ABCMdb

PMID: 24352041

Pomozi V, Brampton C, Fulop K, Chen LH, Apana A, Li Q, Uitto J, Le Saux O, Varadi A
Analysis of pseudoxanthoma elasticum-causing missense mutants of ABCC6 in vivo; pharmacological correction of the mislocalized proteins.
J Invest Dermatol. 2014 Apr;134(4):946-53. doi: 10.1038/jid.2013.482. Epub 2013 Nov 11., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC6 p.Arg1114Pro ABCC6 p.Arg1314Trp ABCC6 p.Gln1347His ABCC6 p.Ser1121Trp However, 4-PBA, a drug approved for clinical use, restored the plasma membrane localization of four ABCC6 mutants (R1114P, S1121W, Q1347H, and R1314W), suggesting that allele-specific therapy may be useful for selected patients with PXE and GACI. Login to comment 29 ABCC6 p.Arg1314Trp Indeed, we have identified one such mutant, R1314W, whose cellular localization was normalized using the chemical chaperone sodium 4-phenylbutyrate (4-PBA) in both in vitro and in vivo experiments. Login to comment 39 ABCC6 p.Arg1141* In addition to 10 missense mutants, R1141X was also used as a negative control in certain experiments. Login to comment 47 ABCC6 p.Gly1321Ser ABCC6 p.Val1298Phe Two missense variants (V1298F and G1321S) showed very little, if any, transport activity (Ilia &#b4;s et al., 2002). Login to comment 48 ABCC6 p.Arg1339Cys The R1339C was found to be unstable in Sf9 cells, similar to that shown earlier (Le Saux et al., 2011), and its transport activity could not be assayed. Login to comment 50 ABCC6 p.Arg1141* The nonsense R1141X mutant was not expressed in Sf9 cells. Login to comment 51 ABCC6 p.Arg1141* Subcellular localization in vitro Each mutant (with the exception of R1141X) was individually expressed in MDCKII cells, which were then grown either on plastic wells as nonpolarized cells or on Transwell filters, which ensures development of monolayers of polarized cells. Login to comment 55 ABCC6 p.Val1298Phe ABCC6 p.Ser1121Trp In nonpolarized cells, only S1121W and the transport-deficient V1298F were targeted to the plasma membrane. Login to comment 56 ABCC6 p.Arg1138Gln ABCC6 p.Arg1114Pro ABCC6 p.Thr1301Ile In addition to these, three other mutants, R1114P, R1138Q, and T1301I, were also found in the basolateral plasma membrane when expressed in polarized MDCKII cells (Figure 2, columns 1 and 3). Login to comment 64 ABCC6 p.Val1298Phe ABCC6 p.Arg1459Cys The R1459C and the transport- inactive V1298F mutants were found in subcellular localization identical to the wt, whereas all the other mutants showed various degrees of intracellular localization, as illustrated in Figure 3, column 1. Login to comment 65 ABCC6 p.Ser1121Trp Interestingly, S1121W, which showed wt-like plasma membrane localization in both MDCKII expression systems, was mostly intracellular in mouse liver, similar to delABCC6. Login to comment 66 ABCC6 p.Arg1138Gln ABCC6 p.Arg1114Pro It is worth noting that R1114P and R1138Q mutants were found both intracellularly and in the plasma membrane. Login to comment 67 ABCC6 p.Arg1141* The R1141X mutant was not expressed in the mouse liver. Login to comment 72 ABCC6 p.Arg1141* Next, the injection of the nonsense R1141X mRNA showed that this mutant was ineffective in rescuing the morpholino-mediated