Terminal sialylation is altered in airway cells with impaired CFTR-mediated chloride transport

Dianne Kube, Lynn Adams, Aura Perez, and Pamela B. Davis

Department of Pediatrics, Case Western Reserve University at Rainbow Babies and Children's Hospital, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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; Delta F508 CFTR, which is misprocessed, wild-type CFTR; or empty vector. Reduced cAMP-stimulated chloride transport is seen in the R domain and Delta F508 transfectants. These two cell lines had consistent, significantly reduced binding of elderberry bark lectin, which recognizes terminal sialic acid in the alpha -2,6 configuration. Binding of other lectins, including Maakia amurensis lectin, which recognizes sialic acid in the alpha -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; Delta F508; regulatory domain; lectins; fluorescent imaging; immunogold labeling; 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 Delta 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 Delta 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), Delta F508 CFTR (pCEP-Delta 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 Delta F508 CFTR as well as with WT CFTR to assess the effect of the misprocessing known to occur with Delta 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 Delta F508 CFTR in a human airway epithelial cell line that normally expresses CFTR is sufficient to produce abnormal glycosylation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-Delta F508 (vector with Delta 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 Delta 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 Delta 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 min-1) 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 an alpha -2,6 linkage to galactose (Gal) or N-acetyl-D-galactosamine present in N- or O-linked glycoproteins and glycolipids (26). MAL I binds to terminal sialic acid residues in an alpha -2,3 linkage to galactose(beta 1right-arrow4)N-acetyl-D-glucosamine (GlcNAc) of glycoproteins (14, 22). ECL binds terminal galactose(beta 1right-arrow4)GlcNAc, a substrate for both alpha -2,6- and alpha -2,3-linked sialic acid, but sialic acid substitution on this structure interferes with lectin binding (27). Because WGA is able to bind N-acetylneuraminic acid as well as cell surface glycoconjugates containing GlcNAc and its beta 1right-arrow4 oligomers and SWGA binds only glycoconjugates containing GlcNAc and does not bind to sialic acid, using a combination of WGA and SWGA allows an estimation of total membrane sialic acid (6). PNA binds preferentially to galactosyl(beta 1right-arrow3)N-acetyl-D-galactosamine, common to O-linked oligosaccharides and glycosphingolipids including asialo-GM1 (12). PNA does not bind to its recognition sugar sequence if it is substituted for by sialic acid, and even sialic acid not bound to the receptor sequence itself may inhibit lectin binding. The B subunit of cholera toxin recognizes the glycosphingolipid GM1 (23).

Cells were plated on vitrogen-coated Permanox four-well tissue culture chamber slides (Lab-Tek, Naperville, IL) and used within 2-4 days. At near confluence, the cells were rinsed two times in cold phosphate-buffered saline (PBS; 2 mM KH2HPO4, 6 mM Na2HPO4, 2 mM KCl, and 136 mM NaCl) and fixed with 4% paraformaldehyde, 1% glutaraldehyde, and 3% sucrose in PBS for 2 h. Fixed cells were rinsed two times with PBS and incubated with 20-100 µg of FITC-conjugated lectin in 300 µl of PBS for 0.5 h except for control wells that were incubated only with 300 µl of PBS. The wells were rinsed three times with PBS and air-dried. The slides were then fixed in methanol for 10 min, air-dried, and mounted for fluorescent microscopy with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) and coverslips. Neuraminidase treatment incubation with EBL to demonstrate lectin specificity for alpha -2,6-sialic acid linkages or PNA exposure to demonstrate lectin binding sites was for 0.5 h at 37°C with either 22.5 U/well of Salmonella typhimurium neuraminidase (260-fold kinetic preference for alpha -2,3-sialyl linkages over alpha -2,6-sialyl linkages) in 50 mM sodium citrate and 100 mM NaCl supplemented with 100 µg/ml of BSA or with 5 U/well of Clostridium perfringens neuraminidase (alpha -2right-arrow3,6,8-sialidase) in 50 mM sodium citrate (New England Biolabs, Beverly, MA). Nontreated cells were incubated under the same conditions without neuraminidase. Experiments testing the addition of calcium to the binding buffer for PNA were done in 10 mM HEPES and 0.15 M NaCl with 0.1 M calcium, and the cells were compared with cells incubated in the same buffer without calcium.

