The Relationship of Amphipathic Beta Structures in The Beta-1

§Department of Biochemistry and Molecular Genetics, University of Alabama at ... University of Alabama at Birmingham, 1808 7th Avenue South, BDB-D680...
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Relationship between Amphipathic β Structures in the β1 Domain of Apolipoprotein B and the Properties of the Secreted Lipoprotein Particles in McA-RH7777 Cells Medha Manchekar,† Richa Kapil,† Zhihuan Sun,† Jere P. Segrest,†,‡,§ and Nassrin Dashti*,† †

Department of Medicine, Basic Sciences Section, and Atherosclerosis Research Unit, and ‡Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294, United States § Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States ABSTRACT: Our previous studies demonstrated that the first 1000 amino acid residues (the βα1 domain) of human apolipoprotein (apo) B-100, termed apoB:1000, are required for the initiation of lipoprotein assembly and the formation of a monodisperse stable phospholipid (PL)-rich particle. The objectives of this study were (a) to assess the effects on the properties of apoB truncates undergoing sequential inclusion of the amphipathic β strands in the 700 N-terminal residues of the β1 domain of apoB-100 and (b) to identify the subdomain in the β1 domain that is required for the formation of a microsomal triglyceride transfer protein (MTP)-dependent triacylglycerol (TAG)rich apoB-containing particle. Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. (1) The presence of amphipathic β strands in the 200 N-terminal residues of the β1 domain resulted in the secretion of apoB truncates (apoB:1050 to apoB:1200) as both lipidated and lipid-poor particles. (2) Inclusion of residues 300− 700 of the β1 domain led to the secretion of apoB:1300, apoB:1400, apoB:1500, and apoB:1700 predominantly as lipidated particles. (3) Particles containing residues 1050−1500 were all rich in PL. (4) There was a marked increase in the lipid loading capacity and TAG content of apoB:1700-containing particles. (5) Only the level of secretion of apoB:1700 was markedly diminished by MTP inhibitor BMS-197636. These results suggest that apoB:1700 marks the threshold for the formation of a TAG-rich particle and support the concept that MTP participates in apoB assembly and secretion at the stage where particles undergo a transition from PL-rich to TAG-rich. 2575 ± 25, residues 2572 ± 25 to 4100 ± 100, and residues 4100 ± 100 to 4550 ± 50, respectively. On the basis of sequence homology between the N-terminal 1000-residue domain of apoB and lipovitellin (LV),11,12 Segrest et al.10,13 suggested that this domain might be a lipid-binding pocket in apoB similar to that in LV.14,15 In the liver, the assembly of apoB-100 into a bona fide VLDL particle proceeds in two-steps. The first step involves the formation of a small particle in the density range of highdensity lipoprotein (HDL). The second step involves recruitment of bulk lipid, primarily TAG, leading to core expansion and VLDL formation.3 Microsomal triglyceride transfer protein (MTP) has an obligatory role in the assembly and secretion of apoB-containing lipoproteins.5,16 ApoB acquires lipids cotranslationally; i.e., while the C-terminal portion is still being synthesized, the N-terminal portion assembles into a small lipoprotein particle.3−5 This step requires disulfide-dependent folding of portions of the N-terminal domain of apoB.17 Numerous investigators have examined the lipid binding

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polipoprotein B (apoB) is required for the formation of TAG-rich lipoproteins in the liver and intestine and plays a fundamental role in their transport and metabolism.1 There is only one apoB molecule per lipoprotein particle;2 hence, its concentration in plasma reflects the number of potential atherogenic lipoproteins. ApoB exists in two forms in humans, apoB-100 (the full-length protein), which is essential for the assembly and secretion of hepatic very low-density lipoproteins (VLDL), and apoB-48 (the N-terminal 48% of the protein), which is required for the assembly and secretion of intestinal chylomicrons.3−5 ApoB-100 is one of the largest monomeric proteins known; it consists of 4536 amino acid residues and is the predominant protein component of the atherogenic lowdensity lipoproteins (LDL).3 ApoB is highly insoluble in aqueous solutions; therefore, it has been difficult to identify the structural motifs responsible for its binding to various lipids.6,7 Sequence analysis of full-length apoB-100 by Segrest and colleagues8−10 led to the currently accepted pentapartite structure, NH2-βα1-β1-α2-β2-α3-COOH. The N-terminal βα1 domain of apoB-100, i.e., the first 1000 amino acid residues of the mature protein, is a mixture of amphipathic β strands and amphipathic α helices.9,10 The β1, α2, β2, and α3 domains encompass residues 1001−2000 ± 25, residues 2075 ± 25 to © XXXX American Chemical Society

Received: November 18, 2016 Revised: July 12, 2017 Published: July 13, 2017 A

DOI: 10.1021/acs.biochem.6b01174 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry properties of truncated forms of apoB18−22 and the potential role of MTP in their formation.23−26 However, systematic studies to establish the structure−function relationship of apoB, based on analysis of the properties of sequences of the protein, are sparse. Our previous comprehensive studies in stable transformants of McA-RH7777 cells expressing logically designed truncated forms of human apoB-10027−30 provided support for the “lipid pocket” hypothesis. Collectively, results demonstrated the following. (i) All 1000 amino acid residues of the βα1 domain of apoB-100 (here termed apoB:1000) are necessary for the completion of the lipid pocket and initiation of apoBcontaining lipoprotein assembly. (ii) Complementary charged residues 717−720 and 997−1000 play key roles in the formation of the lipid pocket, perhaps via a hairpin-bridge mechanism. (iii) ApoB:1000 is secreted as a monodisperse particle, with HDL3 density. (iv) ApoB:1000-containing particles are rich in phospholipid (PL). (v) In marked contrast to apoB:1000, apoB:1200, containing the βα1 domain and 200 residues of the β1 domain, is secreted primarily as a lipid-poor particle. We also provided strong evidence that the early addition of PL to apoB:1000 leading to the formation of the primordial apoB-containing lipoprotein particle occurs in a manner independent of MTP activity.31,32 The results described above raised two questions. (i) What are the effects on the properties of truncated apoB undergoing sequential inclusion of the amphipathic β strands in the 700 Nterminal residues of the β1 domain? (ii) What is the minimum structural domain in the β1 domain that is required for the formation of a MTP-dependent TAG-rich apoB-containing particle? To this end, we made sequential truncated constructs between apoB:1700 (the βα1 domain and the 700 N-terminal residues of the β1 domain) and apoB:1000 to produce apoB:1050, apoB:1100, apoB:1150, apoB:1200, apoB:1300, apoB:1400, apoB:1500, and apoB:1700 and stably expressed them in McA-RH7777 cells. Analyses of the secreted particles indicated that not all amphipathic β strands in the 700 Nterminal residues of the β1 domain have the same effect on the stability and lipid binding specificity of the truncated forms of apoB. Results demonstrated that the MTP-dependent assembly and secretion of the TAG-rich apoB-containing lipoprotein particle require an apoB size no smaller than apoB:1700.

