From the
Hepatic lipase (HL) and lipoprotein lipase (LpL) are
structurally related lipolytic enzymes that have distinct functions in
lipoprotein catabolism. In addition to its lipolytic activity, LpL
binds to very low density lipoproteins and promotes their interaction
with the low density lipoprotein receptor-related protein (LRP)
(Chappell, D. A., Fry, G. L., Waknitz, M. A., Muhonen, L. E., Pladet M.
W., Iverius, P. H., and Strickland, D. K. (1993) J. Biol. Chem. 268, 14168-14175).
In vitro binding assays revealed
that HL also binds to purified LRP with a K
Hepatic lipase (HL)
HL is secreted by hepatocytes and
becomes bound to both hepatocytes (Marteau et al., 1988;
Belcher et al., 1988; Hornick et al., 1992) and
endothelial cells that line hepatic sinusoids (Kuusi et al.,
1979a). Accumulated evidence has implicated HL in the metabolism of
high density lipoproteins (HDL), intermediate density lipoproteins
(IDL), and chylomicron remnants (Shirai et al., 1991; Laboda
et al., 1986; Nilsson et al., 1987; Jensen et
al., 1982; Shafi et al., 1994). Deficiencies of this
enzyme in either HL-null transgenic mice (Homanics et al.,
1995) or human patients (Hegele et al., 1993; Huff et
al., 1993) result in increased plasma levels of HDL. Some patients
lacking HL accumulate cholesterol-rich,
LRP binds diverse ligands and belongs to a family of
endocytic receptors structurally related to the low density lipoprotein
(LDL) receptor (for reviews, see Krieger and Herz (1994), Moestrup
(1994), and Strickland et al. (1994)). Other mammalian members
of the LDL receptor family include the very low density lipoprotein
(VLDL) receptor, and glycoprotein 330 (gp330). LRP mediates the
cellular internalization of proteinases, such as plasminogen
activators, proteinase-inhibitor complexes, and apoE- and lipoprotein
lipase-enriched lipoproteins. Ligand binding by LRP is blocked by a
39-kDa protein, termed the receptor-associated protein (RAP) (Herz
et al., 1991; Williams et al., 1992). While the
complete physiological role of LRP is not fully appreciated at this
time, it is clear that one important function of LRP is to mediate the
hepatic removal of ligands from the circulation, and LRP is thought to
play an important role, along with the LDL receptor, in the hepatic
removal of chylomicron remnants from the circulation (Willnow et
al., 1994).
Recently, Ji et al. (1994b) transfected a
rat hepatoma cell line with the HL cDNA and demonstrated that the
transfected cells had an increased capacity to internalize chylomicron
remnants. Since the receptor responsible for this process has not yet
been identified, we initiated studies to determine if LRP will bind and
mediate the cellular internalization of HL and to examine the role of
proteoglycans in this process. The results of our studies indicate LRP
binds to HL in vitro and mediates the cellular internalization
of HL leading to its degradation in lysosomes. We also demonstrate that
proteoglycans appear essential for the LRP-mediated catabolism of HL.
In the present study we demonstrate that HL binds to purified
LRP with a K
Since HL catabolism was completely blocked by heparin, we also
investigated the role of cell surface proteoglycans in this process.
For this purpose, cell lines deficient in proteoglycan biosynthesis
were utilized (Esko et al., 1986). The results from these
experiments reveal that cell surface proteoglycans appear essential for
the LRP-mediated uptake of HL and that glycosaminoglycans are important
for serving as the primary cell surface binding site for HL. One
possible explanation of these data is that after binding to cell
surface proteoglycans, HL is then transferred to LRP for subsequent
internalization and degradation. Cell surface proteoglycans are also
known to facilitate the LRP-mediated catabolism of LpL (Chappell et
al., 1993). It seems likely that proteoglycans serve to
concentrate HL or LpL on the cell surface, thereby enhancing their
interaction with LRP. Whether or not the proteoglycans are internalized
along with LRP remains to be determined. The LRP-mediated catabolism of
several other molecules, such as tissue-factor pathway inhibitor
(Warshawsky et al. 1994), and thrombospondin (Mikhailenko
et al. 1995) have recently been demonstrated to also be
facilitated by cell surface proteoglycans.
