© 1995 American Heart Association, Inc.
Articles |
Characterization of Recombinant Human ApoB-48–Containing Lipoproteins in Rat Hepatoma McA-RH7777 Cells Transfected With ApoB-48 cDNA
Overexpression of ApoB-48 Decreases Synthesis of Endogenous ApoB-100
From the Departments of Pathology and Biochemistry (M.M.H., R.K.K.), the Medical College of Pennsylvania, Philadelphia, Pa; the Lipid and Lipoprotein Research Group and Department of Biochemistry (Y.Z., Z.Y.), University of Alberta, Edmonton, Canada; and COR Therapeutics (B.D.B.), South San Francisco, Calif.
Correspondence to Dr M. Mahmood Hussain, Departments of Pathology and Biochemistry, The Medical College of Pennsylvania, 2900 Queen Ln, Philadelphia, PA 19129.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract We studied the effect of overexpression of apolipoprotein (apo) B-48 on the synthesis and secretion of endogenous apoB-100 in rat hepatoma McA-RH7777 cell lines stably transfected with human apoB-48 cDNA under the control of the cytomegalovirus promoter. Three cell lines that secrete 40 to 60 ng human apoB · mg cell protein-1 · h-1 were used. The recombinant human apoB-48 exhibited physicochemical characteristics (buoyant density, 1.06 to 1.21 g/mL; ß-electrophoretic mobility and diameters, 16 to 20 nm) indistinguishable from those of endogenous rat apoB-48. Overexpression of the recombinant human apoB-48 resulted in a 50% decrease in the secretion of endogenous apoB-100 but did not affect the secretion of apoE or apoA-I. Several possible mechanisms for the decreased secretion of apoB-100 were evaluated. First, recruitment of lipids into lipoproteins was shown to be unaffected since no major changes in the physicochemical properties of apoB-100–containing lipoproteins were observed. Second, the intracellular degradation of apoB-100 was not altered as the intracellular retention half-time and secretion efficiency remained unaffected by apoB-48 overexpression. Third, the posttranslational regulatory mechanisms for apoB-100 remained normal, as demonstrated by a twofold increase in apoB-100 secretion after supplementation with oleic acid. Unexpectedly, a 35% to 50% decrease in the steady-state synthesis of endogenous apoB-100 was observed in apoB-48–transfected cells compared with control cells. These data suggested that decreased secretion of apoB-100 was secondary to decreased synthesis. The decreased apoB-100 synthesis was not due to decreased steady-state levels of rat apoB-100 mRNA. These results suggest that overexpression of recombinant human apoB-48 may interfere with posttranscriptional events, possibly at the translation-translocation level, and decrease translational yield of apoB-100. These posttranscriptional events prior to the complete synthesis of the apoB-100 polypeptide can be important in the control of apoB-100 secretion.
Key Words: apoB-48 • rat hepatoma • apolipoproteins • lipoproteins • apoB-100
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In humans, the synthesis and secretion of triglyceride-rich lipoproteins by the liver and intestine, respectively, depend on the tissue-specific expression of apolipoprotein (apo) B-100 and apoB-48.1 Both proteins are translational products of a single apoB gene. ApoB-48, the smaller form consisting of the amino-terminal 48% of the apoB-100, is synthesized in the intestine after a novel posttranscriptional modification of the apoB mRNA at nucleotide 6666, resulting in the generation of a premature termination codon at 2153.2 3 4 In contrast, livers of rats, mice, dogs, and horses express apoB mRNA editing activity5 and consequently synthesize and secrete apoB-48–containing lipoproteins.5 6 7 8 The synthesis and secretion of apoB-containing lipoproteins by the rat liver have been studied using liver perfusions and primary hepatocyte cultures.6 7 8 9 10 11 Rat hepatocytes secrete apoB-containing lipoproteins that float at densities corresponding to plasma VLDL and HDL.6 7 8 9 ApoB-100 was found predominantly associated with VLDL, whereas apoB-48 was present in both lipoprotein classes.8
The production of apoB-containing lipoproteins is controlled at different levels. There is some evidence for an increase in apoB mRNA levels,12 13 but it is generally believed that increased transcription of the gene is not a major mechanism of control of apoB synthesis.14 15 16 17 Under certain metabolic conditions, the rate of apoB translation has been shown to be decreased.18 19 The major mechanism for the synthesis and secretion of apoB, however, is the regulated degradation of nascent polypeptide in the endoplasmic reticulum.16 20 Numerous experimental results11 14 16 20 21 22 have indicated that a significant portion of the nascent hepatic apoB molecules are degraded rather than secreted. The intracellular degradation is often decreased after supplementation with oleic acid, resulting in increased secretion of apoB.21 Thus, the amount of apoB secreted does not necessarily reflect the amount of apoB synthesized.
Although hepatic apoB-100 and apoB-48 are synthesized from the same gene, production of these two polypeptides is regulated differently. Increased amounts of apoB-48 and decreased amounts of apoB-100 are secreted in the postprandial compared with the fasting state.6 9 The intracellular retention of apoB-48 is significantly longer than apoB-100 in primary rat hepatocytes.11 22 n-3 Fatty acids induce intracellular degradation of apoB polypeptides to different extents.11
Changes in apoB mRNA editing activity also affect the synthesis of apoB-100 and apoB-48. Fasting decreases apoB mRNA editing and decreases apoB-48 synthesis.13 23 Almost invariably, an increase in apoB mRNA editing in the liver results in increased synthesis and secretion of apoB-48.13 24 25 However, decreases in apoB-100 synthesis and secretion were observed in some13 24 but not all25 studies. Decreased synthesis of apoB-100 has been attributed to a decrease in the population of unedited apoB mRNA, but whether the ratio between production of apoB-100 and apoB-48 is solely determined by apoB mRNA editing is unclear. For example, the effect of increased synthesis of apoB-48 on the synthesis and secretion of apoB-100 has not been addressed. To better understand the relations between hepatic apoB-100 and apoB-48 production, we studied the effect of overexpression of apoB-48 on the synthesis and secretion of apoB-100–containing lipoproteins using rat hepatoma McA-RH7777 cells transfected with recombinant human apoB-48. The expression plasmid encoding the recombinant human apoB-48 was engineered such that a stop codon was placed at codon 2153 of the apoB message; thus, the production of apoB-48 was independent of apoB mRNA editing. Our results demonstrate that synthesis of apoB-48 decreases the synthesis and secretion of apoB-100 without decreasing the abundance of the apoB-100 mRNA.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Construction of Recombinant Human ApoB-48 Expression Plasmids
The expression plasmid pRc/CMV-B48 (pB48; Fig 1A
), was derived from pB53L-L (prepared in pCMV5) that
contained an engineered Mlu I restriction site at nucleotide
7011 of the apoB cDNA.26 To prepare the pB48 plasmid, a
cDNA fragment extending from nucleotide 6507 (an EcoRI site)
to nucleotide 6677 was amplified by polymerase chain reaction. The 3'
polymerase chain reaction primer was designed to change the nucleotide
6666 from cytosine to thymidine (which converts codon 2153 from
glutamine to a stop codon) and to incorporate an Mlu I site
immediately 3' to the stop codon. The amplified
EcoRI–Mlu I fragment was then ligated with
pB53L-L that had been partially digested with EcoRI and
completely digested with Mlu I. In the resulting plasmid, an
Apa I linker was introduced at the Sma I site
(located in the 3' end of the polylinker region of pCMV5). To generate
pB48, the entire apoB-48 coding sequences were excised by digestion of
the resulting construct with Not I and Apa I and
inserted into the pRc/CMV expression vector (Invitrogen), that
contained a gene conferring resistance to neomycin analogues. Sequences
that contained the junctions between the apoB insert and the vectors
and that contained the C-to-T mutation at nucleotide 6666 of the apoB
cDNA were validated in pB48 by double-stranded DNA sequencing with
Sequenase according to the manufacturer's instructions (United States
Biochemical Corp). The plasmid DNA was purified prior to use by
centrifugation twice in a CsCl gradient.
