© 1999 American Heart Association, Inc.
Original Contributions |
Identification of Megalin/gp330 as a Receptor for Lipoprotein(a) In Vitro
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein of unknown physiological function. The mechanism of Lp(a) atherogenicity as well as its catabolic pathways are only incompletely understood at present. In this report, we show that the low density lipoprotein receptor (LDLR) gene family member megalin/glycoprotein (gp) 330 is capable of binding and mediating the cellular uptake and degradation of Lp(a) in vitro. A mouse embryonic yolk sac cell line with native expression of megalin/gp330 but genetically deficient in LDLR-related protein (LRP) and a control cell line carrying a double knockout for both LRP and megalin/gp330 were compared with regard to their ability to bind, internalize, and degrade dioctadecyltetramethylindocarbocyanine perchlorate (DiI)-fluorescence–labeled Lp(a) as well as equimolar amounts of 125I-labeled Lp(a) and LDL. Uptake and degradation of radiolabeled Lp(a) by the megalin/gp330-expressing cells were, on average, 2-fold higher than that of control cells. This difference could be completely abolished by addition of the receptor-associated protein, an inhibitor of ligand binding to megalin/gp330. Mutual suppression of the uptake of 125I-Lp(a) and of 125I-LDL by both unlabeled Lp(a) and LDL suggested that Lp(a) uptake is mediated at least partially by apolipoprotein B100. Binding and uptake of DiI-Lp(a) resulted in strong signals on megalin/gp330-expressing cells versus background only on control cells. In addition, we show that purified megalin/gp330, immobilized on a sensor chip, directly binds Lp(a) in a Ca2+-dependent manner with an affinity similar to that for LDL. We conclude that megalin/gp330 binds Lp(a) in vitro and is capable of mediating its cellular uptake and degradation.
Key Words: megalin • glycoprotein 330 • lipoprotein(a) • LDL • LDLR gene family
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Lipoprotein(a) [Lp(a)] is a lipoprotein particle in human plasma with close structural relationship to LDL. Complexed with lipids, the major structural protein component of both LDL and Lp(a) is apolipoprotein (apo) B100. Lp(a) differs from LDL in that the former contains apo(a) as an additional apolipoprotein that is covalently linked to apoB100 (for a review, see References 1 and 21 2 ). Apo(a) is a highly polymorphic glycoprotein that shares sequence homology with plasminogen, owing to the presence of cysteine-rich protein motifs, the so-called "kringles," and an inactive protease domain that is common to several serine proteases of the blood clotting and fibrinolytic cascades.3 Genetic variation in the number of kringle repeats within the apo(a) gene results in an apo(a) size polymorphism4 and largely controls plasma concentrations of Lp(a) that display considerable interindividual differences.5
Although the physiological function of Lp(a) is unknown, numerous epidemiological studies have shown that high plasma levels of Lp(a) represent an independent risk factor for the development of coronary heart disease as well as for peripheral atherosclerosis and stroke.6 7 8 9 Furthermore, in support of the epidemiological data, Lp(a) was detected in the arterial wall of atherosclerotic patients.10 In addition to its role in cardiovascular disease, high plasma levels of Lp(a) have been described to be associated with renal pathology, such as end-stage renal disease.11 12 13
The catabolic fate of Lp(a), including the precise sites and mechanisms of its elimination from plasma, are not understood to date. Recently, an in vivo approach by Kronenberg at al14 provided the first evidence for a potential role of the kidney in Lp(a) catabolism. Furthermore, apo(a) fragments, most likely originating from plasma and ranging in size from <50 kDa to >200 kDa, have been found in human urine, without providing a detailed understanding of the underlying molecular mechanisms.15 16 17
The high structural similarity between LDL and Lp(a) has prompted numerous investigations into a potential role for the LDL receptor (LDLR) in Lp(a) catabolism. In vitro studies using different cell types as well as in vivo approaches with both transgenic mouse models and human probands initially yielded conflicting results.18 19 20 21 22 23 24 In a recent study we came to the conclusion that Lp(a) constitutes a poor ligand for both the LDLR and the LDLR-related protein (LRP) in vitro.25 Taken together, the prevailing view today is that the LDLR is capable of binding Lp(a) with low affinity but most likely does not represent a catabolic pathway of major importance in vivo.
Of all known LDLR gene family members, the VLDL receptor (VLDLR) is closest in structure to that of LDLR. On the basis of adenovirus-mediated LDLR and VLDLR overexpression in fibroblasts, Lp(a) has recently been shown to bind not only to the LDLR but also to the VLDLR.26
Megalin/glycoprotein 330 (gp330) was first identified as the major autoantigen in Heymann's nephritis, a rat model for membranous glomerulonephritis.27 28 Subsequent structural and functional work led to its classification into the LDLR gene family29 30 31 32 and to the characterization of a multitude of heterogeneous ligands for this 600-kDa endocytotic receptor. Ligands include elements of lipoprotein metabolism, of the blood clotting and fibrinolytic systems, calcium, polybasic drugs, and others,33 34 35 36 37 38 39 40 41 42 43 44 including a receptor-associated protein (RAP).35 RAP associates intracellularly with megalin/gp330 soon after receptor synthesis and functions as a molecular chaperone not only for megalin/gp330 but also for LRP and the VLDLR.45 46 47 RAP tightly binds to these 3 receptors and potently inhibits the binding of other ligands. In contrast, the interaction of RAP with the LDLR is comparatively weak.48
Megalin/gp330 is expressed in a number of resorptive, often polarity-differentiated epithelia that are heavily engaged in receptor-mediated endocytosis. Among others, these include proximal tubule epithelia in the kidney; type II pneumocytes of the lung; epithelial cells of the thyroid and parathyroid; and ependymal cells and choroid plexus in the brain, mammary gland, inner ear, retina, and yolk sac.46 49 50 Little is known at present about the physiological function of megalin/gp330. Megalin/gp330-knockout mice die perinatally, suggesting a vital role for the receptor in early development.51
Megalin/gp330 represents the only member of the LDLR gene family that has been shown to bind plasminogen as well as apoB100.33 41 These binding properties, in combination with high-abundance expression of megalin/gp330 in the kidney and the recent data reporting renal catabolism of Lp(a), prompted us to undertake the current study. We used a mouse embryonic yolk sac cell line with native expression of megalin/gp330 and a control cell line genetically deficient in megalin/gp330 to compare them with regard to their ability to interact with Lp(a).
