ABCMdb

PMID: 19774621

Penmatsa H, Frederick CA, Nekkalapu S, Conoley VG, Zhang W, Li C, Kappes J, Stokes DC, Naren AP
Clinical and molecular characterization of S1118F-CFTR.
Pediatr Pulmonol. 2009 Oct;44(10):1003-9., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Ser1118Phe Results: S1118F-CFTR makes less than 10-15% of mature CFTR (band C) compared to WT-CFTR. Login to comment 9 ABCC7 p.Ser1118Phe The mRNA levels of S1118F-CFTR and WT-CFTR are comparable. Login to comment 10 ABCC7 p.Ser1118Phe S1118F-CFTR is functional but shows about 10-15% of WT-CFTR activity. Login to comment 11 ABCC7 p.Ser1118Phe S1118F-CFTR shows impaired maturation and CF-correctors can increase the amount of mature and functional CFTR by three- to fourfold. Login to comment 12 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe Conclusion: S1118F-CFTR shows impaired maturation and an individual with S1118F-CFTR paired with DF508-CFTRexhibits atypical CF symptomswith intermediate sweat chloride level and meconium ileus despite documented pancreatic sufficiency. Login to comment 21 ABCC7 p.Gly542* The Class I mutations constitute nonsense, splice and frame shift mutants that encode truncated forms of CFTR (e.g., G542X and W1242X). Login to comment 39 ABCC7 p.Ser1118Phe This class of mutations includes C-terminal truncated CFTR which form unstable mature protein with five- to sixfold faster degradation rate than WT-CFTR.2-7 We present herein a clinical case of a CF patient with intermediate sweat chloride levels, pancreatic sufficiency, meconium ileus and mutations in the CFTR gene (DF508 and S1118F). Login to comment 40 ABCC7 p.Ser1118Phe The objective of this study is to characterize S1118F-CFTR mutation at protein level to help understand the associated CF-phenotype. Login to comment 42 ABCC7 p.Ser1118Phe MATERIALS AND METHODS Genotyping Genotyping was performed at Ambry Genetics (Aliso Viejo, CA) which showed DF508/I1027Ton one chromosome and S1118F on the other. Login to comment 45 ABCC7 p.Ser1118Phe Site-directed mutagenesis was performed by a single point mutation in eukaryotic expression vector (pcDNA3) containing WT-CFTR cDNA using Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and appropriate primers to make S1118F-CFTR mutant. Login to comment 50 ABCC7 p.Ser1118Phe Pulse Chase Labeling HEK-293 cells transiently transfected with WTor S1118F-CFTR cDNA were incubated in MEM medium (Invitrogen) without methionine and cysteine for 60 min at 378C. Login to comment 56 ABCC7 p.Ser1118Phe Probe designed by Universal ProbeLibrary (Roche, Indianapolis, IN) was used and TATA-box binding protein (TBP) was used as the house keeping gene.10 Iodide Efflux HEK-293 cells were transfected with 2 mg of pcDNA3 WT-CFTR, pcDNA3 S1118F-CFTR or empty vector (pcDNA3) cDNA. Login to comment 65 ABCC7 p.Ser1118Phe RESULTS An Individual Harboring S1118F-CFTR Exhibits Atypical CF Symptoms A Caucasian infant was born at 376=7 weeks by cesarean section in Mississippi (USA). Login to comment 72 ABCC7 p.Ile1027Thr ABCC7 p.Ser1118Phe Genotyping was sent to Ambry Genetics which showed DF508/I1027T on one chromosome and S1118F on the other. Login to comment 81 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe S1118F-CFTR Makes Less Than 10-15% of Mature CFTR (band C) Compared to WT-CFTR Previous studies in Xenopus oocytes have shown that S1118F-CFTR is a mutant with altered permeation and gating characteristics compared to WT-CFTR.14 It is therefore likely that this mutant belongs to Class IV CFTR mutations. Login to comment 83 ABCC7 p.Ser1118Phe Using Quickchange method, we generated S1118F mutation on pcDNA3-CFTR background. Login to comment 87 ABCC7 p.Ser1118Phe The data shows that S1118F-CFTR appears as a 180 kDa immunoreactive band (band C; the mature CFTR) but is less than 10-15% of that for WT-CFTR (Fig. 1A). Login to comment 89 ABCC7 p.Ser1118Phe Iodide efflux measurements were performed using HEK-293 cells transiently transfected with WTor S1118F-CFTR cDNA. Login to comment 90 ABCC7 p.Ser1118Phe As shown in Figure 1B and C, S1118F-CFTR is functional but has much lower activity (10-15%) compared to WT-CFTR. Login to comment 92 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe In an attempt to define which class this mutation might belong to, we compared S1118F-CFTR with other well-defined classes of