ABCMdb

PMID: 10764788

Van Oene M, Lukacs GL, Rommens JM
Cystic fibrosis mutations lead to carboxyl-terminal fragments that highlight an early biogenesis step of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2000 Jun 30;275(26):19577-84., 2000-06-30 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Pro574His ABCC7 p.Ala455Glu ABCC7 p.Ser549Arg ABCC7 p.Tyr563Asn Analysis of cystic fibrosis-associated missense mutations in the first nucleotide binding domain (NBD1), including A455E, S549R, Y563N, and P574H, revealed reduced levels of mature CFTR with elevated levels of carboxyl-terminal polypeptide fragments of 105 and 90 kDa. Login to comment 41 ABCC7 p.Pro574His ABCC7 p.Ala455Glu ABCC7 p.Ser549Arg ABCC7 p.Tyr563Asn EXPERIMENTAL PROCEDURES Construction of CFTR Expression Plasmids-The CFTR mutants A455E, S549R, P574H, and Y563N were generated from pBQ6.2 (34) as described previously (35). Login to comment 84 ABCC7 p.Pro574His ABCC7 p.Ala455Glu ABCC7 p.Ser549Arg ABCC7 p.Tyr563Asn Analysis of the S549R mutant showed measurable but intermediate levels of band C, whereas A455E, Y563N, and P574H mutants showed markedly reduced levels using both tagged and untagged CFTR. Login to comment 88 ABCC7 p.Ala455Glu Predominant levels of CHA-CFTRwt appear at the cell surface, in contrast to CHA-⌬F508 and CHA-A455E, as described in alternate reports with untagged versions (18, 44). Login to comment 90 ABCC7 p.Ser549Arg Consistent with the observed steady state glycosylation pattern, CHA-S549R shows both cell surface and ER-like staining. Login to comment 92 ABCC7 p.Ala455Glu ABCC7 p.Ser549Arg Identification of Carboxyl CFTR Derivatives-In addition to full-length CFTR forms, both the M3A7 and the anti-HA antibodies revealed products with relative molecular masses of 105 and 90 kDa that were most prominent in detergent extracts of HEK293 cells expressing the A455E and S549R mutants (Fig. 1, B and C, lanes 4 and 5). Login to comment 97 ABCC7 p.Ala455Glu The first involved an analysis of the extract preparation to examine the compartmentalization of CFTR, the second involved analysis of the intermediates at reduced expression levels with the A455E mutant, and the third involved expression in alternate cell types. Login to comment 100 ABCC7 p.Ala455Glu Immunoblot analysis indicated that less than 10% of total immunoreactive band B or carboxyl-terminal polypeptides were present in the original CHA-A455E pellet (Fig. 3A). Login to comment 102 ABCC7 p.Ala455Glu The occurrence of the 90-kDa band, most prominent with the A455E mutant, does suggest that it is relatively less soluble. Login to comment 105 ABCC7 p.Ala455Glu ABCC7 p.Ser549Arg Representative COS-7 cells transfected with (A) CHA-CFTRwt, (B) CHA-⌬F508, (C) CHA-A455E, and (D) CHA-S549R are shown. Login to comment 106 ABCC7 p.Ala455Glu Indirect immunofluorescence revealed a uniform cell surface staining with the monoclonal anti-HA antibody for the wt protein in contrast to the perinuclear staining of the ⌬F508 and A455E mutants. Login to comment 107 ABCC7 p.Ser549Arg CHA-S549R also shows obvious cell surface staining consistent with the mature protein identified in immunoblot analyses. Login to comment 119 ABCC7 p.Ala455Glu 1/20 the amount of the CHA-A455E expression plasmid (Fig. 3C). Login to comment 122 ABCC7 p.Ala455Glu Transient transfections were carried out with CHA-tagged CFTRwt, ⌬F508, and A455E in COS-7, CHO-duk- , and CF bronchial epithelium IB3-1 cell lines. Login to comment 123 ABCC7 p.Ala455Glu Immunoblot analysis of detergent extracts indicated that although the levels varied between the cell types, CHA-A455E consistently revealed the most prominent accumulation of the 105-and 90-kDa bands (Fig. 4, A-C). Login to comment 127 ABCC7 p.Ala455Glu The increased sensitivity clearly shows the accumulation of the 105-kDa intermediate in both the wt and A455E extracts. Login to comment 136 ABCC7 p.Ala455Glu In contrast, no mature forms were detected for the A455E mutation (right panel) or with prolonged pulse and/or chase periods (data not shown). Login to comment 137 ABCC7 p.Ala455Glu The 105-and 90-kDa carboxyl-terminal fragments were evident for both CFTRwt and A455E as early as the completion of the pulse (15 min) and rapidly disappeared during the chase. Login to comment 138 ABCC7 p.Ala455Glu These untagged proteins were detectable with a CFTR antibody mixture (M3A7 and L12B4) and