We herein show that megalin/gp330 is capable of binding and mediating the cellular uptake and degradation of Lp(a). In addition, we provide evidence for a direct molecular interaction by demonstrating that the purified, immobilized receptor specifically binds Lp(a) in a Ca2+-dependent manner with high affinity.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Generation of Yolk Sac Cell Lines
To generate cell lines 10A and 1461, individual embryos from matings of mice heterozygous for the LRP gene defect (LRP±) were isolated at day 10.5 of gestation, and the embryonic membranes were removed. The yolk sacs were placed in ice-cold, 0.05% trypsin-EDTA solution and kept overnight at 4°C. On the following day, the samples were incubated at 37°C and disaggregated by vigorous pipetting. The cell suspension was plated on a 60-mm culture dish and grown to confluence. Individual cell clones were genotyped by Southern blot analysis to identify those heterozygous (10A) or homozygous (1461) for the LRP gene defect. To obtain cell line 6A3, mice doubly homozygous for the LRP and the megalin gene defect (LRP-/-, megalin-/-) were produced by mating of LRP± and megalin± mice. Embryos from this mating were isolated at day 10.5 of gestation, and individual yolk sac cell clones were isolated. Because we were unable to obtain doubly homozygous–deficient cell lines, we treated line 6A (LRP±, megalin-/-) with Pseudomonas aeruginosa exotoxin A, a ligand for the LRP that can be used as a negative selectable marker against cells expressing LRP.52 Accordingly, Pseudomonas aeruginosa exotoxin A–resistant cell clones were isolated from a pool of 6A cells, and the doubly deficient (LRP-/-, megalin-/-) line 6A3 was identified by Southern blot analysis. To immortalize all yolk sac lines, they were transfected with a plasmid encoding Simian virus 40 large T antigen.
Cell Culture and Protein Preparation
Cells were cultured in Dulbecco's modified Eagle's medium
(DMEM) without glutamine and with 10% FCS. For protein blotting
procedures, cells were solubilized in 1% Triton X-100, 50 mmol/L
Tris, 2 mmol/L CaCl2, and 80 mmol/L
NaCl, pH 8.0. A mix of protease inhibitors (Calbiochem) was
added, including 1 mmol/L pepstatin A, 10 mmol/L chymostatin,
10 mmol/L leupeptin, and 10 mmol/L antipain. Protein
concentrations were determined by standard procedures according to
Lowry et al.53
Gel Electrophoresis and Antibodies
SDS–polyacrylamide gel electrophoresis (PAGE) was
performed according to Neville.54 For
125I-labeled proteins, gels were dried and
exposed to an autoradiography film (Cronex, DuPont).
Nonlabeled proteins were electroblotted to nitrocellulose for
nonspecific protein stain (Ponceau S solution, Serva) and
immunodetection with specific antibodies. Polyclonal antibodies against
megalin/gp330, LRP, and the LDLR were given to us by J. Herz
(University of Texas Southwestern Medical Center, Dallas, Tex), and the
polyclonal anti-VLDLR was kindly provided by M. Gåfvels (Karolinska
Institute, Huddinge, Sweden). Peroxidase-labeled goat anti-rabbit
antibodies (Dianova) were used with chloronaphthol as the substrate for
secondary antibodies. Southern blot analyses were performed
according to standard procedures.55
Lipoprotein Purification
Lp(a) was purified from 500 mL of fresh EDTA-plasma of a healthy
proband undergoing plasmapheresis by sequential 3-step
ultracentrifugation as described
previously.25 The Lp(a) concentration was 52 mg/dL with an
apo(a) isoform of 21, as determined by SDS agarose gel electrophoresis.
Other isoforms from additional donors (S1, S2, and S3, according to the
former nomenclature) were isolated by the same method. LDL was obtained
from the same donor as described before.25
Lipoprotein Characterization and Labeling
Lipoproteins were separated on an agarose gel ready kit (Sebia
GmbH) and stained with Sudan black solution. Each Lp(a) and LDL
preparation was subjected to 5% to 7.5% SDS-PAGE under reducing
conditions, followed by electroblotting to nitrocellulose, and
subsequent Ponceau staining or immunoblotting with an
anti-apo(a) and/or apoB100 antibody56 (data not shown);
only pure preparations were used in the cell assays. For iodination the
ICl method was used.57 The protein content of the
different 125I-labeled preparations was
determined by the Lowry technique.53 Typically, the
specific activity of 125I-Lp(a) was within the
range 150 to 300 counts per minute (cpm)/ng, and that of
125I-LDL, between 50 and 150 cpm/ng. After
iodination, 125I-labeled lipoproteins were
checked again for integrity by SDS-PAGE.
Cellular Uptake and Degradation of 125I-Labeled
Ligands: uPA, tPA, and RAP
Complexes of 125I–urokinase-type
plasminogen activator (uPA)/PPACK and
125I-tissue plasminogen
activator (tPA)/YPACK were prepared as
described.58 PPACK (Phe-Pro-Arg chloromethyl ketone) and
YPACK (Tyr-Pro-Arg chloromethyl ketone) are inhibitors of
uPA and tPA catalytic activity, respectively, and are needed to prevent
uPA and tPA from exerting their protease activity in the cellular
assays. All proteins were radiolabeled by the Iodo-Gen (Pierce)
method.59 Yolk sac cells (2x105 per
well) were seeded into 12-well plates and grown for 24 hours. The
medium was replaced with DMEM (without glutamine) containing 0.2%
(wt/vol) BSA and the indicated iodinated ligands. Cellular
degradation of 125I-labeled proteins was measured
as previously described60 and is expressed as nanograms of
125I-labeled trichloroacetic acid–soluble
(noniodide) material released into the culture medium per milligram of
total cell protein.
Uptake and Degradation of 125I-Labeled
Lipoproteins
Cells (5x104/mL) were seeded routinely on
day 0 in 24-well plates and used as confluent monolayers on day 2. The
experiments were performed in DMEM containing 5% BSA (fraction V,
Sigma) and 0.02 mol/L HEPES (pH 7.4). Aliquots (5 to 70 picomoles of
125I-Lp(a) and 125I-LDL per
milliliter) were added. All data points were obtained in duplicate. For
the differentiation between total, specific, and nonspecific uptake and
degradation, a 12- to 50-fold molar excess of unlabeled lipoprotein was
added. RAP in the form of a recombinant polyhistidine fusion peptide
was kindly provided by J. Gliemann, Aarhus, Denmark, and was added at a
concentration of 30 µg/mL. For the determination of cellular uptake,
incubations were performed for 90 minutes at 37°C. After incubation,
cells were washed with PBS, pH 7.4, with 2 mg/mL BSA, followed by a
short PBS rinse without BSA. Surface-bound lipoproteins were then
released by PBS containing 770 U heparin per milliliter (Liquemin,
Roche). The cells were dissolved in 0.1 mol/L NaOH. Finally,
radioactivity and cell protein of the lysate were determined. Specific
uptake data are expressed as nanograms or femtomoles of ligand protein
per milligram of cell protein. Degradation was measured after
incubation for 90 minutes followed by a 4-hour chase. After the chase,
the media were recovered completely, and
125I-labeled trichloroacetic acid–soluble
material was determined as a direct measure of degraded
125I-apolipoproteins.
