway epithelial cell line that normally expresses CFTR is sufficient
to produce abnormal glycosylation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell lines.
Human tracheal epithelial cells (9/HTEo) were transfected
with LIPOFECTIN reagent (GIBCO BRL, Life Technologies, Gaithersburg, MD) with either empty vector (pCEP4, Invitrogen, San Diego, CA) to give
the control cell line, pCEP-R (vector cloned with the R domain of CFTR)
(17), pCEP-WTCFTR (vector with WT CFTR cDNA), or
pCEP-
F508 (vector with
F508 CFTR cDNA). Cells were grown in
Dulbecco's minimal Eagle's medium supplemented with 10% fetal bovine
serum and 2.5 mM L-glutamine, and all cell lines used in this study were maintained under selection with 80 µg/ml of
hygromycin at 37°C in an atmosphere of 95% air- 5%
CO2.
Western blot of CFTR-containing vesicles. HEK293 cells were transfected with LIPOFECTAMINE Plus (GIBCO BRL) with either pCEP4, pCEP-CFTR, or an equal molar ratio of pCEP-CFTR and pCEP-R. Forty-eight hours after transfection, the cell vesicles were harvested following the procedure of Ma et al. (16) and duplicate 7.5% SDS-PAGE was performed for Western blot analysis with either a R domain antibody (monoclonal antibody 13-1, Genzyme, Cambridge, MA) or a mouse anti-human monoclonal CFTR COOH terminus-specific antibody (amino acids 1377-1480; Genzyme). Quantitation of blots was done with SigmaGel (Jandel Scientific, San Rafael, CA).
RT-PCR.
pCEP-R cells were previously characterized and shown to overexpress R
domain while retaining expression of CFTR (17). Coupled RT-PCR (1) was used to show expression of F508 CFTR in
the 9/HTEo
-transfected cells. The primers chosen
amplified a 359-bp fragment of cDNA. The 5' primer was in exon 9 of
CFTR (5'-GAAAGAGGACAGTTGTTGGCGGTTGC-3'), and the 3' primer crossed the
boundary of exon 11/12 (5'-CAAATCAGCATCTTTGTATACTGCTCTTGC-3'). Primers
(100 ng each) were incubated with 40 U of RNase inhibitor and 4 µg of
total RNA at 65°C for 15 min. To obtain a total reaction volume of
100 µl, 95 µl of the reaction mixture were added for a final
concentration of 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 1.5 mM
MgCl2, 200 nM each deoxynucleotide triphosphate, and 2.5 U
of Taq polymerase (Boehringer Mannheim, Indianapolis, IN)
with and without 200 U of SuperScript reverse transcriptase (GIBCO BRL). Reagents were mixed and overlaid with mineral oil. The
thermocycler protocol was 37°C for 0.5 h and 42°C for 0.5 h for RT followed by PCR that included denaturation at 95°C for 3 min
and 30 cycles consisting of 92°C for 1 min, 59°C for 1 min, and
72°C for 1.5 min and then a 10-min extension at 72°C and a 4°C
hold. Ten microliters of reaction products were size fractionated on a
2.5% Tris-acetate-EDTA agarose gel and visualized with ethidium
bromide. For Southern hybridization, the gel was denatured (0.5 N NaOH
and 1.5 M NaCl) for 20 min and transferred by downward capillary action
onto Immobilon-N transfer membranes (Millipore, Bedford, MA) in 10×
saline-sodium citrate (0.3 M NaCl and 30 mM trisodium citrate). The
filter was prehybridized in Church buffer (1% BSA, 1 mM EDTA, 0.5 M
Na2HPO4, pH 7.5, and 7% SDS) at 55°C for
1 h and hybridized overnight in fresh Church buffer with
[32P]dCTP-tailed antisense probes corresponding to the
region of sequence divergence from the WT, covering the
F508
(5'-AACACCAATGATATTTTCTT-3') mutation. RT-PCR was also used to confirm
expression of transfected WT CFTR cDNA and endogenous CFTR in
pCEP-WTCFTR-transfected cells (data not shown). WT CFTR 5' primer
(5'-TAGTGAACCGTCAGATCTCTAGGAGCTGGG-3') in the 5'-untranslated
region of pCEP vector and 3' primer (5'-ATCTGCATTCCAATGTGATGAAGGCC-3') in exon 3 of CFTR amplified a 586-bp product. Endogenous CFTR expression was confirmed by amplification of a 673-bp product with
primer pairs with the plus primer (5'-TGGAAAGTTGCAGATGAGCTTGGGC-3') in
exon 21/22 of CFTR and the minus primer
(5'-GCCAGGAAGCCATTTATCAAGACCC-3') in the 3'-untranslated region of CFTR
that is not present in the pCEP-WTCFTR construct.
