A Novel Nontruncating APOB Gene Mutation, R463W, Causes Familial Hypobetalipoproteinemia*

John R. BurnettDagger §, Jing Shan§||, Brooke A. Miskie**, Amanda J. WhitfieldDagger , Jane Yuan||DaggerDagger, Khai Tran||, C. James McKnight§§, Robert A. Hegele**¶¶, and Zemin Yao||||||

From the Dagger  Department of Core Clinical Pathology and Biochemistry, Royal Perth Hospital and Department of Pathology, University of Western Australia, Perth WA 6847, Western Australia, Australia, the || Lipoprotein and Atherosclerosis Group, Department of Pathology and Laboratory Medicine, and Department of Biochemistry, Microbiology and Immunology, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7, Canada, the ** Robarts Research Institute, London, Ontario N6A 5K8, Canada, and the §§ Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118-2526

Received for publication, January 9, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Familial hypobetalipoproteinemia (FHBL), an autosomal co-dominant disorder, is associated with reduced plasma concentrations (<5th percentile for age and sex) of apolipoprotein (apo) B and beta -migrating lipoproteins. To date, only mutations in APOB encoding prematurely truncated apoB have been found in FHBL. We discovered a novel APOB gene mutation, namely R463W, in an extended Christian Lebanese FHBL kindred. Heterozygotes for R463W had the typical FHBL phenotype, whereas homozygotes had barely detectable apoB-100. The effect of the R463W mutation on apoB secretion was examined using transfected McA-RH7777 cells that expressed one of two recombinant human apoBs, namely B48 and B17. In both cases, the mutant proteins (B48RW and B17RW) were retained within the endoplasmic reticulum and were secreted poorly compared with their wild-type counterparts. Pulse-chase analysis showed that secretion efficiencies of B48RW and B17RW were, respectively, 45 and 40% lower than those of the wild-types. Substitution of Arg463 with Ala in apoB-17 (B17RA) decreased secretion efficiency by ~50%, but substitution with Lys (B17RK) had no effect on secretion, indicating that the positive charge was important. Molecular modeling of apoB predicted that Arg463 was in close proximity to Glu756 and Asp456. Substitution of Glu756 with Gln (B17EQ) had no effect on secretion, but substitution of Asp456 with Asn (B17DN) decreased secretion to the same extent as B17RW. In co-transfection experiments, the mutant B17RW showed increased binding to microsomal triglyceride transfer protein as compared with wild-type B17. Thus, the naturally occurring R463W mutant reveals a key local domain governing assembly and secretion of apoB-containing lipoproteins.

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INTRODUCTION
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Apolipoprotein (apo)1 B is essential for the formation of triglyceride-rich lipoproteins, namely very low density lipoproteins (VLDL) and chylomicrons (1). In humans, the liver secretes full-length apoB-100 containing 4536 amino acids, whereas the intestine secretes apoB-48 consisting of the amino-terminal 48% of apoB-100 (2). Both forms of apoB are encoded by the APOB gene on chromosome 2, which spans 43 kb and contains 29 exons coding for a 14-kb mRNA (3, 4). ApoB-48 arises from a unique editing process in which cytosine at nucleotide position 6666 is converted to uracil, thereby generating an in-frame stop codon (5). The rat liver produces both apoB-100 and apoB-48, and both forms can assemble VLDL (6).

A pentapartite model for apoB-100 on low density lipoproteins (LDL) has been proposed, in which the apoB polypeptide can be divided into five structurally distinct domains, namely NH2-beta alpha 1-beta 1-alpha 2-beta 2-alpha 3-COOH (7). The amino acid sequence of the beta alpha 1 domain is homologous to lamprey lipovitellin and microsomal triglyceride transfer protein (MTP) (8, 9). The beta alpha 1 domain of human apoB has thus been modeled on the basis of the solved lipovitellin structure, in which 13 beta -strands (amino acids 21-263) form a beta  barrel, followed by a two-layered helical bundle consisting of 17 alpha -helices (amino acids 294-592). In vitro experiments suggest that the beta alpha 1 domain contains multiple MTP binding sites encompassing residues 430-570 (10), 512-721 (11), and probably 2-154 (8). The interaction between the apoB beta alpha 1 domain and MTP appears to be ionic; chemical modification of Lys and Arg residues within LDL or recombinant apoB-18 abolished interactions between apoB and MTP (12). It has been postulated that interaction of MTP and the beta alpha 1 domain of apoB creates a lipid pocket that facilitates lipid recruitment during lipoprotein assembly (9).

Familial hypobetalipoproteinemia (FHBL; MIM 107730), a genetically heterogeneous autosomal co-dominant disorder, is associated with reduced plasma concentrations (<5th percentile for age and sex) of apoB and beta -migrating lipoproteins, such as LDL and VLDL (13). Many nonsense and frameshift mutations in APOB leading to formation of prematurely truncated apoB forms have been reported in FHBL subjects, with predicted sizes ranging from apoB-2 to apoB-89 (13, 14). There is indirect evidence that molecular changes other than truncations with APOB can cause FHBL (15). Truncation variants shorter than apoB-31 are undetectable in plasma. Longer truncation variants typically occur at concentrations <5% of the normal allele product. In vivo apoB turnover studies in FHBL subjects with truncated apoB have shown increased fractional catabolic rates (16, 17), decreased production rates (18, 19), or both (20). Heterozygotes for FHBL are usually asymptomatic with LDL cholesterol and apoB-100 concentrations <50% of those in normal plasma. Homozygotes have extremely low plasma LDL cholesterol and apoB-100 concentrations, and clinical presentation may vary from no symptoms to severe gastrointestinal and neurological dysfunction, similar to abetalipoproteinemia (MIM 200100) (21), depending on the specific mutation (14). However, unlike FHBL, abetalipoproteinemia is an autosomal recessive disorder and is caused by defective MTP (21).