Immunofluorescence
For immunofluorescence studies, Lp(a) was
labeled with
1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate (DiI) according to standard
procedures.61 The integrity of labeled Lp(a) was checked
by agarose gel electrophoresis. Cells were grown on glass coverslips
for 2 days, washed, and incubated with 100 pmol/mL DiI-labeled Lp(a) at
37°C for 20 minutes in DMEM containing 1% BSA. Subsequently,
coverslips were washed, and surface-bound Lp(a) was released by 500
U/mL heparin (Sigma) at 4°C for 15 minutes. Cells were fixed at room
temperature in 4% paraformaldehyde and
permeabilized with methanol at -20°C for 5 minutes.
After extensive washing, blocking was performed at room temperature
with 1% BSA, 10% goat serum, and 20 mmol/L glycine in PBS. Cells
were incubated with a polyclonal antibody against megalin/gp330 from
rabbit at 37°C for 60 minutes, washed twice, and incubated at 37°C
for 45 minutes with DTAI
5-([4,6-dichlorotriazin-2-yl]amino)fluorescein–conjugated
goat anti-rabbit immunoglobulins from Dianova. Coverslips were mounted
on a glass slide with a drop of Mowiol (Calbiochem). Confocal laser
scanning microscopy was performed using a Leica TCS 4D (Leica
Lasertechnik) instrument based on an inverted Leitz DMIRBE microscope
interfaced with an Ar-Kr laser adjusted to 488 and 568 nm.
Biosensor Measurements
All measurements were performed on a BIAcore 2000 instrument
(Biosensor) equipped with CM5 sensor chips maintained at 20°C. A
continuous flow of HBS buffer (10 mmol/L HEPES, pH 7.4; 3.4
mmol/L EDTA; 0.15 mol/L NaCl; and 0.005% surfactant P20) passing over
the sensor surface was maintained at 5 µL/min. The carboxylated
dextran matrix of the sensor chip was activated by injection of
60 µL of a solution containing 0.2 mol/L
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide
and 0.05 mol/L N-hydroxysuccinimide in water.
LDLR was injected at a concentration of 40 µg/mL in 10 mmol/L
sodium acetate, pH 4.0 (total volume of 300 µL per flow cells 1
and 2). Megalin was at a concentration of 10 µg/mL in 10 mmol/L
sodium acetate, pH 4.5 (total volume of 350 µL per flow cells 3 and
4). The remaining binding sites were blocked by injection of 35 µL of
1 mol/L ethanolamine, pH 8.5. The immobilized protein in
flow cells 2 and 4 was then reduced by injection of 100 µL of 0.5%
DTT in 6 mol/L guanidine HCl, 5 mmol/L EDTA, and 50 mmol/L
Tris, pH 8.0, into each flow cell. The surface plasmon resonance signal
from immobilized LDLR generated 3107 BIAcore response units
(RUs), equivalent to 19 fmol LDLR per mm2,
and immobilized megalin generated 10 343 BIAcore RUs,
equivalent to 17 fmol megalin per mm2.
Screening of the LDL and Lp(a) samples was performed by injecting
aliquots of 80 µL at concentrations of 10 to 100 µg/mL through all
flow cells at a flow rate of 10 µL/min. The samples were dissolved in
10 mmol/L HEPES, pH 7.4; 150 mmol/L NaCl; 1.5 mmol/L
CaCl2; 1 mmol/L EDTA; and 0.005% surfactant
P20. The same buffer was used as the running buffer. The BIAcore
response is expressed in relative RUs, ie, the difference in response
between the immobilized protein flow cell and the parallel
reduced-protein flow cell. Regeneration of the sensor chip after each
analysis cycle was performed by injecting 5 µL of 0.05% SDS.
Dissociation constants were calculated from fitted curves by means of
the BIAcore software program version 4.0.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Generation of Cell Lines 1461 (LRP-/-) and 6A3 (LRP-/-, Megalin/gp330-/-)
Mouse embryonic yolk sac cell lines 1461 and 6A3 were generated for functional analysis of megalin/gp330. Because many ligands recognized by megalin/gp330 can also bind to LRP, it was mandatory to rule out potential interference from the LRP. For this purpose, both alleles of the LRP gene were disrupted in cell lines 1461 and 6A3 (Figure 1A
, lanes 1 through 3) while the alleles of the
megalin/gp330 gene were left intact in 1461 but disrupted in the
negative control cell line 6A3 only (Figure 1A, lanes 4 and 5).
Western blot analysis confirmed the complete absence of LRP
protein expression by both cell lines as well as exclusive expression
of megalin/gp330 by 1461 cells, according to their genotype
(Figure 1B, lanes 1 through 6). LDLR was expressed by cell line
1461 as well as by 6A3 cells. The amount of immunodetectable protein
without (data not shown) and after LDLR stimulation, induced by
incubation with lipoprotein-deficient serum for 2 days, was slightly
higher for 1461 cells (Figure 1B, lanes 7 through 9). VLDLR
protein could not be detected in either cell line (Figure 1B, lanes 10 through 12).
|
Functional Integrity of Megalin/gp330 Expressed by Cell Line
1461
To ensure that megalin/gp330 was functionally intact in 1461 cells
and absent in 6A3 cells, we compared 1461 and 6A3 cell lines with
regard to their ability to degrade established ligands of
megalin/gp330. Over a time course of >20 hours, GST-RAP and uPA were
efficiently degraded by megalin/gp330-expressing 1461 cells but were
degraded only to a very minor degree by the megalin/gp330-deficient
control cell line 6A3 (Figure 2A and 2B). tPA has been
reported to be degraded by LRP but not by megalin/gp330.58
Irrespective of megalin/gp330 expression, no tPA degradation was
observed by the LRP-negative cell lines 1461 and 6A3 (Figure 2C), whereas tPA was efficiently degraded by an
LRP(±)-expressing (cf Figure 1A
, lane 1) control cell line,
designated 10A.