Chloride efflux assays.
The presence of a cAMP-stimulated chloride efflux in the pCEP and
pCEP-WTCFTR clones was assayed by 36Cl efflux
(28). Cells were plated on vitrogen-coated 35-mm dishes, grown to confluence, and then loaded with 5 µCi of 36Cl
for 1 h. The cells were washed, and 1 ml of HEPES-buffered Ringer
solution was added to the dishes. The buffer was sampled and replaced
every 30 s during the 10-min assay for scintillation counting.
Theophylline (1 mM) and forskolin (10 µM) were added at 240 s.
The cells were lysed with 0.1 N HCl and 1 N NaOH at the end of the
experiment to determine the 36Cl remaining in the cells.
The apparent rate constant (r; in min1) was
calculated at each efflux interval with the equation r = [ln(C1)
ln(C2)]/(t2
t1), where C1 and C2 are
the percentages of counts remaining in the cell layer at times
t1 and t2, respectively.
SPQ assays. SPQ assays were performed following the protocol of Perez et al. (17). Briefly, cells on aclar-coated coverslips were loaded with 5 mM SPQ and studied cell by cell with an upright Zeiss epifluorescence microscope. The images were quantified with Image-1 F1 Software (Universal Imaging, West Chester, PA). Successive images were taken at a rate of ~1 every 5 s. The cells were continually perfused at 37°C with either chloride-containing or chloride-free solution in the presence and absence of 10 µM isoproterenol. The results are expressed as rates (relative fluorescence units per minute).
Fluorescent lectin binding. All fluorescent lectins [elderberry bark lectin (EBL), Erythrina cristagalli lectin (ECL), Maackia amurensis lectin I (MAL I), peanut agglutinin (PNA), succinylated wheat germ agglutinin (SWGA), and wheat germ agglutinin (WGA)] were purchased from Vector Laboratories (Burlingame, CA). FITC-conjugated cholera toxin B subunit was purchased from List Biochemicals (Campbell, CA).
EBL binds specifically to sialic acid in anFluorescence microscopy. The slides were viewed with a Zeiss Axiovert 45 inverted microscope with a ×40 water objective and a numerical aperture of 0.75 (Zeiss, Thornwood, NY) and a FITC filter set (excitation wavelength 495 nm, dichroic wavelength 515 nm, emission wavelength 535 nm; Omega, Brattleboro, VT) and 75-W xenon lamp for fluorescence. Digital images were acquired with a cooled charge-coupled device camera model CH250 (Photometrics, Tucson, AZ) and quantified with a Nu 2000 camera controller board (Photometrics) with a Macintosh configuration. Data were processed with Oncor Image software (Oncor Imaging, Rockville, MD) and were recorded on an optical disk storage system.