Analysis of naturally occurring truncations has been informative in identifying functional domains of apoB. In vivo studies in human FHBL subjects with carboxyl-terminal-truncated apoB variants (22) and in vitro expression studies of truncated forms of human apoB (23-25) have shown the importance of apoB length as a determinant of VLDL assembly and secretion. However, factors in addition to apoB length are involved, including adequate supply of lipid (26) and important amino acid sequences that reside within the amino-terminal 48% of the apoB-100 molecule (27). Here we report a novel nonsynonymous nontruncating APOB gene mutation, namely R463W found in an extended Christian Lebanese FHBL kindred, in which the mutation appears to impair the secretion of apoB and apoB-containing lipoproteins.

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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The R463W Kindred-- The proband (Fig. 1A, subject III:6) was referred to a lipid disorders clinic with marked hypocholesterolemia detected on a routine lipid screening (28). This kindred was extended from the original eight to a total of 36 members that included two homozygotes, 14 heterozygotes, and 20 unaffected subjects. Blood samples were obtained from each family member after an overnight fast. Blood (10 ml) was collected into a plain tube and a tube containing EDTA (1 mg/ml) to separate serum and plasma, respectively, by centrifugation at 2500 rpm for 15 min at 20 °C. The protease inhibitor aprotinin (100 kilounits/ml) was added to plasma.

Lipid, Lipoprotein, Apolipoprotein, and Biochemical Analyses-- Plasma total cholesterol, direct high density lipoprotein (HDL) cholesterol, triglyceride, direct LDL cholesterol, and serum liver enzymes, including alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1), and gamma -glutamyltransferase (EC 2.3.2.2) were measured enzymatically using Roche Diagnostics GmbH reagents on a Hitachi 917 analyzer. Plasma apoB-100 and apoA-I were measured using Dade Behring Marburg GmbH, Marburg, Germany, reagents on a Behring BN-II nephelometer. Serum alpha -tocopherol and retinol were determined by reverse-phase, high performance liquid chromatography using a C-18 column. Serum 25-dihydroxy vitamin D was determined using the INCSTAR/ DiaSorin radioimmunoassay (Stillwater, MN).

APOE Genotype Analysis-- Genomic DNA was extracted from peripheral blood leukocytes by a standard Triton X-100 procedure. Genotype analysis of APOE was performed by PCR amplification followed by digestion with the restriction enzyme HhaI as described (29).

ApoB Immunoblot Analysis-- Delipidated plasma samples were subjected to polyacrylamide gel electrophoresis (5% gel) containing 0.1% SDS (SDS-PAGE) and transferred to a nitrocellulose membrane (Bio-Rad) as described (30). The membrane was incubated with mAb 1D1 (a gift of Dr. R. Milne, University of Ottawa Heart Research Institute) that recognizes an epitope of apoB in amino acids 401-582.

DNA Sequence Analysis-- Genomic DNA was isolated from whole blood (Puregene, Gentra Systems, Minneapolis, MN). The entire APOB coding region, including the 5'-flanking region, all exons, and at least 100 base pairs of intronic sequence at each intron-exon boundary, were amplified using PCR. Amplification products were run on 1.5% agarose gels and purified using QIAEX II gel extraction kit (Qiagen Inc.). Purified DNA fragments were directly sequenced in two directions using the amplification primers. Sequences were detected on the 377 Prism DNA Sequencer (Applied Biosystems Inc., Mississauga, ON, Canada) and results were analyzed with Sequence Navigator software (Applied Biosystems). All sequence variants were detected on electropheretograms of sequencing reactions, and each was seen in both directions. The R463W mutation was confirmed by using a second, independent amplification of the affected genomic region from the proband and re-sequencing in both directions. Genotype analysis of family members and normolipidemic control subjects was performed by direct sequencing of exon 11 from genomic DNA.

Preparation of ApoB-48 and ApoB-17 Expression Plasmids-- The cDNA fragment that encompassed nucleotides 20-6666 of the human apoB cDNA was excised from the plasmid pRc/CMV-B48 (31) by digestion with NotI and MluI and inserted into pCMV5 expression plasmid (32). The resulting pB48wt was used as template to introduce R463W substitution using the QuikChangeTM mutagenesis kit (Stratagene Inc., La Jolla, CA) with mutagenesis primers shown in Table I. To prepare apoB-17 expression plasmids, the cDNA fragment encoding the NH2-terminal 782 amino acids of apoB followed by a stop codon was inserted into pCMV5 to generate pB17wt. The R463W, R463K, R463A, D456N, and E756Q substitutions were introduced by QuikChangeTM mutagenesis with the mutagenesis primers shown in Table I. The expression plasmids were purified by centrifugation twice in a CsCl gradient, and the APOB coding regions were authenticated by sequencing.


                              
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Table I
Oligonucleotides used for mutagenesis

Cell Culture and Transfection-- McA-RH7777 and COS-7 cells were obtained from the American Type Culture Collection and were cultured under conditions as described previously (33). Transfection of McA-RH7777 cells with B48 or B17 expression plasmids was achieved using the calcium phosphate precipitation method as described previously (34) or using the FuGENE 6 Transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. Transient expression of apoB-17 together with human MTP in COS-7 cells was performed as described previously (33). Expression of recombinant apoB and MTP was verified by immunoblotting using mAb 1D1 (32) and a polyclonal anti-MTP antibody, respectively. The anti-MTP antibody was a gift of Dr. C. Shoulders (Hammersmith Hospital, London, United Kingdom).