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a gift from D. Gordon and J. R. Wetterau (Bristol-Myers Squibb Co.). Construction of the Truncated ApoB Expression Plasmid. The cDNA for truncated forms of human apoB100, including apoB:1000, apoB:1050, apoB:1100, apoB:1150, apoB:1200, apoB:1300, apoB:1400, apoB:1500, and apoB:1700, was prepared from pB100L-L33 as a polymerase chain reaction template and appropriate primers as previously described in detail.27 Because of the nature of our studies, only clones with 100% correct sequence, identified by standard cloning procedures, were used. The apoB fragments were ligated into the mammalian expression vector, the Molony murine leukemia virus-based retrovirus LNCX,34 and were used for transformation. Clones containing the apoB gene with the correct orientation were identified by restriction enzyme digestion and confirmed by nucleotide sequencing. Cell Culture and Transfection. Rat hepatoma McARH7777 (henceforth termed McA-RH) cells were obtained from American Type Culture Collection (Rockville, MD). Clonal stable transformants of McA-RH cells expressing the truncated forms of apoB, i.e., apoB:1000, apoB:1050, apoB:1100, apoB:1150, apoB:1200, apoB:1300, apoB:1400, apoB:1500, and apoB:1700, denoting N-terminal amino acid residues 1−1000, 1−1050, 1−1100, 1−1150, 1−1200, 1−1300, 1−1400, 1−1500, and 1−1700 of the mature protein, respectively, were developed as previously described.27 Cells were routinely grown in DMEM containing 20% HS, 5% FBS, and 0.2 mg/mL G418. All experiments were conducted with 3−4-day-old cells as previously described.27 De Novo Synthesis and Secretion of ApoB-Containing Particles. Clonal stable transformants of McA-RH cells expressing the truncated forms of apoB were grown in sixwell dishes. At the start of each experiment, the maintenance medium was removed, cells were washed with phosphatebuffered saline (PBS), serum-, methionine-, and cysteine-free DMEM containing [35S]Met/Cys (70 μCi/mL of medium) was added, and cells were incubated for 3.5 h or overnight as indicated for each experiment.27 The 35S-labeled conditioned medium was collected, and a preservative mixture27 was added to prevent oxidative and proteolytic damage. The cell monolayer was washed with PBS; lysis buffer containing a preservative cocktail27 was added, and cells were processed as previously described.27 The 35S-labeled apoB in the medium and cell lysate was isolated by immunoprecipitation using the polyclonal antibody to human apoB-100, which has negligible cross-reactivity with intrinsic rat apoB. Labeled proteins were resolved by 4 to 12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE)35 or 4 to 20% nondenaturing polyacrylamide gel electrophoresis (NDGGE). After electrophoresis, proteins were detected by Western blot analysis36 using the biotinylated antibody to human apoB-100 as previously described27 or by autoradiography as described in the figure legends. To determine the lipid component of secreted particles, cells were incubated in serum-free DMEM containing 3H-labeled glycerol (7 μCi/mL of medium) and either 0.75% BSA (control) or 0.4 mM oleic acid bound to 0.75% BSA. After overnight (17−20 h) incubation, labeled conditioned medium was collected and processed as described above. The secreted 3 H-labeled apoB-containing particles were isolated by NDGGE or by immunoprecipitation under nondenaturing conditions as previously described.28,30 Lipids were extracted with chloroform/methanol and processed using to the Folch method.37

MATERIALS AND METHODS

Materials. Reagents for cell culture, including Dulbecco’s modified Eagle’s medium (DMEM), trypsin, and G418, were purchased from Mediatech, Inc. (Herndon, VA). Fetal bovine serum (FBS), horse serum (HS), antibiotic-antimycotic, and Tris-glycine gels were obtained from Invitrogen-Novex (Carlsbad, CA). All reagents for standard techniques were from Sigma Chemical Co. (St. Louis, MO). Protein GSepharose CL-4B and Amplify were from Amersham Pharmacia Biotech (Piscataway, NJ). The Immobilon PVDF transfer membrane and Centriprep Centrifugal Filter Devices (YM-30) were purchased from Millipore Corp. (Bedford, MA). TRAN35S-LABLE [35S]methionine/cysteine ([35S]Met/Cys) and 3H-labeled glycerol were from MP Biomedicals, Inc. (Irvine, CA). The affinity-purified polyclonal antibody to human apoB-100 was prepared in our laboratory and was biotinylated at Brookwood Biomedical (Birmingham, AL). The apoB-100 cDNA was a gift from Z. Yao (University of Ottawa Heart Institute, Ottawa, ON). MTP inhibitor BMS-197636 was B