An average of 7 fields/well (range 4-14) with between 16 and 23 cells/field were randomly chosen with the use of transmitted light, and both transmitted and fluorescent images of the same field were recorded. Camera exposure times for each lectin were kept constant between cell lines. The brightest image of each lectin binding experiment was used to determine a camera exposure time that used ~80% of the image dynamic range. Background and autofluorescence values were determined from control wells for background subtraction. The images were represented by histogram plots that displayed the number of pixels in the image at a particular intensity value. Each experiment was analyzed by combining the histograms of the individual images, and average pixel intensities were obtained from this plot. However, the shape of the histogram provided additional information on the distribution or pattern of fluorescence, which was important because the lectin binding images showed clusters of bright fluorescence, or a punctate appearance, rather than uniform fluorescence throughout the cell. Therefore, for each experiment, two statistical comparisons were made. 1) We determined the overall average pixel intensity to assess the total quantity of fluorescent lectin bound to the different cell lines and applied Student's t-test for significance. 2) We assessed the distribution of fluorescence by using the Kolmogorov-Smirnov (K-S) nonparametric statistical test for analysis of the histograms (30). The fluorescence distribution of the images was assessed on the original images that were contrast compressed without any contrast enhancement to make use of the full range of values within the image. For K-S analysis, these histograms were converted to a cumulative frequency or probability distribution curve. The maximum vertical displacement (D) or difference between sample and control curves was then computed. From this "D value," a K-S statistic was derived, and significance was tested by comparison of D against critical table values. The lower threshold was set at the intensity value below which 97% of the pixels fell in control wells, and an upper threshold eliminated very high intensity values (bright artifacts). Therefore, this analysis was done only on the area of the image that represented actual lectin binding.Confocal microscopy. Cells prepared for fluorescence microscopy as in Fluorescent lectin binding were examined under a Bio-Rad MRC600 confocal microscope with a ×40, 1.3-numerical aperture objective. Z stacks of ~20 images with 0.5-µm spacing were collected. For EBL, most of the fluorescence was at the cell surface, but for the other lectins, substantial cytoplasmic fluorescence was observed as well (confocal images not shown).
Electron microscopy. The DAMP method for cytochemical visualization of acidic compartments was a modification of the technique of Anderson and Pathak (3). pCEP and pCEP-R cells were plated on 60-mm vitrogen-coated cell culture dishes, and at 95% confluence, the cells were rinsed and 2 ml of fresh medium with 50 µM DAMP (Oxford Biomedical Research, Oxford, MI) were added and incubated at 37°C for 0.5 h. The cells were rinsed twice in medium and incubated for an additional 10 min. As a control for DAMP accumulation, 25 µM monensin (Oxford Biomedical Research) was added to one set of dishes. The cells were rinsed twice in PBS and fixed in 3% paraformaldehyde, 1% glutaraldehyde, and 3% sucrose for 2 h. The cells were postfixed in 1% OsO4 and embedded in Polybed 812 (Epon). Thin sections were mounted on Formvar-coated nickel grids. For immunogold labeling, the sections were first etched by floating the grids section side down over 50-µl drops of a saturated solution of NaIO3 for 20-40 min and then rinsing three times by floating the grids over fresh drops of 0.2-µm filtered distilled water. The grids were then floated for 1 h over 50-µl drops of buffer B (20 mM Tris · HCl, pH 9.0, 200 mM NaCl, and 1% BSA) followed by incubation in a humidified chamber at 4°C overnight (16-18 h) over 25-µl drops of 15 µg/ml of anti-N-dinitrophenyl (Oxford Biomedical Research) in buffer C (20 mM Tris · HCl, pH 9.0, 200 mM NaCl, and 0.1% BSA). The grids were subsequently incubated over 25-µl drops of 10 µg/ml of affinity-purified rabbit anti-mouse IgG (Sigma, St. Louis, MO) in buffer C for 2 h. Bound antibodies were visualized by floating sections for 1 h over 25-µl drops of a 1:40 dilution of goat anti-rabbit IgG gold conjugate (10 nm; Sigma) in buffer C. Between all treatments, the grids were rinsed by floating them over large drops of buffer C twice for 10 min. Finally, the grids were washed by either 45-s rinses with a stream of 0.2-µm filtered distilled water or dipping them repeatedly in water for 45 s and were air-dried. All incubations except for the primary antibody were carried out at room temperature. The grids were counterstained with uranyl acetate and lead citrate. Two separate DAMP-labeling experiments were performed, and multiple grids from two separate immunogold labeling treatments from each experiment were analyzed.