Metabolic Labeling-- Stably transfected cells were labeled with [35S]methionine/cysteine for 2 h in Dulbecco's modified Eagle's medium (DMEM) containing 20% serum and 0.4 mM oleate. At the end of labeling, the media were fractioned into VLDL1 (Sf > 100), VLDL2 (Sf 20-100), and other lipoproteins by cumulative rate flotation in a KBr density gradient (35). The 35S-apoB was immunoprecipitated and analyzed by SDS-PAGE (3-15% gradient gel) as described previously (27).

Pulse-Chase Analysis-- Stably transfected cells were pulse-labeled with [35S]methionine/cysteine for 1 h in methionine-free DMEM and chased up to 2 h in normal DMEM. Both pulse and chase media contained 20% serum and 0.4 mM oleate. The medium and cell-associated apoB (B48 and B17) or apoA-I were immunoprecipitated at different chase times, and were subjected to SDS-PAGE and visualized by fluorography. Radioactivity associated with 35S-apoB or 35S-apoA-I was quantified by scintillation counting. The recovery of medium and cell 35S-apoB or 35S-apoA-I during chase was presented as percent of initial counts that associated with cell apoB or apoA-I at the end of pulse.

Immunocytochemistry-- Indirect double immunofluorescence co-localization studies were performed as described previously (36) using transiently transfected cells. The mAb 1D1 was used to probe the recombinant human apoB with goat anti-mouse IgG Alexa Fluro 488 conjugate (Molecular Probes, number A-6440) as the secondary antibody. The endoplasmic reticulum (ER) and the Golgi apparatus were probed with anti-calnexin and anti-alpha -mannosidase II antibodies, respectively, with Alexa Fluro 594-conjugated anti-rabbit IgG (Molecular Probes, number R-6394) as the secondary antibody. The images were captured using the MRC-1024 laser scanning confocal imaging system (Bio-Rad).

Subcellular Fractionation-- Subcellular fractionation of intracellular microsomes was performed as described previously (36). In brief, two dishes of cells (100 mm) were homogenized using a ball-bearing homogenizer. Postnuclear supernatant was subjected to fractionation by centrifugation in a Nycodenz gradient (37, 38). After centrifugation, 15 fractions (0.8 ml each) were collected from the top of the tube, and an aliquot of each fraction (50 µl) was resolved by SDS-PAGE (3-15% gradient gel). The proteins were transferred onto nitrocellulose membranes, and probed with mAb 1D1, anti-Hsp47 antibody (for ER), anti-alpha -mannosidase II antibody (for cis/medial Golgi), and anti-TGN38 antibody (for distal Golgi), respectively. On the basis of marker protein localization determined by immunoblotting, fractions 1-3, 4-8, and 9-15 were designated distal Golgi, cis/medial Golgi, and ER, respectively. Polyclonal anti-alpha -mannosidase II was a gift of Dr. M. G. Farquhar (University of California, San Diego, CA). The mAb recognizing proteins containing the KDEL motif (Hsp47) and the anti-TGN38 antibody were purchased from StressGen and Affinity Bioreagents, respectively.

Lipid Assay-- Metabolic labeling of lipid using [3H]glycerol was performed as described (35).

Molecular Modeling-- The molecular model of B17 was created using the program MODELLER (39). Residues 19-766 of human apoB (40) were aligned with residues 18-758 of lamprey lipovitellin (41) with the assistance of the program BLAST (42). This alignment, along with the coordinates from the crystal structure of lamprey lipovitellin (43), was then used as input for MODELLER. Five disulfide bonds were constrained between cysteine residues in the apoB sequence during the modeling. The modeling did not include the region corresponding to residues 689-728 of lipovitellin (residues 676-737 of apoB) that is missing from the electron density in the crystal structure.

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The R463W Kindred-- The proband (III:6) and her daughter (IV:15) had a plasma total cholesterol of ~1 mM, with barely detectable LDL cholesterol and apoB-100. The presence of reduced plasma concentrations of apoB-containing lipoproteins in first-degree relatives confirmed the diagnosis of homozygous FHBL in the proband (Fig. 1A). The individual lipid, lipoprotein, and apolipoprotein values for the R463W kindred are shown in Table II. Heterozygotes (n = 14) had mean plasma total cholesterol, LDL cholesterol, and apoB concentrations of 2.60 mM, 0.93 mM, and 0.31 g/liter, respectively. Heterozygotes had a significant mean decrease in total cholesterol, LDL cholesterol, and apoB-100 concentrations of 44, 67, and 67%, respectively, as compared with unaffected family members (n = 20). No family member had the apoepsilon 2 isoform, ruling out this variant as a possible contributor to hypolipoproteinemia. Despite low lipid and lipoprotein concentrations of the affected subjects, none had any developmental problems, malabsorption, or neurological deficits. When compared with unaffected family members, heterozygotes had significant mean increases in the liver enzymes alanine aminotransferase, aspartate aminotransferase, and gamma -glutamyltransferase of 3.8-, 1.6-, and 3.0-fold, respectively (Table III). Consistent with these results, a 2.9-fold increase in serum ferritin was observed. Furthermore, serum ferritin was positively correlated with alanine aminotransferase (r = 0.76; p < 0.001). Serum alpha -tocopherol concentrations were the lowest in the homozygotes, intermediate in heterozygotes, and the highest in unaffected subjects. Serum alpha -tocopherol was positively correlated with both plasma total cholesterol (r = 0.87; p < 0.001) and apoB (r = 0.90; p < 0.001), reflecting the known relationship between vitamin E and plasma lipid concentrations. Serum retinol and 25-dihydroxy vitamin D levels did not differ between the subject groups.


