From Inserm Unit 391, 35043 Rennes, France and Genset
SA, 75000 Paris, France
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ABSTRACT |
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The lipolysis-stimulated receptor (LSR) is a
lipoprotein receptor primarily expressed in the liver and activated by
free fatty acids. Antibodies inhibiting LSR functions showed that the
receptor is a heterotrimer or tetramer consisting of 68-kDa ( Chylomicrons transport, in plasma, dietary triglycerides
(TG)1 and liposoluble
vitamins absorbed by the intestine after a meal (1). Lipoprotein lipase
(LPL), which is anchored to the surface of capillary endothelium,
hydrolyzes chylomicron TG into free fatty acids (FFA) that are targeted
to the underlying muscles and adipose tissue. The residues of
chylomicrons are then released from the endothelium and taken up by the
liver. Both the low density lipoprotein (LDL) receptor and the
LDL receptor-related protein (LRP) contribute to this process
(2, 3). Studies using anti-LDL receptor antibodies or mice with a
deficiency of the apoE gene suggest that the LDL receptor accounts
for up to half of the clearance of chylomicrons (4, 5). However, human
subjects deficient for the LDL receptor clear chylomicron remnants
normally (6). In addition, mice with CRE-loxP-mediated selective
disruption of the LRP gene in the liver are not hyperlipidemic (7). If LRP-deficient mice are cross-bred with LDL receptor-deficient mice,
apoB48, the main chylomicron apolipoprotein, accumulates in the plasma
(7), but plasma TG concentrations in these mice are not dramatically
increased. This is in contrast with the effect of the 39-kDa
receptor-associated protein, a known inhibitor of LRP activity, which
induces a massive increase of plasma TG and cholesterol when
overexpressed in mice (8).
We have reported the characterization of a lipoprotein receptor that is
inhibited by receptor-associated protein at concentrations similar to
those achieved in the receptor-associated protein overexpression study
(9). This receptor was originally identified by its binding of LDL in
the presence of FFA and is hereafter referred to as the
lipolysis-stimulated receptor (LSR). LSR binds apoB and apoE, displays
the greatest affinity for TG-rich lipoprotein (chylomicrons and very
low density lipoprotein (VLDL)), and does not bind We now report the cloning and characterization of a new gene, primarily
expressed in the liver, which encodes a multimeric receptor that binds
lipoproteins in the presence of FFA. We propose that LSR represents a
rate-limiting step for the clearance of dietary TG from the circulation.
Materials
Na125I and [35S]methionine/cysteine
(Promix) were obtained from Amersham Pharmacia Biotech (Les Ulis,
France); [33P]dCTP was purchased from NEN Life Science
Products (Paris, France). Oleic acid, bovine serum albumin (A2153)
(BSA), 1,2-cyclohexanedione, n-octyl glucopyranoside, and
the 5'-nucleotidase kit were obtained from Sigma (St. Quentin,
Fallavier, France). Sodium suramin was a generous gift from Bayer
Pharmaceuticals (Puteaux, France), and sodium heparin was purchased
from Choay Laboratories (Gentilly, France). Pronase and
1,1'-dioctadecyl-3, 3,3',3'-tetramethyl indocarbocyanine perchlorate
(DiI) were obtained from Calbiochem (Meudon, France) and Molecular
Probes, Inc. (Eugene, OR), respectively. Fetal bovine serum,
Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, trypsin, penicillin/streptomycin, and glutamine were purchased from
Life Technologies, Inc. (Eragny, France). Methionine- and cysteine-free
RPMI medium was obtained from BioWhittaker (Gagny, France). Secondary
antibodies conjugated to alkaline phosphatase and transfection reagent
Superfect were purchased from Immunotech (Marseille, France) and Qiagen
(Courtaboeuf, France), respectively.
Animals
Male Sprague-Dawley rats were purchased from R. Janvier Breeding
Center (Le Genest St. Isle, France) and housed in an animal care
facility approved and monitored by the French Ministries of Health and Agriculture.
