From the 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
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ABSTRACT |
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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 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- 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 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.
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
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.
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- 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- 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.
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 apo 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).
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
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.
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.
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.
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 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 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 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
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.
-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-
1-
2-
2-
3-COOH (7). The amino acid
sequence of the
1 domain is homologous to lamprey
lipovitellin and microsomal triglyceride transfer protein (MTP)
(8, 9). The
1 domain of human apoB has thus been modeled on the
basis of the solved lipovitellin structure, in which 13
-strands
(amino acids 21-263) form a
barrel, followed by a two-layered
helical bundle consisting of 17
-helices (amino acids 294-592).
In vitro experiments suggest that the
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
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
1 domain of apoB
creates a lipid pocket that facilitates lipid recruitment during
lipoprotein assembly (9).
-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).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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
-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).
Oligonucleotides used for mutagenesis
-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).
-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-
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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
-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
-tocopherol concentrations were the lowest in the homozygotes, intermediate in heterozygotes, and the
highest in unaffected subjects. Serum
-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.
Plasma lipid and apoprotein concentrations, and APOE genotype in the
R463W kindred
Serum fat-soluble vitamins, liver enzymes, and ferritin concentrations
in the R463W kindred
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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
helices (
1,
2, and
3) and
strands (
1, and
2)
shown. The bottom line shows apoB48. An arrowhead
indicates the mutation at R463 within the
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.
-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
-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-
-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), 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.
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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 -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,
-mannosidase
II.
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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.
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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 ( 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 (
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.
-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
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
1 domain were unable to be
secreted (46) or else secreted poorly (27). Mutagenesis studies have
identified critical disulfide linkages within the
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
1 domain.
-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
-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.
-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.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. R. Milne, M. G. Farquhar,
and Dr. C. Shoulders for mAb 1D1, polyclonal anti--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.
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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