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Fig. 1.   The R463W mutation within human apoB-100. A, pedigree of the RW kindred. The Roman numerals designate the generation numbers, whereas the individual subjects in each generation are identified by Arabic numerals. The arrow indicates the proband. The carrier status of the individual subjects is indicated in the key to the figure. Although not tested, subjects II:3, II:4, and II:5 are predicted to be obligate heterozygotes based on the autosomal co-dominant inheritance pattern. B, electropheretogram tracings of exon 11 sequence from genomic DNA templates in a normal subject, and a homozygote and heterozygote for APOB R463W. Single letter amino acid codes are shown in bold, along with codon numbers and nucleotide sequences. The position of the mutant nucleotide is indicated by an arrow for both homozygote and heterozygote tracings.


                              
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Table II
Plasma lipid and apoprotein concentrations, and APOE genotype in the R463W kindred


                              
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Table III
Serum fat-soluble vitamins, liver enzymes, and ferritin concentrations in the R463W kindred

Identification of R463W Mutation-- The above plasma lipid and apoB-100 data indicated a co-dominant inheritance in the FHBL kindred, which ruled out the possibility of abetalipoproteinemia. To determine whether or not the FHBL was caused by premature truncation within apoB-100, we analyzed the plasma apoB-100 of the proband by SDS-PAGE followed with immunoblot analysis. Invariably, the full-length apoB-100 was the major or sole form detectable, and there were no truncated forms of apoB presented in the plasma (data not shown). We then performed DNA sequence analysis of the entire human APOB gene and found a single thymine peak instead of cytosine at the first nucleotide of codon 463 in exon 11 in the homozygote proband (Fig. 1B). This predicted a missense mutation whereby the wild-type arginine (CGG) was replaced by tryptophan (TGG) at residue 463 of the mature protein (R463W). Obligate heterozygotes showed both cytosine and thymine peaks at this position. The mutation co-segregated completely with the clinical phenotype in the original nuclear and subsequently in the extended family (LOD score = 9.63 at 0% recombination fraction). Further screening of APOB in 122 control nondyslipidemic subjects revealed complete absence of this mutation in exon 11. One additional single nucleotide polymorphism in exon 4 was found through genomic sequencing, but this was a previously identified common single nucleotide polymorphism in the general population with National Center for Biotechnology Information reference number rs1367117 (chromosome 2 position 21170203A>G, trivial name T71I). This single nucleotide polymorphism did not co-segregate with the FHBL phenotype.

Reduced Secretion of B48RW from Transfected McA-RH7777 Cells-- We postulated that the R463W mutation was the cause of FHBL by impairing apoB secretion. To test this hypothesis, we examined the effect of R463W substitution on the secretion of apoB-48 (Fig. 2A), a naturally occurring apoB form that has the ability to form VLDL. McA-RH7777 cells were transfected with either wild-type B48 (B48wt) or the mutant form B48RW. Pulse-chase experiments with the stably transfected cells showed that in contrast to B48wt, B48RW was poorly secreted (Fig. 2B). In four independent cell lines examined, secretion efficiency of B48RW was decreased by ~45% when compared with that of B48wt (Fig. 2C). The secreted B48wt from stable cells (cultured in exogenous oleate) was associated with particles with the density of VLDL and HDL-like particles. Secretion of B48RW associated with VLDL and HDL-like particles was decreased compared with that of B48wt. Thus, the majority of B48RW was secreted as HDL-like particles, whereas a trace was associated with VLDL (Fig. 3, A and B). The percent distribution of total secreted B48RW among different lipoproteins, however, was unchanged as compared with that of B48wt (Fig. 3B, inset). Secretion of endogenous rat apoB-100 as VLDL from B48RW-transfected cells was markedly decreased in comparison to B48wt-transfected cells (Fig. 3B, bottom), suggesting a dominant-negative effect of B48RW expression on normal apoB secretion. The inhibitory effect of B48RW expression on endogenous rat apo-B100 secretion was also observed in pulse-chase experiments (Fig. 3, C and D). In these experiments, a polyclonal antibody was used for immunoprecipitation that recognized both human and rat apoBs. At the end of the 4-h chase, the secretion efficiency of rat apo-B100 was decreased by >60% from B48RW-transfected cells as compared with that from B48wt-transfected cells (Fig. 3D, left). The diminished secretion of B100-VLDL and B48-VLDL from B48RW-transfected cells was not attributable to low levels of lipid availability. Lipid analysis showed accumulation of triglyceride within the cell and decreased triglyceride secretion (by 30%) into the medium in B48RW-transfected cells as compared with those in B48wt-transfected cells (Fig. 3E).