Methods
Purification of LSR--
Rat liver total membranes were prepared
from overnight-fasted 350-g Sprague-Dawley rats, followed by
solubilization in 125 mM n-octyl glucoside (20 mg of membrane protein/ml of detergent) in 20 mM Tris-HCl
containing 2 mM EDTA, pH 7.4, and proteolytic inhibitor
mixture, as described previously (12, 17). Solubilized membrane protein
was then separated under nondenaturing conditions on preparative
4-12% gradient polyacrylamide gels (35-40 mg/gel). A strip of gel
was cut and transferred to nitrocellulose membrane, which was incubated
with 0.8 mM oleate and 40 µg/ml 125I-LDL and
then washed extensively in phosphate-buffered saline (PBS) containing
0.5% (v/v) Triton X-100 (12). The 240-kDa band exhibiting the ability
to bind 125I-LDL in the presence of oleate was then excised
from the remaining gel, electroeluted, and used for rabbit immunization.
Production of Polyclonal Anti-LSR Protein and Synthetic Peptide
Antibodies--
New Zealand rabbits were injected with partially
purified LSR proteins emulsified with Freund's complete adjuvant and
subsequently with incomplete adjuvant following the protocol described
by Harlow and Lane (18). Sera were collected prior to injections
(preimmune IgG) and at various times from 2 to 12 months after the
injections. IgG were purified using protein A-Sepharose (Amersham
Pharmacia Biotech) following the manufacturer's instructions.
Anti-LSR synthetic peptide with a sequence corresponding to LSR Preparation of Rat Liver Plasma Membranes--
For isolation of
livers, overnight-fasted animals were anesthetized with ether, and the
livers were perfused through the portal vein with ice-cold
Hepes-buffered saline solution (150 mM NaCl containing 5 mM Hepes and 2 mM EDTA, pH 7.4). The livers
were then immediately excised, and plasma membranes were prepared
according to the procedure described previously (19). Plasma membranes were stored at Measurement of LSR Activity in Rat Liver Plasma
Membranes--
Oleate-induced binding of 125I-LDL to rat
liver plasma membranes was measured as reported previously (9, 12) with
some modification. Briefly, aliquots of membranes (100 µg of
protein/tube) were incubated at 37 °C for 30 min in the absence or
presence of the 0.8 mM oleate adjusted to a final volume of
250 µl with 0.1 M phosphate buffer containing 350 mM NaCl and 2 mM EDTA, pH 8.0 (buffer A). The
membranes were then washed by six series of centrifugation (35,000 × g, 15 min, 4 °C) and resuspended into 250 µl of
buffer A by brief sonication (Bioblock Scientific Vibracell, power 1.0, 90% pulse, 5 s). At the final wash, the membrane pellets were
resuspended into 200 µl of buffer A, and the membranes were incubated
at 4 °C for 60 min with the indicated concentrations of an
irrelevant IgG or anti-LSR IgG. 125I-LDL (5 µg/ml) was
then added, and the membranes were further incubated for 1 h at
4 °C in a final volume of 250 µl. At the end of the incubation, 25 µl/tube of PBS containing 2% (w/v) BSA was added. Membrane-bound
125I-LDL was separated from unbound 125I-LDL by
layering a 200-µl aliquot over a 600-µl cushion of 5% (w/v) BSA in
buffer A and centrifuging (35,000 × g, 20 min,
4 °C). After careful aspiration of the supernatants, the bottoms of
the tubes containing the membrane pellets were cut and counted in a
Binding, Uptake, and Degradation Studies--
For these studies,
primary cultures of rat hepatocytes were used 48 h after plating
(10). Oleate-induced 125I-LDL binding, uptake, and
degradation was measured as described previously with the following
modifications (10). Hepatocytes were preincubated for 30 min at
37 °C with 20 ng/ml mouse recombinant leptin (20), followed by 30 min at room temperature with the indicated concentrations of IgG.
125I-LDL was then added, and the cells were further
incubated for 4 h at 37 °C in the presence or absence of 0.5 mM oleate, followed by analysis of the amount of
125I-LDL bound, internalized, and degraded.
Immunoprecipitation--
Primary cultures of rat hepatocytes (48 h after plating) were incubated with 35S-Promix in
methionine- and cysteine-free RPMI medium and then lysed in PBS
containing 1% Triton X-100. Immunoprecipitates were prepared and
separated on SDS-polyacrylamide gels as described by Oukka et
al. (21).
