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First published online 5 August 2008
doi: 10.1242/jcs.028951

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Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator

¹ Mayo Clinic College of Medicine, Mayo Clinic Arizona, Scottsdale, AZ 85259, USA
² Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA
³ Cystic Fibrosis Research Center, University of North Carolina, Chapel Hill, NC 27599, USA
⁴ Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599, USA
⁵ Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599, USA

View larger version (19K): [in this window] [in a new window]   Fig. 1. Comparison of the sequences coding for the two potential N-glycosylation sites, 894 and 900, in extracellular loop 4 of CFTR. Sequences of mammalian CFTRs were aligned using FASTA. Amino acids fitting the consensus N-X-S/T for asparagine-linked glycosylation are highlighted. X can be any amino acid residue other than proline (Imperiali and Hendrickson, 1995).

View larger version (30K): [in this window] [in a new window]   Fig. 2. Unglycosylated CFTR does not bind to calnexin but functions as a chloride channel. (A) Western blot of CFTR and unglycosylated CFTR created either by mutation of the glycosylation sites (N894D/N900D) or by expressing CFTR in tunicamycin-treated cells (+ Tunicamycin). CFTR and CFTR N894D/N900D variants were stably (left panel) or transiently (right panel) expressed in BHK-21 cells. Tunicamycin (5 µg/ml) was added directly after transient transfection to achieve complete inhibition of N-linked glycosylation. CFTR was detected in the microsomal membrane vesicle fraction by western blotting using mouse monoclonal anti-CFTR antibody 596. (B) CFTR N894D/N900D does not interact with calnexin. CFTR was immunoprecipitated by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads (as indicated on the left). CFTR and calnexin (CNX) were detected by western blotting using mouse monoclonal antibody 596 for CFTR or rabbit polyclonal antibody SPA860 (Stressgen) for calnexin (as indicated on the right). Molecular weight marker positions (kDa) are indicated on the left. (C) The channel properties of unglycosylated CFTR are similar to wild-type CFTR. Single-channel measurements were performed using membrane vesicles prepared from stably transfected BHK-CFTR, BHK-N894D/N900D cells or from cells transiently transfected with CFTR that were treated with tunicamycin. Tunicamycin (5 µg/ml) was added directly after transient transfection to achieve complete inhibition of N-linked glycosylation.

View larger version (60K): [in this window] [in a new window]   Fig. 3. F508 N894D/N900D cannot escape ER quality control. (A) Western blot of CFTR, CFTR N894D/N900D, F508 and F508 N894D/N900D expressed in BHK-21 cells. Lysates were separated by SDS-PAGE and CFTR was detected after transfer to nitrocellulose by mouse monoclonal anti-CFTR antibody 570. (B) F508 N894D/N900D does not interact with calnexin. F508 N894D/N900D was immunoprecipitated by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads. CFTR and calnexin (CNX) were detected by western blotting using mouse monoclonal antibody 596 for CFTR or rabbit polyclonal antibody SPA860 for calnexin. Molecular weight marker positions (kDa) are indicated on the left. (C) Immunofluorescence microscopy of CFTR and F508 glycosylation variants in permeabilized cells. Immunostaining was performed on permeabilized BHK-21 cells using anti-CFTR mouse monoclonal antibody 570, followed by goat anti-mouse IgG Alexa Fluor 488 conjugate. Calnexin was stained to visualize the ER compartment using rabbit anti-calnexin antibodies followed by goat anti-rabbit IgG Alexa Fluor 568 conjugate. (D) Visualization of cell-surface CFTR on non-permeabilized cells by applying antibody HA11 to detect the external HA epitope in EL2 of CFTR variants. Cells were grown at 37°C or incubated at 27°C for 48 hours in the presence of 2 mM sodium butyrate to promote cell-surface expression of Extope-F508 CFTR. (E) cAMP-stimulated 36Cl– efflux measurements of stably expressing BHK-CFTR cells. Stimulation cocktail was added (+) at time 0. Each point represents the average of three independent samples and standard deviations are indicated. (F) Immunostaining of CFTR and F508 glycosylation variants in virally transduced well-differentiated primary human airway epithelial cells. All pools of intracellular CFTRs were stained on frozen sections of cultures grown at 37°C with HA11 antibody followed by goat anti-mouse IgG Alexa 488 conjugate. (G) Labeling of apical CFTR in virally transduced well-differentiated primary human airway epithelial cells. Cultures were incubated at 27°C for 48 hours and the apical pool of CFTR variants was labeled with HA11 antibody; cultures were then frozen in OCT and frozen sections labeled with goat anti-mouse Alexa 488 conjugate. Scale bars: 10 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 4. EDEM binds CFTR and accelerates its degradation, but neither the inhibition of EDEM interaction nor of calnexin interaction can rescue F508 CFTR. (A) CFTR was immunoprecipitated from cells transiently expressing HA-tagged EDEM by anti-CFTR monoclonal antibody 596 crosslinked to Dynabeads. EDEM was detected by western blotting using monoclonal antibody HA11 and CFTR was detected by antibody 596. (B) CFTR was expressed transiently in BHK-21 cells alone or together with EDEM using pcDNA3 CFTR and pCMV-SPORT2 EDEM, respectively. In the case of CFTR expression alone, similar amounts of empty control vector were co-transfected with the CFTR plasmid. (C) Immunofluorescence microscopy of F508 CFTR expressed in BHK-21 cells that were treated with various glycosidase inhibitors or tunicamycin. CAS (castanospermine, 0.2 mM) inhibits glucosidase I; DMM (1-deoxymannojirimycin, 0.5 mM) and kifunensine (0.2 mM) are inhibitors of mannosidase I; and tunicamycin (10 µg/ml) completely blocks N-glycosylation. The activity of these compounds was confirmed (supplementary material Fig. S3; Fig. 2A). Inhibitors were added to the growth media for 18 hours at the concentrations indicated and F508 CFTR was visualized on permeabilized cells using anti-CFTR monoclonal antibody 596 followed by goat anti mouse IgG Alexa Fluor 488 conjugate. Scale bar: 10 µm. (D) Cell-surface ELISA showing that glycosidase inhibitors do not rescue F508. BHK-21 cells expressing an externally tagged F508 CFTR (Gentzsch et al., 2004) were seeded in a 96-well plate at 40,000 cells per well. Twenty-four hours after seeding, cells were treated for 20 hours with CAS (0.4 mM), DMM (0.5 mM), KIF (kifunensine, 0.2 mM) or tunicamycin (10 µg/ml). Corrector compounds C3 (VRT-325) and C4 (corr-4a), which have been reported to partially rescue F508 CFTR (Loo et al., 2005; Pedemonte et al., 2005; Van Goor et al., 2006), were used as positive control at 20 µM each. Cells were fixed, labeled with HA11 antibody that recognizes the external tag, followed by goat anti-mouse IgG conjugated to X-Sight 761 and scanned with an infrared imaging system. A significant increase in the cell-surface pool of F508 CFTR was observed on treatment with C3 and C4 (*P<0.0001). Neither treatment with glycosidase inhibitors nor complete inhibition of N-glycosylation by tunicamycin had an effect that was significantly different from the no-drug control. Data represent the average of eight wells; bars indicate s.e.m. Statistical significance was determined using an unpaired Student's t-test. The blue line indicates the baseline level of the no-drug control.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Mutagenesis and enzymatic deglycosylation of individual CFTR N-glycosylation sites. (A) Western blot of CFTR variants N-glycosylated at both, either, or neither of the two native sites. 1% SDS lysates of stably expressing CHO cells were resolved on 6% polyacrylamide gels, transferred to nitrocellulose and probed with mouse monoclonal antibody M3A7. (B) Endoglycosidase H treatment of CFTR individual glycosylation site mutants. Total cell lysates were incubated with (+) or without (–) endoglycosidase H at 37°C for 4 hours and analyzed by western blotting. The arrowheads indicate core-glycosylated protein or protein deglycosylated by endoglycosidase H. (C) Individual glycosylation site variants have different sensitivities towards deglycosylation by N-glycanase. Lysates of stably expressing CHO cells were incubated with 1 U N-glycosidase F at 37°C for 0 (lane 1), 10 minutes (lane 2), 20 minutes (lane 3) or 24 hours (lane 4) and subjected to SDS-PAGE and western blotting.