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Fig. 2.   Expression and secretion of B48wt and B48RW. A, schematic diagram of the apoB-48 construct. The top line shows apoB-100, with locations of amphipathic alpha  helices (beta alpha 1, alpha 2, and alpha 3) and beta  strands (beta 1, and beta 2) shown. The bottom line shows apoB48. An arrowhead indicates the mutation at R463 within the beta alpha 1 domain. B, pulse-chase analysis of apoB-48 secretion. Stably transfected cells expressing B48wt or B48RW were pulse-labeled with [35S]methionine/cysteine (200 µCi/ml) for 30 min in methionine-free DMEM and chased in normal DMEM. Both pulse and chase media contained 20% fetal bovine serum and 0.4 mM oleate. The 35S-B48 was recovered from cell (left) and medium (right) by immunoprecipitation at the indicated times. The apoB proteins were resolved by electrophoresis on SDS-PAGE (5% gel), and visualized by fluorography (insets). Radioactivity associated with 35S-B48 was quantified by scintillation counting. Results are expressed as percent of the initial radiolabeled protein recovered at the end of chase. C, comparison of secretion efficiency of B48 (left) and apoA-I (right) between B48wt- and B48RW-transfected cells. Cells were pulse-labeled for 30 min and chased for 2 h. The secretion efficiency for B48wt, B48RW, and apoA-I was determined as in B. Secretion of B48RW and apoA-I from B48RW-transfected cells is presented as percent of B48wt and apoA-I from B48wt-transfected cells. Data are mean ± S.D. derived from pulse-chase experiments using four different stable cell clones.


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Fig. 3.   Distribution of B100 and B48 in medium lipoproteins. A, stably transfected cells were labeled with [35S]methionine/cysteine (200 µCi/ml) for 2 h in methionine-free DMEM containing 20% fetal bovine serum and 0.4 mM oleate. After labeling, the media were fractionated into VLDL1 (Sf > 100), VLDL2, (Sf 20-100), and other lipoproteins by rate flotation, and 35S-B48 and 35S-B100 in each fraction were immunoprecipitated using a polyclonal antibody that recognized both recombinant human B48 and the endogenous rat apoB-100. The apoB proteins were resolved by SDS-PAGE and visualized by fluorography. B, quantification of radioactivity associated with 35S-B48 and 35S-B100 that were secreted from B48wt- or B48RW-expressing cells. Inset, the percentage of total 35S-B48 secreted as VLDL (VLDL1 + VLDL2), IDL/LDL, and HDL. C, pulse-chase analysis of apoB-48 secretion. Stably transfected cells expressing B48wt (left) or B48RW (right) were pulse-labeled with [35S]methionine/cysteine for 30 min and chased for up to 4 h. Both pulse and chase media contained 20% fetal bovine serum and 0.4 mM oleate. The endogenous rat 35S-B100 and the recombinant human 35S-B48 were recovered from cell and medium by immunoprecipitation. The apoB proteins were resolved by electrophoresis on SDS-PAGE (5% gel) and visualized by fluorography. D, radioactivity associated with 35S-B100 (left) and 35S-B48 (right) was quantified by scintillation counting. Results are expressed as percent of the initial radiolabeled protein recovered at the end of chase. E, cells were labeled with [3H]glycerol (3 µCi/ml) for 2 h in DMEM containing 20% fetal bovine serum and 0.4 mM oleate. At the end of labeling, lipids were extracted from cells and medium, respectively, and resolved by thin-layer chromatography. Radioactivity associated with [3H]triglyceride (TG) and [3H]phosphatidylcholine (PC) was quantified by scintillation counting. Each value is the average of duplicate samples with the indicated deviation. cpm indicates counts per minute.

The above pulse-chase experiments (Fig. 2B) suggested that the reduced B48RW secretion was accompanied by prolonged intracellular retention. To identify the cellular compartments corresponding to the retained B48RW, we performed indirect double immunofluorescence co-localization studies. Confocal images showed that the overall intensity of B48RW was invariably stronger than that of B48wt expressed in multiple transiently transfected cells (Fig. 4, panels labeled with apoB). These data, although semiquantitative in nature, are in agreement with the observation of prolonged intracellular retention of B48RW in stably transfected cells (Fig. 3A). Merging fluorescence derived from apoB with that from calnexin or alpha -mannosidase II showed co-localization of the majority of apoB with the ER (Fig. 4). To confirm that B48RW was indeed accumulated within the ER as suggested qualitatively by immunocytochemistry studies, we performed subcellular fractionation experiments in which the ER, cis/medial Golgi, and distal Golgi compartments were separated by centrifugation using a Nycodenz density gradient (Fig. 5). Distribution of marker proteins for ER, cis/medial Golgi, and distal Golgi is shown in Fig. 5A. The radioactivity of B48RW in the ER fractions was 2-fold higher than that of B48wt, whereas the distribution in the Golgi compartment was identical between B48RW and B48wt (Fig. 5B). These data together suggest that the mutant B48RW may have a reduced exit rate from the ER.


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Fig. 4.   Confocal images of apoB, calnexin, and alpha -mannosidase II. McA-RH7777 cells (in 6-well plates) were transfected with B48wt or B48RW expression plasmids (2 µg of DNA) using FuGENE 6 transfection reagent. Two days after transfection, the cells were subjected to immunocytochemistry experiments as described under "Materials and Methods." Human apoB was probed with mAb 1D1 (visualized green), and ER and cis/medial Golgi were probed with anti-calnexin (Cnx) and anti-alpha -mannosidase II (Man II) antibodies, respectively (visualized red).


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Fig. 5.   Intracellular distribution of B48. A, distribution of marker proteins. Fractionation of subcellular microsomes by Nycodenz gradient centrifugation was achieved as described under "Materials and Methods." Proteins of the fractionated samples were immunoblotted with various antibodies. A, Hsp47 (an ER residence protein containing the KDEL motif), alpha  mannosidase II (Man II), and TGN38; B, B48wt and B48RW. Quantification of radioactivity associated with 35S-B48 in various fractions is shown at the bottom.