|
Cell Culture and Transfection
The McA-RH7777 cells were obtained from American Type Culture
Collection and grown in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine and 10% horse sera (Life Technologies,
Inc). Stably transfected McA-RH7777 cell lines were
obtained,27 28 and mock transfection was performed by
using plasmid pRc/CMV. Three stable transformants expressing
recombinant human apoB-48 (B48-13, B48-15, and B48-16) were selected in
culture medium containing 500 µg/mL G418 and were maintained in
medium containing 200 µg/mL G418. All three clones were used in
different experiments. No differences between these clones were
observed.
Immunoblot Analysis of ApoB-Containing Lipoproteins in the
Medium
Lipoproteins in the conditioned medium were adsorbed onto fumed
silica (Cab-O-Sil, Sigma Chemical Co),28 and aliquots of
protein samples derived from equal amounts of cell proteins (0.2 mg)
were resolved by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE). After transfer to nitrocellulose membranes,
the recombinant human apoB-48 and endogenous rat apoB-100 and
apoB-48 were detected by using specific antibodies. Immunocomplexes
were visualized by the ECL system (Amersham) according to the
manufacturer's instructions or by 125I-labeled protein A
followed by fluorography.
For determination of the density distribution of apoB proteins, the conditioned medium (16 mL from two dishes) was fractionated in a salt gradient by ultracentrifugation.29 Twenty fractions were collected from the bottom of the centrifuge tubes; lipoproteins in each fraction were concentrated with Cab-O-Sil, resolved by SDS-PAGE, transferred to nitrocellulose membranes (125 V, -15°C, 6 hours), and immunoblotted for apolipoproteins.30
Nondenaturing PAGE and Agarose Gel Electrophoresis
The conditioned medium was concentrated 10-fold by using a
Centricon-10 concentrator (Amicon Inc), and aliquots (5 or 10 µL)
were applied to gradient (3% to 10%) polyacrylamide gels and
electrophoresed under nondenaturing conditions. The electrophoresis was
performed (125 V, room temperature, 24 hours) with a recirculating
buffer solution that contained 90 mmol/L Tris base, 90 mmol/L boric
acid, and 2 mmol/L EDTA, pH 8.0. Human LDL, thyroglobulin, ferritin,
and catalase were used as size markers. For agarose gel
electrophoresis, the concentrated conditioned medium was applied to
Beckman Paragon agarose gels (Beckman Instruments Inc) and
electrophoresed according to the manufacturer's instructions. Human
LDL, rat VLDL, rat LDL, and rat HDL were used as lipoprotein standards.
The resulting gels were transferred to nitrocellulose membranes, and
apolipoproteins were detected by using specific antibodies.
Quantification of Secreted Recombinant Human ApoB-48 by
Enzyme-Linked Immunoassay
For quantification of secreted recombinant human apoB-48,
96-well plates (Dynatech Laboratories Inc) were coated overnight at
4°C with 100 µL monoclonal antibody (1D1; 10 µg/mL) and washed
three times with phosphate-buffered saline (PBS), pH 7.4, containing
0.05% Tween 20. Conditioned medium (100 µL) was added to each well
and incubated for 2 hours at room temperature. The wells were washed
and incubated with sheep anti–human apoB polyclonal antibodies (1:2000
dilution; Boehringer Mannheim) for 1 hour. After washing, the wells
were incubated with purified rabbit anti-sheep immunoglobulin G labeled
with alkaline phosphatase (1:1000 dilution; Cappel) at room temperature
for 1 hour. Finally, the wells were washed twice with PBS-Tween, PBS,
10 mmol/L ethanolamine, containing 0.5 mmol/L MgCl2,
pH 9.5, and incubated with 100 µL p-nitrophenyl
phosphate (1 mg/mL in 10 mmol/L ethanolamine, containing 0.5 mmol/L
MgCl2, pH 9.5) for 30 minutes. The reaction was
stopped by the addition of 10 µL of 0.5 mol/L EDTA, and the
absorbance at 405 nm was determined by using a Dynatech micro
enzyme-linked immunosorbent assay reader. Human LDL (0 to 10 ng
protein/well) was used as a standard.
Negative Staining and Electron Microscopy
Lipoproteins (d=1.08 to 1.21 g/mL) were isolated by
ultracentrifugation from serum-free conditioned medium of
pB48-transfected cells, concentrated, and adjusted to 0.125 mol/L
ammonium acetate, 2.6 mmol/L ammonium bicarbonate, and 0.26 mmol/L
EDTA, pH 7.2. A drop of the sample was applied on Formvar-coated grids,
and excess solution was blotted with filter paper. Lipoproteins were
then stained with 2% phosphotungstate, pH 7.4, for 30 seconds and
viewed by using an electron microscope.31 In some
experiments, the d=1.08 to 1.21 g/mL fraction (100 µL) was
incubated with 10 µL anti–rat apoA-I antibody for 1 hour. Protein A
agarose beads (40 µL of a 10% stock) were added and incubated for
another 1 hour. The protein A agarose beads were removed by
centrifugation, and the soluble lipoproteins were analyzed by negative
staining and electron microscopy.
Metabolic Labeling of Proteins and Kinetic Studies of ApoB and
ApoA-I Secretion
For determining the kinetics of apolipoprotein secretion, stably
transfected cells (60-mm Primaria dishes) were incubated for 30 minutes
in 1 mL methionine-free and serum-free DMEM containing 200 µCi of
[35S]methionine (1100 Ci/mmol; Tran35S-label,
ICN Biomedicals, Inc). After labeling, the medium was replaced with
nonradioactive medium containing 2 mmol/L methionine and chased for up
to 4 hours. ApoB or apoA-I was immunoprecipitated and
analyzed.30 Gel slices containing apolipoproteins were
dissolved in 30% hydrogen peroxide at 70°C and quantified by liquid
scintillation spectrometry in Hionic-Fluor solution (Canberra-Packard).