Fluorescence 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 1.   Expression of regulatory (R) domain does not interfere with cystic fibrosis transmembrane conductance regulator (CFTR) processing. Shown are Western blots of membrane vesicles isolated from HEK293 cells transfected with equal molar amounts of plasmids expressing either empty vector (pCEP) plus CFTR (vector:CFTR; lanes 1 and 4), R domain (pCEP-R) plus CFTR (R:CFTR; lanes 2 and 5); or pCEP-CFTR (CFTR; lanes 3 and 6). Left, blot probed with a COOH-terminal antibody (C-term Ab) for CFTR; right, blot probed with an R domain Ab. Quantitation of CFTR by densitometry showed that band C constitutes 79.08 (lane 2), 76.5 (lane 3), 86.9 (lane 5), and 77.4% (lane 6) of total CFTR. Band B was negligible by densitometry in lanes 1 and 4 so percentage of band C is not meaningful. The appearance of fully glycosylated CFTR in lanes 2 and 5 in quantities not reduced compared with control level demonstrates that coexpression of the R domain does not inhibit processing. Nos. at right, molecular mass in kDa.

Expression of Delta F508 and WT CFTR. Figure 2A shows the results of a coupled RT-PCR demonstrating the presence of mRNA for Delta F508 (Fig. 2A, lane 1) in pCEP-Delta F508 cells, which was probed with the appropriate 32P-tailed antisense oligo for the Delta 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).


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2.   Coupled RT-PCR showing presence of Delta F508 message in transfected 9/HTEo- cells (pCEP-Delta F508 clone 1). A: 2.5% Tris-acetate-EDTA agarose gel stained with ethidium bromide (ETBr). B: results of Southern hybridization of filter from same gel as A probed with an antisense oligo corresponding to the pCEP-Delta F508 mutation of CFTR. Lane 1, 2 µg of total RNA from pCEP-Delta F508 clone 1 cells with RT-Taq polymerase; lane 2, 2 µg of total RNA with Taq and without RT; lane 3, 2 µg of total RNA treated with RNase 1 before RT-PCR, with both RT and Taq present; lane 4, same as lane 3 but without RT; lane 5, all reagents but no RNA; lane 6, molecular weight marker; lane 7, pCEP-CFTR plasmid DNA [wild-type CFTR (WTCFTR)] with RT-Taq.

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-Delta F508 cells. They also failed to respond to isoproterenol with an increased rate of chloride efflux. Basal chloride efflux rate was reduced in pCEP-Delta 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-Delta 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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   6-Methoxy-N-(3-sulfopropyl)quinolinium (SPQ) assays of pCEP and pCEP- Delta F508 clones. Chloride efflux was measured in the absence (-) and presence (+) of 10 µM isoproterenol (ISO) for pCEP and the 2 clones (clones 1 and 9) of pCEP-Delta F508 used in this study. n, No. of cells assayed. The basal rate of chloride was reduced in pCEP-Delta F508 clone 9 compared with that in pCEP, whereas there was no significant difference in basal efflux rates in pCEP-Delta F508 clone 1. * Significant difference from pCEP control. ISO increased chloride efflux rate in pCEP-transfected cells but did not increase the rate of chloride efflux in pCEP-Delta F508 transfected cells, P < 0.05 by Student-Newman-Keuls method of pairwise comparisons.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Comparison of pCEP and pCEP-WTCFTR. A: apparent rate coefficient (r) for 36Cl efflux from pCEP and pCEP-WTCFTR. Each symbol represents a different experiment of the same group. Arrow, addition of theophylline (1 mM) and forskolin (10 µM). B: comparison of FITC-elderberry bark lectin (EBL) binding to pCEP and pCEP-WTCFTR. There was no significant difference in EBL binding between pCEP and pCEP-WTCFTR lines by either t-test or Kolmogorov-Smirnov (K-S) test. * Significantly reduced pixel intensity in pCEP-R cells, P < 0.05.

Quantitative fluorescence microscopy. Using two statistical methods of quantitation, we found that sialic acid in an alpha -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 Delta 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.

Based on previous reports (5, 9), we expected that on CF-phenotypic cells, sialic acid in the alpha -2,6 linkage that could be measured with EBL binding would be reduced and that there would be either a commensurate reduction in total sialic acid or a compensatory increase in sialic acid in the alpha -2,3 configuration and in asialo-GM1. The most consistent difference in fluorescent lectin binding between the control and experimental cell lines was observed with EBL (Table 1). A typical experiment (Fig. 5) shows a significant reduction (59%) in the mean pixel intensity of EBL binding to pCEP-R cells and a 45% reduction in mean pixel intensity of the pCEP-Delta F508 images compared with that in control cells. EBL specifically recognizes alpha -2,6-sialic acid linkages. Clostridium perfringens neuraminidase, specific for alpha -2,6-sialic acid, significantly reduced EBL binding almost to background values (Fig. 5), whereas incubation with a sialidase with preference to the alpha -2,3 linkage from Salmonella typhimurium did not significantly alter the binding of EBL (data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Lectin binding summary



