Rescue of functional delF508-CFTR channels in cystic fibrosis epithelial cells by the alpha-glucosidase inhibitor miglustat

In the disease cystic fibrosis (CF), the most common mutation delF508 results in endoplasmic reticulum retention of misfolded CF gene proteins (CFTR). We show that the alpha-1,2-glucosidase inhibitor miglustat (N-butyldeoxynojirimycin, NB-DNJ) prevents delF508-CFTR/calnexin interaction and restores cAMP-activated chloride current in epithelial CF cells. Moreover, miglustat rescues a mature and functional delF508-CFTR in the intestinal crypts of ileal mucosa from delF508 mice. Since miglustat is an orally active orphan drug (Zavesca) prescribed for the treatment of Gaucher disease, our findings provide the basis for future clinical evaluation of miglustat in CF patients.     

Mild processing defect of porcine DeltaF508-CFTR suggests that DeltaF508 pigs may not develop cystic fibrosis disease

Recent efforts have made significant progress in generating transgenic pigs with the ΔF508-CFTR mutation to model the lung and pancreatic disease of human cystic fibrosis. However, species differences in the processing and function of human, pig and mouse ΔF508-CFTR reported recently raise concerns about the phenotypic consequence of the gene-targeted pig model. The purpose of the present study was to characterize the ΔF508 mutant of porcine CFTR to evaluate the severity of its processing defect. Biochemical and immunofluorescence analysis in transfected COS7 and FRT cells indicated that pig ΔF508-CFTR efficiently targets to the plasma membrane and is present mainly as the mature glycosylated protein. Functional characterization in stably transfected FRT cells by fluorometric and electrophysiological assays supported efficient plasma membrane targeting of pig ΔF508-CFTR. The mild cellular processing defect of pig ΔF508-CFTR suggests that its gene-targeted pig model may not develop the lung and pancreatic phenotypes seen in CF patients.     

Beta-oestradiol rescues DeltaF508CFTR functional expression in human cystic fibrosis airway CFBE41o- cells through the up-regulation of NHERF1

Background information. CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, ΔF508 (deletion of Phe‐508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na⁺/H⁺‐exchanger regulatory factor 1) in CF airway cells induced both a redistribution of ΔF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)‐dependent activation of ΔF508CFTR‐dependent chloride secretion. In view of the potential importance of the targeted up‐regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o⁻, with subsequent rescue of apical ΔF508CFTR chloride transport activity. Results. We found that CFBE41o⁻ cells do express ERs (oestrogen receptors) in the nuclear fraction and that β‐oestradiol treatment was able to significantly rescue ΔF508CFTR‐dependent chloride secretion in CFBE41o⁻ cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the ΔF508CFTR translocated to the apical membrane can function as a cAMP‐responsive channel, with a significant increase in chloride secretion noted at 1 nM β‐oestradiol and a maximal effect observed at 10 nM. Importantly, knock‐down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the β‐oestradiol‐dependent increase in ΔF508CFTR protein expression levels and completely prevented the β‐oestradiol‐dependent rescue of ΔF508CFTR transport activity. Conclusions. These results demonstrate that β‐oestradiol‐dependent up‐regulation of NHERF1 significantly increases ΔF508CFTR functional expression in CFBE41o⁻ cells.     

Defective formation of PKA/CnA-dependent annexin 2-S100A10/CFTR complex in DeltaF508 cystic fibrosis cells

Cystic fibrosis (CF) is characterised by impaired epithelial ion transport and is caused by mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/PKA and ATP-regulated chloride channel. We recently demonstrated a cAMP/PKA/calcineurin (CnA)-driven association between annexin 2 (anx 2), its cognate partner –S100A10 and cell surface CFTR. The complex is required for CFTR and outwardly rectifying chloride channel function in epithelia. Since the cAMP/PKA-induced Cl⁻ current is absent in CF epithelia, we hypothesized that the anx 2–S100A10/CFTR complex may be defective in CFBE41o cells expressing the commonest F508del-CFTR (ΔF-CFTR) mutation. Here, we demonstrate that, despite the presence of cell surface ΔF-CFTR, cAMP/PKA fails to induce anx 2–S100A10/CFTR complex formation in CFBE41o− cells homozygous for F508del-CFTR. Mechanistically, PKA-dependent serine phosphorylation of CnA, CnA–anx 2 complex formation and CnA-dependent dephosphorylation of anx 2 are all defective in CFBE41o− cells. Immunohistochemical analysis confirms an abnormal cellular distribution of anx 2 in human and CF mouse epithelia.

Thus, we demonstrate that cAMP/PKA/CnA signaling pathway is defective in CF cells and suggest that loss of anx 2–S100A10/CFTR complex formation may contribute to defective cAMP/PKA-dependent CFTR channel function.     

Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o-

The CFBE41o- cell line was generated by transformation of cystic fibrosis (CF) tracheo-bronchial cells with SV40 and has been reported to be homozygous for the DeltaF508 mutation. A systematic characterisation of these cells, which however, is a pre-requisite for their use as an in vitro model, has not been undertaken so far. Here, we report an assessment of optimal culture conditions, the expression pattern of drug-transport-related proteins and the stability/presence of the CF transmembrane conductance regulator (CFTR) mutation in the gene and gene product over multiple passages. The CFBE41o- cell line was also compared with a wild-type airway epithelial cell line, 16HBE14o-, which served as model for bronchial epithelial cells in situ. The CFBE41o- cell line retains at least some aspects of human CF bronchial epithelial cells, such as the ability to form electrically tight cell layers with functional cell-cell contacts, when grown under immersed (but not air-interfaced) culture conditions. The cell line is homozygous for DeltaF508-CFTR over multiple passages in culture and expresses a number of proteins relevant for pulmonary drug absorption (e.g. P-gp, LRP and caveolin-1). Hence, the CFBE41o- cell line should be useful for studies of CF gene transfer or alternative treatment with small drug molecules and for the gathering of further information about the disease at the cellular level, without the need for primary culture.     

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Dysfunction of mitochondria Ca uptake in cystic fibrosis airway epithelial cells

In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca²⁺ mobilization is abnormally increased. Because the uptake of Ca²⁺ by mitochondria during Ca²⁺ influx or Ca²⁺ release from ER stores may be crucial for maintaining a normal Ca²⁺ homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (ΔΨ_mit) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2–AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca²⁺. The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the ΔΨ_mit of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca²⁺ uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca²⁺ uptake.     

Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the DeltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene

A satisfactory model describing the airway surface fluid (ASF) in the airways of persons with cystic fibrosis (CF) remains to be established due to theoretical challenges to both the "Hydration Hypothesis" and the "Salt Hypothesis." Irrespective of these models, inhaled hypertonic saline is often used to facilitate clearance of inspissated secretions. Hypertonicity induces interleukin-8 (IL-8) expression, a potent chemokine for neutrophils. The objectives of this study were: (i) to determine the relative contribution of three potential cis-regulatory elements in the regulation of NaCl-induced IL-8 production in BEAS-2B human bronchial epithelial cells, (ii) to compare NaCl-induced IL-8 expression in IB3-1 bronchial epithelial cells, which have the DeltaF508/W1282X mutation of the CF transmembrane conductance regulator (CFTR) gene, with that in C38 cells, which are IB3-1 cells stably transfected with a truncated but functional CFTR gene, and (iii) to compare equal osmolar concentrations of NaCl and D-sorbitol in the induction of IL-8 in all three cell types. In human bronchial epithelial cells, binding sites for NFkappaB, AP-1, and NF-IL6 in the 5'-flanking region of the IL-8 promoter are necessary for optimal NaCl induction of IL-8. Human bronchial epithelial cells with the DeltaF508/W1282X CFTR mutation produce an exaggerated amount of basal and NaCl-induced IL-8.     

Characterisation of chloride currents across the proximal colon in CftrTgH(neoim)1Hgu congenic mice

It was the aim of the present study to investigate chloride secretion across the proximal colon of Cftr ^{TgH(neoim)1Hgu} congenic mice. Stripped epithelia were incubated in Ussing chambers and the electrophysiological data were compared between cystic fibrosis (CF) animals and wild type (WT) animals. In comparison with the control animals, all Cftr ^{TgH(neoim)1Hgu} congenic mice had a distinctly reduced basal chloride secretion and a reduced chloride secretion after stimulation with carbachol and forskolin. When comparing chloride secretion across the proximal colon between WT animals, all mice showed a comparable pattern of response to carbachol and forskolin but quantitative differences, BALB/c exhibiting the highest and HsdOla:MF1 exhibiting the lowest increase in Cl current. Likewise, all CF animals showed the same reaction pattern to carbachol and forskolin, but there was no distinct difference that lasted for the whole measurement. To investigate interferences between Ca- and cyclic adenosine monophosphate-activated pathways of Cl secretion in CF animals, we studied epithelia from CF/3CF/1F1 animals with a mixed background. In these animals, the levels of the carbachol or forskolin-induced chloride currents did not depend on the prestimulation with the respective other secretagogue. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, which blocks calcium-activated chloride channels, reduced the current response to carbachol by about 23%. This result, obtained in BALB/c-Cftr ^{TgH(neoim)1Hgu} mice, indicates that alternative chloride channels might be present in the proximal colon of these mice. In contrast, there was no evidence for alternative chloride conductances in BALB/c WT animals, but we cannot exclude that in WT mice a higher chloride secretion via Cftr-channels may have masked an alternative chloride secretion.     