Intracellular retention half-time for apoB-100 was
calculated.11
In some experiments, 0.1 mmol/L oleate conjugated with 0.1% bovine serum albumin (BSA) was included in the culture medium during incubation. Cells (1.0 mg cell protein/dish) were incubated with [35S]methionine (200 µCi · mL-1 · dish-1) for 2 hours in a methionine-deficient (100 µmol/L methionine) medium supplemented with either 0.1 mmol/L oleate/0.1% BSA or 0.1% BSA alone.
Isolation of RNA and RNase Protection Assay
Total RNA was prepared from confluent transfected McA-RH7777
cells (T-75 flasks) using RNAzol (Tel-Test, Inc). Total RNA (25 µg)
was hybridized to a 32P-labeled anti-sense rat apoB RNA
probe encompassing nucleotides 1 through 425 of the rat apoB clone rb9E
corresponding to nucleotides 10 691 through 11 116 of human apoB
cDNA.32 This probe, derived from sequences 3' downstream
of the apoB-48 cDNA, does not react with the recombinant human apoB-48
mRNA sequences. In vitro transcription was performed by using T7
polymerase33 and [32P]UTP (New England
Nuclear). Total RNA (5 µg) was used for the quantification of the
glyceraldehyde phosphate dehydrogenase (GAPDH) mRNAs by using a probe
obtained by in vitro transcription of a linearized plasmid (Ambion).
The hybrids were digested with ribonuclease T1, and the protected
fragments were separated on a 6% polyacrylamide sequencing gel and
autoradiographed. Radioactivity associated with the individual
fragments was quantified by scintillation counting (Packard).
Other Methods
Rat or human plasma was obtained from fasted subjects, and
lipoprotein standards (VLDL, d<1.006; LDL,
d=1.006 to 1.063; and HDL, d=1.063 to 1.21 g/mL)
were isolated by sequential flotation. Protein was determined either by
the microtiter plate BCA assay or Coomassie Plus reagent according to
the manufacturer's protocol (Pierce Chemical Co) using BSA as a
standard.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Generation of Stable Transformants of McA-RH7777 Cells Expressing Recombinant Human ApoB-48
A human apoB-48 cDNA construct (pB48) was prepared in the expression plasmid pRc/CMV (Fig 1A
). It contained a stop codon (instead
of Gln) at position 2153 and lacked the 3' nucleotide sequences of the
editing site required for apoB mRNA editing; thus, the synthesis of
apoB-48 was independent of apoB mRNA editing. The plasmid was stably
transfected into the rat hepatoma cell line McA-RH7777, and three
clones (designated B48-13, B48-15, and B48-16) that secreted apoB-48
were obtained. Immunoblot analysis using monoclonal antibody 1D1,
which is specific for human apoB and recognizes residues 474 through
539,34 detected apoB-48 in the medium of all three cell
lines but did not react with proteins present in the medium
obtained from neotransfected cells (Fig 1B). As expected, the antibody
also reacted with human apoB-100 in LDL (Fig 1B). Other recombinant
human apoB-48 specific antibodies, eg, antibody 2110 (data not shown)
and 2140 (Fig 1C), which were raised against synthetic peptides
(residues 2110 through 2122 and 2140 through 2151, respectively) of
apoB-100,35 also reacted specifically with the recombinant
human apoB-48 proteins. The level of recombinant human apoB-48
secretion as determined by enzyme-linked immunoassay was similar in all
three cell lines: B48-13, 60.5; B48-15, 41.4; and B48-16, 53.5 ng
apoB-48 · h-1 · mg cell
protein-1.
Characterization of Lipoproteins Secreted by Stably Transfected
McA-RH7777 Cells
The flotation properties of the secreted lipoproteins were
determined by ultracentrifugation in a salt density gradient.
Immunoblot analysis demonstrated that recombinant human apoB-48 was
associated with lipoproteins with a broad density range of
d=1.06 to 1.21 g/mL (Fig 2A), similar to that
of endogenous rat apoB-48 (Fig 2B and 2C). The endogenous rat apoB-100
was confined to the lipoproteins of d=1.03 to 1.05 g/mL.
Expression of recombinant human apoB-48 had no effect on the buoyant
density of endogenous apoB-100– and apoB-48–containing lipoproteins,
but it reduced the amount of secreted apoB-100 (compare Fig 2B with
2C).
|
The secreted lipoproteins of d=1.08 to 1.21 g/mL, which do
not contain apoB-100–containing lipoproteins, were spherical as
examined by negative staining and electron microscopy (Fig 3A). The average diameter of these particles was 18±6
nm (n=214). Since the d=1.08 to 1.21 g/mL fraction also
contained apoA-I–containing particles, the samples were subjected to
immunoaffinity chromatography to remove apoA-I–containing particles
and reexamined. The average diameter of the purified
apoB-48–containing particles was 21±11 nm (n=161), which was
significantly larger than those of total HDL (14±8 nm; n =183) (Fig 3B).
|
Apolipoproteins present in apoB-48–containing lipoproteins were
examined by agarose gel electrophoresis followed by immunoblot
analysis (Fig 4). Lipoproteins containing
recombinant apoB-48 exhibited ß-mobility similar to that of human LDL
(Fig 4A) and endogenous apoB-48 (data not shown). Probing the same blot
with anti-apoE (Fig 4B) or anti–apoA-I (Fig 4C) antibodies revealed
that apoE and apoA-I were associated mainly with particles of pre–ß-
or 
-electrophoretic mobilities, suggesting that apoB-48–containing
lipoproteins contained minimal amounts of these exchangeable
apolipoproteins. Overexpression of recombinant human apoB-48 did not
affect the amount of apoE or apoA-I in the conditioned medium (Fig 4B and 4C). Size determination of lipoproteins by native gradient PAGE
demonstrated that the apparent diameter of particles containing
recombinant human apoB-48 was 16 nm (Fig 4D), whereas the apparent
diameters of endogenous apoB-48– and apoB-100–containing
particles were 16 and 20 nm, respectively (Fig 4E). Again, markedly
decreased apoB-100 was observed in the apoB-48–transfected cells
compared with control cells (Fig 4E).
|
2 mg cell protein/60-mm dish, overnight) was
concentrated, and aliquots of samples were electrophoresed on either
(A, B, and C) agarose gels or (D and E) gradient (3%-10%)
polyacrylamide gels under nondenaturing conditions. After
electrophoresis, lipoproteins were transferred to nitrocellulose
membranes, and apolipoproteins were detected with appropriate
antibodies as indicated. Positions of ß-, pre–ß-, or
In summary, recombinant human apoB-48 produced by the transfected McA-RH7777 cells exhibited physicochemical properties (size, buoyant density, and electrophoretic mobility) indistinguishable from those of endogenous apoB-48. While overexpression of recombinant human apoB-48 had no effect on the secretion of endogenous apoE or apoA-I, it decreased the secretion of rat apoB-100. We performed the following experiments to quantify the effect of apoB-48 expression on rat apoB-100 synthesis and secretion.
