Missense mutation in APOC3 within the C-terminal lipid binding domain of human ApoC-III results in impaired assembly and secretion of triacylglycerol-rich very low density lipoproteins: evidence that ApoC-III plays a major role in the formation of lipid precursors within the microsomal lumen

Abstract

Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL(1), the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys(58) with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys(58) and Lys(60) with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.

FIGURE 1.

The K58E is a loss-of-function mutation that abolished TAG-rich VLDL₁ secretion. A, the six α-helices of human apoC-III in helix wheel diagram are shown. The secondary structure assignment was based on the apoC-III structure (PDB ID 2jq3) in SDS-bound state as determined by NMR (26). Hydrophobic residues are denoted in green, acidic residues are in red, basic residues are in blue, hydrophilic residues are in gray, and Lys⁵⁸ is marked with a red arrow. B, shown is an immunoblot of recombinant human apoC-III expressed in McA-RH7777 cells. C3wt, wild type apoC-III; C3KE, the K58E variant. C, cells (1.8 × 10⁶ cells/60 mm dish, in triplicate) were labeled with [³H]glycerol (5 μCi/ml) for 2 h in DMEM containing 20% FBS and 0.4 mm oleate. Lipids were extracted from media and cells at the end of labeling and resolved by TLC. Radioactivity associated with [³H]TAG was quantified by scintillation counting. Radioactivity associated with the [³H]TAG in the media is shown. ***, p < 0.001 (Student's t test of C3wt versus neo or C3KE versus neo; n = 3). D, cells were labeled with [³H]glycerol for 2 h in the presence of 20% serum and 0.4 mm oleate. At the end of labeling, media were subjected to cumulative rate flotation ultracentrifugation. Lipids were extracted from each fraction and separated by TLC. Radioactivity associated with [³H]TAG was quantified by scintillation counting. E, cells were labeled with [³⁵S]methionine/cysteine for 2 h in the presence of 20% serum and 0.4 mm oleate. At the end of labeling, media were subjected to cumulative rate flotation ultracentrifugation. ApoB-100 was recovered from each fraction by immunoprecipitation and resolved by SDS-PAGE and visualized by fluorography (top two panels). Radioactivity associated with ³⁵S-apoB100 was quantified by scintillation counting and plotted (bottom panel).

FIGURE 2.

Impaired VLDL₁-apoC-III secretion from C3KE cells. A, C3wt and C3KE cells were labeled with [³⁵S]methionine/cysteine for 2 h in the presence of 20% serum and 0.4 mm oleate. The conditioned media were fractionated by cumulative rate flotation ultracentrifugation, and immunoprecipitated ³⁵S-apoC-III was resolved by SDS-PAGE and visualized by fluorography. B, conditioned media were fractionated by size exclusion chromatography using FPLC and apoC-III in fractions 14–40 were recovered by immunoprecipitation, resolved by SDS-PAGE, and visualized by immunoblotting. C, cells were treated as in A, and the conditioned media were subjected to sucrose density gradient ultracentrifugation. The immunoprecipitated ³⁵S-apoA-I proteins were resolved by SDS-PAGE and visualized by fluorography. D, the ³⁵S-labeled conditioned media (−heparin, top panels or +heparin (100 units/ml), bottom panels) were subjected to sucrose density gradient ultracentrifugation, and the immunoprecipitated apoE proteins were resolved by SDS-PAGE and visualized by fluorography. E, the radioactivity associated with ³⁵S-apoE was quantified using scintillation counter (only data were obtained under minus heparin conditions are shown). The density (g/ml) of each fraction, which is the mean value of seven density gradients determined gravimetrically, is shown on top of the fluorograms in panels C and D. Repetition of the experiments yielded similar results.

FIGURE 3.

Hepatic VLDL₁ production was impaired in apoc3-null mice expressing apoC3KE protein. The apoc3-null mice (10 weeks old) fed with a high fat diet for 2 weeks were infected with adenovirus encoding C3wt or C3KE. Three days after infection, mice were fasted for 16 h. Blood samples were collected before or 1 and 2 h after P407 injection. A, immunoblots of apoC-III in plasma of infected mice are shown. B and C, shown are immunoblots of apoB-100 and apoB48 in fractionated plasma lipoproteins (only the top six fractions of cumulative rate flotation centrifugation are shown). The plasma samples were pooled from three mice infected with adv-C3wt (B) or adv-C3KE (C). Samples were collected before (top two panels) and 1 h (middle two panels) or 2 h (bottom two panels) after P407 injection. D, TAG mass in the top six fractions of cumulative rate flotation centrifugation (i.e. VLDL₁, VLDL₂, and IDL/LDL) of plasma samples pooled from three adv-C3wt- and three adv-C3KE-infected mice before (top panel), 1 h (middle panel), or 2 h (bottom panel) after P407 injection.

FIGURE 4.