|
Characterization of Purified Lp(a) and LDL
Freshly isolated human Lp(a) and LDL from a healthy human subject
with the slowly migrating, large apo(a) isoform 21 were checked for
purity and integrity before and after 125I
radiolabeling. Figure 3A shows the agarose gel electrophoresis
pattern of the major lipoproteins from the donor. To ensure that only
intact lipoprotein preparations were used, SDS-PAGE of unlabeled
(Figure 3B) and radiolabeled (Figure 3C) Lp(a) and LDL
was routinely performed in addition. These tests demonstrated that the
preparations contained neither significant amounts of degradation
products nor contamination by other proteins. In particular, to
check for the presence of other receptor-binding apolipoproteins,
higher-percentage gels (12% to 15%) of Lp(a) and LDL were
immunoblotted against apoE and apoAI. Neither one was
detectable (data not shown).
|
Megalin/gp330-Mediated Cellular Uptake and Degradation of
125I-Lp(a)
We measured the cellular uptake and degradation
of125I-Lp(a) by the cell line 1461, which
expresses native amounts of megalin/gp330. When these cells were
incubated with increasing amounts of 125I-Lp(a),
cellular degradation of 125I-Lp(a) appeared to be
saturable in a characteristic fashion (Figure 4A) as is observed
for specific receptor-ligand interactions, such as LDL degradation via
the LDLR pathway. Strikingly, specific degradation of
125I-Lp(a) made up <50% of total degradation,
leaving a substantial portion of cellular degradation whose exact
nature remains unclear at the moment. We were interested in the
specific and thus, presumably receptor-mediated portion of uptake, in
particular, asking the question whether this specificity was due to
megalin/gp330 interaction with Lp(a); therefore, we performed another
set of comparative uptake and degradation assays including the
megalin/gp330-deficient control cell line 6A3. These experiments were
performed with the addition of RAP as a highly competent and specific
inhibitor of ligand binding to megalin/gp330. Uptake
(Figure 4B) and degradation (Figure 4C) of
125I-Lp(a) were consistently higher on
megalin/330-expressing 1461 cells compared with the control 6A3 cells.
On average, uptake and degradation of 125I-Lp(a)
by the megalin/gp330-negative control cells were 
50% and 20% of
1461 cells, respectively. Furthermore, specific uptake and degradation
by megalin/gp330-expressing cells was inhibitable by RAP to a residual
40% to 50%, whereas on megalin/gp330-deficient control cells, uptake
and degradation occurred via pathways that were clearly not
RAP-sensitive (Figure 4B and 4C).
|
Owing to the biological variability that is inherent to experimental systems, the absolute values varied considerably for all radioactive assays. However, the relative differences as expressed in the figures were observed consistently, irrespective of the absolute values.
Cellular Uptake of DiI-Fluorescence–Labeled Lp(a)
Fluorescence studies confirmed the results obtained with
125I-radiolabeled Lp(a), in that uptake of
DiI-labeled Lp(a) appeared to be much more efficient on 1461 cells than
on 6A3 controls. Immunofluorescence incubations of
the 2 cell lines with a polyclonal anti-megalin/gp330 antibody resulted
in a distinct punctate signal on 1461, whereas it yielded only a
background stain on 6A3 cells (green in Figure 5A and 5D). For
fluorescence tracing of endocytosed ligands, we used 100
pmol/mL DiI-Lp(a). Uptake was allowed to continue for 20 minutes at
37°C, resulting in a clear-cut difference of intensity in the red
fluorescence signal originating from internalized DiI-Lp(a)
between the 2 cell lines. A perinuclear endosomal staining pattern was
easily detectable on 1461 cells, whereas under the same conditions, 6A3
cells took up hardly any DiI-Lp(a) (Figure 5B and 5E).
Superimposition of images 5A and 5B as well as of 5D and 5E
demonstrates that significant uptake of Lp(a) occurred only on
megalin/gp330-expressing cells (Figure 5C and 5F).
|
Direct Binding of Lp(a) to Immobilized Megalin/gp330
To obtain experimental evidence for a direct molecular interaction
between megalin/gp330 and Lp(a) as suggested by the results of the cell
assays, we analyzed the binding of Lp(a) to megalin/gp330 and
the LDLR and compared it to the binding of equimolar amounts of LDL
(BIAcore system). Purified receptors were immobilized on
flow-cell sensor chips at a concentration of 17 (for megalin/gp330) and
19 (for LDLR) fmol/mm2, and flow cells were
injected with equimolar amounts of Lp(a) and LDL over a concentration
range of 10 to 100 pmol/mL, corresponding to the concentrations used in
the various cell assays. As shown in Figure 6A, Lp(a) as well as
LDL strongly bound to megalin/gp330 in a calcium-dependent manner. The
binding of Lp(a) (Kd 1.5 nmol/L) occurred
with slightly higher affinity than did LDL binding
(Kd 3.4 nmol/L). Binding of both Lp(a) and
LDL was abolished by withdrawal of calcium (ie, in the presence of
EDTA). For the LDLR, the difference between LDL and Lp(a) binding was
much more pronounced: LDL bound strongly to the LDLR
(Kd 1.9 nmol/L) in the presence but not in
the absence of calcium. In contrast to LDL, Lp(a) binding to the LDLR
was hardly measurable and was independent of the presence of
calcium.
|
Cellular Uptake of Lp(a) and LDL by Wild-Type and
LDLR-Negative Fibroblasts
The BIAcore data show that Lp(a) can bind, if only very weakly, to
the LDLR. Because both cell lines 1461 and 6A3 express the LDLR to some
extent, we therefore aimed to rule out potential interference from the
LDLR in the Lp(a) uptake and degradation assays with 1461 and 6A3
cells. We therefore performed uptake experiments with cells more
suitable for analyzing the functional importance of the LDLR. Wild-type
human fibroblasts were compared with LDLR-negative fibroblasts derived
from a patient with familial
hypercholesterolemia (FH) with regard to their
ability to take up 125I-LDL and
125I-Lp(a). As expected, a drastic difference in
the uptake of LDL between wild-type and familial
hypercholesterolemia fibroblasts was observed
(Figure 7); however, uptake of Lp(a) did not differ much and was
considerably lower, reaching 30% of LDL uptake at maximum. These data
reflect and confirm the low affinity of Lp(a) to the LDLR (cf Figure 6B).