Calumenin contributes to ER-Ca2+ homeostasis in bronchial epithelial cells expressing WT and F508del mutated CFTR and to F508del-CFTR retention

Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca²⁺) homeostasis. In this study, we discovered that the Ca²⁺ binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca²⁺ calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca²⁺ concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR.     

Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells

The most common mutation in cystic fibrosis, Delta F508, results in a cystic fibrosis transmembrane conductance regulator (CFTR) protein that is retained in the endoplasmic reticulum (ER). Retention is dependent upon chaperone proteins, many of which require Ca(++) for optimal activity. Interfering with chaperone activity by depleting ER Ca(++) stores might allow functional Delta F508-CFTR to reach the cell surface. We exposed several cystic fibrosis cell lines to the ER Ca(++) pump inhibitor thapsigargin and evaluated surface expression of Delta F508-CFTR. Treatment released ER-retained Delta F508-CFTR to the plasma membrane, where it functioned effectively as a Cl(-) channel. Treatment with aerosolized calcium-pump inhibitors reversed the nasal epithelial potential defect observed in a mouse model of Delta F508-CFTR expression. Thus, ER calcium-pump inhibitors represent a potential target for correcting the cystic fibrosis defect.     

Relation of exaggerated cytokine responses of CF airway epithelial cells to PAO1 adherence

In many model systems, cystic fibrosis (CF) phenotype airway epithelial cells in culture respond to P. aeruginosa with greater interleukin (IL)-8 and IL-6 secretion than matched controls. In order to test whether this excess inflammatory response results from the reported increased adherence of P. aeruginosa to the CF cells, we compared the inflammatory response of matched pairs of CF and non CF airway epithelial cell lines to the binding of GFP-PAO1, a strain of pseudomonas labeled with green fluorescent protein. There was no clear relation between GFP-PAO1 binding and cytokine production in response to PAO1. Treatment with exogenous aGM1 resulted in greater GFP-PAO1 binding to the normal phenotype compared to CF phenotype cells, but cytokine production remained greater from the CF cell lines. When cells were treated with neuraminidase, PAO1 adherence was equalized between CF and nonCF phenotype cell lines, but IL-8 production in response to inflammatory stimuli was still greater in CF phenotype cells. The polarized cell lines 16HBEo-Sense (normal phenotype) and Antisense (CF phenotype) cells were used to test the effect of disrupting tight junctions, which allows access of PAO1 to basolateral binding sites in both cell lines. IL-8 production increased from CF, but not normal, cells. These data indicate that increased bacterial binding to CF phenotype cells cannot by itself account for excess cytokine production in CF airway epithelial cells, encourage investigation of alternative hypotheses, and signal caution for therapeutic strategies proposed for CF that include disruption of tight junctions in the face of pseudomonas infection.     

Intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells

Cystic fibrosis is a common genetic disorder characterized by a severe lung inflammation and fibrosis leading to the patient's death. Enhanced angiogenesis in cystic fibrosis (CF) tissue has been suggested, probably caused by the process of inflammation, as similarly described in asthma and chronic bronchitis. The present study demonstrates an intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells. Microarray experiments showed that CF airway epithelial cells expressed several angiogenic factors such as VEGF-A, VEGF-C, bFGF, and PLGF at higher levels than control cells. These data were confirmed by real-time quantitative PCR and, at the protein level, by ELISA. Conditioned media of these cystic fibrosis cells were able to induce proliferation, migration and sprouting of cultured primary endothelial cells. This report describes for the first time that cystic fibrosis epithelial cells have an intrinsic angiogenic activity. Since excess of angiogenesis is correlated with more severe pulmonary disease, our results could lead to the development of new therapeutic applications.     

Fucosylation in cystic fibrosis airway epithelial cells

Altered glycosylation is a phenotypic characteristic of cystic fibrosis (CF), and some of the alterations are summarized. The lungs are the site of the lethal pathology of the disease. Therefore, two of the characteristics were examined in CF and non-CF immortalized airway epithelial cell lines (AEC). The activity of -l-fucosidase was elevated (280%) in CF AEC when compared with non-CF AEC, whereas the activities of the other lysosomal enzymes which were examined were similar in both cell types. -l-Fucosidase activity was transiently increased in the non-CF cells after treatment with Brefeldin A (BFA) for 6 h. Thus BFA caused the normal cells to express a phenotypic characteristic of CF. Glycopeptides from the CF and non-CF AECs metabolically labeled withl-[³H]fucose were examined for binding to lentil lectin-Sepharose. A higher percentage of CF glycopeptides bound to lentil lectin, 43% compared with 23% for non-CF control. In addition a higher percentage of CF glycopeptides were bound tightly to lentil lectin and required 0.2M -methylmannoside to be eluted. This species of tightly bound glycopeptides increased dramatically to 77% from 46% when the CF AEC were treated with BFA. In contrast, the non-CF cell glycopeptides had a minor decrease in tightly bound glycopeptides to 26% from 33% after BFA treatment. Thus, the CF AEC showed fucosylation alteration observed previously for other CF cells and tissue.     