Gold-labeled grids were examined under a JEOL 100CX transmission electron microscope at 80 kV and photographed at ×20 magnification. Final magnification of the micrographs that were printed for the counting of gold particles was approximately ×60,000. A transparent grid was placed over the micrograph to determine the area of the intracellular organelles and background regions, and the number of gold particles in the region was counted. Background gold counts were obtained by evaluating areas of the cytoplasm without identifiable membrane-bound organelles. Significance was determined by t-test or nonparametric Mann-Whitney rank sum analysis when normality testing failed. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of R domain.
pCEP-R cells have been previously characterized (17). They
express the exogenous R domain mRNA (confirmed by RT-PCR) and protein
(shown by immunoprecipitation and/or phosphorylation assay as well as
by immunofluorescence) as well as the mRNA for endogenous CFTR. During
the course of these experiments, R domain expression was verified
periodically by immunoprecipitation of the cell supernatant with
monoclonal antibody 13-1 (Genzyme) directed against the R domain of
CFTR, phosphorylation with PKA, and SDS-PAGE. Figure 1 demonstrates that coexpression of equal
molar amounts of CFTR and R domain in a heterologous expression system
(HEK293 cells) results in little change in the ratio of band C (fully
processed) to band B (core-glycosylated) CFTR, indicating no
interference by the R domain in CFTR processing. This suggests that the
lack of cAMP-activated chloride channel activity in pCEP-R cells is not
due to increased misprocessing of CFTR. Although this heterologous, high-level expression system is not identical to the low-level CFTR
expression in the 9/HTEo cell line, there is no reason to
suspect qualitatively different protein-protein interaction in airway
epithelial cells.
|
Expression of F508 and WT CFTR.
Figure 2A shows the results of
a coupled RT-PCR demonstrating the presence of mRNA for
F508 (Fig.
2A, lane 1) in pCEP-
F508 cells, which was
probed with the appropriate 32P-tailed antisense oligo for
the
F508 mutation (Fig. 2B, lane 1) to confirm
that the message detected was not endogenous CFTR. WT CFTR does not
bind the allele-specific probe (Fig. 2B, lane 6).
Total RNA was DNase treated, and the negative controls were samples
that were further treated with RNase I before RT-PCR to confirm that
the reaction product originated from mRNA, not from transfected cDNA.
RT-PCR was also used to confirm the expression of transfected WT CFTR
cDNA (data not shown).
|
Chloride efflux assays.
The cAMP-mediated stimulation of the rate of chloride efflux,
characteristic of functioning CFTR chloride channels, has previously been shown to be markedly reduced in pCEP-R cells as measured by SPQ
fluorescence (17). As reported previously
(17), the two pCEP-R clones used in this study,
clones F and E, failed to respond to 10 µM
isoproterenol with an increased rate of chloride efflux. The control
cell line transfected with the empty vector pCEP responded to the cAMP
agonist with an approximately twofold increase in rate of chloride
efflux. The basal rate of chloride efflux (without isoproterenol
stimulation) was also significantly reduced in the pCEP-R clones.
Responses of these clones were checked periodically and new vials were
opened if the chloride transport response changed. Figure
3 shows the results of the SPQ assays with two clones (clones 1 and 9) of pCEP-F508
cells. They also failed to respond to isoproterenol with an increased
rate of chloride efflux. Basal chloride efflux rate was reduced in
pCEP-
F508 clone 9 but not in clone 1 compared
with pCEP control clone 3. Assays of 36Cl efflux
in pCEP-WTCFTR cells showed that this cell line, like cells transfected
with empty vector (pCEP), could be stimulated by a combination of the
cAMP agonists theophylline and forskolin (Fig.
4A). Thus the control
cell line transfected with empty vector (pCEP) and the line transfected
with WT CFTR both express functioning CFTR channels, whereas pCEP-R-
and pCEP-
F508-transfected cell lines lack cAMP-stimulated chloride
effluxes. Because the responses of pCEP and pCEP-WTCFTR were so
similar, pCEP was the standard control cell line.
|
|
Quantitative fluorescence microscopy.
Using two statistical methods of quantitation, we found that sialic
acid in an -2,6 linkage (as assessed by EBL binding) showed no
difference between the control cell lines pCEP-WTCFTR and pCEP (Fig.