Experiments in the
present study also confirm that HL is catabolized by a second pathway
that does not involve LRP. This conclusion is supported by the fact
that RAP or LRP antibodies are unable to completely block
The significance of the interaction of HL
with LRP likely centers around the important role HL plays in
lipoprotein catabolism. HL is a lipolytic enzyme that has both
triglyceride lipase and phospholipase activities and participates in
the metabolism of remnants, IDL, and HDL particles. Remnant lipoprotein
particles are generated from chylomicrons and VLDL during lipolysis of
their triglycerides by LpL. The smaller, cholesterol-rich remnant
particles are then removed from the circulation by liver parenchymal
cells. This process is thought to occur by an initial interaction of
the lipoproteins with cell surface proteoglycans, followed by
internalization via a receptor-mediated process (Ji et al. 1994a). Efficient catabolism of lipoproteins requires apoE to be
present on the particle surface and can be mediated by the LDL
receptor. However, since these lipoproteins are efficiently catabolized
in patients with homozygous familial hypercholesterolemia who have
severe deficiencies of LDL receptor activity (Goldstein and Brown,
1989), another receptor must assume this function in these patients.
Recent studies in mice in which the LDL receptor expression was
prevented by gene targeting suggest that LRP is a candidate for this
receptor (Willnow et al., 1994). It is important to point out
that LRP may not directly bind these remnant particles; in vitro experiments document that both remnant-like particles, and native
VLDL require ``activation'' in order to bind to LRP. This can
occur either by the addition of exogenous apoE (Kowal et al. 1989; Beisiegel et al., 1989) or LpL (Chappell et
al., 1993). A secretion-recapture model for remnant uptake in the
liver has been proposed (Brown et al. 1991; Ji et al. 1994a) in which remnants entering the sinusoidal space of the
liver are exposed to high concentrations of proteoglycan-bound apoE
that has been secreted by hepatocytes. After enrichment of the remnants
with apoE, they are active ligands for LRP, which can then mediate
their internalization and subsequent degradation. HL and to a lesser
extent LpL are also present in the hepatic sinusoids, and thus these
molecules may also play an important role in remnant catabolism.
The
potential role of LpL in lipoprotein catabolism was first suggested by
Felts et al. (1975) who proposed that LpL promotes lipoprotein
catabolism by providing a binding site for an endocytic receptor to
internalize LpL-lipoprotein complexes. Using cross-linking experiments,
Beisiegel et al. (1991) demonstrated that LpL could be
cross-linked to a large cell surface receptor thought to be LRP. LpL
was subsequently shown to bind to LRP and promote the LRP-mediated
internalization and degradation of
It is possible that HL also
promotes the LRP-mediated cellular internalization of lipoproteins by a
similar mechanism. HL is abundant on the luminal surface of endothelial
cells and hepatocytes within hepatic sinusoids and can be released from
proteoglycan attachment sites by intravenous heparin. Injection of HL
antibodies into animals resulted in an increase in remnant particles
(Kuusi et al., 1979b; Goldberg et al., 1982; Daggy
and Bensadoun, 1986; Shafi et al., 1994), suggesting a role
for HL in remnant clearance. Infusion of HL into rabbits fed a high
cholesterol diet reduced the levels of
In summary,
we have identified LRP as a primary endocytic receptor for HL in HepG2
cells, fibroblasts, and CHO cells. Our studies indicate that
proteoglycans are critical for initial binding of HL to the cell
surface. Studies are underway to determine if HL bound chylomicron
remnants are also endocytosed in association with HL via LRP.
of 52 n
M. Its binding to LRP is inhibited by the 39-kDa
receptor-associated protein (RAP), a known LRP antagonist, and by
heparin.
I-Labeled HL is rapidly internalized and
degraded by HepG2 cell lines, and approximately 70% of the cellular
internalization and degradation is blocked by either exogenously added
RAP or anti-LRP IgG. Mouse fibroblasts that lack LRP display a greatly
diminished capacity to internalize and degrade HL when compared to
control fibroblasts. These data indicate that LRP-mediated cellular
uptake of HL accounts for a substantial portion of the internalization
of this molecule. Proteoglycans have been shown to participate in the
clearance of LpL, and consequently a role for proteoglycans in HL
clearance pathway was also investigated. Chinese hamster ovary cell
lines that are deficient in proteoglycan biosynthesis were unable to
internalize or degrade
I-HL despite the fact that these
cells express LRP. Thus, the initial binding of HL to cell surface
proteoglycans is an obligatory step for the delivery of the enzyme to
LRP for endocytosis. A small, but significant, amount of
I-HL was internalized in LRP deficient cells indicating
that an LRP-independent pathway for HL internalization does exist. This
pathway could involve cell surface proteoglycans, the LDL receptor, or
some other unidentified surface protein.
