ABCMdb

PMID: 16417523

Loo TW, Bartlett MC, Wang Y, Clarke DM
The chemical chaperone CFcor-325 repairs folding defects in the transmembrane domains of CFTR-processing mutants.
Biochem J. 2006 May 1;395(3):537-42., 2006-05-01 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro To test if CFcor-325 promoted correct folding of the TMDs (TM domains), we selected two of the CL4 mutants (Q1071P and H1085R) for disulphide cross-linking analysis. Login to comment 5 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Pairs of cysteine residues that were cross-linked in mature wild-type CFTR were introduced into mutants Q1071P and H1085R. Login to comment 6 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro The cross-linking patterns of the Q1071P or H1085R double cysteine mutants rescued with CFcor-325 were similar to those observed with mature wild-type double cysteine proteins. Login to comment 21 ABCC7 p.Leu1065Pro ABCC7 p.His1085Arg ABCC7 p.Leu1077Pro ABCC7 p.Arg1066His ABCC7 p.Trp1098Arg ABCC7 p.His1054Asp ABCC7 p.Gly1061Arg ABCC7 p.Gln1071Pro EXPERIMENTAL Construction and expression of mutants The cDNAs of wild-type and CL4 mutants (H1054D, G1061R, L1065P, R1066H, Q1071P, L1077P, H1085R and W1098R) were inserted into pcDNA3 vector (Invitrogen, Oakville, ON, Canada) as described previously [2]. Login to comment 22 ABCC7 p.Thr351Cys Construction of CFTR double cysteine mutants M348C(TM6)/T1142C(TM12), T351C- (TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) was described previously [10]. Login to comment 23 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Site-directed mutagenesis was used to introduce the Q1071P or H1085R mutations into the double cysteine mutants as described previously [11]. Login to comment 31 ABCC7 p.His1085Arg ABCC7 p.Thr1142Cys ABCC7 p.Gln1071Pro Disulphide cross-linking analysis The cDNAs of double cysteine mutants M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) constructed in wild-type, mutant Q1071P or mutant H1085R backgrounds were expressed in HEK-293 cells in the presence or absence of 3 µM CFcor-325. Login to comment 37 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Briefly, cells expressing wild-type, mutant Q1071P or H1085R CFTR were grown with or without 3 µM CFcor-325. Login to comment 51 ABCC7 p.Leu1065Pro ABCC7 p.His1085Arg ABCC7 p.Leu1077Pro ABCC7 p.Arg1066His ABCC7 p.Trp1098Arg ABCC7 p.His1054Asp ABCC7 p.Gly1061Arg ABCC7 p.Gln1071Pro Accordingly, HEK-293 cells were transfected with mutants H1054D, G1061R, L1065P, R1066H, Q1071P, L1077P, H1085R or W1098R cDNAs. Login to comment 55 ABCC7 p.Arg1066His In contrast, the amount of mature protein in all the processing mutants except mutant R1066H was less than 5% (Figure 2B). Login to comment 56 ABCC7 p.Arg1066His The amount of mature CFTR in mutant R1066H was 10%. Login to comment 57 ABCC7 p.His1085Arg ABCC7 p.Leu1077Pro ABCC7 p.Arg1066His ABCC7 p.Trp1098Arg ABCC7 p.His1054Asp ABCC7 p.Gly1061Arg ABCC7 p.Gln1071Pro Expression of mutants H1054D, G1061R, R1066H, Q1071P, L1077P, H1085R and W1098R in the presence of 3 µM CFcor-325, however, induced expression of the 190 kDa mature protein. Login to comment 59 ABCC7 p.His1085Arg ABCC7 p.Arg1066His ABCC7 p.Trp1098Arg ABCC7 p.Gln1071Pro The chemical chaperone most efficiently rescued mutants R1066H, Q1071P, H1085R and W1098R as the amount of mature CFTR was approx. Login to comment 61 ABCC7 p.Leu1077Pro ABCC7 p.His1054Asp ABCC7 p.Gly1061Arg Lower levels of rescue were observed with mutants H1054D, G1061R and L1077P (5-15% mature CFTR protein). Login to comment 62 ABCC7 p.Leu1065Pro Mutant L1065P was the only mutant that could not be rescued with CFcor-325 (Figure 2B). Login to comment 73 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Two