4B). Thus 9/HTEo
cells transfected with the
empty vector or WT CFTR cDNA retain cAMP-stimulated chloride efflux and
have similar cell surface lectin binding properties. Simultaneous
studies of the control pCEP line and two clones of both R domain- and
F508-transfected 9/HTEo
cells were performed for six
FITC-conjugated lectins and the B subunit of cholera toxin at
concentrations that minimize low-affinity binding.
|
|
|
|
DAMP accumulation in intracellular organelles.
To determine whether the sialylation changes in pCEP-R cells were
associated with the higher pH in the TGN, we used DAMP and subsequent
immunogold labeling to assess the pH change across the membranes
delimiting intracellular compartments. Because prior measurements with
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, a
proton-sensitive fluorescent dye, showed no difference in the cytoplasmic pH between pCEP and pCEP-R cells (13), DAMP
labeling directly compared the pH in the organelles in pCEP and pCEP-R cells. Figure 8 illustrates that DAMP
accumulated in acidic vesicles, shown in pCEP-R (A) and pCEP
(B) cells as the clustering of many gold particles in the
electron-dense or gray organelles and few particles in the cytoplasm or
background label. We observed no apparent differences in cellular
morphology, including size and structure of intracellular organelles,
between pCEP-R (Fig. 8C) and pCEP-R (Fig. 8D)
cells. We identified intracellular organelles by their distinctive
morphology and ability to accumulate DAMP. Gold granules were counted
in mitochondria, identified by a round to elongated shape and lamellar
folds of inner membrane; nuclei; multivesicular bodies, identified as
membrane-bound organelles with internal membrane structures resembling
vesicles; and uniformly gray or electron-dense organelles that may be
late endosomes or lysosomes. Although the electron-dense vesicles shown
in Fig. 8, A (pCEP-R) and B (pCEP), are of
different sizes, there was no overall difference in size of this
lysosomal or endosomal compartment when these organelles from many
cells were quantitated. Gold particles in the TGN were identified in
vesicles near the classically defined Golgi stacks (at least three
visible organized stacks) and the area of the TGN included the region
of cytoplasm encompassing those vesicles. In two separate DAMP
experiments, each with two separate immunolabeling procedures of
multiple grids, we found no significant difference in the pH of
intracellular organelles in pCEP and pCEP-R cells (Fig.
9). Grids from monensin-treated samples showed no accumulation of gold particles in intracellular vesicles, and grids immunolabeled without primary antibody were void of
gold (data not shown).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results show that reduced function of CFTR in a human airway
epithelial cell line, whether achieved by overexpression of the R
domain in the face of persistent CFTR expression or by overexpression
of F508 CFTR, is associated with altered sialylation in the cell.
Binding of EBL, which recognizes sialic acid in the
-2,6 linkage, is
reduced in two separate clones of each transfectant in multiple
experiments, and the results were quantitative, consistent, and highly
significant. These cell lines were transfected with an episomal vector
(pCEP) so that they could not be subject to insertional mutagenesis or
unusual phenomena from the chromosomal placement of transfected DNA and
could be closely matched in properties except for the genes added via
the vector. Both the total amount and the distribution of EBL
fluorescence was altered in the pCEP-R and pCEP-
F508 lines compared
with both the pCEP and pCEP-WTCFTR lines.
9/HTEo cells transfected with the R domain continued to
express the mRNA for endogenous WT CFTR but lacked cAMP-stimulated chloride conductance. As discussed above, this impairment is most likely due to direct interaction of the R domain with CFTR (15, 16), which continues to be transcribed (17) and
processed normally. This is demonstrated by our results in HEK293 cells where transfection of both the R domain and CFTR results in no change
in CFTR maturation compared with cells transfected with CFTR only. The
abnormal sialylation in these cells must thus be attributed to reduced
CFTR function, not to misprocessed mutant CFTR. We also tested whether
cells transfected with the R domain have a higher pH in intracellular
compartments compared with that in cells transfected with empty vector
but could not demonstrate significant differences in pH in any
organelle. Our experiments achieved an intensity of DAMP label in
intracellular vesicles, after background subtraction, comparable to
that reported by Barasch et al. (5) for SV40-transformed
airway epithelial cells, who concluded from distribution of DAMP in
these cells that TGN pH is increased in CF cells. Thus differences
between our findings and theirs are probably not due to inefficient
DAMP label penetration in our cells. Our results are in agreement with
those of Seksek et al. (25), who found a normal pH in the
intracellular compartments in CF cells using fluorescent labels.