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Biochemistry Extracted lipids were applied to a thin layer chromatography (TLC) plate, and bands corresponding to PL, diacylglycerols (DAG), and TAG were isolated and analyzed as previously described.28,30,38 The cell monolayer was used to determine the protein content by the method of Lowry et al.39 Lipoprotein Isolation and Density Gradient Ultracentrifugation. Cells were incubated with unlabeled serumfree DMEM containing either 0.75% BSA or 0.4 mM oleic acid bound to 0.75% BSA. After overnight (17−20 h) incubation, conditioned medium was collected and processed as described above. The density was adjusted to 1.23 g/mL, and lipoproteins (d < 1.23 g/mL) were isolated by centrifugation for 45 h at 50000 rpm. The lipoprotein fractions and infranatants (d > 1.23 g/mL) were dialyzed against PBS and concentrated using Centriprep YM-30. The concentrated conditioned medium and lipoprotein fractions were analyzed by NDGGE and immunoblotting as described above. In density gradient ultracentrifugation experiments, conditioned medium from ten 100 mm dishes was concentrated 10-fold and processed according to the method of Chung et al.40 as previously described in detail.28,30 After centrifugation, 40 fractions of 1.0 mL each were collected from the bottom of the centrifuge tube and, after their densities had been measured, were analyzed by NDGGE and immunoblotting. Calculation of the Number of Lipid Molecules per Particle. Calculations were performed essentially as described by Carraway et al.18 and previously reported.28

Figure 1. Floatation properties of the secreted apoB-containing particles containing apoB:1000 through apoB:1200. Stable transformants of McA-RH cells were incubated with serum-free DMEM containing 0.75% BSA (control) or 0.4 mM oleic acid bound to 0.75% BSA (oleate) for 20 h. Lipoprotein (d < 1.23 g/mL) and infranatant (d > 1.23 g/mL) fractions were isolated and processed as described in Materials and Methods. Conditioned medium (lane 1), lipoprotein fractions (lane 2), and infranatant (lane 3) were analyzed by 4 to 20% NDGGE and immunoblotting using the polyclonal antibody to human apoB100 (A−E). In panel F, cells were incubated with [3H]glycerol and [14C]oleic acid (0.4 mM bound to 0.75% BSA). Aliquots of concentrated conditioned medium were subjected to 4 to 20% NDGGE and analyzed for labeled lipids in apoB-containing lipoproteins by autoradiography. Lipid-rich particles are labeled as “p”. Results are representative of four separate experiments in panels A−E and three experiments in panel F.

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RESULTS Effects of Short Sequences in the 200 N-Terminal Residues of the β1 Domain of ApoB-100 on the Floatation Properties and Density Distribution of Secreted ApoB-Containing Particles. Our previous studies27,28 demonstrated that within the fragment of 200 residues between apoB:1000 and apoB:1200, the particles change from being relatively lipid-rich and stable to being lipid-poor and collapsed. To identify the putative region that causes this change, the properties of the secreted particles containing apoB:1000, apoB:1050, apoB:1100, apoB:1150, and apoB:1200 were assessed. Consistent with our previous study,30 apoB:1000 was secreted as a monodisperse particle (Figure 1A, lane 1); a significant proportion of these particles floated at d = 1.23 g/ mL (Figure 1A, lane 2), indicating that they are relatively rich in lipids. ApoB:1050 containing only 50 residues of the β1 domain formed two distinct particle species (Figure 1B, lane 1); a major proportion of the large particles floated at d = 1.23 g/ mL (Figure 1B, lane 2), indicating that they are relatively rich in lipids. ApoB:1100 formed one small and several large particles (Figure 1C, lane 1); only the large particles were recovered in the d < 1.23 g/mL lipoprotein fraction (Figure 1C, lane 2). ApoB:1150 was distributed almost equally between the large and small particles (Figure 1D, lane 1); a significant proportion of the large particles were recovered in the d < 1.23 g/mL fraction (Figure 1D, lane 2). In contrast to the truncated apoB described above, the small lipid-poor particles were the major form of apoB:1200 (Figure 1E, lane 1). Only a minor fraction of apoB:1200 formed large lipidated particles that floated at d = 1.23 g/mL (Figure 1E, lane 2). The small particles formed by all truncated forms of apoB were recovered entirely in the d > 1.23 g/mL fraction (Figure 1B−E, lane 3), indicating that they are lipid-poor. Autoradiogram analysis of 3H- and 14C-labeled particles confirmed that only the large forms of apoB:1050, apoB:1100, apoB:1150, and apoB:1200 contained a significant

amount of lipids (Figure 1F). These results suggest that addition of amino acid residues 1150−1200 might cause a conformational change in the protein, hindering its lipidation. Consistent with our previous study,30 oleic acid, which increases the TAG content of apoB-containing particles (hence the size of the particles), had no effect on the floatation property of apoB:1000-containing particles (Figure 1A), indicating that they do not undergo core expansion. Similarly, oleic acid did not have a major effect on the floatation properties of apoB:1050 (Figure 1B) or apoB:1200 (Figure 1E). Oleic acid caused a moderate increase in the relative proportion of the apoB:1100- and apoB:1150-containing particles that floated at d = 1.23 g/mL particles (Figure 1C,D), suggesting a small increase in the TAG content of these particles. Thus, particles containing apoB:1050 through apoB:1200 did not have the capacity to recruit TAG. We previously demonstrated30 that apoB:1000 formed predominantly monodisperse lipidated particles with a peak hydrated density of 1.208 g/mL, which is within the HDL3 density range. In this study, we assessed the effect of sequential addition of local sequences in the first 200 residues of the β1 domain on the density gradient distribution of the particles containing the truncated apoB. Results shown in Figure 2 demonstrate that the large particles formed by apoB:1050, apoB:1100, apoB:1150, and apoB:1200 had a peak hydrated density of 1.197 g/mL; the smaller particle had a similar peak hydrated density ranging from 1.23 to 1.25 g/mL. Interestingly, both the large and small forms of these particles appeared to have a progressively wider density range, and this was more striking with small apoB:1200-containing particles (Figure 2). These data confirmed the results of the floatation experiments (Figure 1) and in addition provided information about the C

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Figure 3. Intracellular stability of apoB:1050, apoB:1100, apoB:1150, and apoB:1200. McA-RH cells expressing the truncated forms of apoB were pulse-labeled for 10 min in serum-, methionine-, and cysteinefree DMEM containing [35S]Met/Cys (100 μCi/mL of medium), and the labeled proteins were chased for the indicated time interval. 35Slabeled truncated forms of apoB were isolated by immunoprecipitation with the polyclonal antibody to human apoB-100; proteins were resolved by 4 to 12% SDS−PAGE and visualized by autoradiography. The intensities of the labeled proteins were determined by computerassisted image processing. The percent recovery of the labeled protein at 180 min chase was calculated by dividing the total labeled apoB (cells and medium) at the 180 min chase time by the total at the 20 min chase time, when apoB appears in the medium.