were consistently most prominent for the A455E mutant. Login to comment 146 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu HEK293 cells were transfected with pCMV (vector alone), CHA-A455E, or with mixtures of pCMV and CHA-A455E to maintain constant plasmid-lipid ratios for transfection. Login to comment 155 ABCC7 p.Ala455Glu The half-life of the 105-kDa intermediate of both CFTRwt and A455E was determined to be very similar to that of band B (t1/2 ϳ30-40 min) (16, 17) emphasizing that this fragment is susceptible to proteolysis. Login to comment 157 ABCC7 p.Ser549Arg Results with CHA-S549R at steady state expression are shown (Fig. 6) as the 105-and 90-kDa intermediates as well as both core-and complex-glycosylated full-length protein forms are detectable. Login to comment 158 ABCC7 p.Ser549Arg The treatment of CHA-S549R with N-glycosidase F (left panel) shifted both complex-glycosylated (band C) and core-glycosylated (band B) CFTR to the unglycosylated form (band A), as expected. Login to comment 159 ABCC7 p.Ser549Arg Digestion with endoglycosidase H (right panel) removed only the high mannose-type asparagine-linked glycans from the band B form of CHA-S549R with minimal effect on band C forms of the protein (18, 46). Login to comment 161 ABCC7 p.Ala455Glu Epitope-tagged versions of wt and the A455E mutant gave consistent results, as did radioactively labeled and immunoprecipitated untagged CFTR polypeptides (data not shown). Login to comment 172 ABCC7 p.Ala455Glu RIPA soluble CFTR peptides were immunoprecipitated demonstrating that the inhibitors had no effect on the prevalence of the 105-and 90-kDa polypeptides formed during A455E biosynthesis (Fig. 7B). Login to comment 181 ABCC7 p.Ala455Glu ABCC7 p.Ser549Arg It is imposed on the wt protein but is more prominent in the presence of missense mutations such as A455E or S549R. Login to comment 188 ABCC7 p.Ala455Glu The relative levels of the 105-kDa band with CFTRwt, A455E, and ⌬F508 were determined by phosphorimage analysis. Login to comment 193 ABCC7 p.Ser549Arg RIPA soluble protein extracts of CHA-S549R were treated with N-glycosidase F (N-Glyc F) and endoglycosidase H (Endo H). Login to comment 196 ABCC7 p.Ala455Glu Further, it is evident that for enhanced proteolytic activity, the mutated amino acid need not be present in the resulting carboxyl fragments as size estimation would preclude this for at least the A455E mutant. Login to comment 209 ABCC7 p.Ala455Glu ABCC7 p.Ser549Arg It would therefore be interesting to closely examine its interaction with the S549R and A455E CFTR mutants. Login to comment 228 ABCC7 p.Ala455Glu ABCC7 p.Ser549Arg The proximity of the amino acid deletion to a proteolytic site may account for this; however, analysis of cells expressing CFTR versions with both the ⌬F508 mutation and either S549R or A455E mutations retain the formation of prominent levels of the carboxyl-terminal fragments (data not shown). Login to comment 235 ABCC7 p.Ser549Arg In attempting to correlate the abundance of the carboxyl degradation bands with the formation of band C, it was evident from the S549R mutation that moderate levels of complex-glycosylated CFTR can be generated even though the production of 90- and 105-kDa products is prominent. Login to comment 237 ABCC7 p.Ala455Glu This is consistent with the findings of the wt protein but less discernible for some mutations such as A455E. Login to comment 238 ABCC7 p.Pro574His ABCC7 p.Ala455Glu ABCC7 p.Tyr563Asn The A455E, Y563N, and P574H mutations do appear to be able to achieve at least nominal levels of chloride conduction at the cell surface based both on the presenting phenotype in CF patients (54) and on single channel and whole cell current measurements (55) in heterologous expression systems. Login to comment 239 ABCC7 p.Pro574His ABCC7 p.Ala455Glu ABCC7 p.Tyr563Asn In contrast to the Y563N and P574H mutations, where low levels of mature band C forms were detectable with long exposure and/or long label incorporation times in HEK293 cells (data not shown), fully glycosylated protein could not be detected for A455E using our assay systems. Login to comment 240 ABCC7 p.Ala455Glu Of the NBD1 missense mutations surveyed, A455E degradation products appeared at the highest levels relative to full-length nascent CFTR such that immature band B may be too low to lead to detectable levels of band C. Login to comment