In addition, we found that transfecting F508 CFTR into
9/HTEo
cells resulted in a marked decrement of endogenous
CFTR activity, whereas transfection of WT CFTR had little effect on
chloride transport. We had expected that endogenous WT CFTR would
continue to be produced and processed and function at the cell surface, but this was not the case. It is possible that transfection of a
misprocessed CFTR allele increased the proportion of WT CFTR that is
retained and degraded in the endoplasmic reticulum, but we speculate
that it is more likely that the reduction in activity comes from
dimerization of WT with mutant CFTR. Recent structural studies
(10, 32) indicated that CFTR may exist and function in
membranes as two closely associated molecules. In this case, an excess
of mutant molecules would result in pairing of the few WT molecules
with mutant partners. If both members of a pair must be functional for
normal activity, then the greater the excess of mutant molecules, the
greater the reduction in activity. Because endogenous CFTR expression
in 9/HTEo
cells occurs at too low a level to be tracked
by immunofluorescence, it is not possible to determine its level of
expression or subcellular location in
F508-CFTR-transfected cells.
However, again, CFTR activity was paralleled by EBL binding, which was
reduced in the
F508 cells compared with the WT transfectants. These
data, taken together with the R domain-transfected data, provide
further support for the hypothesis that CFTR activity, not processing
per se, is most closely associated with cell surface alterations.
However, this explanation is probably not applicable to the studies of glycosylation in cells heterologously expressing either WT or
F508
CFTR (9) in which
F508 CFTR expression led to
abnormalities in glycosylation even compared with untransfected control
cells. Moreover, Zar et al. (31) reported that primary
cultures of airway epithelial cells from patients with CF homozygous
for the
F508 mutation, but not other mutations, have excess
binding of P. aeruginosa.
However, binding of P. aeruginosa cannot be equated directly
to the lectin binding changes we measured, and primary cultures of
cells from multiple different patients might, of course, differ by more
than the specific CFTR allele. Our cell lines, isogenic except for the
transfected genes, are advantageous for comparison purposes. In another
study (9), overexpression of F508 CFTR, but not of the
WT, in a cell line that does not express endogenous CFTR resulted in
lectin binding changes, suggesting that overexpression of
F508 CFTR
in itself is sufficient to produce abnormal sialylation in cells.
However, chloride transport was not measured in these cells.
Reduced chloride transport through CFTR appears to affect sialylation
whether it is caused by overexpression of F508 CFTR, a processing
mutant, or by blocking the CFTR channel with an excess of the R domain
that does not increase CFTR misprocessing. This is not explained in
these cells by changes in pH in membrane-bound compartments as
suggested earlier by Barasch et al. (5). Although it is
possible that misprocessing of mutant CFTR disrupts processing of other
molecules, including sialyltransferases, this is unlikely to account
for the results in the R domain cells where the only CFTR is WT.
Alterations in overall sialic acid uptake should affect sialic acid
available for addition in an
-2,3 as well as in an
-2,6 linkage,
and this is not the case. It is possible that the sialyltransferases
are mislocalized in CF (29) for unknown reasons. It is
also possible that sialyltransferase function somehow requires functional CFTR. Alternatively, transcriptional regulation of these
enzymes may be abnormal in CF. Further experiments are required to
allow us to choose among these possibilities.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Cathy Silski for making the pCEP-F508 and
pCEP-wild-type cystic fibrosis transmembrane conductance regulator cell lines, Yoshie Hervey for excellent cell culture care, and Dave Fletcher
for the 36Cl efflux assays. Also, this work has benefited
from the helpful discussions and critical reviews of Ulrich Hopfer,
Mitch Drumm, Tom Ferkol, and Alice Prince.