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   EBL binding experiment. Cell monolayers were treated with and without Clostridium perfringens (alpha -2,6-neuraminidase) before EBL binding. Neuraminidase (22.5 U/well)-treated cells were incubated for 0.5 h at 37°C before binding with 200 µg/ml of EBL for 0.5 h. Values are means ± SE of digital pixel intensity. Mean values before treatment were 1,619.29 ± 194.0 for pCEP, 666.37 ± 53.8 for pCEP-R, and 905.99 ± 35.4 for pCEP-Delta F508. For both pCEP-R and Delta F508, mean pixel intensities were significantly reduced from pCEP values (P < 0.001 and P = 0.01, respectively, by Student's t-test). Mean values of neuraminidase-treated wells were 477.33 ± 26.5 for pCEP, 344.19 ± 14.7 for pCEP-R, and 350.23 ± 17.0 for pCEP-Delta F508. Background pixel intensity value for this experiment was 252. Neuraminidase-treated wells were significantly reduced for each cell line, P <=  0.01 compared with respective untreated monolayers by t-test.

However, there was no significant change in overall total sialic acid (as assessed by ECL binding as well as by WGA and SWGA) or in alpha -2,3-linked sialic acid (as assessed by MAL I binding). Moreover, we detected no difference in asialo-GM1 using the binding of PNA, although both the pCEP-R and pCEP-Delta F508 cells used in this study, when assessed by specific antibody binding, showed increases in asialo-GM1 and also increased binding of Pseudomonas aeruginosa compared with those in the control pCEP cell line (8). However, when the cells were incubated with an alpha -2,3-specific sialidase, the pCEP-R and pCEP-Delta F508 cells showed significant increases in binding PNA, whereas pCEP cells did not (K-S test results comparing untreated vs. neuraminidase treated: pCEP, P > 0.1; pCEP-R, P < 0.05; pCEP-Delta F508, P < 0.001; data not shown). Thus asialo-GM1 sites may be inaccessible to lectin unless the surrounding molecules are modified to better expose the receptor similar to the increased binding of Pseudomonas observed after incubation with PAO1 supernatant (20, 21).

Table 1 summarizes the results of comparisons of the total intensity of fluorescent lectin binding (t-test) to pCEP-R or pCEP-Delta F508 cells compared with that in pCEP control cells. Separate analysis of individual experiments allows both internal experimental comparisons and assessment of experimental variability as well as quantitative determination of significance. All of the EBL binding experiments showed a reduction in mean pixel intensity or a reduction in the intensity of the overall fluorescence in the experimental cell lines, and most reached significance (P < 0.05). Four of the experiments comparing pCEP-R cells with pCEP cells showed a significant decrease in mean pixel intensity ranging from 14 to 60% of control values, and 4 of 6 experiments showed a significant reduction in EBL binding to pCEP-Delta F508 cells of 16-61% of the quantitated mean pixel intensities of the control cells. For the other lectins, there were no consistent changes in the intensity of overall fluorescence in experimental compared with control lines.

We also compared the distribution of fluorescence intensity in the various cell lines using the K-S test. For this analysis, the absolute intensity values are ignored, and the distribution of bright fluorescence within an image is considered. Figure 6 illustrates one experiment of EBL binding. Figure 6A, inset, shows the average pixel intensities of the original individual images from which the mean pixel intensities of the experiment were obtained. Figure 6A shows these data combined. The average pixel intensity obtained from the histogram is the same value as the mean pixel intensity shown in Fig. 6A, inset. Figure 6B represents the same images as in Fig. 6A converted for analysis of distribution of fluorescence by the K-S nonparametric test for analysis of histograms. Figure 6B, inset, shows the first step in the statistical analysis, conversion of the histogram to probability distribution functions from which the K-S values are derived. Figure 6 shows that there are both more pixels at lower intensity values and fewer pixels at higher intensity values for both the pCEP-R cell line and the Delta F508 cell line compared with those in the control line. Thus in the region that represents specific EBL binding, there is less EBL bound to pCEP-Delta F508 and pCEP-R cells than to the control line pCEP (Table 1). Figure 7 shows a representative experiment.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6.   Representative plots of EBL binding experiments. A: histograms of the 12-bit images (0-4,095 intensity range) from each cell line were combined into 1 histogram plot representing the experiment. The average pixel intensities computed from the histograms is the same as the mean intensities of the fields shown in the inset (pCEP, 1,073.08 ± 40.8; pCEP-R, 921.22 ± 29.02; pCEP-Delta F508, 883.05 ± 28.6). B: conversion of the histograms in Fig. 5A to a 0- to 256-pixel intensity scale used for K-S test analysis. Inset, probability distribution curve derived from the histograms. Shaded areas, background values or autofluorescence determined for each experiment. Arrow, maximum vertical displacement ("D" value, -0.181317 between pCEP and pCEP-R) from which a K-S value (1.884297) was derived and compared with critical table values to determine level of significance for the K-S test, P < 0.005. For this experiment comparing pCEP-Delta F508 with pCEP, D = 0.245462, K-S = 2.550918, and P < 0.001.