(
)
is a member of a
family of structurally and functionally related enzymes that include
pancreatic lipase and lipoprotein lipase (LpL) (Ben-Zeev et
al., 1987) (for reviews, see Nilsson-Ehle et al. (1980)
and Olivecrona and Bengtsson-Olivecrona (1990)). The three-dimensional
crystallographic structure of human pancreatic lipase (Winkler et
al., 1990) shows that this molecule consists of two domains; an
N-terminal domain (amino acids 1-335), which contains the active
site of the enzyme that is dominated by a central parallel
-sheet
structure, and a smaller C-terminal domain (amino acids 336-449)
that forms a
sandwich structure. The overall folding patterns of
HL and LpL are thought to be similar to that of pancreatic lipase
(Derewenda and Cambillau, 1991).
very low density
lipoproteins (
-VLDL) which are abnormal lipoproteins that are
enriched in apolipoprotein E (apoE). On the other hand, overexpression
of HL in transgenic rabbits results in a lowering of plasma HDL and IDL
levels (Fan et al., 1994). It has been speculated that HL may
serve to promote hepatic clearance of apoE-containing lipoproteins by
either hydrolyzing/remodeling them to a form that exposes an apoE
receptor binding site or by binding to lipoproteins and serving as a
recognition site for hepatic receptors (Ji et al., 1994b).
This latter model is similar to a putative role played by LpL, which
binds to the low density lipoprotein receptor-related protein (LRP) and
promotes the interaction of lipoproteins with this receptor (Beisiegel
et al., 1991; Chappell et al. 1992, 1993; Nykjaer
et al., 1993). The ability of LpL to promote the LRP-mediated
cellular internalization and degradation of lipoproteins may not
require the catalytic site of LpL, since the carboxyl-terminal domain
of LpL, which lacks this site, binds to LRP and promotes the
LRP-mediated catabolism of lipoproteins (Williams et al.,
1994).
Proteins
LRP was isolated from human placenta as
described by Ashcom et al. (1990). Human RAP, expressed in
bacteria as a fusion protein with glutathione S-transferase,
was prepared and purified as described previously (Williams et
al., 1992). HL was labeled with [I]iodine
to a specific activity ranging from 2 to 10 µCi/µg of protein
using Iodogen (Pierce Chemical Co., Rockford, IL). Bovine serum
albumin, fraction V, and ovalbumin were purchased from Sigma.
Production and Purification of Recombinant Rat
HL
Roller cultures of Chinese hamster ovary (CHO)-K1 cells
stably transformed with HL (Komaromy and Reed, 1991) were grown in
Dulbecco's modified Eagle's medium supplemented with 1%
Nutridoma (Boehringer Manneheim) until subconfluent. The medium was
replaced with 30 ml of induction medium (Dulbecco's modified
Eagle's medium containing Nutridoma, 30 µ
M
ZnSO, and 10 units/ml heparin) for 14 days, harvesting, and
replacing the medium every 24 h. Harvested medium was spun at 1200
g for 20 min and stored at -80 °C until
utilized. Rat HL was purified by a four-step procedure utilizing
octyl-Sepharose, heparin-Sepharose, hydroxylapatite, and dextran
sulfate-Sepharose columns. Elution and loading steps were linked to
maximize enzyme recovery and prevent loss of activity. Briefly, rapidly
thawed CHO medium (1 liter) was loaded onto an octyl-Sepharose column
(80-ml bed volume) which had been previously equilibrated with 5
m
M barbital, 0.35
M NaCl, 20% glycerol, pH 7.4
(Buffer A). Following a wash step to remove unbound proteins, the
column was eluted with 200 ml of 1.2% Triton N-101 in Buffer A. This
material was applied immediately to a column of heparin-Sepharose
(50-ml bed volume), washed, and eluted with a 0-1
M NaCl
step gradient. Fractions containing HL were then applied to a 2-ml
hydroxylapatite column, washed with 0.1
M NaPO
, pH
7.4, and eluted with 0.3
M NaPO
. The eluate was
applied directly to a dextran sulfate-Sepharose column (6 ml), washed
with Buffer A, and eluted with a linear NaCl gradient (0.35-1.5
M). This procedure resulted in isolation of a homogeneous band
as determined by silver staining of SDS-polyacrylamide gel
electrophoresis gels (data not shown). The specific activity was
indistinguishable from HL purified from rat liver perfusates (Ben-Zeev
et al., 1987). Typically, 1 mg of recombinant HL was obtained
from 2 liters of CHO cell medium.
