ABCMdb

PMID: 24045840

Degiorgio D, Corsetto PA, Rizzo AM, Colombo C, Seia M, Costantino L, Montorfano G, Tomaiuolo R, Bordo D, Sansanelli S, Li M, Tavian D, Rastaldi MP, Coviello DA
Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction.
Eur J Hum Genet. 2014 May;22(5):633-9. doi: 10.1038/ejhg.2013.214. Epub 2013 Sep 18., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCB4 p.Leu481Arg Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. Login to comment 3 ABCB4 p.Tyr403His According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. Login to comment 7 ABCB4 p.Tyr403His On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile. Login to comment 31 ABCB4 p.Tyr403His The nucleotide change c.1207T4C that encodes the mutant protein characterized by p.(Y403H) was previously described at homozygous state in the child of family B and at heterozygous state in the other members of family B and C.14 To evaluate the expression of MDR3 in vitro, we used two plasmids: one containing the full-length cDNA of ABCB4 wild type on a pcDNA3.1/ Hygro( &#fe; ) backbone (pcDNA3.1/Hygro( &#fe; )/ABCB4) and the second containing the b-galactosidase gene (pcDNA3.1.V5/His-lacZ). Login to comment 33 ABCB4 p.Tyr403His ABCB4 p.Leu481Arg The plasmid pcDNA3.1/Hygro(&#fe; )/ABCB4 was also used to obtain clones harboring the p.(Y403H) or the p.(L481R) mutation. Login to comment 36 ABCB4 p.Leu481Arg Bioinformatic analysis The three-dimensional models of the human MDR3 and of the p.Leu481Arg mutant were built by homology based on the structure of the murine multidrug resistance protein 1A (gene name, Abcb1a), determined with crystallographic methods (PDB: 3g5u).16 The models were built with an automated procedure with the SwissModel web server (http://www. Login to comment 54 ABCB4 p.Leu481Arg RESULTS Molecular analysis and in silico evaluation The sequencing analysis of ABCB4 gene in the members of family A showed a new heterozygous mutation, c.1442T4G, which causes the p.(L481R) amino-acid change. Login to comment 56 ABCB4 p.Leu481Arg The p.(L481R) replaces the hydrophobic Leu side chain with a bulkier and hydrophilic Arg residue in the first NBD. Login to comment 77 ABCB4 p.Leu481Arg (b) Region surrounding the p.Leu481Arg mutation, with the neighboring residues represented in ball-and-stick. Login to comment 80 ABCB4 p.Tyr403His ABCB4 p.Tyr403His ABCB4 p.Leu481Arg ABCB4 p.Leu481Arg Wild-type cells secreted more PC and Chol than the mutant ones; PC: 3.417 nmol/mg proteins versus 1.410 for p.L481R and 2.346 for p.Y403H and Chol: 4.476 nmol/mg proteins versus 3.193 for p.L481R and 3.811 for p.Y403H (Figures 4a and b; Supplementary Table 1). Login to comment 82 ABCB4 p.Tyr403His ABCB4 p.Leu481Arg Although wild-type cells and p.Y403H mutant showed similar fold increases in PC excretion, p.L481R mutant showed higher sensitivity to NaTC reaching 11-fold increase in PC secretion at 3 mM compared with the absence of NaTC (Supplementary Table 1). Login to comment 85 ABCB4 p.Tyr403His In particular, p.Y403H displayed higher Chol excretion compared with wild-type cells at 3.0 mM NaTC: 9.04 versus 6.05 by referring at parameter 'fold increase` and 34.438 versus 27.074 (nmol/mg proteins) by referring at parameter 'absolute amount`. Login to comment 92 ABCB4 p.Tyr403His ABCB4 p.Leu481Arg Lanes 5 and 6 show the stable expression of p.L481R-ABCB4 and p.Y403H, respectively. Login to comment 94 ABCB4 p.Tyr403His The PC/Chol ratio showed a similar trend in absence or with 1.0 and 3.0 mM of NaTC; it was reduced in both mutants, particularly in Y403H cells, whereas an inverted trend was shown after treatment with 0.5 mM NaTC. Login to comment 96 ABCB4 p.Tyr403His As reported in Figure 4c, in mutant Y403H cells low values of PC efflux are associated with high Chol into the culture medium. Login to comment 98 ABCB4 p.Tyr403His ABCB4 p.Leu481Arg In this study, besides reporting the identification of a novel ABCB4 mutation (p.(L481R)) within the Q-loop in three siblings suffering from juvenile cholelithiasis, we demonstrate that this mutation, stably transfected in HUH28 cells, as well as the mutation p.(Y403H), previously described by our group,14 do not prevent protein targeting to the plasma membrane but induce an abnormal efflux of PC and Chol. Login to comment 102 ABCB4 p.Tyr403His ABCB4 p.Leu481Arg The p.(L481R) mutation was identified in three out of seven affected siblings belonging to family A, whereas the already characterized p.(Y403H) mutation6,14 was identified in four affected members belonging to other two families. Login to comment 104 ABCB4 p.Tyr403His In particular, the infant bearing the homozygous p.(Y403H) mutation was diagnosed as PFIC-3 at the age of 3 months, and at the age of 3 years presented compensated cirrhosis with portal hypertension; (ii) a single mutant allele can be associated with hepatobiliary diseases with less significant and highly variable clinical features. Login to comment 111 ABCB4 p.Tyr403His ABCB4 p.Leu481Arg Analysis of PC (a) and Chol (b) secretion, and PC-Chol correlation (c) in MDR3 wild-type, MDR3 p.Y403H and MDR3 p.L481R cells. Login to comment 118 ABCB4 p.Tyr403His As for biliary Chol, it is widely documented that in mice it is excreted into the canalicular lumen through different pathways; among them, a relevant rate of efflux is because of the direct activity of the specific ABC hemitransporters G5 and G8 (ABCG5 and ABCG8).27 Our data show that, in absence or low concentration of NaTC, mutant cells have less Chol efflux ability than wild-type cells, and this direct proportionality between reduced excretion of biliary PC and Chol was originally described in Mdr2/ mice.7 Instead, higher concentrations of NaTC seem to increase Chol efflux by mutant cells, with a maximum value obtained in presence of the p.Y403H mutation so that the ratio between PC and Chol is inverted. Login to comment