Functional analysis of the missense APOC3 mutation Ala23Thr associated with human hypotriglyceridemia

Abstract

We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [(3)H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [(3)H]TAG from C3AT cells was markedly decreased. The lowered [(3)H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL(1). Moreover, [(3)H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [(3)H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [(3)H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL(1) maturation. Furthermore, lumenal [(35)S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL(1) maturation.

Fig. 1.

Expression of Ala23Thr mutant in McA-RH7777 cells fails to stimulate [³H]TAG or [³⁵S]apoB-100 secretion under lipid-rich conditions. A: Cells expressing wild-type apoC-III (C3wt) or Ala23Thr mutant (C3AT) or neo control were labeled with [³H]glycerol for 2 h in DMEM containing 20% FBS and 0.4 mM oleate. Lipids were extracted from cells and media, respectively, at the end of labeling and separated by TLC. Radioactivity associated with [³H]TAG and [³H]PC was quantified by scintillation counting. The values are expressed as cpm/mg of cell protein. Statistical significance *** P < 0.001, *P < 0.05 (Student t-test of C3wt versus Neo, or C3wt versus C3AT as indicated). Error bars indicate average ± SD (n = 4 dishes of cells). B: The cells were continuously labeled with [³⁵S]methionine/cysteine for 1 h or 2 h with methionine/cysteine-free DMEM supplemented with 20% FBS and 0.4 mM oleate. [³⁵S]apoB-100 and [³⁵S]apoE were recovered from the medium by immunoprecipitation, and subjected to SDS-PAGE. Representative fluorograms of [³⁵S]apoB-100 and [³⁵S]apoE are shown. Quantification of radioactivity associated with [³⁵S]apoB-100 was achieved by scintillation counting (bottom panels) and the values are expressed as cpm/mg of cell protein. Statistical significance *** P < 0.001 (Student t-test of C3wt versus neo or C3wt versus C3AT as indicated). Error bars indicate average ± SD (n = 3 dishes of cells). apo, apolipoprotein; C3AT, Ala23Thr; C3wt, wild-type apoC-III; PC, phosphatidylcholine; TAG, triacylglycerol.

Fig. 2.

Expression of Ala23Thr mutant in McA-RH7777 cells fails to promote VLDL₁ assembly. Cells expressing wild-type apoC-III (panel A) or Ala23Thr mutant (panel B) were labeled with [³⁵S]methionine/cysteine for 60 or 90 min in the presence of 20% serum and 0.4 mM oleate. Cells were homogenized at the end of labeling, and the microsomes were isolated from respective cells by ultracentrifugation. The microsome vesicles were treated with Na₂CO₃ to release the lumenal content, which was further separated into the indicated lipoprotein fractions by cumulative rate flotation centrifugation. The [³⁵S]apoB-100 in each fraction was recovered by immunoprecipitation and subjected to SDS-PAGE. Representative fluorograms of [³⁵S]apoB-100 in the lipoprotein fractions are shown on top. Radioactivities associated with [35S]apoB-100 were quantified by scintillation counting (bottom panels). apo, apolipoprotein; C3AT, Ala23Thr; C3wt, wild-type apoC-III.

Fig. 3.

Expression of Ala23Thr mutant in McA-RH7777 cells does not affect lipid partitioning into microsomes. The C3wt and C3AT cells were labeled with [³H]glycerol (15 µCi/ml medium) for 30 min or 60 min. At the end of labeling, cells were homogenized and separated into cytosol and microsomes. The microsome vesicles were treated with Na₂CO₃, followed by ultracentrifugation to separate the membranes and lumen. Lipids were extracted from the respective samples, resolved by TLC, and the radioactivity associated with [³H]TAG (top) and [³H]PC (bottom) of microsomes (panel A), microsomal membranes (panel B), microsomal lumen (panel C), and cytosol (panel D) was quantified by liquid scintillation counting. Each bar represents average values from two dishes of cells. apo, apolipoprotein; C3AT, Ala23Thr; C3wt, wild-type apoC-III; PC, phosphatidylcholine; TAG, triacylglycerol.

Fig. 4.