Reduced Secretion of B17RW from Transfected McA-RH7777 Cells-- We next examined the effect of R463W mutation on the secretion of apoB-17 (Fig. 6A), a truncated form of apoB that associates poorly with lipids but has the ability to interact with synthetic liposomes (44) and MTP (45). In transfected McA-RH7777 cells, secretion efficiency of B17RW, as determined by pulse-chase experiments, was decreased by 40% as compared with that of B17wt (Fig. 6B). Immunocytochemistry experiments revealed that as what was observed for B48RW, the mutant B17RW also exhibited enhanced accumulation within the ER (Fig. 7, panels labeled with apoB). Thus, the R463W mutation in apoB-17 also resulted in reduced secretion and impaired ER exit.


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Fig. 6.   Effect of mutations at Arg463 on B17 secretion. A, schematic diagrams of the recombinant B17 constructs. The top two lines represent wild type B48 and B17, respectively. Three mutant B17 constructs that contain single amino acid substitutions at Arg463 (RW, RK, and RA) are depicted. B, comparison of secretion efficiency of B17 (left) and apoA-I (right) between B17wt- and B17RW-transfected cells. Cells were pulse-labeled for 30 min and chased for 2 h. The secretion efficiency for B17wt, B17RW, and apoA-I was determined as described in the legend to Fig. 2C. Secretion of B17RW and apoA-I secreted from B17RW-transfected cells is presented as percent of B17wt and apoA-I from B17wt-transfected cells. Data are mean ± S.D. derived from pulse-chase experiments using two stable B17wt-transfected clones and four B17RW-transfected clones.


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Fig. 7.   Confocal images of B17, calnexin, and alpha -mannosidase II. The experiments were performed essentially the same as described in the legend to Fig. 4, except that the cells were transfected with B17wt or B17RW expression plasmids. Cnx, calnexin; ManII, alpha -mannosidase II.

The Positive Charge of Arg463 Is Important for ApoB-17 Secretion, but Not for Its Interaction with MTP-- Knowing that the R463W mutation affected secretion of apoB-17, we used apoB-17 as a model to define further the mechanism by which one amino acid substitution has such a profound effect on apoB secretion. We substituted Arg463 with either Lys (B17RK) or Ala (B17RA) (Fig. 6A) to test whether the charge or the bulkiness of the side chain at residue 463 was important for apoB secretion. Data from a typical pulse-chase experiment using cells transiently transfected with B17wt, B17RW, B17RK, or B17RA are shown in Fig. 8A, and quantification of medium (right panel) and cell-associated (left panel) apoB is presented in Fig. 8B. The secretion efficiency of B17RK was normal as compared with that of B17wt, but the secretion of B17RA was decreased to a level similar to that of B17RW. Thus, the positive charge at residue 463, rather than the size of the side chain, is important for efficient secretion of apoB-17.


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Fig. 8.   Pulse-chase analysis of B17RK and B17RA secretion. A, McA-RH7777 cells were transfected with expression plasmids encoding B17wt, B17RW, B17RK, or B17RA, respectively. Two days after transfection, the cells were pulse-labeled with [35S]methionine/cysteine (200 µCi/ml) for 30 min and chased in normal DMEM as described in the legend to Fig. 2B. At the indicated times, the 35S-B17 was recovered from cell and medium by immunoprecipitation, and analyzed by SDS-PAGE and fluorography. B, radioactivity associated with 35S-B17 was quantified by scintillation counting. Results are expressed as percent of the initial radiolabeled protein recovered at the end of chase.

Previous studies showed that apoB-17 could bind to MTP in vitro (45), and the interaction appeared to be mediated by Lys and Arg residues within apoB (12). To examine whether or not the R463W mutation affected apoB/MTP interaction, we co-transfected apoB-17 and MTP into COS-7 cells and determined their interaction within the cells by co-immunoprecipitation experiments (Fig. 9). The COS-7 cell line was chosen for its lack of endogenous apoB or MTP expression. In transiently transfected cells, both B17wt and B17RW were expressed similarly (Fig. 9A). The cell-associated B17wt and B17RW were both decreased by co-transfection of MTP, probably because the additional MTP cDNA attenuated apoB-17 transfection. As was the case in McA-RH7777 cells, B17RW was poorly secreted compared with B17wt from transfected COS-7 cells. Co-expression of MTP, as expected (33), had no effect on B17wt or B17RW secretion (Fig. 9B). The interaction between apoB-17 and MTP was determined by immunoprecipitation with anti-MTP antibody (to recover apoB·MTP complex) and then probing apoB with an anti-apoB antibody on a Western blot (Fig. 9C). The intensity of the B17RW signal detected on the Western blot was noticeably stronger that that of B17wt. The level of MTP expression was similar between B17wt- and B17RW-transfected COS-7 cells (Fig. 9D). These results suggest that the R463W mutation did not diminish, but rather enhanced apoB-17/MTP interaction in the transfected cells.


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Fig. 9.   Co-expression of B17 and MTP in COS-7 cells. COS-7 cells were transfected with 10 µg of B17wt or B17RW cDNAs ± 10 µg of MTP cDNA. Two days after transfection, the cells were lysed under nondenaturing conditions, and the intracellular B17 proteins were immunoprecipitated with a polyclonal anti-apoB antibody (alpha B), resolved by SDS-PAGE, and visualized by fluorography using mAb 1D1 (panel A). The medium was collected and the secreted B17 proteins were recovered by immunoprecipitation and visualized by fluorography using mAb 1D1 (panel B). An aliquot of the cell lysate was mixed with a polyclonal anti-MTP antibody (alpha MTP). The immunocomplex was resolved by SDS-PAGE, proteins were transferred onto a nitrocellulose membrane, and probed with mAb 1D1 to detect B17 that was co-immunoprecipitated with MTP (panel C). An aliquot of the cell lysate was resolved by SDS-PAGE and probed with the anti-MTP antibody (panel D). Repetition of the experiments yielded identical results.

