Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction

Abstract

The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of 'toxic bile'. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. 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. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. 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.

Phosphorylation of ABCB4 impacts its function: Insights from disease-causing mutations

“…A similar conclusion was drawn for another missense mutation associated with intrahepatic cholestasis of pregnancy . Recently, a study demonstrated that two mutations found in patients with biliary diseases, Y403H and L481R, decreased ABCB4 activity without altering its membrane targeting . These mutations were localized in the first nucleotide‐binding domain and therefore may affect ABCB4 function by interfering with ATP binding and/or hydrolysis.…”

A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3

“…We have shown that the I541F mutation identified in a PFIC3 patient caused misfolding and retention of the protein in the endoplasmic reticulum . Subsequent studies have reported some mutations that reduced PC secretion or affected ABCB4 protein expression . The aim of these studies was primarily to correlate ABCB4 mutations with patients' phenotypes.…”

“…(14,15) Subsequent studies have reported some mutations that reduced PC secretion or affected ABCB4 protein expression. (12,(16)(17)(18) The aim of these studies was primarily to correlate ABCB4 mutations with patients' phenotypes.…”

Lipid flopping in the liver

“…The lipids accumulated in the culture medium (presumably bound to an acceptor in the added calf serum) and were quantified by thin layer chromatography. Whereas stable cells have been used to characterize suspected disease-associated ABCB4 mutants, this is probably impractical for the large number of idiopathic non-synonymous single nucleotide polymorphisms (SNPs) that have been reported [50]. To develop a high(er) throughput analytical system to relate genotype and phenotype at the protein level required an understanding of the link between PC flopping and PS flipping.…”