![]() |
FOOTNOTES |
---|
This work is supported by National Heart, Lung, and Blood Institute Grants R01-HL/DK-49003 and T32-HL-07415; National Institute of Diabetes and Digestive and Kidney Diseases Grant P30-DK-27651; and a Research Development Program grant from the Cystic Fibrosis Foundation.
Address for reprint requests and other correspondence: D. Kube, Pediatric Pulmonary Division, CWRU School of Medicine, BRB 835, 2109 Adelbert Rd., Cleveland, OH 44106-4948 (E-mail: dmk8{at}po.cwru.edu).
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.
Received 2 February 2000; accepted in final form 6 October 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aatsinki, JR,
Lakkakorpi JT,
Pietila EM,
and
Rajaniemi HJ.
A coupled one-step reverse transcription PCR procedure for generation of full-length open reading frames.
Biotechniques
16:
283-288,
1994.
2.
Al-Awqati, Q,
Barasch J,
and
Landry D.
Chloride channels of intracellular organelles and their potential role in cystic fibrosis.
J Exp Biol
172:
245-266,
1992
3.
Anderson, RGW,
and
Pathak RK.
Vesicles and cisternae in the trans-Golgi apparatus of human fibroblasts are acidic compartments.
Cell
40:
635-643,
1985[ISI][Medline].
4.
Barasch, J,
and
Al-Awqati Q.
Defective acidification of the biosynthetic pathway in cystic fibrosis.
J Cell Sci Suppl
17:
229-233,
1993.
5.
Barasch, J,
Kiss B,
Prince A,
Saiman L,
Gruenert D,
and
Al-Awqati Q.
Defective acidification of intracellular organelles in cystic fibrosis.
Nature
352:
70-73,
1991[ISI][Medline].
6.
Bhavanandan, VP,
and
Katlic AW.
The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid.
J Biol Chem
254:
4000-4008,
1979[ISI][Medline].
7.
Boucher, RC,
Stutts MJ,
Knowles MR,
Cantley L,
and
Gatzy JT.
Na+ transport in cystic fibrosis respiratory epithelia.
J Clin Invest
78:
1245-1252,
1986[ISI][Medline].
8.
Bryan, R,
Kube D,
Perez A,
Davis P,
and
Prince A.
Overproduction of the CFTR R domain leads to increased levels of asioloGM1 and increased Pseudomonas aeruginosa binding by epithelial cells.
Am J Respir Cell Mol Biol
19:
269-277,
1998
9.
Dosanjh, A,
Lencer W,
Brown D,
Ausiello DA,
and
Stow JL.
Heterologous expression of F508 CFTR results in decreased sialylation of membrane glycoconjugates.
Am J Physiol Cell Physiol
266:
C360-C366,
1994
10.
Eskandari, S,
Wright EM,
Kremar M,
Starace DM,
and
Zampighi GA.
Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy.
Proc Natl Acad Sci USA
95:
11235-11240,
1998
11.
Gabriel, SE,
Clark IL,
Boucher RC,
and
Stutts MJ.
CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship.
Nature
363:
263-266,
1993[ISI][Medline].
12.
Goldstein, IJ,
and
Hayes CE.
The lectins: carbohydrate-binding proteins of plants and animals.
Adv Carbohydr Chem Biochem
35:
127-340,
1978[Medline].
13.
Kashyap, R.
pHi Regulation Differs in Normal and CF Phenotype Pancreatic Ductal Cells (MSc thesis). Cleveland, OH: Case Western Reserve University, 1995.
14.
Knibbs, RN,
Goldstein IJ,
Ratcliffe RM,
and
Shibuya N.
Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis: comparison with other sialic acid-specific lectins.
J Biol Chem
266:
83-88,
1991
15.
Ma, JJ,
Tasch JE,
Tao T,
Zhao JY,
Xie JX,
Drumm ML,
and
Davis PB.
Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein.
J Biol Chem
271:
7351-7356,
1996
16.
Ma, J,
Zhao J,
Drumm ML,
Xie J,
and
Davis PB.
Function of the R domain in the CFTR chloride channel.
J Biol Chem
272:
28133-28141,
1997
17.
Perez, A,
Risma KA,
Eckman EA,
and
Davis PB.