|
Cross-Competition of Lp(a) and LDL in Megalin/gp330-Mediated
Cellular Uptake
The cellular uptake of Lp(a) via megalin/gp330 could be mediated
by either apo(a) or apoB100 binding to the receptor. Because apoB100
has been described as a ligand for megalin/gp330 before, we
investigated whether the cellular uptake of
125I-Lp(a) could be inhibited by an excess of
unlabeled LDL and vice versa. First, we compared the uptake of
125I-LDL by the cell lines 1461 and 6A3. These
assays were performed under the same experimental conditions as the
Lp(a) uptake assays (cf Figure 4) and yielded surprisingly
similar results (Figure 8A). LDL uptake by
megalin/gp330-expressing 1461 cells was RAP-sensitive and twice as
high, on average, as uptake by the control cells, which in turn was not
inhibitable by the addition of RAP. These results suggest that a
similar or an identical cellular mechanism is responsible for the
internalization of LDL and Lp(a), a finding that is in agreement with
the comparable binding of both ligands to purified megalin/gp330
(Figure 6A). Finally, the addition of a 50-fold molar excess of
unlabeled Lp(a) and LDL to the incubation medium with either
125I-Lp(a) or 125I-LDL
resulted in a similar pattern of inhibition for both ligands on
megalin/gp330-expressing 1461 cells (Figure 8B). In the case of
125I-LDL uptake, owing to the presence of the
LDLR, the inhibition by LDL was more efficient than that by Lp(a).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In this study a cell system derived from LRP- and megalin/gp330-knockout mice embryos was used for the functional analysis of megalin/gp330 with regard to its ability to bind and mediate the cellular uptake and degradation of Lp(a) in vitro. ApoB100 has been previously described as a ligand for megalin/gp33041 ; however, receptor binding of apoB100 does not necessarily imply that the respective receptor will bind Lp(a), as shown for the LDLR, which has been under discussion as a potential Lp(a) receptor in numerous studies.18 19 20 21 22 23 24 25 Megalin/gp330 is known to be the only member of the LDLR gene family to bind the apo(a) homologue plasminogen,33 36 thus suggesting that Lp(a) might bind to megalin/gp330 by either apoB100, apo(a), or both apolipoproteins. Taking several different methodological approaches, we compared the interaction of Lp(a) and LDL with megalin/gp330 and the LDLR and demonstrated that megalin/gp330 constitutes a specific receptor for Lp(a) in vitro. We also confirmed previous data obtained by several groups, including our own,25 that came to the conclusion that the LDLR is able to bind Lp(a) but that the interaction is weak and of a smaller order of magnitude than that of the LDL-LDLR interaction. A mouse embryonic yolk sac cell line with native expression of megalin/gp330 but genetically deficient for LRP was compared with a control cell line carrying a double knockout for both megalin/gp330 and LRP. We made use of these 2 cell lines to examine whether megalin/gp330 was capable of binding and mediating the cellular uptake and degradation of Lp(a). Lp(a) was specifically taken up and degraded by 1461 cells in a saturable manner, with average values being twice as high as for control cells. The pathway on 1461 cells was sensitive to the addition of RAP, a potent inhibitor of ligand binding to megalin/gp330, suggesting that the difference between the cell lines was mediated by megalin/gp330. It is of note that there was specific uptake and degradation of Lp(a) by the control cell line, occurring via a pathway that was not RAP-sensitive and for which the mechanism remains unclear at present. However, this did not have any bearing on the presumably megalin/gp330-mediated difference between the cell lines. To confirm the results obtained with 125I-Lp(a) and to rule out potential artifacts inherent to the radiolabel, we followed the fate of DiI-labeled Lp(a) by means of fluorescence microscopy and again observed a difference in uptake between the cell lines. The indirect evidence for Lp(a) interaction with gp330/megalin obtained from the cell studies was further elaborated by binding studies investigating the direct molecular interaction of Lp(a) with immobilized, purified megalin/gp330. Experiments employing the BIAcore system enabled us to quantify the binding of equimolar amounts of Lp(a) and LDL to immobilized megalin/gp330 and LDLR, revealing that binding of Lp(a) to megalin/gp330 occurred with similar affinity as LDL binding. The binding was calcium dependent in both cases. In contrast, binding of Lp(a) to the LDLR was hardly measurable, providing further evidence that Lp(a) constitutes a poor ligand for the LDLR.25 62 63 However, because the yolk sac cell line 1461 was shown to express slightly higher amounts of LDLR than did control cells (Figure 1B
), it was essential to make sure that the weak
Lp(a) binding to the LDLR was of negligible influence within the assay
system used in this study. Therefore, LDLR-negative FH fibroblasts were
used. Uptake of Lp(a) was only 30% less than by wild-type
fibroblasts, demonstrating that even a functional null mutation of the
LDLR gene does not provoke differences as high as those observed
between the yolk sac cell lines. Therefore, the difference in uptake
and degradation of Lp(a) between the yolk sac cell lines can only be
explained by megalin/gp330. In this study, isoform 21 was used as a
standard for all experimental approaches. In cellular assays, other
isoforms behaved similarly, suggesting that the binding to
megalin/gp330 is not an isoform-specific phenomenon. However, whether
this holds true for the entire spectrum of apo(a) isoforms remains
unknown at this point and will have to be elucidated by a broader, more
systematic approach. Uptake experiments with
125I-LDL revealed the same RAP-sensitive
difference between megalin/gp330-expressing and control cell lines as
observed for 125I-Lp(a). Because apoB100 has been
described as a ligand for megalin/gp330 before,41 this
finding was not surprising but raised the question as to the mechanism
of Lp(a) binding to megalin/gp330. From the displacement studies with
unlabeled Lp(a) and LDL, showing that the megalin/gp330-mediated uptake
of Lp(a) was competitively inhibitable by both Lp(a) and LDL, we
conclude that the megalin/gp330-Lp(a) interaction involves apoB100.