Antibodies
Rabbit polyclonal antibodies to the
515-kDa heavy chain of LRP (R777) and to a synthetic peptide from the
cytoplasmic domain of the 85-kDa light chain of LRP (R704) have been
described elsewhere (Kounnas et al., 1992b). R777 antibodies
were affinity selected on a column of LRP-Sepharose. R704 antibodies
were purified on protein G-Sepharose (Pharmacia Biotech Inc.). Both of
the rabbit antibody preparations were dialyzed against isotonic PBS
prior to use in cell assays.
Solid-phase Binding Assays
Homologous and
heterologous ligand displacement assays were performed as described by
Williams et al. (1992). Microtiter wells were coated with LRP
or ovalbumin (5 µg/ml) in Tris-buffered saline, pH 8.0, 5
m
M CaCl, and nonspecific sites were blocked with
0.5% ovalbumin, Tris-buffered saline, pH 8.0, 5 m
M
CaCl
. Coated wells were incubated with radiolabeled ligand
(2 n
M) in 0.5% ovalbumin, Tris-buffered saline, 5 m
M
CaCl
, 0.05% Tween-20 in the presence of varying
concentrations of unlabeled competitor for 18 h at 4 °C. Binding
data were analyzed by using the computer program LIGAND (Munson and
Rodbard, 1980).
Cells
Human hepatocellular carcinoma (HepG2, ATCC
HB 8065) cells were obtained from the American Type Culture Collection
(ATCC, Rockville, MD) and were grown in Eagle's minimal essential
medium containing 10% bovine calf serum (Hyclone Labs, Logan, UT), 100
units/ml penicillin (Life Technologies, Inc.), 100 µg/ml
streptomycin (Life Technologies, Inc.), and 1%
L-glutamine
(Life Technologies, Inc.). Three types of CHO cells were used in these
studies. CHO 745 provided by Dr. J. Esko (University of Alabama,
Birmingham, AL) are deficient in all types of proteoglycans (Esko
et al., 1986), CHO C16 (ATCC CCL 61) are a wild type cell
line, and CHO 13-5-1 are LRP-deficient cells prepared by toxin-mediated
selection of mutagenized CHO C16 cells.(
)
CHO
cells were grown to approximately 80% confluence in Ham's F-12
medium supplemented with 5% bovine calf serum-optimized for CHO cells
(Hyclone), 100 units/ml penicillin, 100 µg/ml streptomycin, and 1%
L-glutamine. A normal mouse embryo fibroblast (MEF) line and a
derivative of this cell line (Willnow and Herz, 1994) that is deficient
in LRP biosynthesis (PEA13) were obtained from Dr. J. Herz (University
of Texas Southwestern Medical Center, Dallas, TX) and maintained as
described by Willnow and Herz (1994).
Cell-mediated Ligand Internalization and Degradation
Assays
Cells were transferred to 12-well dishes (Corning,
Corning, NY) at 2-3 10
cells/well and allowed
to grow for 24 h at 37 °C, 5% CO
. Prior to performing
assays, cultured cells were washed with medium and incubated in medium
containing 1% Nutridoma (Boehringer Manneheim), 20 m
M Hepes,
penicillin/streptomycin, and 1.5% bovine serum albumin (assay medium).
For assays with HepG2 cells, radiolabeled HL (4 n
M) in assay
medium was incubated with cells for 6 h at 37 °C, 5% CO
in the presence of increasing concentrations of RAP
(0.2-1350 n
M) or heparin (Sigma, 0.1-300 mg/ml).