CL4 mutants, Q1071P and H1085R, were selected for cross-linking analysis because they were efficiently rescued with CFcor-325. Login to comment 74 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Expression of mutants Q1071P or H1085R in the presence of CFcor-325 increased the amount of mature CFTR from less than 5% (in the absence of corrector) of total to more than 30% (Figures 2A and 2B). Login to comment 77 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Mature 190 kDa CFTR, however, contains complex carbohydrate groups that are resistant to endo H but not to PNGase F. Accordingly, cells expressing wild-type, mutant Q1071P or mutant H1085R CFTR and grown in the presence or absence of CFcor-325 were extracted with SDS and then treated with endoglycosidases. Login to comment 78 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Figure 3 shows that the 170 kDa immature CFTRs but not the 190 kDa mature CFTRs of wild-type, mutant Q1071P or mutant H1085R were sensitive to digestion with endo H. Login to comment 79 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Both the 190 kDa mature and 170 kDa immature CFTRs were sensitive to digestion with PNGase F. Therefore expression of mutant Q1071P and H1085R in the presence of CFcor-325 promoted maturation and trafficking of the proteins from the ER to the Golgi where complex carbohydrates are added to the protein. Login to comment 81 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Cells expressing wild-type, mutant Q1071P or mutant H1085R CFTRs were grown in the presence or absence of 3 µM CFcor-325. Login to comment 82 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro The cells Figure 3 Endoglycosidase digestion Whole cell SDS extracts of HEK-293 cells expressing wild-type, mutant Q1071P or mutant H1085R in the absence (-) or presence (+) of 3 µM CFcor-325 were treated with endo H (H), PNGase F (F) or no endoglycosidase (-). Login to comment 85 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Figure 4 Cell surface labelling of CFTR (A) BHK cells (Control) or BHK cells stably expressing wild-type (Wild-type), mutant Q1071P or mutant H1085R CFTRs were grown in the presence (+) or absence (-) of 3 µM CFcor-325. Login to comment 93 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Figure 4(A) Figure 5 Measurement of cAMP-stimulated iodide efflux activities Iodide efflux assays were performed on BHK cells stably expressing wild-type, mutant Q1071P or mutant H1085R CFTRs that were grown in the presence (+CFcor-325) or absence (untreated) of CFcor-325 as described in the Experimental section. Login to comment 94 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro The activity of mutant H1085R grown without CFcor-325 was similar to that of mutant Q1071P (untreated) and is omitted for clarity. Login to comment 97 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Similarly, labelled mature 190 kDa CFTR proteins were detected in mutants Q1071P and H1085R only after rescue with CFcor-325. Login to comment 98 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Figure 4(B) shows that there was good correlation between the amount of biotinylated CFTR proteins in mutants Q1071P and H1085R with the amount of mature CFTRs detected in Figure 2(B) (63 and 50% of wild-type respectively). Login to comment 99 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Next, we tested whether mutant Q1071P or H1085R was active at the cell surface after rescue with CFcor-325. Login to comment 100 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Mutants Q1071P and H1085R CFTRs were stably expressed in BHK cells because adherent cells are essential for the iodide efflux assays. Login to comment 102 