phenotype (4.8%). Login to comment 74 ABCC6 p.Val1298Phe ABCC6 p.Ser1121Trp The efficacy of S1121W was somewhat better (7.9%), whereas that of the V1298F was significantly higher (32.0%). Login to comment 78 ABCC6 p.Gly1321Ser ABCC6 p.Val1298Phe ABCC6 p.Arg1459Cys Note that this type of experiment was not performed in vivo with the two inactive mutants, V1298F and G1321S, or with R1459C, as this mutant was found in the plasma membrane without 4-PBA treatment. Login to comment 80 ABCC6 p.Gln1347His ABCC6 p.Arg1459Cys For instance, mutants Q1347H and R1459C were found mostly intracellularly in nonpolarized and in polarized MDCKII cells, yet the 4-PBA treatment resulted in plasma membrane localization only when these two variants were expressed in nonpolarized cells. Login to comment 82 ABCC6 p.Arg1138Gln ABCC6 p.Arg1138Gln ABCC6 p.Arg1138Gln ABCC6 p.Arg1114Pro ABCC6 p.Arg1114Pro ABCC6 p.Arg1314Trp ABCC6 p.Arg1314Trp ABCC6 p.Gln1347His ABCC6 p.Gln1347His ABCC6 p.Gly1321Ser ABCC6 p.Gly1321Ser ABCC6 p.Val1298Phe ABCC6 p.Ser1121Trp ABCC6 p.Ser1121Trp ABCC6 p.Arg1339Cys ABCC6 p.Thr1301Ile ABCC6 p.Thr1301Ile ABCC6 p.Thr1301Ile ABCC6 p.Arg1459Cys ABCC6 p.Arg1459Cys No 4-PBA-induced plasma membrane rescue was observed for R1138Q and T1301I in mouse liver, whereas Extracellular Walker B Q-loop Signature Missense mutations Intercellular 140 ABCC6 wt V1298F G1321S R1114P R1138Q R1314W R1459C S1121W T1301I Q1347H delABCC6 120 100 80 60 40 20 0 LTC4 transport activity (%) TMD0 L0 TMD1 L1 TMD2 R1114P S1121W R1138Q T1301I R1314W G1321S R1339C R1459C Q1347H Figure 1. Login to comment 92 ABCC6 p.Arg1314Trp As for the R1314W mutant, 4-PBA treatment resulted in plasma membrane targeting both in polarized and nonpolarized MDCKII cells, as well as in mouse liver. Login to comment 93 ABCC6 p.Gln1347His 4-PBA treatment was also effective in the case of Q1347H in vivo, while it facilitated plasma membrane targeting only in nonpolarized MDCKII cells (but not in polarized cell cultures). Login to comment 94 ABCC6 p.Arg1339Cys No plasma membrane targeting was achieved in the case of mutant R1339C irrespective of which experimental system was used. Login to comment 101 ABCC6 p.Arg1339Cys Of the three remaining mutants, only two displayed decreased transport function (Figure 1b), whereas the other mutant could not be stably expressed in Sf9 cells (R1339C). Login to comment 105 ABCC6 p.Arg1339Cys We achieved a good level of expression of the R1339C mutant in this culture model. Login to comment 113 ABCC6 p.Arg1138Gln ABCC6 p.Arg1114Pro ABCC6 p.Arg1314Trp ABCC6 p.Gln1347His ABCC6 p.Gly1321Ser ABCC6 p.Val1298Phe ABCC6 p.Ser1121Trp ABCC6 p.Arg1339Cys ABCC6 p.Thr1301Ile ABCC6 p.Arg1459Cys MDCKII cell line in vitro Nonpolarized - 4-PBA 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m 20 &#b5;m Wild type R1114P S1121W R1138Q V1298F T1301I R1314W G1321S R1339C Q1347H R1459C deltaABCC6 - 4-PBA + 4-PBA + 4-PBA Not determined Polarized intended to correct their cellular localization, as described previously (Le Saux et al., 2011). Login to comment 114 ABCC6 p.Gln1347His ABCC6 p.Arg1459Cys For both mutants Q1347H and R1459C, the chemical chaperone was effective in correcting its cellular trafficking