Preparation of Lipoproteins--
Human LDL (1.025 < density (d) < 1.055 g/ml) were purified from plasma
obtained from the local blood bank exactly as described previously and
stored under N2 and in the dark for not more than 15 days
prior to use (10, 11). Human VLDL (d < 1.006) and high
density lipoprotein (1.085 < d < 1.21) were
obtained from overnight-fasted normolipidemic volunteers by sequential
ultracentrifugation (11, 12). Chylomicrons were obtained by
catheterization of the abdominal lymphatic duct of rats weighing
between 150 and 200 g after force feeding of a fat meal with a
composition similar to that described above for mice (11). The
chylomicrons were separated from the lymph by two consecutive
centrifugations (200,000 × g, 1 h, 15 °C, SW41
Beckman rotor) at d = 1.006 g/ml.
Pronase treatment and 1,2-cyclohexanedione modification were performed
as described by Bihain and Yen (11) and Shepherd and Packard (22),
respectively. The inability of cyclohexanedione-modified LDL to bind to
the LDL receptor was verified in control experiments using normal human
fibroblasts. LDL was labeled with DiI according to the method described
by Via and Smith (23). Lipoprotein-deficient (d > 1.21 g/ml) fetal bovine serum was used as a source of cholesterol ester
transfer protein. Radioiodinations of LDL were based on the McFarlane
procedure modified by Bilheimer et al. (24). Radiolabeled lipoproteins were filtered (0.2 µm) on the day of the experiment and
used within 1 week of preparation.
Western Blotting--
Nitrocellulose membranes were incubated 30 min at room temperature with PBS containing 3% (w/v) BSA and then
washed three times for 10 min each in PBS containing 0.5% (v/v) Tween
20. The strips were incubated for 1 h at room temperature with a
1:400 dilution of anti-LSR serum or 75 µg/ml anti-LSR peptide 170 IgG (immunoglobulin was purified by protein A affinity column
chromatography (Amersham Pharmacia Biotech)) in PBS containing 0.5%
(v/v) Tween 20. After washing three times for 10 min in PBS containing
0.5% Tween 20, the membranes were incubated 1 h at room
temperature with goat anti-rabbit IgG that was either 1) radiolabeled
with 125I at 20,000 cpm/ml (Iodobeads; Pierce) according to
the manufacturer's instructions) (Fig. 1) or 2) conjugated to alkaline
phosphatase (Fig. 6). Protein bands were revealed by 1) exposing on a
phosphor screen and image analysis on a PhosphorImager (Molecular
Dynamics, Inc., Sunnyvale, CA) or 2) coloration, using alkaline
phosphatase substrates.
Library Screening--
A 5' Rapid Amplification of cDNA Ends PCR--
Rat liver
mRNA was purified using Dynabeads oligo(dT)25 (Dynal,
Compiègne, France) following the manufacturer's instructions. cDNA were synthesized at 50 °C using Superscript II (Life
Science Technologies) with primer T12VN where V stands for
A, C, or G and N for the four nucleotides according to the
manufacturer's protocol. Double strand DNA was obtained by the
replacement technique, and recessed termini were repaired by T4 DNA
polymerase as described by Sambrook et al. (27). Double
strand adapters included the NotI site and were prepared by
hybridization of the two modified oligonucleotides (AD1,
5'-phosphate-GCGGCCGCAT-NH2-3'; AD2,
5'-GCTATCTGAGCGATCGACATGCGGCCGC-3') and ligated to cDNA with T4
ligase (26). Nested PCRs were then performed with 5'-RA
(5'-GCTATCTGAGCGATCGAC-3') and LSR 10 (5'-TGGGTCACTGGCTGGAACAGTATCACTACG-3') and LSR 12 (5'-CGATGAATTCGAGACACACGAAGACCACGGTA-3'), which introduced an
EcoRI site. PCR products were cloned into the
NotI and EcoRI sites of pBluescript (Stratagene, Ozyme).