Overexpression of R domain eliminates cAMP-stimulated Cl secretion in 9/HTEo
cells in culture.
Am J Physiol Lung Cell Mol Physiol
271:
L85-L92,
1996
18.
Pilewski, JM,
and
Frizzell RA.
How do cystic fibrosis transmembrane conductance regulator mutations produce lung disease?
Curr Opin Pulm Med
1:
435-443,
1995[Medline].
19.
Puchelle, E,
de Bentzmann S,
and
Zahm JM.
Physical and functional properties of airway secretions in cystic fibrosistherapeutic approaches.
Respiration
62:
2-12,
1995[ISI][Medline].
20.
Saiman, L,
Cacalano G,
Gruenert D,
and
Prince AL.
Comparison of adherence of Pseudomonas aeruginosa to respiratory epithelial cells from cystic fibrosis patients and healthy subjects.
Infect Immun
60:
2808-2814,
1992[Abstract].
21.
Saiman, L,
and
Prince A.
Pseudomonas aeruginosa pili bind to asialoGM1 which is increased on the surface of cystic fibrosis epithelial cells.
J Clin Invest
92:
1875-1880,
1996.
22.
Sata, T,
Lackie PM,
Taatjes DJ,
Peumans WJ,
and
Roth J.
Detection of the NeuAc (2,3) Gal (
1,4) GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid in
2,3 and
2,6 linkage.
J Histochem Cytochem
37:
1577-1588,
1989[Abstract].
23.
Schengrund, CL,
and
Ringler NJ.
Binding of Vibrio cholera toxin and the heat-labile enterotoxin of Escherichia coli to GM1, derivatives of GM1, and nonlipid oligosaccharide polyvalent ligands.
J Biol Chem
264:
13233-13237,
1989
24.
Schwiebert, EM,
Egan ME,
Hwang TH,
Fulmer SB,
Allen SS,
Cutting GR,
and
Guggino WB.
CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.
Cell
81:
1063-1073,
1995[ISI][Medline].
25.
Seksek, O,
Biwersi J,
and
Verkman AS.
Evidence against defective trans-Golgi acidification in cystic fibrosis.
J Biol Chem
271:
15542-15548,
1996
26.
Shibuya, J,
Goldstein IJ,
Broekaert WF,
Nsimba-Lubaki M,
Peeters B,
and
Peumans WJ.
The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac (2,6) Gal/GalNAc sequence.
J Biol Chem
262:
1596-1601,
1987
27.
Teneberg, S,
Angstrom J,
Jovall PA,
and
Karlsson KA.
Characterization of binding of Gal 4GlcNAc-specific lectins from Erythrina cristagalli and Erythrina corallodendron to glycosphingolipids. Detection, isolation, and characterization of a novel glycosphingolipid of bovine buttermilk.
J Biol Chem
269:
8554-8563,
1994
28.
Venglarik, CJ,
Bridges RJ,
and
Frizzell RA.
A simple assay for agonist-regulated Cl and K+ conductances in salt-secreting epithelial cells.
Am J Physiol Cell Physiol
259:
C358-C364,
1990
29.
Weyer, P,
Barasch J,
Al-Awqati Q,
Ausiello DA,
and
Brown D.
Immunolocalization of two sialyltransferases is altered in polarized LLC-PK1 epithelial cells expressing F508 CFTR (Abstract).
Pediatr Pulmonol Suppl
12:
238,
1995.
30.
Young, IT.
Proof without prejudice: use of the Kolmogorov-Smirnov test for analysis of histograms from flow systems and other sources.
J Histochem Cytochem
25:
935-941,
1977[Abstract].
31.
Zar, H,
Saiman L,
Quittell L,
and
Prince A.
Binding of Pseudomonas aeruginosa to respiratory epithelial cells from patients with various mutations in the cystic fibrosis transmembrane regulator.
J Pediatr
126:
230-233,
1995[ISI][Medline].
32.
Zerhusen, G,
Zhao J,
Xie J,
Davis PB,
and
Ma J.
A single conductance pore for chloride ion formed by two CFTR molecules.
J Biol Chem
274:
7627-7630,
1999