only in nonpolarized cultures. Login to comment 126 ABCC6 p.Arg1138Gln ABCC6 p.Arg1114Pro ABCC6 p.Arg1314Trp ABCC6 p.Gln1347His ABCC6 p.Gly1321Ser ABCC6 p.Val1298Phe ABCC6 p.Ser1121Trp ABCC6 p.Arg1339Cys ABCC6 p.Thr1301Ile ABCC6 p.Arg1459Cys Mouse liver in vivo - 4-PBA Not determined Not determined Not determined Wild type R1114P S1121W R1138Q V1298F T1301I R1314W G1321S R1339C Q1347H R1459C deltaABCC6 + 4-PBA Figure 4. Login to comment 133 ABCC6 p.Arg1141* (d) Animals injected with morpholino and with ABCC6 R1141X mRNA. Login to comment 137 ABCC6 p.Val1298Phe Minimal level of rescue was provided by the disease-causing mutants (07.9%) irrespective of their transport activity or their cellular localization, with the exception of V1298F (32.0%; Table 1). Login to comment 139 ABCC6 p.Val1298Phe We hypothesized that V1298F probably possesses some residual transport activity of the endogenous zebrafish substrate. Login to comment 140 ABCC6 p.Arg1459Cys ABCC6 p.Arg1459Cys In addition, it is noteworthy that the PXE-associated mutant R1459C (Chassaing et al., 2005) was found to be an active transporter, and it localized in the plasma membrane in mouse hepatocytes, suggesting that R1459C could be a neutral polymorphism rather than a disease-causing mutant. Login to comment 142 ABCC6 p.Arg1459Cys The case of R1459C illustrates the necessity of using multiple model systems in parallel to study the functional consequences of ABCC6 mutations. Login to comment 143 ABCC6 p.Arg1114Pro ABCC6 p.Arg1314Trp ABCC6 p.Gln1347His ABCC6 p.Ser1121Trp The major finding of our study was that 4-PBA treatment restored the plasma membrane localization of three transport-competent missense mutants, R1114P, S1121W, and Q1347H in mouse liver, in addition to R1314W, which served as a positive control in the present study. Login to comment 150 ABCC6 p.Arg1141* ABCC6 p.Arg1138Gln ABCC6 p.Arg1114Pro ABCC6 p.Gln1347His ABCC6 p.Gly1321Ser ABCC6 p.Val1298Phe ABCC6 p.Ser1121Trp ABCC6 p.Arg1339Cys ABCC6 p.Thr1301Ile ABCC6 p.Arg1459Cys Summary of the characterization and rescue of disease-causing ABCC6 mutants Localization in mouse liver Localization in MDCKII cell line Nonpolarized Polarized ABCC6 variant Sf9 transport activity Without treatment After 4-PBA treatment Without treatment After 4-PBA treatment Without treatment After 4-PBA treatment Zebrafish &#fe; mRNA rescue (%) Wild type Active PM1 PM PM PM PM PM 90.6 R1114P Active IC4PM PM (rescue) ICoPM PM (rescue) PM PM 0.0 S1121W Active IC4PM PM (rescue) PM PM PM PM 7.9 R1138Q Active IC4PM IC4PM (no effect) IC PM (rescue) PM PM 1.8 V1298F o20% PM ND PM PM PM ND 32.0 T1301I Active IC4PM IC4PM (no effect) IC4PM PM (rescue) PM PM 5.1 R1314W1 Active IC4PM PM (rescue) IC PM (rescue) IC4PM PM (rescue) 0.0 G1321S o20% IC ND IC4PM IC4PM (no effect) IC IC (no effect) 0.0 R1339C Not stable IC IC (no effect) IC IC (no effect) IC IC (no effect) 0.0 Q1347H Active IC4PM PM (rescue) IC4PM PM (rescue) IC &#bc; PM IC&#bc; PM (no effect) 0.8 R1459C Active PM ND IC &#bc; PM PM (rescue) ICoPM ICoPM (no effect) 0.0 delABCC6 o20% IC IC IC IC IC IC ND R1141X Stop ND ND ND ND ND ND 4.8 Abbreviations: IC, intracellular; ND, not determined; PM, plasma membrane. Login to comment