The regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) contains consensus phosphorylation sites for cAMP-dependent protein kinase (PKA) that are the basis for physiological regulation of the CFTR chloride channel. A short peptide segment in the R domain with a net negative charge of B9 (amino acids 817-838, NEG2) and predicted helical tendency is shown to play a critical role in CFTR chloride channel function. Deletion of NEG2 from CFTR completely eliminates the PKA dependence of channel activity. Exogenous NEG2 peptide interacts with CFTR to exert both stimulatory and inhibitory effects on the channel function. The NEG2 peptide with sequence scrambled to remove helical tendencies also inhibits channel function, but does not stimulate. Similar results are found for a NEG2 peptide whose helical structure is disrupted by a proline residue. When six of the negatively charged carboxylic acid residues are replaced by their cognate amides, reducing net negative charge to B3, but increasing helical propensity as assessed by circular dichroism, the peptide stimulates CFTR channel function, but does not inhibit. We speculate that the NEG2 region interacts with other cytosolic domains of CFTR to control opening and closing transitions of the chloride channel.Defects in CFTR, 1 a chloride channel located in the apical membrane of epithelial cells, are associated with the common genetic disease, cystic fibrosis (1-3). CFTR is a 1480-amino acid protein that is a member of the ATP binding cassette transporter family (4). The general structure of these membrane proteins includes two membrane spanning domains, each consisting of six transmembrane segments, and two nucleotide binding folds (NBF1 and NBF2). Most members of the ATP binding cassette family use the free energy of ATP hydrolysis to actively transport substrates across the membrane (5). However, unlike the other members of this family, CFTR contains a unique regulatory (R) domain, and encodes a cAMP-regulated chloride channel (6 -8).The R domain of CFTR contains several consensus PKA phosphorylation sites (9 -11) that are the basis for physiological regulation of this chloride channel. CFTR channel opening requires phosphorylation of serine residues in the R domain, and ATP binding and hydrolysis at the nucleotide binding folds (7, 12, 13). Phosphorylation adds negative charges to the R domain, and introduces global conformational changes reflected by a reduction in the ␣-helical content of the R domain protein (14). Thus, electrostatic and/or allosteric changes mediated by phosphorylation are likely responsible for interactions between the R domain and other CFTR domains that regulate channel function (15,16). Rich et al. (17) showed that deletion of amino acids 708 -835 from the R domain (⌬R-CFTR), which removes most of the PKA consensus sites, allows the CFTR chloride channel to open without phosphorylation. The open probability of ⌬R-CFTR is one-third that of the wild type (wt) CFTR channel and does not increase upon PKA phosp...
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; DeltaF508 CFTR, which is misprocessed, wild-type CFTR; or empty vector. Reduced cAMP-stimulated chloride transport is seen in the R domain and DeltaF508 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.
MethodsIn this 26-week, multicenter, double-blind study, patients ≥ 40 years with COPD (forced expiratory volume in 1 second [FEV 1 ] ≥40% to <80% predicted and no history of exacerbations in the previous year) were randomised (1:1) to QVA149 110/50 mg or SFC 50/500 mg. In this post hoc analysis, we report the rate of mild, moderate or severe COPD exacerbations during 26 weeks of treatment with QVA149 or SFC. Preventing Streptococcus pneumoniae lung infections will have substantial health benefits, yet existing polysaccharide vaccines are not effective against pneumonia. Identifying the mechanisms of naturally-acquired immunity to S. pneumoniae lung infections could indicate alternative preventative strategies. Human intravenous immunoglobulin (IVIG) preparations pooled from >1000 donors prevent respiratory infections in patients with hypogammaglobuniaemia. IVIG therefore provides a tool to investigate the naturally-acquired antibody responses to S. pneumoniae within a population. We have used mouse models and in vitro assays to assess the efficacy of IVIG for preventing S. pneumoniae lung infections and to identify the immunodominant target antigens. ResultsIn a mouse pneumonia model, IVIG treatment was highly protective against bacteraemia (17% septicaemia v. 100% in controls, P = 0.015) and partially protected against lung infection (lung CFU log 10 3.7 for IVIG v. 6.3 for controls, P = 0.041) but not against nasopharyngeal colonisation. Depletion of phagocyte subsets demonstrated that IVIG-mediated protection required neutrophils and macrophages for lungs and blood respectively. Flow-cytometry assays demonstrated that IgG within IVIG preparations opsonised S. pneumoniae effectively. Importantly, IgG opsonisation was reduced by pre-treatment of bacteria with pronase to remove bacterial surface proteins but not by depletion of anti-capsular antibody. Furthermore, in vitro assays demonstrated that IVIG facilitated phagocytosis, growth impairment and bacterial agglutination of capsule-deficient S. pneumoniae mutants, in mice IVIG depleted of anti-capsular antibody remained protective against lung infection and septicaemia. These results demonstrate that surface proteins rather than the capsule are targets for naturally-acquired adaptive immunity to S. pneumoniae. The potential S. pneumoniae protein antigen targets in IVIG were assessed using a semi-quantitative assay against 18 recombinant pneumococcal proteins. The results demonstrated significant IgG responses to the conserved pneumococcal protein antigens PhtD, PspC, PspA and PsaA. Interestingly, antibody titres to some of these antigens were reduced in sera from elderly compared to younger subjects, potentially identifying people at higher risk of S. pneumoniae infection. Our data demonstrate that the accepted paradigm that naturally-acquired immunity to S. pneumoniae depends on anti-capsular antigen is inaccurate, and instead antibody to proteins is dominant. These data will allow better evaluation of those at risk of S. pneumoniae infection and improved v...
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