Taking into account that the apo(a) homologue plasminogen
binds to megalin/gp330,33 it is still tempting to
speculate that apo(a) might be partially responsible for the mediation
of Lp(a) binding, perhaps in a concerted action with apoB100. However,
the addition of plasminogen alone did not have any effect
on Lp(a) uptake by either cell line (data not shown). Based on these
data, further studies will be needed to completely elucidate the nature
of the binding mechanism of Lp(a) to megalin/gp330. At its epithelial
expression sites in vivo, megalin/gp330 faces a specialized milieu,
such as the cerebrospinal fluid, seminal fluid, or primary urinary
filtrate.46 49 50 As of yet, there are no reports of the
existence of intact Lp(a) in these fluids. Therefore, the question
whether megalin/gp330-mediated uptake of Lp(a) can play a
physiologically important role remains open at
present. To address these questions, further investigations will
clearly be needed. In conclusion, we have identified megalin/gp330 as a specific receptor for Lp(a) in vitro as demonstrated by the combination of several methodological approaches. The cellular binding, uptake, and degradation of Lp(a) via megalin/gp330 is at least partially mediated by apoB100.
|
Acknowledgments |
|---|
This work was supported by the Austrian Science Foundation (P12358) to Hans Dieplinger and by the Deutsche Forschungsgemeinschaft (DFG) in the form of a Clinical Research Group to Ulrike Beisiegel. Andreas Niemeier was supported by the "GRK336 Molecular Endocrinology and Metabolism" grant from the DFG. We wish to thank Joachim Herz from the University of Texas Southwestern Medical Center, Dallas, and Mats Gåfvels from the Karolinska Institute, Huddinge, Sweden, for providing the antibodies. The expertise and helpful advice of Jörg Heeren (University of Hamburg, Hamburg, Germany) in the immunofluorescence studies are greatly appreciated.
Received May 19, 1998; accepted August 7, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Utermann G. Lipoprotein(a). In: Scriver CR, Beaudet AL, Sly WS, eds. The Metabolic Basis of Inherited Disease. New York, NY: McGraw-Hill Inc; 1993.
2. Kronenberg F, Steinmetz A, Kostner GM, Dieplinger H. Lipoprotein(a) in health and disease. Crit Rev Clin Lab Sci. 1996;33:495–543.[Medline] [Order article via Infotrieve]
3. McLean JW, Tomlinson JE, Kuang WJ, Eaton DL, Chen EY, Fless GM, Scanu AM, Lawn RM. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature. 1987;330:132–137.[Medline] [Order article via Infotrieve]
4. Lackner C, Boerwinkle E, Leffert CC, Rahmig T, Hobbs HH. Molecular basis of apolipoprotein (a) isoform size heterogeneity as revealed by pulsed-field gel electrophoresis. [published erratum appears in J Clin Invest.. 1991;88:723]. J Clin Invest. 1991;87:2153–2161.
5. Kraft HG, Kochl S, Menzel HJ, Sandholzer C, Utermann G. The apolipoprotein (a) gene: a transcribed hypervariable locus controlling plasma lipoprotein (a) concentration. Hum Genet. 1992;90:220–230.[Medline] [Order article via Infotrieve]
6. Kostner GM, Avogaro P, Cazzolato G, Marth E, Bittolo BG, Qunici GB. Lipoprotein Lp(a), and the risk for myocardial infarction. Atherosclerosis. 1981;38:51–61.[Medline] [Order article via Infotrieve]
7. Scanu AM. Lipoprotein(a): a potential bridge between the fields of atherosclerosis and thrombosis. Arch Pathol Lab Med. 1988;112:1045–1047.[Medline] [Order article via Infotrieve]
8. Sandholzer C, Boerwinkle E, Saha N, Tong MC, Utermann G. Apolipoprotein(a) phenotypes, Lp(a) concentration and plasma lipid levels in relation to coronary heart disease in a Chinese population: evidence for the role of the apo(a) gene in coronary heart disease. J Clin Invest. 1992;89:1040–1046.
9. Sigurdsson G, Baldursdottir A, Sigvaldason H, Agnarsson U, Thorgeirsson G, Sigfusson N. Predictive value of apolipoproteins in a prospective survey of coronary artery disease in men. Am J Cardiol. 1992;69:1251–1254.[Medline] [Order article via Infotrieve]
10. Rath M, Niendorf A, Reblin T, Dietel M, Krebber HJ, Beisiegel U. Detection and quantification of lipoprotein(a) in the arterial wall of 107 coronary bypass patients. (published erratum appears in Arteriosclerosis. 1990;10:1147). Arteriosclerosis. 1989;9:579–592.
11. Dieplinger H, Lackner C, Kronenberg F, Sandholzer C, Lhotta K, Hoppichler F, Graf H, Konig P. Elevated plasma concentrations of lipoprotein(a) in patients with end-stage renal disease are not related to the size polymorphism of apolipoprotein(a). J Clin Invest. 1993;91:397–401.
12.
Kronenberg F, Konig P, Lhotta K, Ofner D, Sandholzer C,
Margreiter R, Dosch E, Utermann G, Dieplinger H. Apolipoprotein(a)
phenotype-associated decrease in lipoprotein(a) plasma
concentrations after renal transplantation. Arterioscler
Thromb. 1994;14:1399–1404.
13. Kronenberg F, Utermann G, Dieplinger H. Lipoprotein(a) in renal disease. Am J Kidney Dis. 1996;27:1–25.[Medline] [Order article via Infotrieve]
14. Kronenberg F, Trenkwalder E, Lingenhel A, Friedrich G, Lhotta K, Schober M, Moes N, Konig P, Utermann G, Dieplinger H. Renovascular arteriovenous differences in Lp[a] plasma concentrations suggest removal of Lp[a] from the renal circulation. J Lipid Res. 1997;38:1755–1763.[Abstract]
15. Mooser V, Marcovina SM, White AL, Hobbs HH. Kringle-containing fragments of apolipoprotein(a) circulate in human plasma and are excreted into the urine. J Clin Invest. 1996;98:2414–2424.[Medline] [Order article via Infotrieve]
16. Mooser V, Seabra MC, Abedin M, Landschulz KT, Marcovina S, Hobbs HH. Apolipoprotein(a) kringle 4-containing fragments in human urine: relationship to plasma levels of lipoprotein(a). J Clin Invest. 1996;97:858–864.[Medline] [Order article via Infotrieve]
17.
Kostner KM, Maurer G, Huber K, Stefenelli T, Dieplinger
H, Steyrer E, Kostner GM. Urinary excretion of apo(a) fragments: role
in apo(a) catabolism. Arterioscler Thromb Vasc Biol. 1996;16:905–911.
18. Maartmann MK, Berg K. Lp(a) lipoprotein enters cultured fibroblasts independently of the plasma membrane low density lipoprotein receptor. Clin Genet. 1981;20:352–362.[Medline] [Order article via Infotrieve]
19. Havekes L, Vermeer BJ, Brugman T, Emeis J. Binding of LP(a) to the low density lipoprotein receptor of human fibroblasts. FEBS Lett. 1981;132:169–173.[Medline] [Order article via Infotrieve]
20. Krempler F, Kostner GM, Roscher A, Haslauer F, Bolzano K, Sandhofer F. Studies on the role of specific cell surface receptors in the removal of lipoprotein (a) in man. J Clin Invest. 1983;71:1431–1441.