CFTR-mutations such as DF508-CFTR (Class II), G551D-CFTR (Class III) and R117H-CFTR (Class IV) and the data is shown in Figure 1D. It is likely that S1118F-CFTR is a trafficking mutant with impaired maturation. Login to comment 93 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe S1118F-CFTR Shows Impaired Maturation To test if the reduced expression level of S1118F-CFTR is due to reduced maturation efficiency, we performed pulse chase experiments by treating HEK293 cells expressing S1118F-CFTR or WT-CFTR with a brief pulse for 30 min. Login to comment 96 ABCC7 p.Ser1118Phe However, the maturation is not efficient for S1118F-CFTR and only a small portion of band B was converted to band C (impaired maturation). Login to comment 97 ABCC7 p.Ser1118Phe These biochemical studies are consistent with a mild CF-phenotype observed for individuals harboring S1118F-CFTR as it is a trafficking mutant with impaired maturation efficiency. Login to comment 98 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe To investigate if S1118F-CFTR mRNA has reduced stability, real-time PCR was performed to compare mRNA Pediatric Pulmonology expression levels in HEK-293 cells transfected with equal amounts of WTor S1118F-CFTR cDNA. Login to comment 99 ABCC7 p.Ser1118Phe The results showed that the mRNA expression level for S1118F-CFTR is not reduced compared to WT-CFTR (Fig. 3). Login to comment 100 ABCC7 p.Ser1118Phe It is therefore reasonable to propose that S1118F-CFTR does not belong to the class V mutations. Login to comment 101 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe CF-Correctors Can Increase the Amount and Function of S1118F-CFTR Several Folds To test if S1118F-CFTR can be rescued by using CF-correctors, HEK-293 cells expressing WTor S1118F-CFTR was incubated with CF-corrector C1 [6-(1H- benzomidazol-2-ylsulfanylmethyl)-2-(6-methoxy-4-methyl- quinazolin-2-ylamino)-pyrimidin-4-ol] or C3 [(4-cyclohexyloxy-2-{1-[4-(4-methoxy-benzensulfonyl)-piperazin- 1-yl]-ethyl}-quinazoline], lysed, immunoprecipitated and probed for CFTR. Login to comment 102 ABCC7 p.Ser1118Phe The result showed that treatment of HEK-293 cells expressing S1118F-CFTR with these CF-correctors did increase the mature form of CFTR. Login to comment 105 ABCC7 p.Ser1118Phe Iodide efflux experiments were performed by using HEK-293 cells expressing S1118F-CFTR. Login to comment 106 ABCC7 p.Ser1118Phe As shown in Figure 4B,C, in the presence of CF-Corrector C3, the function of S1118F-CFTR increases two- to fourfold. Login to comment 107 ABCC7 p.Ser1118Phe This result is consistent with the Western Blotting result suggesting that S1118F-CFTR mutant can be functionally rescued by using CF-correctors. Login to comment 108 ABCC7 p.Ser1118Phe DISCUSSION S1118F-CFTR is a serine-to-phenylalanine mutation in the 11th transmembrane-spanning domain (TMD) of Pediatric Pulmonology Fig. 1. Login to comment 109 ABCC7 p.Ser1118Phe Characterization of S1118F-CFTR. Login to comment 110 ABCC7 p.Ser1118Phe A: HEK-293 cells were transiently transfected with pcDNA3 containing WT-, S1118F- or DF508-CFTR cDNA, lysed after 48 hr, immunoprecipitated and western blotted for CFTR (a 24-1 mab). Login to comment 111 ABCC7 p.Ser1118Phe The experiments were performed 4 times. B: Iodide efflux assay in HEK-293 cells transiently transfected with WTor S1118F-CFTR cDNA. Login to comment 112 ABCC7 p.Ser1118Phe PKA activating agonist cocktail (containing 10 mM forskolin, 100 mM IBMX and 200 mM cpt-cAMP) was added to activate CFTR. Line graph is Iodide efflux (nmol/min) as mean of 3 (WT and Control) or 5 (S1118F-CFTR) experiments with standard error mean (SEM). Login to comment 114 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ser1118Phe D: HEK-293cells weretransiently transfected with pCDNA3 containing WT-, S1118F-, R117H-, G551D- or DF508-CFTR cDNA, lysed 48 hr later, immunoprecipitated (a 24-1 mab) and western blotted for CFTR (a 24-1 mab). Login to comment 119 ABCC7 p.Ser1118Phe expressed S1118F-CFTR mutant in stage V Xenopus oocytes and studied the channel properties electrophysiologically. Login to comment 122 ABCC7 p.Ser1118Phe S1118F-CFTR shows impaired maturation. Login to comment 123 ABCC7 p.Ser1118Phe A: Pulse-chase experiments to monitor the formation of mature protein over a period of 2 hr in HEK-293 cells transfected with WTor S1118F-CFTR