For assays using antibodies, cultured cells were preincubated for 1 h
at 37 °C, 5% CO
with assay medium containing various
concentrations of antibodies. Fresh medium containing the
I-labeled ligand and antibodies was then added and
incubated with the cells for 6 h at 37 °C, 5% CO
. For
CHO cell assays,
I-HL (4 n
M) was incubated with
cells in the presence of unlabeled heparin (100 µg/ml) or LRP
antibodies (100 µg/ml) for the indicated time intervals. In assays
utilizing cultured fibroblasts,
I-HL (4 n
M) was
added in assay medium either alone or in the presence of RAP (450
n
M), heparin (100 µg/ml), or chloroquine (0.1 m
M,
Sigma). Radioactivity appearing in the cell culture medium that was
soluble in 10% trichloroacetic acid was taken to represent degraded
ligand. Total ligand degradation was corrected for the amount of
degradation that occurred in control wells lacking cells. The amount of
radioligand that was bound to the cell surface or that was internalized
by cells was determined as described previously (Chappell et
al., 1992). Briefly, following incubation with the radioligand,
cells were washed with phosphate-buffered saline and treated with a
trypsin-EDTA, proteinase K solution. Surface-bound material was defined
as the amount of radioactive ligand released by this treatment, and the
amount of internalized ligand was defined as the amount of
radioactivity which remained associated with the cell pellet following
the treatment.
Hepatic Lipase Binds to LRP
To determine if HL
can bind to LRP in vitro, homologous ligand displacement
binding assays were conducted. The results of these experiments, shown
in Fig. 1 ( Panel A), demonstrate that I-HL bound
to purified LRP immobilized on microtiter plastic wells, but not to
ovalbumin-coated microtiter wells. Its binding was prevented by excess
cold HL, which is consistent with specific binding. Quantitative
information from these data was derived using the program LIGAND, and
the binding was adequately described by a model containing a single
class of sites with a K
of 52
n
M. This value is slightly weaker than the
K
of 20 n
M measured for the
interaction of LpL with LRP (Chappell et al., 1993) using a
similar assay. RAP, a known antagonist of ligand binding to LRP,
inhibited the binding of HL to LRP (Fig. 1, Panel B). HL
is known to bind heparin, and consequently the effect of heparin on its
interaction with LRP was also measured. The results (Fig. 1,
Panel B) revealed that heparin almost completely blocked the
binding of
I-HL to LRP.
Figure 1:
Binding of I-HL to LRP.
In Panel A,
I-HL (2 n
M) was incubated
for 18 h at 4 °C with LRP-coated wells ( closed circles) or
ovalbumin-coated wells ( open circles) in the presence of
increasing concentrations of unlabeled HL. Following incubation, wells
were washed and counted. The data were analyzed using the program
LIGAND and the curve represents the best-fit data to a single class of
sites with a K of 52 n
M. In Panel B,
I-HL (2 n
M) was incubated as in Panel A in the absence of competitor or in the presence of either 450
n
M RAP, or 100 µg/ml heparin. Plotted values represent
means of duplicate experiments.
To investigate whether or not LRP is
able to mediate the cellular internalization of HL, we utilized a MEF
cell line, and a derivative of this cell line that is LRP-deficient
(PEA13) (Willnow and Herz, 1994). As shown in Fig. 2,
LRP-expressing MEF cells efficiently internalized and degraded
I-HL Degradation Is Greatly Diminished in
LRP-deficient Fibroblasts
I-HL. Approximately 80% of the degradation of
I-HL was inhibited by RAP, while heparin completely
blocked the degradation of this ligand. Chloroquine, a drug which
effectively prevents lysosomal degradation by altering the pH of
lysosomes, also blocked the degradation of
I-HL in these
cells, confirming that the degradation of this ligand requires
lysosomal enzymes. In contrast,
I-HL was degraded
relatively poorly in LRP-deficient mouse fibroblasts (PEA13 cells) to a
level of 20% of that found in MEF cells (Fig. 2, right four
bars).
I-HL degradation in PEA13 cells was largely
insensitive to RAP, but was inhibited by heparin and chloroquine. The
data indicate that LRP is the predominant mediator of HL degradation in
fibroblasts. Since a small amount of degradation was observed in PEA13
cells, it does appear that an LRP-independent pathway for HL clearance
exists in these cells.
Figure 2:I-HL degradation by
LRP-deficient mouse fibroblasts. Control LRP-expressing fibroblasts
(MEF cells, closed bars) and LRP-deficient fibroblasts (PEA13
cells, open bars) were incubated with
I-HL (4
n
M) for 8 h in the absence of competitor or in the presence of
either RAP (450 n
M), heparin (100 µg/ml), or chloroquine
(0.1 m
M). The levels of radioligand degradation were
determined as described under ``Experimental Procedures.''
