ABCMdb

PMID: 23317763

van Meegen MA, Terheggen SW, Koymans KJ, Vijftigschild LA, Dekkers JF, van der Ent CK, Beekman JM
CFTR-mutation specific applications of CFTR-directed monoclonal antibodies.
J Cyst Fibros. 2013 Sep;12(5):487-96. doi: 10.1016/j.jcf.2012.12.005. Epub 2013 Jan 11., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* Mutant CFTR was detected in HEK293 cells transiently expressing the mutations; G542X, R1162X, F508del, N1303K, G551D, R117H, A455E. Login to comment 5 ABCC7 p.Asn1303Lys Using immunofluorescence, some antibodies can better detect mutant forms of CFTR (F508del and N1303K by mAbs 596 and 769), or display lower aspecific detection by Western blot analysis (mAbs 432, 450, 769 and 596) or immunofluorescence (mAbs 432, 450, 570 and 769). Login to comment 14 ABCC7 p.Gly542* ABCC7 p.Arg1162* Class I mutations such as G542X and R1162X encode for premature stop codons and affect CFTR protein translation [6]. Login to comment 15 ABCC7 p.Asn1303Lys The most common mutant CFTR allele contains a phenylalanine deletion at position 508 (F508del)[7], and is classically recognized as a class II mutation (like N1303K) that leads to incorrect folding and retention in the endoplasmic reticulum (ER) from where it is degraded. Login to comment 17 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Class III (G551D) and IV (R117H) mutations affect gating and chloride conductivity, respectively, but display normal trafficking [6,10,11]. Login to comment 22 ABCC7 p.Ala455Glu http://dx.doi.org/10.1016/j.jcf.2012.12.005 functional CFTR is observed for class V (A455E) and VI mutations, due to impaired biosynthesis and decreased stability of the CFTR protein, respectively [5,12]. Login to comment 23 ABCC7 p.Ala455Glu However, many CFTR mutations cannot be strictly classified into single classes as was shown for A455E and F508del [12-14]. Login to comment 76 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* We selected wt-CFTR, F508del (class II), G542X (I), R1162X (I), G551D (III), R117H (IV), A455E (V), and N1303K (II) that were ectopically expressed in HEK293 cells. Login to comment 79 ABCC7 p.Gly542* ABCC7 p.Arg1162* G542X could only be detected by L12B4 (50 kDa) and R1162X was detected around 110 kDa but not using NBD2 and C-terminal-specific mAbs. Login to comment 81 ABCC7 p.Asn1303Lys We observed for F508del lower expression levels of B and C-band compared to N1303K. Login to comment 82 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Expression of G551D (class III) and R117H (class IV) was comparable to wt-CFTR. Login to comment 83 ABCC7 p.Ala455Glu For class V mutant A455E, the majority of the protein was core-glycolysated band B and only limited complex-glycosylated CFTR (C-band) was observed. Login to comment 95 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Asn1303Lys ABCC7 p.Arg1162* CFTR Mutants F508del R1162X G551D R117H A455E N1303K 596, 769, 24.1 , 570, 432,450 570, 432, 450 570, 432, 450, 24.1, 769, 596 570, 570, 596, 769, 24.1, 432, 450 596, 769, 24.1, 432, 450 769, 596, 570, 24.1, 432, 450 is likely still preserved and bound to interacting proteins that may affect the binding to specific mAbs. Login to comment 111 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* G551D was expressed at levels comparable to wt-CFTR, expression of R117H, N1303K, A455E and R1162X was intermediate, and low levels were observed for F508del and G542X. Login to comment 112 ABCC7 p.Arg117His ABCC7 p.Asn1303Lys Expression of R117H and N1303K by mAb 570 was higher compared to the other R-domain mAbs. Login to comment 113 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys The NBD2 selective mAbs 769 and 596 indicated higher expression of F508del and N1303K and lower expression of G551D compared to the R-domain antibodies. Login to comment 114 ABCC7 p.Gly542* ABCC7 p.Arg1162* As expected, expression levels for R1162X were similar to empty vector, however, some expression was consistently observed for G542X, possibly due to production of an alternative transcript from this cDNA construct downstream of position 542. Login to comment 118 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys Some mAbs preferentially recognized CFTR mutants as indicated by strong recognition of F508del and N1303K by mAbs 596 and 769, and high recognition of G551D by R-domain selective mAbs. Login to comment 145 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* Western blots containing 25 bc;g of total protein of HEK293 cells transiently transfected with pcDNA3 containing; CFTR-wt, F508del, G542X, R1162X, G551D, R117H, A455E, N1303K or empty vector. Login to comment 149 ABCC7 p.Asn1303Lys We compared the phosphorylation-sensitive mAb 450 which displayed high wt-CFTR specificity, mAb 596 that preferentially recognized F508del and N1303K, and mAb 660 that displayed poor CFTR-specific detection in HEK293 cells. Login to comment 151 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Asn1303Lys Monoclonal antibody 450 detected wt-CFTR, G551D, R117H and N1303K (Fig. 4C). Login to comment 152 ABCC7 p.Ala455Glu ABCC7 p.Arg1162* Limited detection was observed for R1162X and A455E. Login to comment 153 ABCC7 p.Gly542* Mutant CFTR G542X and F508del could not be detected by mAb 450. Login to comment 154 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Asn1303Lys The NBD2-selective mAb 596 could recognize wt-CFTR, G551D, R117H and N1303K equally (Fig. 4C). Login to comment 155 ABCC7 p.Ala455Glu ABCC7 p.Gly542* ABCC7 p.Arg1162* Detection of CFTR was limited for F508del and A455E and CFTR mutants G542X and R1162X were not detectable. Login to comment 184 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys Importantly, we found selectivity of some mAbs for specific mutant forms of CFTR, as the R-domain-specific antibodies poorly recognized F508del and N1303K compared to the NBD2-directed antibodies, but excelled in recognition of G551D (Table 1B). Login to comment 194 ABCC7 p.Asn1303Lys In contrast, preferential recognition of CFTR mutations F508del and N1303K by NBD2 antibodies 596 and 769 was observed by flow cytometry. Login to comment 213 ABCC7 p.Asn1303Lys We observed for mutant N1303K considerable C-band expression (complex-glycosylated CFTR) by Western blot analysis, and relative high CFTR protein levels by flow cytometry compared to F508del. Login to comment 214 ABCC7 p.Asn1303Lys This may suggest that the N1303K mutation also affect gating or conductivity as this mutant is associated with severe CF (http://cftr2.org). Login to comment 216 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Furthermore, we confirmed [31,32] and expanded data on mutant G551D and R117H by demonstrating with Western blot and flow cytometry that expression and trafficking were not affected. Login to comment 217 ABCC7 p.Ala455Glu Interestingly, for mutant A455E we demonstrated CFTR protein levels slightly higher than mutant F508del, with comparable B-C ratio (quantification not shown), suggesting that trafficking is impaired as well. Login to comment 218 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Asn1303Lys A455E is considered to be a mild mutation as it is associated with pancreas sufficiency (http://cftr2.org), indicating that the limited levels of A455E C-band have increased activity as compared to N1303K and F508del, as previously noted [33], or that some A455E B-band may have activity at the plasma membrane as has been described for F508del [34]. Login to comment 220 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Asn1303Lys Furthermore, we provide more insight on the impact of CFTR mutations N1303K, A455E, G551D and R117H on CFTR expression. Login to comment