Northern Blot Analysis--
Rat multiple tissue Northern blots
(CLONTECH) were hybridized at 42 °C for 16 h with a [33P]dCTP-labeled
XbaI-XbaI fragment from the LSR candidate
cDNA and a [33P]dCTP-labeled Primers for RT-PCR--
Primer sequences for the analysis
of the mRNA by RT-PCR were as follows (Fig. 3c): a,
5'-GTTACAGAATTCGCCGCGATGGCGCCGGCG-3'; b, 5'-GCCAGGACAGTGTACGCACT-3'; c,
5'-ACCTCAGGTGTCCCGAGCAT-3'; d, 5'-GAAGATGACTGGCGATCGAG-3'; e,
5'-ACCTCTATGACCCGGACGAT-3'; b', 5'-CACCACCCTGACAGTGCGTA-3'; c', 5'-
CTGGGGGCATAGATGCTCGG-3'; d', 5'-GCCCTGGAAGGCCTCGATCG-3'; e',
5'-AAGTCCCTAGGATCGTCCG-3'; f',
5'-CGTCACGAATTCCGTGGATCAGACGTC-3'. The complete coding sequences corresponding to LSR 1893 and LSR 2097 were obtained by RT-PCR using
primers a and f' and cloned in pcDNA3 (Invitrogen, Leek, The Netherlands).
Synthetic Peptide--
A synthetic peptide, peptide 170, with
the sequence EEGQYPPAPPPYSET was obtained commercially, conjugated to
KLH, and used to immunize rabbits (Eurogentec, Seraing, Belgium).
Transient Transfection Studies--
Chinese hamster ovary cells
(CHO-K1, CCL-61, ATCC, Rockville, MD) were plated in six-well plates
(Falcon) at 2.5-2.75 × 105 cells/well. After 24-h
culture in Ham's F-12 medium containing 10% (v/v) fetal bovine serum,
2 mM glutamine, and 100 units/ml each of penicillin and
streptomycin, 2 µg of plasmid/well were transfected using Superfect
(Qiagen) according to the manufacturer's instructions (10 µl of
Superfect/well, 2 h at 37 °C in serum-free Ham's F-12 medium).
The plates were then washed in PBS to remove the transfection reagent,
and the cells were further grown in serum-containing Ham's F-12
medium. LSR activity was then measured as described previously (10, 11)
48 h after transfection.
Preparation of Recombinant Mouse Leptin--
The leptin cDNA
was obtained from mouse C57BL/6J (R. Janvier Breeding Center) adipose
tissue mRNAs by reverse transcription-PCR. The PCR 5' primer
introduced an initiation codon after the signal sequence, which was
deleted, and a sequence coding a hexahistidine tag. The modified mouse
leptin coding sequence was cloned into the pSE280 expression vector
(Invitrogen, France) and expressed in E. coli (TG1). DNA
sequencing of the plasmid confirmed the sequence of the coding open
reading frame. Cells were grown at 37 °C, and the protein synthesis
was induced by 1 mM
isopropyl-
To test the activity of recombinant leptin, ob/ob and
db/db C57BL/Ks (R. Janvier Breeding Center) mice were
injected intraperitoneally with 25 µg of recombinant leptin or
physiological saline (n = 3 for each condition). This
caused a 32% (p < 0.005) decrease in the amount of
food ingested by the ob/ob strain (6.9 ± 0.15 g
(saline) versus 4.7 ± 0.87 g (leptin) food
intake/24 h) but no change in the food intake of db/db mice
(4.33 ± 0.15 g (saline) versus 4.5 ± 0.46 g (leptin) food intake/24 h). To rule out the possibility
that the leptin-induced body weight reduction did not result from
bacterial contaminants, lysates from E. coli transfected with empty plasmid were subjected to the same purification procedure. These preparations had no detectable effects on the body weight of
ob/ob or db/db mice or on the LSR activity in
cultured cells.
Protein Determinations--
Protein concentrations were
determined using a modified Lowry assay as described previously (11),
using BSA as a standard.
Statistical Analysis--
Results were analyzed using an
unpaired Student's t test.
Ligand blotting experiments were performed to identify the protein
responsible for LSR activity. As described previously, three main bands
of apparent molecular mass ~240, 115, and 90 kDa bound
125I-LDL after incubation with oleate (Fig.
1). Polyclonal antibodies were prepared
against the ~240-kDa band obtained by preparative electrophoresis.