Plotted values represent means of duplicate experiments ±
S.E.
To characterize
the cellular catabolism of HL further, we next investigated this
process in HepG2 cells. These cells were observed to rapidly
internalize and degrade I-HL Degradation in HepG2 Cells Is
Partially Mediated by an LRP-independent Pathway
I-HL. To identify the receptor(s)
that are responsible for this process in these cells, the effect of RAP
and heparin on
I-HL catabolism were investigated. As
shown in Fig. 3 A, heparin inhibited the surface binding
of
I-HL to HepG2 cells, while RAP slightly increased HL
binding to HepG2 cell surfaces. However, concentrations of RAP
sufficient to prevent binding of
I-HL to LRP (Fig. 1,
Panel B) blocked approximately 55% of the internalization and
60% of the degradation of
I-HL in these cells
(Fig. 3, Panels B and C). Exogenously added
heparin completely blocked both processes. Experiments using both RAP
and heparin together as competitors of HL binding, uptake, and
degradation showed results similar to those obtained with heparin alone
( data not shown). Because RAP prevented HL binding to cells
without preventing its surface binding, while heparin completely
blocked both processes, the data suggest that HL binding to cells is
largely independent of LRP and is perhaps mediated by cell surface
proteoglycans.
Figure 3:
RAP and
heparin inhibit I-HL uptake and degradation by HepG2
cells. HepG2 cells were incubated for 6 h at 37 °C with
I-HL (4 n
M) in the presence of increasing
concentrations of unlabeled RAP (0.6-1350 n
M) or heparin
(0.11-300 µg/ml). Panels show binding ( A),
internalization ( B), and degradation ( C) data,
respectively. Plotted values represent means of duplicate
experiments.
Since RAP can also block LDL receptor-mediated
catabolism (Medh et al., 1995), additional experiments were
performed using antibodies that specifically inhibit LRP function.
Surface binding of I-HL to HepG2 cells was not inhibited
by LRP antibodies or control antibodies (Fig. 4, Panel
A). LRP-antibodies did, however, effectively block 80% of
I-HL internalization (Fig. 4, Panel B) and
60% of its degradation (Fig. 4, Panel C). Control
antibodies had little or no effect on
I-HL binding,
internalization, or degradation by HepG2 cells (Fig. 4). When
assays were performed which included both heparin and LRP antibodies as
competitors of
I-HL binding, uptake, and degradation, the
results paralleled those observed using heparin alone. These data are
consistent with the observation that heparin competes for HL binding to
LRP in vitro (Fig. 1, Panel B) as well as for
HL binding to cell surface proteoglycans, a likely primary binding site
for HL on the cell surface, and support the hypothesis that HL binds
initially to cell surface proteoglycans and that its subsequent
internalization and degradation are substantially mediated by LRP.
Figure 4:
LRP-antibodies inhibit I-HL
uptake and degradation by HepG2 cells. Wells containing 3
10
HepG2 cells were incubated for 6 h at 37 °C with
I-HL (4 n
M) in the presence of increasing
concentrations of affinity purified anti-LRP IgG (0.11-300
µg/ml), control IgG against the cytoplasmic domain of LRP
(0.11-300 µg/ml), or heparin (0.11-300 µg/ml).
Panels show binding ( A), internalization ( B), and
degradation ( C) data, respectively. Plotted values represent
means of duplicate experiments.
Proteoglycans Are Required for Cell-mediated Catabolism
of HL
To further study the individual roles of LRP and
proteoglycans in the catabolism of HL, cellular assays with
I-HL were performed with normal CHO cells (C16),
LRP-deficient CHO cells (13-5-1), or proteoglycan-deficient CHO cells
(745) . The degradation of
I-HL by normal CHO
cells (Fig. 5, Panel A), was inhibited by LRP antibodies
(65%) and heparin (100%), while control antibodies had no effect. In
the LRP-deficient cells (Fig. 5, Panel B),
I-HL degradation is not inhibited by LRP antibodies but
was blocked by heparin. In the proteoglycan-deficient cells
(Fig. 5, Panel C) only a small amount of degradation of
I-HL was observed. These data indicate that proteoglycans
are required for HL catabolism. In addition, it is apparent that, like
LRP-deficient mouse fibroblasts, the LRP-deficient CHO cells possess an
alternative pathway for HL clearance, albeit somewhat less than that
mediated by LRP. It is interesting to note that the alternative pathway
seems to play a greater role in the CHO cells when compared to either
HepG2 or fibroblasts. This is evidenced by comparing the percent of
total HL internalized and degraded in the presence and absence of LRP
antagonists in the individual cell lines. Likely this reflects that
fact that CHO cells contain far fewer LRP molecules than HepG2 cells or
fibroblasts.