Figure 2. Density gradient ultracentrifugation distribution of particles containing apoB:1000 and the 200 N-terminal residues of the β1 domain of apoB-100 secreted by McA-RH cells. Cells were incubated with serum-free DMEM for 20 h. The conditioned medium was prepared for gradient ultracentrifugation as previously described40 and outlined in Materials and Methods. Forty fractions were collected and analyzed by NDGGE and immunoblotting with the polyclonal antibody to human apoB-100. Results are representative of three separate experiments.

density distribution and peak hydrated density of the individual apoB-containing particles. ApoB:1050, ApoB:1100, ApoB:1150, and ApoB:1200 Are Not Susceptible to Intracellular Degradation. Studies by Ginsberg and co-workers41,42 have suggested that the initial appearance of a portion of the lipid-binding β1 sheet domain of apoB-100 increases its susceptibility to proteasomal degradation. Therefore, we assessed the potential intracellular degradation of apoB, the predominant regulatory mechanism for apoB production,3,43 by pulse−chase experiments. Results demonstrated that, similar to apoB:1000,30 apoB:1050, apoB:1100, apoB:1150, and apoB:1200 appeared to be stable because 85−95% of the 35S-labeled apoB that was lost from the cells during the chase period was recovered in the medium (Figure 3). These results indicate that the amphipathic β strands in the 200 N-terminal residues of the β1 domain of apoB do not render the protein susceptible to intracellular degradation. Results also rule out the possibility that the small lipid-poor particles might have been formed by fragments of apoB. Lipid Composition of Secreted Particles Containing Truncated Forms of ApoB. We previously demonstrated that the [3H]glycerol-labeled apoB:1000-containing particles secreted in the absence of oleic acid and isolated by NDGGE were PL-rich and contained 71−74% PL, 8% DAG, and 18− 21% TAG.28 The calculated numbers of PL, DAG, and TAG molecules per apoB:1000 particle were 50, 7, and 12, respectively; the surface:core lipid ratio was approximately 4:1, and oleic acid had no effect on this composition.30 Here, we assessed the potential effects of the first 200 residues of the β1 domain on the lipid composition of the secreted particles. Results listed in Table 1 demonstrated that in the absence of oleic acid, the lipid composition and stoichiometries of apoB:1050-, apoB:1100-, and apoB:1150-containing particles

were similar to those we reported for apoB:1000-containing particles;30 i.e., they were also PL-rich. These particles contained 60−66% PL, 12−15% DAG, and 19−26% TAG; the calculated numbers of PL, DAG, and TAG molecules per particle were 50−55, 11−15, and 14−20, respectivley, and their surface:core lipid ratios ranged from 3:1 to 4:1 (Table 1). The lipid composition and stoichiometry of apoB:1200-containing particles were markedly different from those containing shorter truncated forms of apoB. These particles contained 82% PL, 9% DAG, and 9% TAG; the calculated numbers of PL, DAG, and TAG molecules per particle were 80, 11, and 8, respectively, resulting in a surface:core lipid ratio of 11:1 (Table 1). We previously demonstrated28 that apoB:1000-containing particles possess a maximum capacity of 50 molecules of PL and 11 molecules of TAG per particle and that this composition was not altered by oleic acid. As shown in Table 1, oleic acid had no effect on the lipid composition of apoB:1050-containing particles but caused a moderate increase in the TAG content of particles containing apoB:1100 and to a greater extent apoB:1150 (Table 1). ApoB:1100 particles contained 52% PL, 19% DAG, and 30% TAG; the calculated numbers of PL, DAG, and TAG molecules per particle were 40, 18, and 21, respectively (Table 1). ApoB:1150 particles contained 51% PL, 11% DAG, and 38% TAG; the calculated numbers of PL, DAG, and TAG molecules per particle were 41, 11, and 27, respectively. The surface:core lipid ratios for apoB:1100- and apoB:1150-containing particles were 3:1 and 2:1, respectively (Table 1). AapoB:1200-containing particles remained PL-rich in the presence of oleic acid and had a surface:core lipid ratio of approximately 6:1 (Table 1). Thus, particles containing apoB:1050, apoB:1100, apoB:1150, and D

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Table 1. Composition of [3H]Glycerol-Labeled Lipids Associated with ApoB:1050-, ApoB:1100-, ApoB:1150-, and ApoB:1200Containing Particles Secreted by Stable Transformants of McA-RH Cells and Isolated by Nondenaturing Gradient Gel Electrophoresisa % lipid composition of particles apoB

addition

B:1050 (n = 14)

control oleate control oleate control oleate control oleate

B:1100 (n = 15) B:l 150 (n = 14) B:1200 (n = 20)

PL 66 69 66 52 60 51 82 73

± ± ± ± ± ± ± ±

DAG 2 2 2 2 2 3 1 2

12 7 15 I9 I3 11 9 I0

± ± ± ± ± ± ± ±

no. of lipid molecules per particle

TAG

1 1 1 1 1 1 1 1

23 24 19 30 26 38 9 I8

± ± ± ± ± ± ± ±

1 1 2 2 2 3 1 2

PL 51 54 55 40 50 41 80 68

± ± ± ± ± ± ± ±

DAG 2 2 2 2 2 3 2 3

11 7 15 18 14 11 11 11

± ± ± ± ± ± ± ±

1 1 1 1 1 1 1 1

TAG 16 18 14 21 20 27 8 14

± ± ± ± ± ± ± ±

1 1 1 1 2 2 1 1

total 79 79 84 78 84 79 99 94

± ± ± ± ± ± ± ±

1 1 1 1 1 2 1 1

a

Cells were incubated with serum-free DMEM with or without 0.4 mM oleic acid bound to 0.75% BSA, and [3H]glycerol-labeled secreted particles were isolated by NDGGE. Lipid identity was determined by thin layer chromatography. Values are means ± the standard error of the indicated number of samples from six to nine experiments.

