Ex vivo biochemical analysis of CFTR in human rectal biopsies

Barneveld, Andrea van; Stanke, Frauke; Ballmann, Manfred; Naim, Hassan Y.; Tümmler, Burkhard

doi:10.1016/j.bbadis.2006.01.007

Cited by 15 publications

(17 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Constitutive endocytic (clathrin-mediated) recycling to and from the plasma membrane efficiently controls the number of channels on the cell surface at steady state because CFTR has a long half-life (Ͼ20 h) in intestinal epithelial cells (12,44,48,55). Endocytic recycling and regulated exocytosis directly modulate CFTR ion transport in the intestine in a cell typespecific manner in endogenous CFTR-expressing epithelial cells and tissues (9,27).…”

mentioning

confidence: 99%

Syntaxin 3 is necessary for cAMP- and cGMP-regulated exocytosis of CFTR: implications for enterotoxigenic diarrhea

Collaco

Marathe

Kohnke

et al. 2010

American Journal of Physiology-Cell Physiology

View full text Add to dashboard Cite

Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal Caco-2(BB)e cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized Caco-2(BB)e cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea.

show abstract

mentioning

confidence: 99%

Syntaxin 3 is necessary for cAMP- and cGMP-regulated exocytosis of CFTR: implications for enterotoxigenic diarrhea

Collaco

Marathe

Kohnke

et al. 2010

American Journal of Physiology-Cell Physiology

View full text Add to dashboard Cite

show abstract

“…1A) consistent with the genetic diagnosis of a heterozygous carrier of a CFTR mutation. The immunoblot [2,5] of the proband's mucosal biopsies revealed three interpretable CFTR immunoreactive bands (Fig. 1B).…”

Section: Case Reportmentioning

confidence: 97%

“…This response is atypical for non-CF probands, but is characteristic for most CF patients with residual chloride secretion [4,14]. (B) T84 cells or rectal biopsies were homogenized [5], lysed with 0.02% SDS for 30 min, followed by 1% (v/v) Triton X-100 and 0.5% (w/v) sodium deoxycholate for 40 min, and centrifuged (16,000 g, 4°C, 20 min). After incubation with protein Asepharose (PAS) for 60 min, the supernatant was immunoprecipitated with rabbit polyclonal antibodies R39, R40 (raised against hCFTR peptide 1465-1480) and R453 (raised against CFTR peptides 1-14 and 1466-1480) [5] in the presence of PAS.…”

Section: Case Reportmentioning

confidence: 99%

“…(B) T84 cells or rectal biopsies were homogenized [5], lysed with 0.02% SDS for 30 min, followed by 1% (v/v) Triton X-100 and 0.5% (w/v) sodium deoxycholate for 40 min, and centrifuged (16,000 g, 4°C, 20 min). After incubation with protein Asepharose (PAS) for 60 min, the supernatant was immunoprecipitated with rabbit polyclonal antibodies R39, R40 (raised against hCFTR peptide 1465-1480) and R453 (raised against CFTR peptides 1-14 and 1466-1480) [5] in the presence of PAS. CFTR immunoreactive bands were detected on 5% SDS-PAGE separated immunoblots with monoclonal antibodies 570 and 596 (raised against linear epitopes in the R domain and NBD2, respectively, 1:1000) [2] and anti-mouse-IgG-HRP from donkey (1:50,000) using ECL Advance (Amersham Bioscience).…”

Section: Case Reportmentioning

confidence: 99%

“…Immediately thereafter the mucosal biopsies are lysed, immunoprecipitated with a cocktail of polyclonal antihCFTR antibodies, and CFTR immunoreactive bands are visualized on immunoblot with monoclonal antibodies against hCFTR (Fig. 1B) [5].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

CFTR protein analysis of splice site mutation 2789+5 G-A

Barneveld¹,

Stanke²,

Claass³

et al. 2008

Journal of Cystic Fibrosis

Self Cite

View full text Add to dashboard Cite

Ex vivo biochemical analysis of rectal biopsies of a carrier of the mild 2789+5 G-A CFTR frameshift splice site mutation revealed mutant truncated CFTR of expected size and an imbalance of more core-glycosylated and less mature full-length CFTR. This first immunoblot analysis of a non-F508del CFTR mutant protein derived from human tissue demonstrates that splice site mutations should not only be investigated at the mRNA, but also at the protein level to properly interpret the associations between genotype, molecular pathology and disease.

show abstract

Functional analysis of F508del CFTR in native human colon

Barneveld

Stanke

Tamm

et al. 2010

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

Self Cite

View full text Add to dashboard Cite

The major cystic fibrosis mutation F508del has been classified by experiments in animal and cell culture models as a temperature-sensitive mutant defective in protein folding, processing and trafficking, but literature data on F508del CFTR maturation and function in human tissue are inconsistent. In the present study the molecular pathology of F508del CFTR was characterized in freshly excised rectal mucosa by bioelectric measurement of the basic defect and CFTR protein analysis by metabolic labelling or immunoblot. The majority of investigated F508del homozygous subjects expressed low amounts of complex-glycosylated mature F508del CFTR and low residual F508del CFTR-mediated chloride secretory activity in the rectal mucosa. The finding that some F508del CFTR escapes the ER quality control in vivo substantiates the hope that the defective processing and trafficking of F508del CFTR can be corrected by pharmacological agents.

show abstract

Ex vivo biochemical analysis of CFTR in human rectal biopsies

Cited by 15 publications

References 24 publications

Syntaxin 3 is necessary for cAMP- and cGMP-regulated exocytosis of CFTR: implications for enterotoxigenic diarrhea

Syntaxin 3 is necessary for cAMP- and cGMP-regulated exocytosis of CFTR: implications for enterotoxigenic diarrhea

CFTR protein analysis of splice site mutation 2789+5 G-A

Functional analysis of F508del CFTR in native human colon

Contact Info

Product

Resources

About