Expression of Ala23Thr mutant results in accumulation of TAG and PC in IDL/LDL fractions in microsomal lumen. The lumenal content obtained in Fig. 3C was further separated into the indicated lipoprotein fractions by cumulative rate flotation centrifugation. Lipids were extracted from each lipoprotein fraction, and the radioactivity associated with [³H]TAG (panel A) and [³H]PC (panel B) was quantified. At each time point, [³H]TAG and [³H]PC that was secreted into the medium was also quantified as shown in panel C and panel D, respectively. C3AT, Ala23Thr; C3wt, wild-type apoC-III; IDL, intermediate density lipoprotein; PC, phosphatidylcholine; TAG, triacylglycerol.

Fig. 5.

Expression of Ala23Thr mutant does not increase Mttp expression or MTP activity. A: The relative Mttp mRNA concentrations (with respect to 18s rRNA) were quantified by real-time RT-PCR. Data are presented as fold changes between C3wt and C3AT cells in comparison to that in neo control (set as 1). B: The TAG transfer activity of MTP was determined using cell lysate obtained from C3wt, C3AT, or neo control and the MTP activity is expressed relative to neo control. Each bar represents average values ± SD obtained from triplicate samples. ***P < 0.001; **P < 0.01 (Student t-test of C3wt versus neo or C3wt versus C3AT as indicated). C3AT, Ala23Thr; C3wt, wild-type apoC-III; MTP, microsomal triglyceride-transfer protein; PC, phosphatidylcholine; TAG, triacylglycerol.

Fig. 6.

Treatment with low dose brefeldin A blocks second step VLDL₁ assembly and results in accumulation of TAG and PC in IDL/LDL fractions in microsomal lumen. A and B: Cells were pretreated with brefeldin A (Bfa; 0.2 µg/ml) or Bfa + MTP inhibitor (iMTP; 0.2 µM) for 30 min prior to labeling with [³⁵S]methionine/cysteine for 60 min. Both pretreatment and labeling media contained 20% FBS and 0.4 mM oleate. C and D: Cells were treated exactly the same as in A and B, and labeled with [³H]glycerol for 60 min. C3AT, Ala23Thr; C3wt, wild-type apoC-III; IDL, intermediate density lipoprotein; MTP, microsomal triglyceride-transfer protein; PC, phosphatidylcholine; TAG, triacylglycerol.

Fig. 7.

Model of Ala23 location in apoC-III peptide and lumenal distribution of Ala23Thr mutant. A: Schematic representation of human apoC-III (adopted from Cohn et al. (14), PDB 2jq3) with six segments of α helices (labeled with h1 through h6) that form an arch with hydrophobic residues, including Ala23, facing inward. Positive residues Lys-17, Lys-21, Lys-24, and His-18 on the polar surface of h1 and h2 are also shown. B: A helical wheel diagram of residues 7-33, with hydrophobic residues highlighted in green circles, acidic residues in red, basic residues in blue, hydrophilic neutral residues in gray, and the helix destabilizing residue Gly in yellow. Residue Ala23 is highlighted in red. C: Lumenal [³⁵S]apoC-III distribution coincides with that of [³H]TAG. The experiments were performed essentially the same as that described in Fig. 2, except that [³⁵S]apoC-III in microsomal lumen of C3wt and C3AT cells was analyzed. Data of [³H]TAG derived from Fig. 4A are plotted as % of total lumenal [³H]TAG for comparison. D: Two-way co-immunoprecipitation analysis of apoB and apoC-III. Cell lysates were subjected to immunoprecipitation with either anti-apoB or anti-apoC-III antibody. The immunocomplexes were resolved by SDS-PAGE, and probed for apoB or apoC-III by Western blot analysis, respectively. Rabbit preimmune serum was used as a negative control. apo, apolipoprotein; C3AT, Ala23Thr; C3wt, wild-type apoC-III; IDL, intermediate density lipoprotein; TAG, triacylglycerol.

Fig. 8.

Model of a possible role of apoC-III in the formation of lipid droplets and in promoting the second step VLDL assembly. The nascent apoB-containing lipoprotein particles are assembled within the ER lumen co- or posttranslationally, and exit ER through coatomer-mediated vesiculation. Lipid substrates synthesized in the ER probably also exit ER through vesiculation, and apoC-III may play a role in the formation of lipid droplets and eventual fusion with VLDL assembly intermediates. The Ala23Thr mutant is unable to promote fusion, resulting in accumulation of lipid substrates similar to the brefeldin A treatment (not shown in the model). apo, apolipoprotein; ER, endoplasmic reticulum.