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro The BHK cells stably expressing mutants Q1071P or H1085R were treated for 48 h with or without 3 µM CFcor-325 and then used in iodide efflux assays. Login to comment 106 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro In contrast, BHK cells stably expressing CFTR mutants Q1071P or H1085R demonstrated iodide efflux activity only when grown in the presence of CFcor-325 (Figure 5). Login to comment 108 ABCC7 p.Gln1071Pro Therefore mutants Q1071P and h1085R were used for cross-linking studies. Login to comment 109 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro Accordingly, M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutations were introduced into a Q1071P or H1085R CFTR background. Login to comment 110 ABCC7 p.His1085Arg ABCC7 p.Thr1142Cys ABCC7 p.Gln1071Pro Mutants Q1071P or H1085R containing M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/ W1145C(TM12) mutations were then transiently expressed in HEK-293 cells in the presence or absence of 3 µM CFcor-325 for 48 h and then treated with the homobifunctional cross-linkers M5M, M8M or M17M. Login to comment 115 ABCC7 p.His1085Arg ABCC7 p.His1085Arg ABCC7 p.His1085Arg ABCC7 p.His1085Arg ABCC7 p.Thr351Cys ABCC7 p.Trp356Cys ABCC7 p.Gln1071Pro ABCC7 p.Gln1071Pro ABCC7 p.Gln1071Pro ABCC7 p.Gln1071Pro ABCC7 p.Gln1071Pro Mature mutant Q1071P/ M348C(TM6)/T1142C(TM12) protein was cross-linked with Figure 6 Disulphide cross-linking analysis of CFTR processing mutants HEK-293 cells expressing mutants Q1071P/M348C(TM6)/T1142C(TM12), Q1071P/T351C- (TM6)/T1142C(TM12) and Q1071P/W356C(TM6)/W1145C(TM12) (A), mutants H1085R/ M348C(TM6)/T1142C(TM12), H1085R/T351C(TM6)/T1142C(TM12) and H1085R/W356C- (TM6)/W1145C(TM12) (B) or wild-type, mutant Q1071P or mutant H1085R (C) were incubated for 48 h with (+) or without (-) 3 µM CFcor-325. Login to comment 119 ABCC7 p.Thr351Cys ABCC7 p.Gln1071Pro ABCC7 p.Gln1071Pro Mature mutant Q1071P/T351C- (TM6)/T1142C(TM12) protein was cross-linked with M8M and to a lesser extent with M17M, while the mature mutant Q1071P/W356C(TM6)/W1145C(TM12) protein was cross-linked with M5M, M8M and M17M (Figure 6A). Login to comment 120 ABCC7 p.His1085Arg Similar results were observed with the H1085R double cysteine mutants (Figure 6B). Login to comment 122 ABCC7 p.His1085Arg ABCC7 p.Gln1071Pro In contrast, wild-type, mature mutant Q1071P or H1085R CFTRs lacking the introduced cysteine residues was not cross-linked by M5M, M8M or M17M (Figure 6C). Login to comment 123 ABCC7 p.Gln1071Pro The immature 170 kDa mutant proteins did not 7 Effect of dithiothreitol on immature CFTR treated with cross-linker HEK-293 cells expressing mutant Q1071P/M348C(TM6)/T1142C(TM12) in the absence of CFcor-325 were treated with 0.2 mM M5M, M8M or M17M cross-linker for 15 min at 20◦C. Samples were then incubated with (+DTT) or without (-DTT) 30 mM dithiothreitol for 5 min at 20◦C. Following SDS/PAGE on a 5.5% gel, the stacking and separating gels were transferred on to a sheet of nitrocellulose and CFTR protein was detected by immunoblot analysis. Login to comment 126 ABCC7 p.Gln1071Pro An example of aggregation is shown for mutant Q1071P/M348C(TM6)/ T1142C(TM12) (Figure 7). Login to comment 129 ABCC7 p.His1085Arg Similar results were observed with mutant H1085R/M348C(TM6)/T1142(TM12) (results not shown). Login to comment 144 ABCC7 p.Gln1071Pro The processing mutation Q1071P traps the protein in the ER in a loosely folded conformation because of altered TM packing (B). Login to comment 152 ABCC7 p.Gln1071Pro The presence of a processing mutation such as Q1071P introduces a thermodynamic hurdle during folding so that the protein is trapped in an immature conformation (Figure 8B). Login to comment