Effect of Overexpression of ApoB-48 on the Secretion of Endogenous
ApoB-100
The relative amounts of apolipoproteins secreted by the
transfected cells were determined by immunoblot analysis (Fig 5). The amount of apoB-100 secreted by
apoB-48–transfected cells was 80% less than that secreted by control
cells as determined by the quantification of 125I-labeled
counts (Fig 5A). In five different experiments the average amount of
apoB-100 secreted by apoB-48–transfected cells was 50% less than
control cells as determined by densitometric scanning of the gels. No
differences were observed in the amounts of secreted apoA-I (n=5) or
apoE (n=5) by apoB-48–transfected and control cells (Fig 5B and 5C).
|
To examine whether the effect of recombinant human apoB-48 was specific
or other apoB polypeptides also decrease endogenous apoB-100 synthesis,
we studied the amount of apoB-100 secreted by McA-RH7777 cells
transfected with human apoB-18 cDNA compared with nontransfected cells
(Fig 5).28 The amount of apoB-100 secreted by apoB-18
transfected cells was 60% less than that secreted by control cells. In
three different experiments the average amount of apoB-100 secreted by
apoB-18–transfected cells was 40% less than that secreted by
nontransfected control cells. Again no differences were observed in the
amounts of apoA-I and apoE secreted by nontransfected and
apoB-18–transfected cells. These data suggested that the
overexpression of apoB-48 or apoB-18 decreased the secretion of
endogenous apoB-100. Several possible reasons for the decreased levels
of apoB-100 in the medium of apoB-48–transfected cells were
considered.
Effect of ApoB-48 Overexpression on the Posttranslational
Processing of Rat ApoB-100
Overexpression of apoB-48 may deplete the intracellular lipid pool
required for the assembly of apoB-100 and thus will result in the
secretion of denser apoB-100–containing lipoproteins. Examination of
the total 35S-labeled apolipoproteins secreted by the
transfected cells showed that apoB-48 transfection had no significant
effect on the density distribution of endogenous apoB-100, apoE, or
apoA-I (Fig 6). Visual inspection of the fluorograms
revealed no significant differences in apoE or apoA-I secretion between
the apoB-48–transfected and control cells. However, markedly reduced
levels of apoB-100 were observed (compare fraction 20 in the top and
bottom panels of Fig 6).
|
Intracellular degradation and kinetics of apoB-100 secretion were
analyzed by pulse-chase experiments (Fig 7). The
intracellular retention half-time (the time required for 50% of the
newly synthesized apoB to disappear from the cells) of apoB-100 in
apoB-48–transfected and neotransfected cells (113 and 129 minutes,
respectively) was similar. The secretion efficiency (the portion of
newly synthesized apoB that was secreted) of apoB-100 was not altered
in the apoB-48–transfected cells compared with control cells (Fig 7C).
|
In the apoB-48–transfected cells, supplementing the medium with
oleate resulted in a twofold increased secretion of
35S-labeled apoB-100 in 2 hours (Fig 8).
However, oleate supplementation had no stimulatory effect on apoB-48.
These data suggested that endogenous apoB-100 was responsive to
intracellular, posttranslational regulatory mechanisms and that the
overexpression of apoB-48 did not affect the posttranslational
processing of endogenous apoB-100.
|
Effect of ApoB-48 Overexpression on the Synthesis of Endogenous
ApoB-100
To further understand the mechanism of decreased secretion of
apoB-100 by apoB-48–transfected cells, the amounts of apoB-100
synthesized at steady state by control and apoB-48–transfected cells
were determined by labeling cells with [35S]methionine
for 30 minutes followed by immunoprecipitation. Preliminary experiments
showed that during this labeling time there was a linear incorporation
of [35S]methionine into apoB in McA-RH7777 cells. Under
these conditions a decreased incorporation in apoB-100 was observed
(data not shown). Secretion of apolipoproteins associated with VLDL and
LDL is decreased in postconfluent cultures of McA-RH7777
cells.36 To eliminate the possibility that decreased
synthesis of apoB-100 was due to differences in cell densities, the
amount of apoB-100 synthesized by apoB-48–transfected cells was
studied in cells plated at different cell densities (Fig 9). At all cell densities the amount of apoB-100
synthesized was decreased compared with neotransfected control cells
(Fig 9A). Such a decrease was not observed for apoA-I (Fig 9B). The
amount of apoB-100 synthesized by apoB-48–transfected cells was 35%
to 50% lower than that synthesized by neotransfected cells (Fig 9C).
These studies suggested that the decreased amount of apoB-100 in the
medium of apoB-48–transfected cells was probably due to decreased
synthesis.
|
The decreased apoB-100 synthesis was not attributable to low
steady-state levels of the apoB-100 mRNA. An RNase protection assay
using a probe that reacted with the 3' end of the apoB-100 message
showed that the rat apoB-100 mRNA levels did not decrease but actually
increased (1.6- to 2.9-fold) in the apoB-48–transfected cells (Fig 10 and the Table). The steady-state
levels of the GAPDH mRNA did not differ between apoB-48–transfected
and control cells.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We overexpressed human recombinant apoB-48 in rat hepatoma McA-RH7777 cells. A thorough characterization of the physicochemical properties of the recombinant human apoB-48 and the apoB-48–containing lipoproteins by density gradient ultracentrifugation, nondenaturing gradient gel electrophoresis, electron microscopy, agarose gel electrophoresis, and immunoblot analysis indicated that the recombinant human apoB-48 synthesized by the transfected cells possessed characteristics indistinguishable from those of endogenous rat apoB-48. Nonetheless, overexpression of apoB-48 resulted in changes in the secretion pattern of apoB: the ratio of apoB-48/apoB-100 secreted by the recombinant human apoB-48–transfected cells resembled that seen in liver perfusion experiments performed on fed rats (which secrete more apoB-48), whereas the ratio of apoB-48/apoB-100 secreted by wild type McA-RH7777 cells resembled that of hepatocytes from fasted rats (which secrete more apoB-100).6 9 Changes in the secretion pattern of hepatic apoB, in which there is an increase in hepatic apoB-48 synthesis, have been correlated with beneficial effects on reducing plasma cholesterol-rich lipoproteins. For example, Greeve et al5 report that high hepatic apoB mRNA editing activity and increased apoB-48 synthesis tend to correlate with relatively low levels of VLDL and LDL.