cDNA. Login to comment 124 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe The experiments were performed 4 times. B: Quantitation of mature(band C) andimmature (bandB) protein in WTand S1118F-CFTR from pulse-chase experiment showing impaired maturation of S1118F-CFTR. Login to comment 125 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe The band B at time ''0`` was considered as 100% for WTand S1118F-CFTR, respectively. Login to comment 127 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe Real-time PCR for comparing mRNA expression levels in HEK-293 cells transfected with equal amounts of WTand S1118F-CFTR cDNA. Login to comment 129 ABCC7 p.Ser1118Phe The change in relative expression levels between WT-CFTR and S1118F-CFTR was found to be insignificant. Login to comment 132 ABCC7 p.Ser1118Phe CF-correctors can increase the amount and function of S1118F-CFTR. Login to comment 134 ABCC7 p.Ser1118Phe The experiments were repeated for 4 times. B: Iodide efflux assay in HEK-293 cells transiently transfected with S1118F-CFTR with or without CF-corrector C3 treatment (5 mM). Login to comment 138 ABCC7 p.Ile1027Thr Based on these observations, they suggested that TMD11 plays a pore-lining role in the functional CFTR channel.14 We present here a clinical case in which a patient harboringS1118F-CFTR on one chromosome and DF508/ I1027T on the other exhibits atypical CF symptoms. Login to comment 142 ABCC7 p.Ser1118Phe To help understand this mild CF-phenotype, we used various biochemical methods to characterize S1118F-CFTR mutant at protein level. Login to comment 146 ABCC7 p.Ser1118Phe Secondly, to investigate if S1118F-CFTR is functional (as a ClÀ channel), we performed iodide efflux experiments. Login to comment 147 ABCC7 p.Ser1118Phe The result showed S1118F-CFTR is functional but has much lower activity compared to WT-CFTR (Fig. 1B,C). Login to comment 148 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe This functional result is consistent with the protein expression data and with Zhang`s result which showed S1118F-CFTR is a functional ClÀ channel yet with altered permeation and gating characteristics compared to WT-CFTR.14 Thirdly, we compared S1118F-CFTR with other well-defined CF-mutations using Western blotting. Login to comment 149 ABCC7 p.Gly551Asp ABCC7 p.Arg117His G551D-CFTR (Class III; regulation mutant) and R117H (Class IV; permeation mutant) are mutations with partial loss of channel function. Login to comment 150 ABCC7 p.Ser1118Phe Based on the observations as described above and Zhang`s result,14 it is therefore reasonable to assume that S1118F-CFTR might be a trafficking mutant with impaired maturation. Login to comment 151 ABCC7 p.Ser1118Phe Pulse chase experiments were employed to test the maturation efficiency of S1118F-CFTR. Login to comment 153 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe Next, to investigate if the impaired maturation is due to the reduced mRNA stability, we performed real-time PCR to compare mRNA expression levels in HEK293 cells transfected with equal amounts of WTor S1118F-CFTR cDNA. Login to comment 154 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe The results showed that the mRNA expression level for S1118F-CFTR is not reduced compared to WT-CFTR suggesting that S1118F-CFTR does not belong to class V mutations (Fig. 3). Login to comment 158 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe The use of pharmacological chaperones (CF-correctors) to promote the maturation of such mutants is one of the promising approaches.19-22 To test if the maturation efficiency for S1118F-CFTR can be improved by using CF-correctors, we treated HEK-293 cells expressing S1118F-CFTR with CF-correctors C1 and C3. Login to comment 160 ABCC7 p.Ser1118Phe In the presence of CF-corrector C3, the mature band C increased by three- to fourfold over untreated cells and the function of S1118F-CFTR increased two- to fourfold as well in iodide efflux experiments. Login to comment 161 ABCC7 p.Ser1118Phe Consistent with a mild CF-phenotype observed for a patient, our studies suggest that S1118F-CFTR is likely to be trafficking mutant with impaired maturation. Login to comment 163 ABCC7 p.Ser1118Phe To our knowledge, this is the first study in which S1118F-CFTR mutant was fully characterized at the protein level. Login to comment