View larger version (76K):
[in this window]
[in a new window]
 
Fig. 7.   Representative images from EBL binding experiments. Top: EBL-FITC fluorescence. Bottom: transmitted light images of the same fields in A. The images were selected from between 4 and 14 fields collected from each well of a chamber slide and were close to the mean pixel intensity of those images digitized for the experiment. For illustrative purposes only, equal contrast enhancement has been applied.

To examine the subcellular accumulation of the fluorescent labeling by lectins in these permeabilized cells, confocal microscopy was performed. More than 80% of the EBL labeling was at the cell surface, but for the other lectins, fluorescence was distributed throughout the cytoplasmic compartment as well (data not shown).

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).


View larger version (130K):
[in this window]
[in a new window]
 
Fig. 8.   Electron micrographs of immunogold labeling of 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) in acidic vesicles. PCEP (A) and pCEP-R (B) cells were incubated with the acidotrophic amine DAMP, fixed, and embedded in Epon. Immunocytochemistry on thin sections labeled the DAMP that accumulated in intracellular vesicles with 10-nm gold. Circles, region that is enlarged at right showing the trans-Golgi network (TGN). Arrows, gold particles in the TGN. N, nucleus; M, mitochondria; MVB, multivesicular bodies. There was no apparent difference in cellular morphology between the cell lines. Final magnification was approximately ×61,300.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 9.   DAMP accumulation in intracellular organelles. pCEP or pCEP-R monolayers were incubated with the acidotrophic amine DAMP, which was subsequently detected with immunogold labeling techniques. Gold particles in intracellular organelles were counted from negatives enlarged to approximately ×60,000. Values are averages ± SE of combined data from 2 separate experiments after background subtraction. There was no significant difference in the size of the organelles in the cell lines. Cells incubated with monensin to dissipate the pH gradients showed gold particles in the vesicles at background levels (data not shown). Nucleus: pCEP, 111.4 µm2 counted; pCEP-R, 20.6 µm2 counted. Mitochondria: pCEP, n = 17; pCEP-R, n = 9. Multivesicular bodies (MV): pCEP, n = 23; pCEP-R, n = 31. Gray, dense organelles (G/D): pCEP, n = 11; pCEP-R, n = 33. trans-Golgi network: pCEP, n = 19; pCEP-R, n = 12. n, No. of vesicles counted. There was no significant difference in the pH of measured intracellular organelles between pCEP and pCEP-R by the nonparametric Mann-Whitney rank sum test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta F508 CFTR, is associated with altered sialylation in the cell. Binding of EBL, which recognizes sialic acid in the alpha -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-Delta 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 Delta 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 Delta F508-CFTR-transfected cells. However, again, CFTR activity was paralleled by EBL binding, which was reduced in the Delta 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 Delta F508 CFTR (9) in which Delta 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 Delta 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 Delta 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 Delta 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 Delta 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 alpha -2,3 as well as in an alpha -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-Delta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

9.   Dosanjh, A, Lencer W, Brown D, Ausiello DA, and Stow JL. Heterologous expression of Delta F508 CFTR results in decreased sialylation of membrane glycoconjugates. Am J Physiol Cell Physiol 266: C360-C366, 1994[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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 fibrosis---therapeutic 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 (alpha 2,3) Gal (beta 1,4) GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid in alpha 2,3 and alpha 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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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 (alpha 2,6) Gal/GalNAc sequence. J Biol Chem 262: 1596-1601, 1987[Abstract/Free Full Text].

27.   Teneberg, S, Angstrom J, Jovall PA, and Karlsson KA. Characterization of binding of Gal beta  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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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 Delta 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[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 280(3):L482-L492
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society