21. Widhalm K, Genser D. Increased lipoprotein(a) levels in children with familial hypercholesterolaemia. [letter]. Lancet. 1988;2:1262.
22. Hofmann SL, Eaton DL, Brown MS, McConathy WJ, Goldstein JL, Hammer RE. Overexpression of human low density lipoprotein receptors leads to accelerated catabolism of Lp(a) lipoprotein in transgenic mice. J Clin Invest. 1990;85:1542–1547.
23.
Utermann G, Hoppichler F, Dieplinger H, Seed M,
Thompson G, Boerwinkle E. Defects in the low density lipoprotein
receptor gene affect lipoprotein (a) levels: multiplicative interaction
of two gene loci associated with premature
atherosclerosis. Proc Natl Acad Sci U S A. 1989;86:4171–4174.
24.
Utermann G. The mysteries of lipoprotein(a).
Science. 1989;246:904–910.
25. Reblin T, Niemeier A, Meyer N, Willnow TE, Kronenberg F, Dieplinger H, Greten H, Beisiegel U. Cellular uptake of lipoprotein[a] by mouse embryonic fibroblasts via the LDL receptor and the LDL receptor-related protein. J Lipid Res. 1997;38:2103–2110.[Abstract]
26. Argraves KM, Kozarsky KF, Fallon JT, Harpel PC, Strickland DK. The atherogenic lipoprotein Lp(a) is internalized and degraded in a process mediated by the VLDL receptor. J Clin Invest. 1997;100:2170–2181.[Medline] [Order article via Infotrieve]
27.
Kerjaschki D, Farquhar MG. The pathogenic antigen of
Heymann nephritis is a membrane glycoprotein of the renal
proximal tubule brush border. Proc Natl Acad Sci U S A. 1982;79:5557–5581.
28.
Kerjaschki D, Ullrich R, Diem K, Pietromonaco S,
Orlando RA, Farquhar MG. Identification of a pathogenic epitope
involved in initiation of Heymann nephritis. Proc Natl Acad Sci
U S A. 1992;89:11179–11183.
29.
Raychowdhury R, Niles JL, McCluskey RT, Smith JA.
Autoimmune target in Heymann nephritis is a glycoprotein
with homology to the LDL receptor. Science. 1989;244:1163–1165.
30.
Chen WJ, Goldstein JL, Brown MS. NPXY, a sequence often
found in cytoplasmic tails, is required for coated pit-mediated
internalization of the low density lipoprotein receptor. J
Biol Chem. 1990;265:3116–3123.
31.
Saito A, Pietromonaco S, Loo AK, Farquhar MG. Complete
cloning and sequencing of rat gp330/"megalin," a distinctive member
of the low density lipoprotein receptor gene family. Proc Natl
Acad Sci U S A. 1994;91:9725–9729.
32. Hjalm G, Murray E, Crumley G, Harazim W, Lundgren S, Onyango I, Ek B, Larsson M, Juhlin C, Hellman P, Davis H, Akerstrom G, Rask L, Morse B. Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential intracellular signaling properties. Eur J Biochem. 1996;239:132–137.[Medline] [Order article via Infotrieve]
33.
Kanalas JJ, Makker SP. Identification of the rat
Heymann nephritis autoantigen (GP330) as a receptor site for
plasminogen. J Biol Chem. 1991;266:10825–10829.
34. Christensen EI, Gliemann J, Moestrup SK. Renal tubule gp330 is a calcium binding receptor for endocytic uptake of protein. J Histochem Cytochem. 1992;40:1481–1490.[Abstract]
35.
Kounnas MZ, Argraves WS, Strickland DK. The 39-kDa
receptor-associated protein interacts with two members of the low
density lipoprotein receptor family, 
2-macroglobulin receptor
and glycoprotein 330. J Biol Chem. 1992;267:21162–21166.
36.
Willnow TE, Goldstein JL, Orth K, Brown MS, Herz J. Low
density lipoprotein receptor-related protein and gp330 bind similar
ligands, including plasminogen
activator-inhibitor complexes and lactoferrin,
an inhibitor of chylomicron remnant clearance. J
Biol Chem. 1992;267:26172–26180.
37.
Kounnas MZ, Chappell DA, Strickland DK, Argraves WS.
Glycoprotein 330, a member of the low density lipoprotein
receptor family, binds lipoprotein lipase in vitro. J Biol
Chem. 1993;268:14176–14181.
38.
Moestrup SK, Nielsen S, Andreasen P, Jorgensen KE,
Nykjaer A, Roigaard H, Gliemann J, Christensen EI. Epithelial
glycoprotein-330 mediates endocytosis of
plasminogen activator-plasminogen
activator inhibitor type-1 complexes.
J Biol Chem. 1993;268:16564–16570.
39.
Kounnas MZ, Loukinova EB, Stefansson S, Harmony JA,
Brewer BH, Strickland DK, Argraves WS. Identification of
glycoprotein 330 as an endocytic receptor for
apolipoprotein J/clusterin. [published erratum appears in J
Biol Chem.. 1995;270:23234]. J Biol Chem.
1995;270:13070–13075.
40. Moestrup SK, Cui S, Vorum H, Bregengard C, Bjorn SE, Norris K, Gliemann J, Christensen EI. Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs. [see comments]. J Clin Invest. 1995;96:1404–1413.
41.
Stefansson S, Chappell DA, Argraves KM, Strickland DK,
Argraves WS. Glycoprotein 330/low density lipoprotein
receptor-related protein-2 mediates endocytosis of low density
lipoproteins via interaction with apolipoprotein B100. J
Biol Chem. 1995;270:19417–19421.
42.
Cui S, Verroust PJ, Moestrup SK, Christensen EI.
Megalin/gp330 mediates uptake of albumin in renal proximal
tubule. Am J Physiol. 1996;271:F900–F907.
43.
Moestrup SK, Birn H, Fischer PB, Petersen CM, Verroust
PJ, Sim RB, Christensen EI, Nexo E. Megalin-mediated endocytosis of
transcobalamin-vitamin-B12 complexes suggests a role of the receptor in
vitamin-B12 homeostasis. Proc Natl Acad Sci U S A. 1996;93:8612–8617.
44.
Zlokovic BV, Martel CL, Matsubara E, McComb JG, Zheng
G, McCluskey RT, Frangione B, Ghiso J. Glycoprotein
330/megalin: probable role in receptor-mediated transport of
apolipoprotein J alone and in a complex with Alzheimer disease
amyloid-ß at the blood-brain and blood-cerebrospinal fluid
barriers. Proc Natl Acad Sci U S A. 1996;93:4229–4234.