4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (−/−) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs.     

Basolateral K channel activated by carbachol in the epithelial cell line T84

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (I _SC), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T₈₄ cells. Carbachol had little effect on I _SC when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent I _SC response to calcium ionophores. Carbachol (100 m) transiently elevated intracellular free calcium ([Ca²⁺] _i ) by 3-fold in confluent cells cultured on glass coverslips with a time course resembling the I _sc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (–54 mV) with pipette solution containing 150 mm KCl (37°C). This rectification persisted when patches were bathed in symmetrical 150 mm KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mm barium. It is referred to as the K_BIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single K_BIC channels, the carbachol-stimulated I _SC was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the K_BIC channel mediates basolateral membrane K conductance in T₈₄ cell monolayers during stimulation by cholinergic secretagogues.We thank Dr. Marcel Crest (Laboratoire de Neurobiologie, CNRS, Marseille) for providing a sample of kaliotoxin. This work was supported by the Canadian Cystic Fibrosis Foundation and the Respiratory Health Network of Centres of Excellence. J.W.H. is a Chercheur-Boursier of the Fonds de la recherche en santé du Québec.     

Low temperature and chemical rescue affect molecular proximity of DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC)

An imbalance of chloride and sodium ion transport in several epithelia is a feature of cystic fibrosis (CF), an inherited disease that is a consequence of mutations in the cftr gene. The cftr gene codes for a Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Some mutations in this gene cause the balance between Cl(-) secretion and Na(+) absorption to be disturbed in the airways; Cl(-) secretion is impaired, whereas Na(+) absorption is elevated. Enhanced Na(+) absorption through the epithelial sodium channel (ENaC) is attributed to the failure of mutated CFTR to restrict ENaC-mediated Na(+) transport. The mechanism of this regulation is controversial. Recently, we have found evidence for a close association of wild type (WT) CFTR and WT ENaC, further underscoring the role of ENaC along with CFTR in the pathophysiology of CF airway disease. In this study, we have examined the association of ENaC subunits with mutated ΔF508-CFTR, the most common mutation in CF. Deletion of phenylalanine at position 508 (ΔF508) prevents proper processing and targeting of CFTR to the plasma membrane. When ΔF508-CFTR and ENaC subunits were co-expressed in HEK293T cells, we found that individual ENaC subunits could be co-immunoprecipitated with ΔF508-CFTR, much like WT CFTR. However, when we evaluated the ΔF508-CFTR and ENaC association using fluorescence resonance energy transfer (FRET), FRET efficiencies were not significantly different from negative controls, suggesting that ΔF508-CFTR and ENaC are not in close proximity to each other under basal conditions. However, with partial correction of ΔF508-CFTR misprocessing by low temperature and chemical rescue, leading to surface expression as assessed by total internal reflection fluorescence (TIRF) microscopy, we observed a positive FRET signal. Our findings suggest that the ΔF508 mutation alters the close association of CFTR and ENaC.     

Terminal glycosylation of cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) has a characteristic glycosylation phenotype usually expressed as a decreased ratio of sialic acid to fucose. The glycosylation phenotype was found in CF/T1 airway epithelial cells (F508/F508). When these cells were transfected and were expressing high amounts of wtCFTR, as detected by Western blot analysis and in situ hybridization, the cell membrane glycoconjugates had an increased sialic acid content and decreased fucosyl residues in 1,3/4 linkage to antennary N[emsp4 ]-acetyl glucosamine (Fuc1,3/4GlcNAc). After the expression of wtCFTR decreased, the amount of sialic acid and Fuc1,3/4GlcNAc returned to levels shown by the parent CF cells. Sialic acid was measured by chemical analysis and Fuc1,3/4GlcNAc was detected with a specific 1,3/4 fucosidase. CF and non-CF airway cells in primary culture also had a similar reciprocal relationship between fucosylation and sialylation. It is possible that the glycosylation phenotype is involved in the pathogenesis of CF lung disease by facilitating bacterial colonization and leukocyte recruitment.