Overexpression of recombinant human apoB-48, under the control of a
heterologous viral promoter and independent of the apoB mRNA editing
mechanism, decreased the secretion of endogenous apoB-100 (Figs 2, 4, 5, and 6). Decreased secretion was due to decreased synthesis of
apoB-100 at steady state (Fig 9), but decreased synthesis was not due
to decreased transcription of the gene (Fig 10). The mechanism by which
overexpression of apoB-48 decreased the synthesis or translation of
endogenous apoB-100 could not be elucidated, but several possibilities
were considered.
Overexpression of apoB-48 had no significant effect on the
intracellular posttranslational processing (degradation, retention
half-time, or secretion efficiency) of endogenous apoB-100 (Fig 7). In
addition, oleic acid treatment increased the secretion of endogenous
apoB-100 by transfected cells (Fig 8). These results agree with several
studies demonstrating that lipid availability affects apoB secretion at
the posttranslational level. Oleic acid treatment decreases
intracellular degradation and increases secretion of apoB-100 in HepG2
cells.21 Three different pools of nascent apoB-100,
tightly bound to membrane, luminal HDL, and luminal LDL/VLDL-like
particles, have been documented in HepG2 cells.37 38 Only
the LDL/VLDL-like particles were shown to be secreted. Boren et
al37 have demonstrated that oleic acid has no effect on
the initiation of translation of apoB but increases the pool of apoB
that is destined for secretion, ie, LDL/VLDL-like particles. In
McA-RH7777 cells, oleic acid has no effect on the translation of apoB
mRNA but increased the secretion of apoB-containing lipoproteins by
reducing presecretory degradation of apoB.39 Other fatty
acids also affect intracellular degradation and secretion of apoB in
rat hepatocytes and McA-RH7777 cells.11 40 Since in the
present studies no effect was observed on the intracellular
degradation of endogenous apoB-100 and oleic acid increased the
secretion of apoB-100, it can be concluded that the regulation of
posttranslational processing of apoB is probably not affected by the
overexpression of apoB-48.
Recombinant human apoB-48 probably does not decrease apoB-100 translation due to insufficient supply of lipids because apoB translation is not affected by the availability of lipids. For example, oleic acid supplementation of HepG2 cells,21 37 McA-RH7777 cells,39 40 and rat hepatocytes11 stimulate triglyceride synthesis but have no effect on apoB synthesis. In all the studies discussed above oleate stimulated the secretion of apoB by affecting posttranslational processes rather than translational yields. Moreover, a genetic defect in microsomal triglyceride transport protein, which is expected to result in decreased availability of lipids during lipoprotein synthesis, does not affect the synthesis of apoB.41 In these patients apoB is synthesized and degraded. This is not to imply that the rate of hepatic apoB synthesis is invariant. ApoB synthesis is decreased in hepatocytes obtained from diabetic rats19 and in insulin-treated HepG2 cells18 ; under these conditions, intracellular lipids are usually increased (for review see Reference 1717 ). Therefore, it appears that lipid availability does not positively correlate with apoB synthesis. Thus, it is very unlikely that the decreased synthesis of apoB-100 in apoB-48–transfected cells is due to decreased lipid availability.
One possible mechanism for the decreased synthesis of apoB-100 in apoB-48–transfected cells is a competition at the translation-translocation level. Like other secretory proteins, apoB-100 is synthesized on ribosomes bound to the endoplasmic reticulum and is cotranslationally translocated across the membranes. The overexpression of apoB-48 most likely interferes with apoB-100 synthesis prior to or during apoB-100 mRNA targeting to specific translation-translocation channels. This is expected to result in decreased synthesis of apoB-100 at steady state. Evidence has been presented for other mRNAs for the existence of domain structures within the endoplasmic reticulum and for the translation-independent targeting of mRNAs to these domains.42 In a recently formulated theoretical "square lattice" model, Chen et al43 have proposed that when the translation-translocation of full-length apoB is retarded by "pause" sequences (which presumably mediate transient stop and restart of apoB translocation44 45 ), overexpression of the short apoB messages might interfere with apoB-100 translation-translocation. This would result in decreased translational yields. Whether apoB-100 translation is inhibited by competition for the translation-translocation channels in the transfected cells needs to be explored further.
The observation that the overexpression of apoB-48 and apoB-18 decreases the secretion of apoB-100 may provide a partial explanation for the lower than expected levels of LDL found in individuals heterozygous for familial hypobetalipoproteinemia.15 46 Heterozygous individuals have apoB-100 concentrations less than half those of normal individuals. Our results suggest that hepatic expression of smaller peptides may decrease the amount of apoB-100 secreted in individuals expressing truncated forms of apoB. Further investigation of the relation between the synthesis of apoB-100 and the truncated apoB species and comparison to other models, such as the HepG2 cells in which one allele of the apoB gene is inactivated by gene targeting,47 may provide insights into the mechanisms responsible for the low levels of LDL cholesterol in heterozygous hypobetalipoproteinemia.
In summary, stably transfected recombinant human apoB-48 in rat hepatoma McA-RH7777 cell lines was obtained, and lipoproteins secreted by these cells were characterized. The overexpression of apoB-48 exerts a beneficial effect by a novel mechanism, ie, by decreasing the synthesis and secretion of apoB-100 independent of apoB mRNA editing activity. This is the first example in which the overexpression of apoB-48 has been demonstrated to decrease the synthesis of apoB-100. ApoB-48 probably competes at the translation-translocation step of apoB synthesis and can be an important regulatory step in the production of apoB-100–containing lipoproteins.
|
Acknowledgments |
|---|
This work was supported in part by Grants-in-Aid from the American Heart Association, National Center and Southeastern Pennsylvania affiliate, the W.W. Smith Trust Foundation (Dr Hussain), and the Medical Research Council of Canada (Dr Yao). Dr Yao is a Research Scholar of the Alberta Heritage Foundation for Medical Research and the Heart and Stroke Foundation of Canada. The authors wish to thank Drs R. Milne, Y. Marcel, R. Davis, T. Innerarity, S. Young, and K. Weisgraber for antibodies; Dr E. Fisher for helpful discussions as well as the PGEM4Z vector containing the rat apoB cDNA; and Dr R. McLeod for various insights into the project.
Received August 4, 1994; accepted January 17, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Kane JP, Havel RJ. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 6th ed. New York, NY: McGraw-Hill Information Services Co; 1989:1139-1164.
2. Powell LM, Wallis SC, Pease RJ, Edwards WH, Knott TJ, Scott J. A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine. Cell. 1987;50:831-840. [Medline] [Order article via Infotrieve]
3.
Chen S-H, Habib G, Yang C-Y, Gu Z-W, Lee BR, Weng S-A,
Silberman SR, Cai S-J, Deslypere JP, Rosseneu M, Gotto AMJ, Li W-H,
Chan L. Apolipoprotein B-48 is the product of a messenger RNA with an
organ-specific in-frame stop codon. Science. 1987;238:363-366.