Asp456 to Asn (B17DN) Substitution Impairs ApoB-17 Secretion-- As mentioned previously, apoB shares sequence homology to lamprey lipovitellin. A molecular model of apoB-17 based on the known crystal structure of lipovitellin (Fig. 10A) predicted that Arg463 was located in an extended alpha -helical region in close proximity to acidic residues Glu756 and Asp456 (Fig. 10B). To determine whether Glu756 or Asp456 may also be important for apoB secretion, we performed Glu756 to Gln (B17EQ) and Asp456 to Asn (B17DN) substitutions (Fig. 11A), and assessed their secretion efficiency in transiently transfected McA-RH7777 cells. Immunoblot analysis showed that accumulation of B17DN in the medium, like B17RW and B17RA, was lower than that of B17wt. In contrast, accumulation of B17EQ was comparable with that of B17wt and B17RK (Fig. 10B).


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Fig. 10.   Charged amino acids near Arg463 in the model of B17. A, charged residues in the vicinity of Arg463 are shown in a space-filling representation. Arg463 (blue), positively charged residues and histidine (cyan), and negatively charged residues (red) are shown. The disulfide bond between Cys451 and Cys486 is indicated in yellow. B, expanded view highlighting the position of residues Arg463, Asp456, and Glu756. This figure was created using MOLMOL (57).


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Fig. 11.   Effect of mutation at Glu756 and Asp456 on B17 secretion. A, schematic diagrams of the mutant B17 constructs that contain a single amino acid substitution, E756Q (B17EQ) or D456N (B17DN). B, secretion of B17 from transiently transfected McA-RH7777 cells. The cells were transfected with various apoB-17 expressing plasmids as indicated. Two days after transfection, the cells were lysed and media were collected, and an aliquot of sample was used for immunoprecipitation using polyclonal anti-apoB antiserum. The immunocomplex was resolved by SDS-PAGE, proteins were transferred onto a nitrocellulose membrane, and the human B17 proteins were detected with the mAb 1D1. C, indicates cell; M, media.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have discovered a novel and rare nonsynonymous, nontruncating APOB gene mutation, R463W, in a Christian Lebanese FHBL kindred. The mutation showed co-dominant segregation with the FHBL phenotype because heterozygotes and homozygotes had, respectively, lower than half the normal and barely measurable plasma apoB concentrations. To date, the only other phenotype that has been associated with naturally occurring missense mutations in APOB is a co-dominant form of hypercholesterolemia (MIM 144010), which results from the LDL receptor binding-defective forms of apoB-100 (i.e. the R3500Q and R3531C variants) (14). The diseases caused by these two kinds of point mutations within the coding sequences of the APOB gene exemplify the critical role that apoB-100 plays in controlling cholesterol homeostasis at both synthesis (i.e. VLDL assembly and secretion) and catabolism (i.e. receptor-mediated endocytosis) levels.

The R463W mutation found in the FHBL kindred specifies a local domain that appears to be critical for the efficient secretion of apoB and for lipid recruitment during lipoprotein assembly. The amino acid 463 is located within the beta alpha 1 domain, which contains sequence elements that are shown to be important for proper folding of apoB, for the physical interaction between MTP and apoB, and for lipoprotein assembly. Transfection studies have suggested that apoB segments containing sequences lacking the beta alpha 1 domain were unable to be secreted (46) or else secreted poorly (27). Mutagenesis studies have identified critical disulfide linkages within the beta alpha 1 domain that are essential for efficient secretion of apoB and for apoB-containing lipoprotein assembly (47-49). In the two truncated forms of apoB (apoB-17 and apoB-48) examined, the R463W mutation resulted in decreased secretion efficiency (by ~45% compared with wild type) regardless of the apoB polypeptide length or its ability to assemble lipoproteins (Figs. 2C and 6B). In the case of apoB-48, the R463W mutation markedly compromised its ability to assemble VLDL and the HDL-like particles (Fig. 3, A and B). Recruitment of bulk lipids during B-48 VLDL assembly occurs post-translationally at a stage where the HDL-like particles are converted to mature VLDL (commonly known as the second step) (50). The diminished secretion of both VLDL and the HDL-like particles (Fig. 3B) suggests strongly that the R463W mutation exerts its effect at the early stage of lipoprotein assembly. The current results thus provide additional evidence for the functional importance of the beta alpha 1 domain.

The mechanism by which the R463W mutation impairs apoB secretion has been investigated using apoB-17 as a model. The proximity of R463W to the putative MTP binding regions within apoB might have explained the FHBL in the study kindred. Mutagenesis experiments with altered amino acid indeed demonstrate the importance of the positive charge associated with Arg463. However, co-immunoprecipitation experiments did not suggest impaired binding of mutant apoB to MTP. Rather, in transfected COS-7 cells, the mutant apoB exhibited increased binding to MTP (Fig. 9C). The enhanced binding to MTP may explain the prolonged ER retention of the mutant proteins as shown by immunocytochemistry (Figs. 4 and 7) and subcellular fractionation experiments (Fig. 5). Perhaps the most surprising observation of the present work is that in stably transfected cells, expression of B48RW resulted in marked reduction in the secretion of endogenous rat apoB-100 (Fig. 3, A-D). It is tempting to speculate that the dominant negative effect of B48RW expression on endogenous rat apoB secretion is attributable to the enhanced MTP binding of the mutant proteins. The mechanism by which expression of the RW mutants affects apoB secretion remains to be defined using apoB-48 and apoB-100 as models.

