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

Chang, Xiu Bao; Mengos, April; Hou, Yue Xian; Cui, Liying; Jensen, Timothy J.; Aleksandrov, Andrei A.; Riordan, John R.; Gentzsch, Martina

doi:10.1242/jcs.028951

Cited by 72 publications

(72 citation statements)

References 84 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The CFTR EL4-FAP construct did not produce a distinct mature band, which suggests that the efficiency of its glycosylation was impaired. The fourth extracellular loop of CFTR is the locus of two asparagine residues (N894 and N900) that acquire N-linked glycosylation and other modifications; therefore, careful consideration was taken to preserve these residues, and the consensus glycosylation sequences (NXS/T) surrounding them, in the FAP fusion construct (29,30). The observed incomplete glycosylation could be due to reduced glycan modification or partial oligosaccharide modification at these sites, which may result from impaired glycosylation enzyme recognition and/or accessibility at these sites when the FAP tag is present.…”

Section: Discussionmentioning

confidence: 99%

“…Because CFTR had been found to tolerate these modifications while retaining functional activity, we chose this location to create an FAP-CFTR chimeric protein. The CFTR EL4-FAP chimera was generated by inserting the FAP in the EL4 between the two asparagine residues required for the posttranslational glycosylation of CFTR (29,30).…”

Section: Development Of Cftr Fap Reporters and Their Labeling At The mentioning

confidence: 99%

See 1 more Smart Citation

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

Holleran

Glover

Peters

et al. 2012

Mol Med

View full text Add to dashboard Cite

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Development Of Cftr Fap Reporters and Their Labeling At The mentioning

confidence: 99%

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

Holleran

Glover

Peters

et al. 2012

Mol Med

View full text Add to dashboard Cite

show abstract

“…On-cell ELISA-On-cell ELISAs were performed according to published protocol with several modifications (26). Briefly, Corning Costar Tissue Culture-treated clear 24-well plates (Corning Costar 3526, Corning) were incubated with 100 g/ml poly-D-lysine (Sigma P0899) for 6 h at room temperature or overnight at 4°C.…”

Section: Internalization Assaysmentioning

confidence: 99%

Constitutive Internalization of the Leucine-rich G Protein-coupled Receptor-5 (LGR5) to the Trans-Golgi Network

Snyder

Rochelle

Lyerly

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Expression of the G protein-coupled receptor LGR5 demarcates adult tissue stem cells in the intestine, stomach, hair follicle, and mammary epithelium. Results:LGR5 is rapidly and constitutively internalized to the trans-Golgi network at steady state. Conclusion: Internalization occurs through a potential phosphorylation domain within the C-terminal tail. Significance: An understanding of LGR5 trafficking dynamics is expected to clarify its role in signaling and stem cell biology.

show abstract

“…(Supplemental data for this article are available online at the American Journal of Physiology-Lung Cellular and Molecular Physiology website.) CFTR immunoprecipitation (IP)-Westerns were performed as previously described (8). Briefly, 12 ϫ 12-mm well-differentiated ALI Millicell CM cultures were lysed in 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris ⅐ HCl, pH 7.4, and 10 mM NaMoO 4 with protease inhibitors (1 g/ml leupeptin, 2 g/ml aprotinin, 50 g/ml Pefabloc, 121 g/ml benzamidine, and 3.5 g/ml E64).…”

Section: Western Blots and Cftr Immunoprecipitation-westerns Antibodmentioning

confidence: 99%

Novel human bronchial epithelial cell lines for cystic fibrosis research

Fulcher¹,

Gabriel²,

Olsen³

et al. 2009

American Journal of Physiology-Lung Cellular and Molecular Phys

View full text Add to dashboard Cite

Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three ΔF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Ω·cm2. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1β, TNF-α, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.

show abstract

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

Abstract: Role of N-linked oligosaccharides in the biosynthetic

Cited by 72 publications

References 84 publications

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

Constitutive Internalization of the Leucine-rich G Protein-coupled Receptor-5 (LGR5) to the Trans-Golgi Network

Novel human bronchial epithelial cell lines for cystic fibrosis research

Contact Info

Product

Resources

About