4. Hospattankar AV, Higuchi K, Law SW, Meglin N, Cartwright IJ, Brewer HB Jr. Identification of a novel in-frame translational stop codon in human apoB mRNA. Biochem Biophys Res Commun. 1987;148:279-285. [Medline] [Order article via Infotrieve]
5. Greeve J, Altkemper I, Dieterich J-H, Greten H, Windler E. Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins. J Lipid Res. 1993;34:1367-1383. [Abstract]
6. Marsh JB, Sparks CE. The effect of fasting on the secretion of lipoproteins and two forms of apo B by perfused rat liver. Proc Soc Exp Biol Med. 1982;170:178-181. [Medline] [Order article via Infotrieve]
7. Fainaru M, Felker TE, Hamilton RL, Havel RJ. Evidence that a separate particle containing ß-apoprotein is present in high density lipoproteins from perfused rat liver. Metab Clin Exp. 1977;26:999-1004.
8. Hussain MM, Zanni EE, Kelly M, Zannis VI. Synthesis, modification, and flotation properties of rat hepatocyte apolipoproteins. Biochim Biophys Acta. 1989;1001:90-101.[Medline] [Order article via Infotrieve]
9. Windmueller HG, Spaeth AE. Regulated biosynthesis and divergent metabolism of three forms of hepatic apolipoprotein B in the rat. J Lipid Res. 1985;26:70-81. [Abstract]
10.
Davis RA, Engelhorn SC, Pangburn SH, Weinstein DB, Steinberg
D. Very low density lipoprotein synthesis and secretion by cultured rat
hepatocytes. J Biol Chem. 1985;254:2010-2016.
11. Wang H, Chen X, Fisher EA. N-3 fatty acids stimulate intracellular degradation of apolipoprotein B in rat hepatocytes. J Clin Invest. 1993;91:1380-1389.
12.
Sorci-Thomas M, Wilson MD, Johnson FL, Williams DL, Rudel LL.
Studies on the expression of genes encoding apolipoproteins B100 and
B48 and the low density lipoprotein receptor in nonhuman primates.
J Biol Chem. 1989;264:9039-9045.
13.
Baum CL, Teng B, Davidson NO. Apolipoprotein B messenger RNA
editing in the rat liver: modulation by fasting and refeeding a high
carbohydrate diet. J Biol Chem. 1990;265:19263-19270.
14. Boren J, White A, Wettesten M, Scott J, Graham L, Olofsson S-O. The molecular mechanism for the assembly and secretion of apoB100-containing lipoproteins. Prog Lipid Res. 1991;30:205-218. [Medline] [Order article via Infotrieve]
15.
Young SG. Recent progress in understanding apolipoprotein B.
Circulation. 1990;82:1574-1594.
16. Yao Z, McLeod RS. Synthesis and secretion of hepatic apolipoprotein B-containing lipoproteins. Biochim Biophys Acta. 1994;1212:152-166. [Medline] [Order article via Infotrieve]
17. Gibbons GF. Assembly and secretion of hepatic very-low-density lipoprotein. Biochem J. 1990;268:1-13. [Medline] [Order article via Infotrieve]
18. Adeli K, Theriault A. Insulin modulation of human apolipoprotein B mRNA translation: studies in an in vitro cell-free system from HepG2 cells. Biochem Cell Biol. 1992;70:1301-1312. [Medline] [Order article via Infotrieve]
19. Sparks JD, Zolfaghari R, Sparks CE, Smith HC, Fisher EA. Impaired hepatic apolipoprotein B and E translation in streptozotocin diabetic rats. J Clin Invest. 1992;89:1418-1430.
20. Dixon JL, Ginsberg HN. Regulation of hepatic secretion of apolipoprotein B-containing lipoproteins: information obtained from cultured liver cells. J Lipid Res. 1993;34:167-179. [Abstract]
21.
Dixon JL, Furukawa S, Ginsberg HN. Oleate stimulates secretion
of apolipoprotein B-containing lipoproteins from Hep G2 cells by
inhibiting early intracellular degradation of apolipoprotein B. J
Biol Chem. 1991;266:5080-5086.
22.
Sparks JD, Sparks CE. Insulin modulation of hepatic synthesis
and secretion of apolipoprotein B by rat hepatocytes. J Biol
Chem. 1990;265:8854-8862.
23. Leighton JK, Joyner J, Zamarripa J, Deines M, Davis RA. Fasting decreases apolipoprotein B mRNA editing and the secretion of small molecular weight apoB by rat hepatocytes: evidence that the total amount of apoB secreted is regulated post-transcriptionally. J Lipid Res. 1990;31:1663-1668. [Abstract]
24.
Davidson NO, Powell LM, Wallis SC, Scott J. Thyroid hormone
modulates the introduction of a stop codon in rat liver apolipoprotein
B messenger RNA. J Biol Chem. 1988;263:13482-13485.
25.
Thorngate FE, Raghow R, Wilcox HG, Werner CS, Heimberg M, Elam
MB. Insulin promotes the biosynthesis and secretion of apolipoprotein
B-48 by altering apolipoprotein B mRNA editing. Proc Natl Acad
Sci U S A. 1994;91:5392-5396.
26.
Yao Z, Blackhart BD, Johnson DF, Taylor SM, Haubold KW,
McCarthy BJ. Elimination of apolipoprotein B48 formation in rat
hepatoma cell lines transfected with mutant human apolipoprotein B cDNA
constructs. J Biol Chem. 1992;267:1175-1182.
27.
Blackhart BD, Yao Z, McCarthy BJ. An expression system for
human apolipoprotein B100 in a rat hepatoma cell line. J Biol
Chem. 1990;265:8358-8360.
28.
Yao Z, Blackhart BD, Linton MF, Taylor SM, Young SG, McCarthy
BJ. Expression of carboxyl-terminally truncated forms of human
apolipoprotein B in rat hepatoma cells: evidence that the length of
apolipoprotein B has a major effect on the buoyant density of the
secreted lipoproteins. J Biol Chem. 1991;266:3300-3308.
29.
Yao Z, Vance DE. The active synthesis of phosphatidylcholine
is required for very low density lipoprotein secretion from rat
hepatocytes. J Biol Chem. 1988;263:2998-3004.
30.
McLeod RS, Zhao Y, Selby SL, Westerlund J, Yao Z.
Carboxyl-terminal truncation impairs lipid recruitment by
apolipoprotein-B100 but does not affect secretion of the truncated
apolipoprotein-B-containing lipoproteins. J Biol
Chem. 1994;269:2852-2862.
31. Anderson LJ, Boyles JK, Hussain MM. A rapid method for staining large chylomicrons. J Lipid Res. 1989;30:1819-1824. [Abstract]
32. Reuben MA, Svenson KL, Doolittle MH, Johnson DF, Lusis AJ, Elovson J. Biosynthetic relationships between three rat apolipoprotein B peptides. J Lipid Res. 1988;29:1337-1347. [Abstract]
33. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Press; 1989.
34.