45.
Biemesderfer D, Dekan G, Aronson PS, Farquhar MG.
Biosynthesis of the gp330/44-kDa Heymann nephritis antigenic complex:
assembly takes place in the ER. Am J Physiol. 1993;264:F1011–F1020.
46.
Zheng G, Bachinsky DR, Stamenkovic I, Strickland DK,
Brown D, Andres G, McCluskey RT. Organ distribution in rats of two
members of the low-density lipoprotein receptor gene family, gp330 and
LRP/2 MR, and the receptor-associated protein (RAP).
J Histochem Cytochem. 1994;42:531–542.[Abstract]
47. Willnow TE, Rohlmann A, Horton J, Otani H, Braun JR, Hammer RE, Herz J. RAP, a specialized chaperone, prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors. EMBO J. 1996;15:2632–2639.[Medline] [Order article via Infotrieve]
48.
Medh JD, Fry GL, Bowen SL, Pladet MW, Strickland DK,
Chappell DA. The 39-kDa receptor-associated protein modulates
lipoprotein catabolism by binding to LDL receptors. J Biol
Chem. 1995;270:536–540.
49. Kounnas MZ, Haudenschild CC, Strickland DK, Argraves WS. Immunological localization of glycoprotein 330, low density lipoprotein receptor related protein and 39 kDa receptor associated protein in embryonic mouse tissues. In Vivo.. 1994;8:343–351.[Medline] [Order article via Infotrieve]
50.
Lundgren S, Carling T, Hjalm G, Juhlin C, Rastad J,
Pihlgren U, Rask L, Akerstrom G, Hellman P. Tissue distribution of
human gp330/megalin, a putative Ca(2+)-sensing
protein. J Histochem Cytochem. 1997;45:383–392.
51.
Willnow TE, Hilpert J, Armstrong SA, Rohlmann A, Hammer
RE, Burns DK, Herz J. Defective forebrain development in mice lacking
gp330/megalin. Proc Natl Acad Sci U S A. 1996;93:8460–8464.
52. Willnow TE, Herz J. Genetic deficiency in low density lipoprotein receptor-related protein confers cellular resistance to Pseudomonas exotoxin A: evidence that this protein is required for uptake and degradation of multiple ligands. J Cell Sci. 1994;107:719–726.[Abstract]
53. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;195:265–275.
54.
Neville DMJ. Molecular weight determination of
protein-dodecyl sulfate complexes by gel electrophoresis in a
discontinuous buffer system. J Biol Chem. 1971;246:6328–6334.
55. Sambrook J, Fritsch EF, Marcovina S. Molecular Cloning: A Laboratory Manual. New York, NY: Cold Spring Harbor Laboratory Press; 1989.
56. Dieplinger H, Gruber G, Krasznai K, Reschauer S, Seidel C, Burns G, Muller HJ, Csaszar A, Vogel W, Robenek H, et al. Kringle 4 of human apolipoprotein[a] shares a linear antigenic site with human catalase. J Lipid Res. 1995;36:813–822.[Abstract]
57. McFarlane AS. Efficient trace labeling of proteins with iodine. Nature. 1958;182:53.[Medline] [Order article via Infotrieve]
58.
Orth K, Madison EL, Gething MJ, Sambrook JF, Herz
J. Complexes of tissue-type plasminogen
activator and its serpin inhibitor
plasminogen-activator inhibitor
type 1 are internalized by means of the low density lipoprotein
receptor-related protein/2-macroglobulin receptor. Proc
Natl Acad Sci U S A. 1992;89:7422–7426.
59.
Fraker PJ, Speck-JC J. Protein and cell membrane
iodinations with a sparingly soluble chloroamide,
1,3,4,6-tetrachloro-3,6-diphrenylglycoluril. Biochem
Biophys Res Commun. 1978;80:849–857.[Medline]
[Order article via Infotrieve]
60. Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260.[Medline] [Order article via Infotrieve]
61.
Pitas RE, Innerarity TL, Weinstein JN, Mahley RW.
Acetoacetylated lipoproteins used to distinguish fibroblasts
from macrophages in vitro by fluorescence microscopy.
Arteriosclerosis. 1981;1:177–185.
62. Armstrong VW, Walli AK, Seidel D. Isolation, characterization, and uptake in human fibroblasts of an apo (a)-free lipoprotein obtained on reduction of lipoprotein(a). J Lipid Res. 1985;26:1314–1323.[Abstract]
63.
Snyder ML, Hay RV, Whitington PF, Scanu AM, Fless GM.
Binding and degradation of lipoprotein(a) and LDL by primary cultures
of human hepatocytes: comparison with cultured human
monocyte-macrophages and fibroblasts. Arterioscler
Thromb. 1994;14:770–779.
This article has been cited by other articles:
 |
|
 |
|
  G. Helguera, M. Eghbali, D. Sforza, T. Y. Minosyan, L. Toro, and E. Stefani Changes in global gene expression in rat myometrium in transition from late pregnancy to parturition Physiol Genomics, January 8, 2009; 36(2): 89 - 97. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Pepe, G. Chimienti, G. M. Liuzzi, B. L. Lamanuzzi, M. Nardulli, F. Lolli, E. Angles-Cano, and S. Mata Lipoprotein(a) in the Cerebrospinal Fluid of Neurological Patients with Blood-Cerebrospinal Fluid Barrier Dysfunction Clin. Chem., November 1, 2006; 52(11): 2043 - 2048. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. J. Cain, J. S. Millar, A. S. Himebauch, U. J. F. Tietge, C. Maugeais, D. Usher, and D. J. Rader Lipoprotein [a] is cleared from the plasma primarily by the liver in a process mediated by apolipoprotein [a] J. Lipid Res., December 1, 2005; 46(12): 2681 - 2691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Fan, M. Challah, H. Shimoyamada, M. Shiomi, S. Marcovina, and T. Watanabe Defects of the LDL receptor in WHHL transgenic rabbits lead to a marked accumulation of plasma lipoprotein[a] J. Lipid Res., June 1, 2000; 41(6): 1004 - 1012. [Abstract] [Full Text]
|
|
|
|
 |
|
 B. Garner, A. H. Merry, L. Royle, D. J. Harvey, P. M. Rudd, and J. Thillet Structural Elucidation of the N- and O-Glycans of Human Apolipoprotein(a). ROLE OF O-GLYCANS IN CONFERRING PROTEASE RESISTANCE J. Biol. Chem., June 15, 2001; 276(25): 22200 - 22208. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||