apoB:1200 contained PL as their major lipid moiety, both in the presence and in the absence of oleic acid. Identification of the Amino Acid Residues in the β1 Domain of ApoB-100 That Markedly Enhance TAG Recruitment and the Formation of a Relatively TAGRich Particle. To identify specific sequences in the β1 domain of apoB-100 beyond residue 1200 that might enhance TAG recruitment, we produced four additional C-terminally truncated apoB constructs, including apoB:1300, apoB:1400, apoB:1500, and apoB:1700. Expression plasmids encoding apoB:1300 (apoB-29.00), apoB:1400 (apoB-30.86), apoB:1500 (apoB-33.07), and apoB:1700 (apoB-37.48) were used to generate clonal stable transformants of McA-RH cells as previously described.28 Despite numerous attempts, we were not successful in obtaining clonal stable McA-RH cells with high expression levels of apoB:1600 necessary for the planned studies. Analysis of the secreted particles demonstrated that apoB:1300 (Figure 4A) and apoB:1400 (Figure 4B) formed one major particle (Figure 4A,B, lane 1). A large proportion of apoB:1400 floated at d = 1.23 g/mL (Figure 4B, lane 2), indicating that they are relatively rich in lipids. ApoB:1500 formed one major larger particle and two minor larger particles (Figure 4C, lane 1), all of which were predominately recovered in the d < 1.23 g/mL fraction (Figure 4C, lane 2). Oleic acid supplementation of cells did not have any major effect on the floatation properties of the secreted apoB:1300-, apoB:1400-, and apoB:1500-containing particles (Figure 4A−C). The floatation properties of apoB:1700 were distinct from those of other shorter truncated forms of apoB shown in Figures 1 and 4. Although apoB:1700 also formed one major particle and two minor particles (Figure 4D, lane 1), unlike apoB:1500containing particles, they were all recovered entirely in the d < 1.23 g/mL fraction, both in the presence and in the absence of oleic acid (Figure 4D, lane 2), suggesting that they are lipidrich. The density gradient distribution of particles containing the larger truncated forms of apoB described above corroborated the results of floatation experiments. As shown in Figure 5, apoB:1300-, apoB:1400-, and apoB:1500-containing particles exhibited a wide range of hydrated density from 1.15 to 1.23 g/ mL. ApoB:1300- and apoB:1400-containing particles had a peak hydrated density of 1.197 g/mL, which is similar to that of the shorter forms of apoB shown in Figure 2. ApoB:1500containing particles had a slightly lower peak hydrated density

Figure 4. Floatation properties of the secreted apoB:1300-, apoB:1400-, apoB:1500-, and apoB:1700-containing particles. McARH cells expressing the truncated forms of apoB were incubated with or without oleic acid, and lipoprotein (d < 1.23 g/mL) and infranatant (d > 1.23 g/mL) fractions were isolated from the conditioned medium as described in the legend of Figure 1. The concentrated conditioned medium (lane 1), lipoprotein fraction (lane 2), and infranatant (lane 3) were analyzed by 4 to 20% NDGGE and immunoblotting using the polyclonal antibody to human apoB-100. Lipid-rich particles are labeled as “p”. Results are representative of four separate experiments.

of 1.176 g/mL (Figure 5). Oleic acid did not alter the peak hydrated densities of particles containing apoB:1300, apoB:1400, and apoB:1500 (Figure 5). ApoB:1700-containing particles were recovered in a narrow density range of 1.14−1.18 g/mL and had a peak hydrated density of 1.153 g/mL (Figure 5). In contrast to all other shorter truncated forms of apoB, oleic acid shifted the hydrated density of apoB:1700-containing particles to a lower range of 1.12−1.17 g/mL and a peak hydrated density of 1.142 g/mL (Figure 5). Identification of the Domain in ApoB That Is Competent To Form TAG-Rich Particles That Are Responsive to the Addition of Oleic Acid. We next determined the composition and stoichiometry of the lipid component of the truncated apoB-containing particles shown in Figure 5. In the absence of oleic acid, the secreted apoB:1300containing particles were PL-rich and contained 68% PL, 10% DAG, and 22% TAG (Table 2). The calculated numbers of PL, DAG, and TAG molecules per apoB:1300 particle were 69, 12, and 20, respectively; the surface:core lipid ratio was 4:1 (Table E

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surface:core lipid ratios of 1.67:1, 1.50:1, and 1.04:1, respectively (Table 2). However, despite moderate differences in the PL:TAG ratios, the total number of lipid molecules per particle remained relatively constant for all three particles (Table 2). The lipid composition and stoichiometry of apoB:1700containing particles were distinct from those observed for the particles containing smaller truncated apoB. First, in the absence of oleic acid, the secreted apoB:1700 particles contained equal levels (47%) of PL and TAG (Table 2). The calculated numbers of PL, DAG, and TAG molecules per apoB:1700 particle were 89, 15, and 80, respectively, and their surface:core lipid ratio was 1.3:1.0 (Table 2). Second, oleic acid markedly increased the TAG content of the secreted apoB:1700 particles. These particles contained 37% PL, 7% DAG, and 56% TAG; the calculated numbers of PL, DAG, and TAG molecules per particle were 64, 15, and 90, respectively, and hence, the surface:core lipid ratio was 0.9:1.0 (Table 2). Third, there was a marked increase in the total number of lipid molecules per apoB:1700-containing particle, when compared to those of the other three particles (Table 2). Notably, apoB:1700-containing particles had nearly double the lipid loading capacity of other particles containing shorter apoB truncates. These results suggest that apoB:1700 might mark the threshold for the acquisition of TAG and, hence, the capacity for particle core expansion. A schematic diagram of changes in the composition of large particles formed by truncated apoB undergoing sequential inclusion of the amphipathic β strands in the 700 N-terminal residues of the β1 domain of apoB-100 is shown in Figure 6. MTP Activity Is Required for the Secretion of ApoB:1700 but Not for Shorter Truncated Forms of ApoB. One of the most important factors in the assembly and secretion of apoB-containing lipoproteins is MTP.44 We previously demonstrated that the initial addition of PL to apoB:1000 and initiation of apoB particle assembly occur in a manner independent of MTP activity.31,32 In this study, the putative role of MTP in the secretion of larger apoB-containing particles was assessed by testing the effect of BMS-197636, a potent inhibitor of MTP activity,45,46 on the secretion of 35Slabeled truncated forms of apoB. The secretion of human and rat endogenous apoB-100 in HepG2 and wild-type McA-RH cells, respectively, served as controls. As expected, BMS-197636 caused a drastic reduction in the level of both the cellular and secreted endogenous apoB-100 in HepG2 and McA-RH cells