Molecular modeling of apoB-17 based on the known crystal structure of lamprey lipovitellin predicts that Arg463 is mostly buried in an extended alpha -helical region and is in close proximity to acidic residues Glu756 and Asp456. Interestingly, substitution of Asp456 with Asn also decreased apoB17 secretion. These results suggest strongly that the alpha -helical region containing Asp456 and Arg463 are important in apoB folding and MTP binding. The molecular mechanism whereby the apoB sequences encompassing Arg463 and Asp456 attain proper folding and MTP interaction also needs to be further defined in the context of apoB-48 and apoB-100.

The low plasma triglyceride concentrations that are typical of FHBL may be due, at least in part, to defective assembly and secretion of triglyceride-rich lipoproteins. Among reported APOB mutations causing premature truncation of apoB, the length of the mutant apoB was correlated with the hepatic secretion of apoB-containing lipoproteins both in vitro (51) and in vivo (13). However, in our previous cell cultures studies, we did not observe impaired secretion efficiency for variously truncated apoB forms (ranging from apoB-15 to apoB-94). In fact, most of the truncated recombinant apoBs were secreted as efficiently as normal apoB-100 or apoB-48 (23, 32). Thus, it is rather unusual that the R463W mutation, which has seemingly a much less severe impact than COOH-terminal truncations of apoB (13, 14), can cause FHBL. In addition to diminished secretion, increased catabolism because of prematurely truncated apoB has also been implicated in causing FHBL (16-20). However, a catabolic defect for apoB R463W seems unlikely, because the preponderance of the mature protein, once secreted would be expected to be intact and qualitatively normal. Nevertheless, the assembly and secretion defect suggested by the current cell culture experiment needs to be further confirmed by in vivo lipoprotein turnover studies using the human FHBL subjects and ultimately in transgenic mice that harbor the R463W mutation in APOB through genetic knock-in techniques.

Lipid analysis showed that stable expression of B48RW was associated with intracellular triglyceride accumulation and decreased triglyceride secretion (Fig. 3E). There is evidence that the hepatobiliary system can be affected in FHBL, with steatosis reported in several kindreds (52-54). The elevated serum transaminases and ferritin in the R463W heterozygotes could represent a subclinical phenotype that is consistent with such previous observations. Sensitive noninvasive imaging studies of the hepatobiliary system in mutation carriers would be of interest. Furthermore, the apparent R463W allele dose-dependent gradient, specifically for serum alpha -tocopherol was consistent with the known relationship between serum vitamin E and plasma lipid and apoB concentrations (55). Whereas homozygotes had no neurological dysfunction clinically, and acanthocytes were only occasionally observed in the blood film, it is possible that evaluation of intermediate subclinical vitamin E-dependent traits may be informative.

In conclusion, the in vitro function consequences of the APOB R463W mutation would be consistent with the defective assembly and secretion of apoB-containing lipoproteins in vivo. The mechanism responsible for the impaired secretion of VLDL involves impaired ER exit and enhanced binding to MTP. This would explain the clinical phenotype of FHBL in carriers of R463W among the extended Christian Lebanese kindred studied herein. Thus, the naturally occurring R463W mutation within the postulated binding site for MTP reveals a key local domain governing assembly and secretion of apoB-containing lipoproteins. The findings represent an example of how analysis of a single kindred with a monogenic dyslipidemia can provide insight into biologically relevant mechanisms. Also, the findings extend the spectrum of phenotypes because of missense mutations in APOB (56). Finally, the findings target this apoB domain for development of strategies to suppress overproduction of apoB-containing lipoproteins in reciprocal conditions such as familial combined hyperlipidemia or polygenic hypercholesterolemia, which are characterized by elevated plasma apoB concentrations and early atherosclerosis.

    ACKNOWLEDGEMENTS

We thank Drs. R. Milne, M. G. Farquhar, and Dr. C. Shoulders for mAb 1D1, polyclonal anti-alpha -mannosidase, and anti-MTP antibody, respectively.

    FOOTNOTES

* This work was supported in part by grants from the Royal Perth Hospital Medical Research Foundation and the Raine Medical Research Foundation (to J. R. B.), National Institutes of Health Grant HL26335 (to C. J. M.), a Canada Research Chair (Tier 1) in Human Genetics (to R. A. H.), and a Heart and Stroke Foundation of Ontario Career Investigator Award (to R. A. H.), grants from the Canadian Genetic Diseases Network, Canadian Institutes for Health Research Grants MT-13430 and MT-15486, Heart and Stroke Foundation of Ontario Grants-in-aid T-4643 and T-4772, and a Scientist Award from the Canadian Institutes for Health Research (to Z. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to the results of this work.

To whom correspondence may be addressed. Tel.: 61-8-9224-3121; Fax: 61-8-9224-2491; E-mail: john.burnett@health.wa.gov.au.

Dagger Dagger Recipient of a John D. Schultz Scholarship from the Heart and Stroke Foundation of Canada.

¶¶ To whom correspondence may be addressed. Tel.: 519-663-3461; Fax: 519-663-3789; E-mail: hegele@rri.on.ca.

|||| To whom correspondence may be addressed. Tel.: 613-798-5555 (ext. 18711); Fax: 613-761-5281; E-mail: zyao@ottawaheart.ca.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M300235200

    ABBREVIATIONS

The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoproteins; LDL, low density lipoproteins; MTP, microsomal triglyceride transfer protein; FHBL, familial hypobetalipoproteinemia; HDL, high density lipoproteins; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; mAb, monoclonal antibody.

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