Milne R, Theolis R Jr, Maurice R, Pease RJ, Weech PK, Rassart
E, Fruchart JC, Scott J, Marcel YL. The use of monoclonal antibodies to
locate the low density lipoprotein receptor-binding domain of
apolipoprotein B. J Biol Chem. 1989;264:19754-19760.
35. Innerarity TL, Young SG, Poksay KS, Mahley RW, Smith RS, Milne RW, Marcel YL, Weisgraber KH. Structural relationship of human apolipoprotein B48 to apolipoprotein B100. J Clin Invest. 1987;80:1794-1798.
36. Tanabe S, Sherman H, Smith L, Yang LA, Fleming R, Hay R. Biogenesis of plasma lipoproteins in rat hepatoma McA-RH7777: importance of diffusion-mediated events during cell growth. In Vitro Cell Dev Biol. 1993;25:1129-1140.
37.
Boren J, Rustaeus S, Wettesten M, Andersson M, Wiklund A,
Olofsson SO. Influence of triacylglycerol biosynthesis rate on the
assembly of apoB-100–containing lipoproteins in Hep G2 cells.
Arterioscler Thromb. 1993;13:1743-1754.
38.
Boren J, Graham L, Wettesten M, Scott J, White A, Olofsson
S-O. The assembly and secretion of apoB100-containing lipoproteins in
Hep G2 cells: apoB 100 is cotranslationally integrated into
lipoproteins. J Biol Chem. 1992;267:9858-9867.
39.
White AL, Graham DL, LeGros J, Pease RJ, Scott J.
Oleate-mediated stimulation of apolipoprotein B secretion from rat
hepatoma cells. J Biol Chem. 1992;267:15657-15664.
40.
Wang H, Yao Z, Fisher EA. The effects of n-3 fatty acids on
the secretion of carboxyl-terminally truncated forms of human
apoprotein B. J Biol Chem. 1994;269:18514-18520.
41.
Wetterau JR, Aggerbeck LP, Bouma M-E, Eisenberg C, Munck A,
Hermier M, Schmitz J, Gay G, Rader DJ, Gregg RE. Absence of microsomal
triglyceride transfer protein in individuals with abetalipoproteinemia.
Science. 1992;258:999-1001.
42. Okita TW, Li X, Roberts MW. Targeting of mRNAs to domains of the endoplasmic reticulum. Trends Cell Biol. 1994;4:91-96. [Medline] [Order article via Infotrieve]
43. Chen L, Zhang J, Chan L. Apolipoprotein B synthesis: a square lattice model. J Lipid Res. 1994;35:84-92. [Abstract]
44. Chuck SL, Yao Z, Blackhart B, McCarthy BJ, Lingappa VR. New variation on the translocation of proteins during early biogenesis of apolipoprotein B. Nature. 1990;346:382-385. [Medline] [Order article via Infotrieve]
45. Chuck SL, Lingappa VR. Pause transfer: a topogenic sequence in apolipoprotein B mediates stopping and restarting of translocation. Cell. 1992;68:9-21. [Medline] [Order article via Infotrieve]
46. Linton MF, Farese RV Jr, Young SG. Familial hypobetalipoproteinemia. J Lipid Res. 1993;34:521-541. [Medline] [Order article via Infotrieve]
47. Farese RV Jr, Flynn LM, Young SG. Modification of the apolipoprotein B gene in HepG2 cells by gene targeting. J Clin Invest. 1992;90:256-261.
This article has been cited by other articles:
 |
|
 |
|
  L. Wang, D. D. O. Martin, E. Genter, J. Wang, R. S. McLeod, and D. M. Small Surface study of apoB1694-1880, a sequence that can anchor apoB to lipoproteins and make it nonexchangeable J. Lipid Res., July 1, 2009; 50(7): 1340 - 1352. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Cheng, J. Iqbal, J. Devenny, C.-H. Chu, L. Chen, J. Dong, R. Seethala, W. J. Keim, A. V. Azzara, R. M. Lawrence, et al. Acylation of Acylglycerols by Acyl Coenzyme A:Diacylglycerol Acyltransferase 1 (DGAT1): FUNCTIONAL IMPORTANCE OF DGAT1 IN THE INTESTINAL FAT ABSORPTION J. Biol. Chem., October 31, 2008; 283(44): 29802 - 29811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Iqbal, L. L. Rudel, and M. M. Hussain Microsomal Triglyceride Transfer Protein Enhances Cellular Cholesteryl Esterification by Relieving Product Inhibition J. Biol. Chem., July 18, 2008; 283(29): 19967 - 19980. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Pan and M. M. Hussain Diurnal Regulation of Microsomal Triglyceride Transfer Protein and Plasma Lipid Levels J. Biol. Chem., August 24, 2007; 282(34): 24707 - 24719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Pan, F. N. Hussain, J. Iqbal, M. H. Feuerman, and M. M. Hussain Inhibiting Proteasomal Degradation of Microsomal Triglyceride Transfer Protein Prevents CCl4-induced Steatosis J. Biol. Chem., June 8, 2007; 282(23): 17078 - 17089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Anwar, H. J. Kayden, and M. M. Hussain Transport of vitamin E by differentiated Caco-2 cells J. Lipid Res., June 1, 2006; 47(6): 1261 - 1273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Rava, G. K. Ojakian, G. S. Shelness, and M. M. Hussain Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins J. Biol. Chem., April 21, 2006; 281(16): 11019 - 11027. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Iqbal and M. M. Hussain Evidence for multiple complementary pathways for efficient cholesterol absorption in mice J. Lipid Res., July 1, 2005; 46(7): 1491 - 1501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Lapierre, D. L. Currie, Z. Yao, J. Wang, and R. S. McLeod Amino acid sequences within the {beta}1 domain of human apolipoprotein B can mediate rapid intracellular degradation J. Lipid Res., February 1, 2004; 45(2): 366 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Iqbal, K. Anwar, and M. M. Hussain Multiple, Independently Regulated Pathways of Cholesterol Transport across the Intestinal Epithelial Cells J. Biol. Chem., August 22, 2003; 278(34): 31610 - 31620. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Sakai, Y. Uchida, K. Ohashi, T. Hibuse, Y. Saika, Y. Tomari, S. Kihara, H. Hiraoka, T. Nakamura, S. Ito, et al. Measurement of fasting serum apoB-48 levels in normolipidemic and hyperlipidemic subjects by ELISA J. Lipid Res., June 1, 2003; 44(6): 1256 - 1262. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Vukmirica, K. Tran, X. Liang, J. Shan, J. Yuan, B. A. Miskie, R. A. Hegele, M. D. Resh, and Z. Yao Assembly and Secretion of Very Low Density Lipoproteins Containing Apolipoprotein B48 in Transfected McA-RH7777 Cells. LACK OF EVIDENCE THAT PALMITOYLATION OF APOLIPOPROTEIN B48 IS REQUIRED FOR LIPOPROTEIN SECRETION J. Biol. Chem., April 11, 2003; 278(16): 14153 - 14161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Burnett, J. Shan, B. A. Miskie, A. J. Whitfield, J. Yuan, K. Tran, C. J. McKnight, R. A. Hegele, and Z. Yao A Novel Nontruncating APOB Gene Mutation, R463W, Causes Familial Hypobetalipoproteinemia J. Biol. Chem., April 4, 2003; 278(15): 13442 - 13452. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Singh, O. A. Batuman, H. O. Akman, M. H. Kedees, V. Vakil, and M. M. Hussain Differential, Tissue-specific, Transcriptional Regulation of Apolipoprotein B Secretion by Transforming Growth Factor beta J. Biol. Chem., October 11, 2002; 277(42): 39515 - 39524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Vukmirica, T. Nishimaki-Mogami, K. Tran, J. Shan, R. S. McLeod, J. Yuan, and Z. Yao The N-linked oligosaccharides at the amino terminus of human apoB are important for the assembly and secretion of VLDL J. Lipid Res., September 1, 2002; 43(9): 1496 - 1507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Nayak, E. H. Harrison, and M. M. Hussain Retinyl ester secretion by intestinal cells: a specific and regulated process dependent on assembly and secretion of chylomicrons J. Lipid Res., February 1, 2001; 42(2): 272 - 280. [Abstract] [Full Text]
|
|
|
|
|
|
Z. Li, Y. Kako, L. Pang, M. W. Freeman, J. M. Glick, X. Wang, and I. J. Goldberg Effects of overexpression of the amino-terminal fragment of apolipoprotein B on apolipoprotein B and lipoprotein production J. Lipid Res., December 1, 2000; 41(12): 1912 - 1920. [Abstract] [Full Text]
|
|
|
|
 |
|
 Y. Zhao, J. B. McCabe, J. Vance, and L. G. Berthiaume Palmitoylation of Apolipoprotein B Is Required for Proper Intracellular Sorting and Transport of Cholesteroyl Esters and Triglycerides Mol. Biol. Cell, February 1, 2000; 11(2): 721 - 734. [Abstract] [Full Text]
|
|
|
|
|
|
Y. Wang, K. Tran, and Z. Yao The Activity of Microsomal Triglyceride Transfer Protein Is Essential for Accumulation of Triglyceride within Microsomes in McA-RH7777 Cells. A UNIFIED MODEL FOR THE ASSEMBLY OF VERY LOW DENSITY LIPOPROTEINS J. Biol. Chem., September 24, 1999; 274(39): 27793 - 27800. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Luchoomun and M. M. Hussain Assembly and Secretion of Chylomicrons by Differentiated Caco-2 Cells. NASCENT TRIGLYCERIDES AND PREFORMED PHOSPHOLIPIDS ARE PREFERENTIALLY USED FOR LIPOPROTEIN ASSEMBLY J. Biol. Chem., July 9, 1999; 274(28): 19565 - 19572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Hussain, A. Bakillah, N. Nayak, and G. S. Shelness Amino Acids 430-570 in Apolipoprotein B Are Critical for Its Binding to Microsomal Triglyceride Transfer Protein J. Biol. Chem., October 2, 1998; 273(40): 25612 - 25615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Reardon, L. Blachowicz, K. M. Watson, E. Barr, and G. S. Getz Association of human apolipoprotein E with lipoproteins secreted by transfected McA RH7777 cells J. Lipid Res., July 1, 1998; 39(7): 1372 - 1381. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. Luchoomun, Z. Zhou, A. Bakillah, H. Jamil, and M. M. Hussain Assembly and Secretion of VLDL in Nondifferentiated Caco-2 Cells Stably Transfected With Human Recombinant ApoB48 cDNA Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 2955 - 2963. [Abstract] [Full Text]
|
|
|
|
|
|
Y. Wang, R. S. McLeod, and Z. Yao Normal Activity of Microsomal Triglyceride Transfer Protein Is Required for the Oleate-induced Secretion of Very Low Density Lipoproteins Containing Apolipoprotein B from McA-RH7777 Cells J. Biol. Chem., May 9, 1997; 272(19): 12272 - 12278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. McLeod, Y. Wang, S. Wang, A. Rusinol, P. Links, and Z. Yao Apolipoprotein B Sequence Requirements for Hepatic Very Low Density Lipoprotein Assembly. EVIDENCE THAT HYDROPHOBIC SEQUENCES WITHIN APOLIPOPROTEIN B48 MEDIATE LIPID RECRUITMENT J. Biol. Chem., August 2, 1996; 271(31): 18445 - 18455. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wang, R. S. McLeod, D. A. Gordon, and Z. Yao The Microsomal Triglyceride Transfer Protein Facilitates Assembly and Secretion of Apolipoprotein B-containing Lipoproteins and Decreases Cotranslational Degradation of Apolipoprotein B in Transfected COS-7 Cells J. Biol. Chem., June 14, 1996; 271(24): 14124 - 14133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Gretch, S. L. Sturley, L. Wang, B. A. Lipton, A. Dunning, K. A. A. Grunwald, J. R. Wetterau, Z. Yao, P. Talmud, and A. D. Attie The Amino Terminus of Apolipoprotein B Is Necessary but Not Sufficient for Microsomal Triglyceride Transfer Protein Responsiveness J. Biol. Chem., April 12, 1996; 271(15): 8682 - 8691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Selby and Z. Yao Level of Apolipoprotein B mRNA Has an Important Effect on the Synthesis and Secretion of Apolipoprotein B–Containing Lipoproteins : Studies on Transfected Hepatoma Cell Lines Expressing Recombinant Human Apolipoprotein B Arterioscler. Thromb. Vasc. Biol., November 1, 1995; 15(11): 1900 - 1910. [Abstract] [Full Text]
|
|
|
|
|
|
M. Pan, J.-s. Liang, E. A. Fisher, and H. N. Ginsberg Inhibition of Translocation of Nascent Apolipoprotein B across the Endoplasmic Reticulum Membrane Is Associated with Selective Inhibition of the Synthesis of Apolipoprotein B J. Biol. Chem., August 25, 2000; 275(35): 27399 - 27405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Hussain, J. C. Obunike, A. Shaheen, M. J. Hussain, G. S. Shelness, and I. J. Goldberg High Affinity Binding between Lipoprotein Lipase and Lipoproteins Involves Multiple Ionic and Hydrophobic Interactions, Does Not Require Enzyme Activity, and Is Modulated by Glycosaminoglycans J. Biol. Chem., September 15, 2000; 275(38): 29324 - 29330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bakillah and M. M. Hussain Binding of Microsomal Triglyceride Transfer Protein to Lipids Results in Increased Affinity for Apolipoprotein B. EVIDENCE FOR STABLE MICROSOMAL MTP-LIPID COMPLEXES J. Biol. Chem., August 10, 2001; 276(33): 31466 - 31473. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||