Figure 5. Density gradient ultracentrifugation distribution of apoB:1300-, apoB:1400-, apoB:1500-, and apoB:1700-containing particles secreted by McA-RH cells. Cells expressing the indicated truncated forms of apoB were incubated with serum-free DMEM with or without oleic acid as described in the legend of Figure 1. The conditioned medium was prepared for density gradient ultracentrifugation as previously described40 and in Materials and Methods. Forty fractions were collected and, after their densities had been measured, were analyzed by NDGGE and immunoblotting with the polyclonal antibody to human apoB-100. Results are representative of three separate experiments.

2). The lipid composition and stoichiometry of apoB:1400containing particles were similar to those of apoB:1300containing particles with the exception of a slightly higher content of TAG (30%), a smaller amount of PL (63%), and, hence, a lower surface:core lipid ratio of approximately 3:1 (Table 2). There was a further increase in the TAG content of apoB:1500-containing lipoproteins; these particles contained 53% PL, 6% DAG, and 41% TAG (Table 2). The calculated numbers of PL, DAG, and TAG molecules per apoB:1500 particle were 61, 9, and 43, respectively; the surface:core lipid ratio was 1.6:1 (Table 2). Oleic acid supplementation of cells resulted in the secretion of apoB:1300-, apoB:1400-, and apoB:1500-containing particles with a lower percent content of PL, a higher percent content of TAG, and, thus, lower

Table 2. Composition of [3H]Glycerol-Labeled Lipids Associated with ApoB:1300-, ApoB:1400-, ApoB:1500-, and ApoB:1700Containing Particles Secreted by Stable Transformants of McA-RH Cells and Isolated by Immunoprecipitationa % lipid composition of particles apoB

addition

B:1300 (n = 12)

control oleate control oleate control oleate control oleate

B:1400 (n = 16) B:1500 (n = 12) B:1700 (n = 12)

PL 68 54 63 48 53 43 47 37

± ± ± ± ± ± ± ±

DAG 3 1 3 1 2 3 2 3

10 7 7 8 6 6 6 7

± ± ± ± ± ± ± ±

no. of lipid molecules per particle

TAG

1 1 1 1 1 1 1 1

22 40 30 44 41 52 47 56

± ± ± ± ± ± ± ±

2 1 3 3 2 3 2 3

PL 69 48 63 44 61 41 89 64

± ± ± ± ± ± ± ±

DAG 5 2 8 4 4 3 5 6

12 7 9 10 9 7 15 15

± ± ± ± ± ± ± ±

1 1 1 2 1 1 2 3

TAG 20 33 27 37 43 46 80 90

± ± ± ± ± ± ± ±

2 1 4 2 3 3 3 5

total 102 89 99 91 113 94 184 169

± ± ± ± ± ± ± ±

3 2 4 2 6 4 3 3

a Cells were incubated in serum-free DMEM with or without 0.4 mM oleic acid bound to 0.75% BSA and 3H-labeled glycerol. The secreted labeled particles were isolated by immunoprecipitation using the polyclonal antibody to human apoB-100. Lipid identity was determined by thin layer chromatography. Values are means ± the standard error of the indicated number of samples from five experiments.

F

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Assessment of the Intracellular Stability of ApoB:1300, ApoB:1400, ApoB:1500, and ApoB:1700. To determine if amphipathic β strands between apoB:1200 and apoB:1700 render the proteins susceptible to intracellular degradation, we performed pulse−chase studies. As shown in Figure 8, only apoB:1500 appeared to be stable as all the 35SFigure 6. Schematic diagram of the composition of particles containing truncated forms of apoB. The composition of particles containing truncated forms of apoB was calculated using the results for the large particles shown in Tables 1 and 2. Data, expressed as the percent of the total mass of the particle, were calculated using the molecular weight of the truncated apoB and the number of molecules of each lipid per particle multiplied by the average molecular weights of PL, DAG, and TAG.

(Figure 7). The synthesis and secretion of PL-rich apoB:1000, apoB:1150, apoB:1300, and apoB:1400 were independent of

Figure 8. Variable intracellular stability of apoB:1300, apoB:1400, apoB:1500, and apoB:1700. McA-RH cells expressing the indicated truncated forms of apoB were pulse-labeled for 10 min with serum-, methionine-, and cysteine-free DMEM containing [35S]Met/Cys (100 μCi/mL of medium) and either 0.75% BSA or 0.4 mM oleic acid bound to 0.75% BSA. During the indicated chase period, medium and cell lysate were collected and 35S-labeled truncate apoB fragments were isolated by immunoprecipitation with the polyclonal antibody to human apoB-100, resolved by 4 to 12% SDS−PAGE, and visualized by autoradiography. The intensities of the labeled proteins were determined by computer-assisted image processing. The autoradiogram is representative of three or four separate experiments.

labeled protein that was lost from the cells during the 180 min chase was recovered in the medium. In contrast, only 30% of apoB:1300, 65% of apoB:1400, and 40% of apoB:1700 that were lost from the cells during the chase were recovered in the medium (Figure 8), demonstrating their intracellular degradation. Oleic acid, which is known to protect apoB-100 from intracellular degradation,3−5,43,47 increased the level of recovery of apoB:1300, apoB:1400, and apoB:1700 to 52, 75, and 65%, respectively, partially rescuing them from intracellular degradation (Figure 8). These results, together with those shown in Figure 3, suggest that not all the amphipathic β strands in the β1 domain of apoB-100 have the same effect on the intracellular stability of apoB.