Impaired LLD formation in C3KE cells. A, shown is a proteomics analysis of LLD-associated proteins. C3wt cells were cultured in DMEM supplemented with 20% serum and 0.4 mm oleate. The cells were homogenized to isolate the microsomes, and the lumenal contents were released from the microsomes by Na₂CO₃ treatment. The content was fractionated by cumulative rate flotation centrifugation to obtain LLD fractions, and the identity of the LLD-associated proteins was determined by liquid chromatography-MS/MS as described under “Experimental Procedures.” The identified proteins are visualized using Cytoscape software (Version 2.6.0) (34). The line width, size, and color intensity of each individual protein is related to its identified peptides. The detailed information of each identified protein is shown in supplemental Table 3. B, apoC-III and apoB-100 distribution within microsomal lumen is shown. C3wt and C3KE cells were labeled with [³⁵S]methionine/cysteine for 60 min in the presence of 20% serum and 0.4 mm oleate. Microsomal content was fractionated as in A. The ³⁵S-apoC-III (top two panels) or ³⁵S-apoB-100 (bottom two panels) in each fraction was recovered by immunoprecipitation, resolved by SDS-PAGE, and visualized by fluorography. C, C3wt and C3KE cells (∼ 6 × 10⁶ cells/100-mm dish, in duplicates) were labeled with [³H]glycerol (20 μCi/ml) in DMEM supplemented with 20% FBS and 0.4 mm oleate for 0.5, 1, and 2 h. At the end of labeling, cells from two dishes were combined, and lumenal content was isolated from the microsomes followed by fractionation as in A. Lipids were extracted from each fraction and resolved by TLC. Radioactivity associated with [³H]TAG (top), [³H]DAG (middle), and [³H]PC (bottom) was quantified and plotted.

FIGURE 5.

Amphipathicity of helix 5 is important for apoC-III function. A and B, increasing quantities (5–100 μg) of the indicated lipid groups spotted on nitrocellulose membranes were incubated with an equal amount of apoC-III collected from conditioned media of stable cell lines expressing the indicated apoC-III variants. The bound apoC-III was detected by immunoblotting, and the intensity of each spot was semiquantified by scanning densitometry (for calculating K_A values for each lipid group). Chol, cholesterol. CE, cholesteryl ester. C, cells (1.8 × 10⁶ cells/60 mm dish, in triplicate) expressing the indicated apoC-III variants were labeled with [³H]glycerol (5 μCi/ml) in DMEM supplemented with 20% FBS and 0.4 mm oleate, and secretion of [³H]TAG and [³H]PC was determined as in Fig. 1C. * p < 0.05; *** p < 0.001 (Student's t test of C3 variants versus neo; n = 3).

FIGURE 6.

Synthesis, secretion, and turnover of lipids in C3wt and C3KE cells. C3wt and C3KE cells (1.8 × 10⁶ cells/60 mm dish, in triplicate) were labeled with [³H]glycerol (5 μCi/ml) for up to 2 h in DMEM containing 20% FBS and 0.4 mm oleate. Lipids were extracted from media and cells, respectively, at the end of labeling and resolved by TLC. Radioactivity associated with ³H-labeled lipid was quantified by scintillation counting. A-C, at each time point, the radioactivity of cell-associated [³H]DAG, [³H]TAG, and [³H]PC are shown. D–F, at each time point, the radioactivity of [³H]DAG, [³H]TAG, and [³H]PC from the conditioned media is shown. G–I, cells were labeled with [³H]glycerol (5 μCi/ml) for 1 h in DMEM supplemented with 20% FBS and 0.4 mm oleate. The cells were replaced with DMEM supplemented with 20% FBS, 0.4 mm oleate, and 40 μm triacsin C and cultured (chase) for up to 8 h. Lipids were extracted from cells and media at the indicated chase time and separated by TLC, and radioactivity associated with [³H]DAG, [³H]TAG, and [³H]PC was quantified. The total counts (media and cell) are expressed as percent of counts at the end of initial 1 h labeling. ***, p < 0.001; **, p < 0.01; *, p < 0.05 (Student's t test of C3wt versus C3KE; n = 3).

FIGURE 7.

Distribution of lipids in microsomes and cytosol are different in C3wt and C3KE cells. The C3wt and C3KE cells (100 mm dishes, in duplicate) were labeled with [³H]glycerol (20 μCi/ml) for up to 2 h in DMEM supplemented with 20% FBS and 0.4 mm oleate. The cells were harvested, and the intracellular components were separated into post-nuclear supernatant and nucleus-mitochondria (pellet) by centrifugation (1000 × g, 10 min, 4 °C). Post-nuclear supernatant was further separated into microsomes (pellet) and cytosol (supernatant) by ultracentrifugation (500,000 × g, 30 min, 4 °C). Lipids were extracted from the conditioned media as well as from all intracellular fractions and separated by TLC, and the radioactivity associated with [³H]DAG (A), [³H]TAG (B), and [³H]PC (C) was quantified. The counts in microsomes and cytosol fractions were plotted as a percent of total (cellular fractions and media).

FIGURE 8.

Lipid staining of apoc3-null mice infected with adv-C3wt or adv-C3KE. The experiments were performed as in Fig. 3. Two hours after P407 treatment, the mice were sacrificed, and the livers were retrieved and processed for lipid staining (panels A–C) and TAG mass quantification (panel D). A, liver of three control mice (apoc3-null mice infected with empty adenovirus vector) is shown. B, liver of three mice expressing C3wt is shown. C, liver of three mice expressing C3KE is shown. Scale bar, 50 μm. CV, central vein. D, TAG mass in the liver is shown. Error bars indicate S.D. of average TAG values of liver samples obtained from three independent mice per group.

FIGURE 9.

A model for the two-domain hypothesis of apoC-III action. The C-terminal lipid binding domain, encompassing the amphipathic α-helix 5, facilitates TAG synthesis and binding to LLD, probably through its strong interaction with glycerophospholipids. The N-terminal domain, near α-helix 2, possesses fusogenic activity in promoting fusion between LLD and VLDL precursors to form VLDL₁. Some apoC-III molecules remain associated with the resultant VLDL₁ after secretion, and the others dissociate from LLD and are secreted as HDL. The K58E mutant (C3KE) is unable to bind LLD or participate in VLDL₁ maturation and is, therefore, secreted as HDL.