Functional characterization of genetic variants affecting the intracellular domains of ATP-binding cassette transporter A1 (ABCA1)

Abstract

The ATP-binding cassette transporter A1 (ABCA1) effluxes cellular cholesterol and phospholipids to extracellular acceptors, mainly apolipoprotein A1, generating high-density lipoprotein (HDL) particles. This is the first step in the anti-atherosclerotic process of transporting excess cholesterol from non-hepatic tissues to the liver. Loss-of-function variants in ABCA1 lead to reduced HDL cholesterol levels in plasma, thus possibly diminishing the atheroprotective effect of HDL. More than 250 missense variants have been reported in the ABCA1 gene, most of which remain to be functionally characterized. In this study, we have characterized 74 variants affecting the intracellular domains of ABCA1 by assessing cholesterol efflux activity and cell surface localization of the protein, thereby shifting the pathogenicity classification of 10 variants from class 3 (uncertain significance) to class 4 (likely pathogenic) or class 2 (likely benign). Consequently, functional characterization contributes to a better understanding of the molecular basis of the pathogenicity of genetic variants in ABCA1, which could also clarify the mechanism of action of the protein.

Keywords: HDL; HEK293 cells; Tangier disease; cholesterol efflux; lipid metabolism; lipoproteins.

Fig. 1

Cholesterol efflux activity of ABCA1 variants. Relative cholesterol efflux from HEK293 cells transiently transfected with 74 ABCA1 variants and three known loss-of-function control variants (striped columns) from four independent experiments, normalized to WT ABCA1 (WT). Variants were assessed to be functionally normal (efflux > 80% of WT, white columns), loss-of-function (efflux < 41% of WT, black columns), or of uncertain significance (efflux 41%–80% of WT, gray columns). Error bars represent 1 SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, t-test versus WT ABCA1.

Fig. 2

Cell surface expression of the 15 loss-of-function variants. Relative amounts of cell surface ABCA1 were evaluated in HEK293 cells transiently transfected with 15 loss-of-function variants (black columns) and two control variants (striped columns), normalized to WT ABCA1 (WT) in four independent experiments. Error bars represent 1 SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, t-test versus WT ABCA1. One representative Western blot showing cell surface ABCA1 detected using a V5-HRP antibody is presented. Dotted lines designate the merger of blots. A: Total cell surface ABCA1. β-actin in cell lysates was used as a loading control. B: Cell surface ABCA1 corrected (corr.) for amounts of ABCA1 in total lysates. ABCA1 in corrected amounts of cell lysates (corr. total ABCA1) was used as a loading control.

Fig. 3

Functional rescue of loss-of-function ABCA1 variants by 4-PBA treatment. Effect of 4-PBA on functionality of 15 loss-of-function variants and three control variants (striped columns), assessed in transiently transfected HEK293 cells after 20 h incubation with 10 mM 4-PBA or an equal volume of dH₂O (−). Error bars represent 1 SD. A: Relative cholesterol efflux normalized to mock-treated WT ABCA1 (WT) in five independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗P < 0.0001, one-tailed t-test versus mock-treatment. B: Cell surface expression of the ATPase deficient control variants K939M and K1952M, and loss-of-function variants with a suspected ATPase deficiency, normalized to mock-treated WT ABCA1 (WT) in three independent experiments (P < 0.05, one-tailed t-test vs. mock-treatment). One representative Western blot is shown. ABCA1 was detected using a V5-HRP antibody, and β-actin in cell lysates was used as a loading control.

Fig. 4

Domain structure of ABCA1 and localization of loss-of-function variants. Each ABCA1 domain structure is distinctly colored for clarity: ECD1 is shown in cyan, ECD2 in purple, TMD1 in yellow, TMD2 in green, NBD1 in red-violet, NBD2 in blue, and the R-domains (R1 and R2) in orange and yellow. The accompanying enhanced image on the right highlights the structural mapping of 14 of the 15 loss-of-function variants specifically located in NBD1 (residues 903–1147), NBD2 (residues 1907–2143), R-domains (residues 1182–1251 and 2155–2220), and the intracellular coupling helices (residues; IH1: 3–20, IH2: 667–673, IH3: 1327–1344 and IH4: 1684–1690). This structural representation is derived from Protein Data Bank entry 7ROQ and was created using PyMOL (The PyMOL Molecular Graphics System, Version 3.1.6.1, Schrödinger, LLC, New York, NY). Because p.N1948 is not depicted in the 7ROQ model, the localization of this residue is shown in supplemental Fig. S4B.

Fig. 5

Comparison of the two ABCA1 nucleotide-binding domains. Comparison of sequence and equivalent variants of the two nucleotide-binding domains (NBD1 and NBD2) of ABCA1. A: Sequence alignment of the two NBDs, specifically from residue 923 to 1121 in NBD1, and from residue 1936 to 2134 in NBD2. The alignment was conducted using Jalview with the MUSCLE algorithm (28) and is visualized using a gray color scheme to highlight the conserved residues. Equivalent variants assessed in this study are indicated. B: Relative cholesterol efflux activity of 16 equivalent NBD variant pairs (NBD1|NBD2), normalized to WT ABCA1 in four independent experiments. Variants were assessed to be functionally normal (efflux > 80% of WT, white column), loss-of-function (efflux < 41% of WT, black columns), or of uncertain significance (efflux 41%–80% of WT, gray columns). Error bars represent 1 SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, t-test versus WT ABCA1. ^†Data already presented in Fig. 1.