Figure 7. Inhibition of MTP lipid transfer activity markedly decreases the level of synthesis and secretion of apoB:1700. McA-RH cells expressing the indicated truncated forms of apoB, HepG2 cells, and parental McA-RH cells secreting endogenous human and rat apoB were incubated for 4 h with serum-, methionine-, and cysteine-free DMEM containing [35S]Met/Cys (100 μCi/mL of medium) in the presence or absence of 0.1 μM BMS-197636. The 35S-labeled apoB in the medium and cell lysate were immunoprecipitated with the antibody to human apoB-100 (hApoB-100) in McA-RH cells expressing truncated forms of apoB and HepG2 cells or the antibody to rat apoB-100 (rApoB-100) in parental untransfected McA-RH cells. Proteins were separated by 4 to 12% SDS−PAGE and visualized by autoradiography. The autoradiogram is representative of three separate experiments.

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DISCUSSION In this study, we have shown that, consistent with our previous work,30 apoB:1000 (B-22.05) is secreted as a monodisperse, relatively lipid-rich particle with HDL3-like hydrated density. ApoB:1050 containing the 50 N-terminal residues of the β1 domain formed two distinct particles, a predominant large lipidcontaining particle and a small lipid-poor particle. The loss of the ability to assemble monodisperse lipidated particles persisted for apoB:1100, apoB:1150, and apoB:1200. This observation suggests that the addition of amphipathic β strands in the 200 N-terminal residues of the β1 domain to the growing peptide may induce instability in particle assembly, so that only a portion of the truncated protein (reaching the minimum in apoB:1200) is secreted in the form of a lipid-containing particle. A plausible explanation for the bimorphic nature of the truncated forms of apoB mentioned above is that the presence

MTP activity (Figure 7). The modest decrease in the level of secretion of apoB:1500 by BMS-197636 was concurrent with its increased level of cellular accumulation, resulting in its unchanged net synthesis and secretion (Figure 7). In contrast, BMS-197636 inhibited the secretion and cellular accumulation of 35S-labeled apoB:1700 by 57 and 14%, respectively, resulting in a 50% reduction in the net level of synthesis and secretion of the protein (Figure 7). These results, together with those shown in Table 2, suggest that MTP is involved in the secretion of TAG-rich apoB-containing lipoproteins. G

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Biochemistry of amphipathic β strands may alter the conformation of the proteins, resulting in their release from the ER membrane before significant lipidation occurs in a fraction of the particles. This possible mechanism is supported by studies by Ginsberg and co-workers41,42 suggesting that the initial appearance of a portion of the lipid-binding β1 sheet domain of apoB-100 hinders the translocation efficiency of the protein. The lipid composition, the stoichiometry, and the total number of lipid molecules per particle of the larger particles formed by apoB:1050, apoB:1100, apoB:1150, and apoB:1200 were similar to those of apoB:1000-containing particles;28,30 i.e., they were all PL-rich and not significantly altered by oleic acid. These results provided compelling evidence that the 200 Nterminal residues of the β1 domain do not have the structural elements necessary for TAG recruitment. Despite numerous studies examining the effects of apoB truncation on its lipid affinity, the structural motifs within the apoB polypeptide that specify TAG recruitment are not fully resolved. Because many domains throughout the entire length of apoB appear to bind lipids,8,9 truncated apoB is expected to form denser particles with a lower lipid content. Accumulated evidence suggests that the size and lipid content of the secreted particles are proportional to the length of the truncated apoB.18,20−22 To identify the motif in the β1 domain of apoB100 beyond residue 1200 that might initiate TAG recruitment for VLDL formation, we assessed the properties of apoB:1300 (apoB-28.67), apoB:1400 (apoB-30.86), apoB:1500 (apoB33.07), and apoB:1700 (apoB-37.48). We found that the inclusion of the 200−700 N-terminal residues of the β1 domain abolished the formation of the small lipid-poor particles observed for apoB:1050 through apoB:1200. In addition, with the exception of apoB:1500, all these larger forms of apoB were susceptible to intracellular degradation and were only partially rescued by oleic acid supplementation. The lipid compositions of apoB:1300- and apoB:1400-containing particles were similar to those formed by shorter apoB truncates; i.e., they were PLrich. Although apoB:1500-containing particles had a higher content of TAG and a lower surface:core lipid ratio than the particles containing apoB:1300 and apoB:1400 did, the apoB:1500-containing particles still contained PL as their major lipid component. Collectively, these results indicate that small motifs in the 500 N-terminal residues of the β1 domain of apoB-100 differentially impact the properties of the host lipoprotein particle, perhaps by inducing transient conformational changes in the protein. More importantly, our findings provide compelling evidence that this domain of apoB does not contain structural elements necessary for marked TAG recruitment. The properties of apoB:1700-containing particles were markedly different from those of particles containing apoB:1050 through apoB:1500 as described below. In contrast to apoB:1500, apoB:1700 was degraded intracellularly by 56% and was only partially rescued by oleic acid. This observation is similar to that reported for intrinsic apoB-100 in HepG2 cells,48 primary rat hepatocytes,49 and McA-RH cells.20 ApoB:1700containing particles were recovered entirely in the d < 1.23 g/ mL fraction and had a lower peak hydrated density of 1.157 g/ mL that was further decreased in response to oleic acid supplementation of cells. There were striking differences in the lipid composition, stoichiometry, and lipid loading capacity of apoB:1700-containing particles, when compared with the values of those containing shorter apoB truncates. Whereas all other particles were rich in PL and had similar lipid loading capacities,