Figure 5:
Proteoglycans are essential for
I-HL catabolism. Normal CHO cells ( Panel A);
LRP-deficient CHO cells ( Panel B); proteoglycan-deficient CHO
cells ( Panel C) were incubated with
I-HL (4
n
M) for selected time intervals in the presence of control IgG
(100 µg/ml), affinity-purified LRP antibodies (100 µg/ml), or
heparin (100 µg/ml). At selected times, the medium was removed and
the amount of radioligand degradation determined as described under
``Experimental Procedures.'' Plotted values represent means
of duplicate experiments.
of 52 n
M. Like all
known LRP ligands, the interaction of HL with LRP is inhibited by RAP.
Heparin, which is known to bind to HL, also prevents its association
with LRP. An interaction of LRP with HL immobilized on nitrocellulose
has been recently reported (Nykjaer et al., 1994) and is
supported by findings in the present investigation. Cellular uptake
experiments show that
I-HL is rapidly internalized and
degraded in mouse fibroblasts, HepG2 cells, and Chinese hamster ovary
cells. Our studies identified LRP as a major receptor responsible for
mediating the cellular internalization and degradation of
I-HL. The role of LRP in HL catabolism is supported by
the fact that, at concentrations known to completely block binding of
HL and other ligands to LRP, RAP reduced the cellular catabolism of
I-HL by 80 and 60%, in mouse fibroblasts and HepG2 cells,
respectively. Further, anti-LRP IgG reduced the extent of cellular
mediated degradation of
I-HL by 60 and 65%, in HepG2
cells and Chinese hamster ovary cell lines, respectively. Finally, two
mutant cell lines that do not express LRP show a greatly diminished
capacity to internalize and degrade
I-HL when compared to
the control cell lines from which they were derived that do express
LRP.
I-HL uptake and degradation in all cell lines examined
and by the observation that
I-HL is degraded in a
heparin-dependent but RAP-independent manner in cell lines which lack
LRP. While the receptor responsible for this second pathway has not
been identified, it is not likely to be gp330 or the VLDL receptor,
since RAP is an effective antagonist of ligand binding by these two
receptors (Kounnas et al., 1992a; Battey et al.,
1994). Further, gp330 is not expressed in any of the cell lines used in
the current investigation. On the other hand, the LDL receptor has weak
affinity for RAP ( K
= 300
n
M) and thus could be responsible for the low levels of uptake
seen in these cells. The LDL receptor is present on HepG2 cells,
fibroblasts, and CHO cells and is known to participate in the uptake of
lipoprotein lipase.(
)
Alternately, another
unidentified receptor may be responsible for the catabolism of HL in
these cell types. This receptor, like LRP, is likely to be dependent on
proteoglycans for serving as the initial binding site for HL, since no
specific uptake and degradation was observed in cells deficient in
proteoglycan biosynthesis.
I-labeled VLDL
(Chappell et al. 1992, 1993; Nykjaer et al., 1993).
Thus, in addition to its capacity to hydrolyze triglycerides on the
surface of lipoproteins, LpL serves as a proteoglycan-anchored ligand
for remnant binding and promotes the interaction of the lipoprotein
with the endocytic receptor, LRP.
-VLDL which had accumulated
in their plasma (Chang and Borensztajn, 1993). In addition, studies
have shown that cells transfected with HL cDNA bind 3-fold more
-VLDL than nontransfected cells (Ji et al. 1994b).
Further, Ji et al. (1994b) demonstrated that transfected cells
displayed an increased capacity to internalize
-VLDL, presumably
via a receptor-mediated process. While the receptor responsible for
this process has not been identified, LRP is a likely candidate since
our studies have confirmed that HL interacts with LRP.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.