View larger version (44K): [in this window] [in a new window]   Fig. 6. Turnover of CFTR glycosylation site variants with a single N-linked oligosaccharide. (A) Glycosylation site variant maturation and turnover as monitored by metabolic pulse-chase labeling. Cells were washed twice with methionine-free MEM and starved for 30 minutes in the same medium. [35S]methionine was added to allow incorporation during a 20-minute pulse. Cells were then maintained in medium supplemented with 1 mM methionine, lysed with RIPA buffer at the times indicated, CFTR protein immunoprecipitated and the eluted proteins resolved on 7% polyacrylamide gels. (B) Effect of brefeldin A on turnover of glycosylation site variants. Wild-type and variant CFTR proteins were labeled in the presence of brefeldin A and pulse-chase experiments performed as described in A and Materials and Methods. (C) The amount of labeled protein remaining after the indicated chase periods was quantified by electronic autoradiography image analysis and is shown as a percentage of the initial label.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Interaction of N894D and N900D single-chain mutants with calnexin and EDEM. (A) Cell lysates from BHK-21 cells stably expressing CFTR, CFTR N894D, CFTR N900D or CFTR N894D/N900D were subjected to immunoprecipitation by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads (IP: CFTR, as indicated on the left). CFTR and calnexin (CNX) were detected by western blotting using mouse monoclonal antibody 596 for CFTR or rabbit polyclonal antibody SPA860 for calnexin (as indicated on the right). Molecular weight marker positions (kDa) are indicated on the left. (B) CFTR was immunoprecipitated from cells that were stably expressing CFTR, CFTR N894D, CFTR N900D or CFTR N894D/N900D and that were also transiently expressing HA-tagged EDEM, by incubation with anti-CFTR monoclonal antibody 596 crosslinked to Dynabeads. EDEM was detected by western blotting using monoclonal antibody HA11 and CFTR using antibody 596.

View larger version (35K): [in this window] [in a new window]   Fig. 8. Individual N-linked oligosaccharides promote different fates in the processing of CFTR. Immediately after addition of the core glycan to the nascent polypeptide chain, the outermost of the three glucose residues is removed by glucosidase I. Subsequently, glucosidase II removes the next glucose residue. The resulting monoglucosylated core glycans bind to calnexin. When glucosidase II removes the remaining glucose residue, the glycoprotein dissociates from calnexin and, if properly folded, is free to leave the ER. If the protein is incompletely folded it is reglucosylated by the UDP-glucose:glycoprotein glucosyltransferase, creating again a monoglycosylated core oligosaccharide that can bind to calnexin. Proteins that have stayed in the ER for too long and are terminally misfolded become a substrate for -mannosidase I. The trimmed oligosaccharide binds to EDEM (ER degradation-enhancing -mannosidase-like protein) and the glycoprotein is consequently directed towards ERAD (ER-associated degradation). The carbohydrate at position 900 in CFTR N894D supports the route to maturation and progress to the Golgi, whereas the oligosaccharide at asparagine residue 894 in N900D promotes degradation.

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