apoB:1700-containing particles had an equal percentage of PL and TAG, a similar number of PL and TAG molecules per particle, a lower surface:core lipid ratio of 1.3:1, and a marked 67% higher number of total lipid molecules per particle. Importantly, the lipid composition and stoichiometry of apoB:1700-containing particles were responsive to oleic acid supplementation of cells. These results indicate that apoB:1700 has the structural elements necessary for TAG recruitment and might mark the threshold for the formation of TAG-rich particles. Two models for the physical assembly of apoB with lipids have been suggested. The first model is the budding oil droplet mechanism.1 In this model, the N-terminal segment of apoB inserts into the inner monolayer of the ER membrane where it desorbs lipids and forms an oil droplet. When apoB synthesis is completed, this oil droplet is released from the ER membrane to form the nascent lipoprotein. Small and co-workers18 have proposed an alternative model and have suggested that the acquisition of lipids by apoB and the formation of the lipoprotein particle make up a gradual process that involves the initial recruitment of PL by the N-terminal region of apoB followed by incorporation of core lipids directed by sequences in the β1 domain at or beyond apoB-29.18 Studies by Shelness et al.50 in nonhepatic COS cells showing that particles formed by the 912 N-terminal amino acid residues of apoB are TAGrich and have a surface:core lipid ratio of ≤1:1 are consistent with the budding oil droplet model.1 Our data demonstrating that particles containing apoB:1000 (apoB-22) through apoB:1400 (apoB-30) secreted by liver-derived McA-RH cells are all rich in PL and have a surface:core lipid ratio between 3:1 and 4:1 are consistent with the model proposed by Small and co-workers18 and further support our conclusion that these truncated forms of apoB do not contain the structural elements required for recruitment of bulk TAG. Our findings provide strong evidence that the formation of a TAG-rich apoBcontaining particle with a surface:core lipid ratio of 1:1 requires sequences between amino acid residues 1500 and 1700 in the β1 domain of apoB-100. Numerous studies have established the obligatory role of MTP in the secretion of VLDL;16,44 however, its relative importance in the two steps of VLDL assembly remains controversial. The requirement for MTP in both the first step, assembly of VLDL,24,46,51,52 and second step, bulk TAG transfer and particle core expansion,53,54 has been reported. In two previous studies,31,32 using BMS-197636 or MTPknockdown McA-RH cells, we demonstrated that MTP activity is not required for the addition of PL to apoB:1000 and the initiation of apoB-containing lipoprotein assembly. We recently demonstrated that this step is, in part, mediated by phospholipid transfer protein (PLTP).55 In this study, we found that inhibition of MTP activity by BMS-197636 did not alter the net level of synthesis or secretion of particles containing apoB:1050 through apoB:1500 but resulted in a 50% reduction in the net level of synthesis and secretion of apoB:1700. On the basis of these results, we conclude that the secretion of particles containing apoB no larger than apoB:1500 (apoB-33) occurs in a manner independent of MTP activity and that only TAG-rich particles containing apoB no smaller than apoB:1700 require MTP for their assembly and secretion. We do not know the molecular basis for the observed effect of MTP on apoB:1700 but offer the following speculative mechanism. ApoB:1000 acquires PL mediated, in part, by PLTP, leading to the initiation of nascent apoB-containing H

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phoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; TAG, triacylglycerol; VLDL, very low-density lipoprotein.

particle assembly. Although apoB:1000 contains the binding sites for MTP,44 we have demonstrated that MTP lipid transfer activity is not required in this early stage of apoB particle assembly.31,32 Translation of multiple amphipathic β-sheets in the β1 domain of apoB-100 induces transient conformational changes allowing gradual acquisition of small increments of lipid molecules. When apoB reaches the critical size of no smaller than apo:1700, it attains competency to assemble substantial amounts of TAG. This step is mediated by MTP activity and results in the transfer of bulk TAG to the growing protein, leading to particle core expansion. In summary, our results suggest that addition of amphipathic β strands in the 200 N-terminal residues of the β1 domain of apoB-100 may induce conformational changes in apoB, so only a portion of the truncated protein is correctly folded for secretion as a relatively lipid-rich particle. We have demonstrated that particles ranging from apoB:1000 to apoB:1500 were all rich in PL, suggesting a small, bilayer-type organization, and that their secretion occurred in a manner independent of MTP lipid transfer activity. In marked contrast, apoB:1700containing particles were rich in TAG and had a surface:core lipid ratio of 1.3:1, indicating a spheroidal HDL3-type organization, and their secretion was dependent on MTP activity. We propose that apoB:1700 marks the threshold for MTP-dependent formation and secretion of a TAG-rich particle that is responsive to lipid supply. This study supports the concept that MTP participates in the assembly and secretion of apoB-containing lipoproteins at the stage at which particles undergo the transition from PL-rich to TAGrich and the idea that this step requires apoB fragments no smaller than apoB:1700.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Department of Medicine, The University of Alabama at Birmingham, 1808 7th Ave. S., BDB-D680, Birmingham, AL 35292-0012. Telephone: 205-975-2159. Fax: 205-975-8079. Email: [email protected]. ORCID

Nassrin Dashti: 0000-0001-9314-7439 Funding

This work was supported by National Institutes of Health Grant HL084685. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Zamin Yao (University of Ottawa Heart Institute) for the gift of apoB100 cDNA and Drs. David Gordon and J. R. Wetterau (Bristol-Myers Squibb Co.) for providing BMS-197636.

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ABBREVIATIONS apo, apolipoprotein; BSA, bovine serum albumin; CE, cholesteryl ester; DAG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; FBS, fetal bovine serum; hAPOB, human apolipoprotein B; HDL, high-density lipoprotein; HS, horse serum; KBr, potassium bromide; LDL, low-density lipoprotein; LV, lipovitellin; MTP, microsomal triglyceride transfer protein; NDGGE, nondenaturing gradient gel electrophoresis; PL, phospholipids; PAGE, polyacrylamide gel electroI

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DOI: 10.1021/acs.biochem.6b01174 Biochemistry XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.biochem.6b01174 Biochemistry XXXX, XXX, XXX−XXX