This molecular mass corresponds to that of the representative example
shown here in Fig. 1. However, the average molecular weight, determined
on the basis of 48 ligand blots, was 230 ± 60 kDa with 3 peaks at
174 ± 16 (46%), 244 ± 20 (31%), and 314 ± 19 (23%), respectively. Despite the fact that the antigen preparation
remained rather crude, the specificity of the antibodies was found
adequate by Western blotting (Fig. 1, lane 4).
After electrophoresis and transfer of total solubilized plasma membrane proteins, positive signals were detected only at bands of apparent molecular masses of 240, 115, and 90 kDa. We next tested the inhibitory effect of these antibodies on LSR activity using two independent assays. Purified IgG directed against the 240-kDa band inhibited oleate-induced binding of 125I-LDL to rat liver plasma
membranes (Fig. 2A) and the
oleate-induced binding, uptake, and degradation of 125I-LDL
in primary cultures of rat hepatocytes (Fig. 2B) by ~60% and >90%, respectively.
) and
56-kDa (
) subunits associated through disulfide bridges. Screening
of expression libraries with these antibodies led to identification of
mRNAs derived by alternate splicing from a single gene and coding
for proteins with molecular masses matching that of LSR
and
.
Antibodies directed against a synthetic peptide of LSR
and
putative ligand binding domains inhibited LSR activity. Western
blotting identified two liver proteins with the same apparent molecular
mass as that of LSR
and
. Transient transfections of LSR
alone in Chinese hamster ovary cells increased oleate-induced binding
and uptake of lipoproteins, while cotransfection of both LSR
and
increased oleate-induced proteolytic degradation of the particles.
The ligand specificity of LSR expressed in cotransfected Chinese
hamster ovary cells closely matched that previously described using
fibroblasts from subjects lacking the low density lipoprotein receptor.
LSR affinity is highest for the triglyceride-rich lipoproteins, chylomicrons, and very low density lipoprotein. We speculate that LSR
is a rate-limiting step for the clearance of dietary triglycerides and
plays a role in determining their partitioning between the liver and
peripheral tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-VLDL isolated
from subjects with type III hyperlipidemia (10, 11). Several
characteristics of LSR suggest that it represents an important step for
the clearance of chylomicrons. Indeed, LSR is expressed in the liver,
and its activity is markedly increased in endocytic vesicles (10). LSR
is inhibited by lactoferrin, a milk protein that, when injected
intravenously, inhibits the uptake of chylomicrons by the liver (10,
12, 13). Also, apoCIII inhibits the binding of triglyceride-rich
lipoprotein chylomicrons and VLDL but not that of LDL to LSR (14),
while apoCIII overexpression in mice induces profound
hypertriglyceridemic effects (15). Finally, in rats, the apparent
numbers of LSR expressed at the surface of hepatocytes correlate
strongly and negatively with plasma TG levels measured in the
postprandial stage (12). The limitations of this model are 2-fold.
First, maximal activation of the receptor requires FFA at
concentrations that exceed albumin-binding capacity. It is our
hypothesis, as yet unproven that large amounts of FFA are released by
hepatic lipase acting upon chylomicrons and VLDL directly in the
environment that bathes the receptors (16). Second, the molecular
characterization of the receptor remained incomplete and relied
entirely on the identification of candidate proteins by ligand blotting
in the presence of oleate (10, 12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
residues 488-502 was obtained commercially. Polyclonal antibodies directed against this synthetic peptide conjugated to KLH were obtained, and the IgG fraction was purified as described above.
80 °C in the presence of a proteolytic inhibitor mixture (19).
-counter (Pharmacia 1470 Wizard).
gt11 rat liver 5' stretch plus
cDNA expression library (CLONTECH, Ozyme,
Montigny Le Bretonneux, France) was screened with polyclonal anti-LSR
antibodies (10 µg/ml purified IgG, 10 mM Tris-HCl, pH 8, containing 150 mM NaCl, and 0.05% Tween 20 (TNT)) in the presence of 5% nonfat dry milk (25, 26). After washing with TNT,
membranes were incubated with an alkaline phosphatase-conjugated affinity-purified F(ab')2 fragment goat anti-rabbit IgG
(Immunotech). Positive clones were isolated and verified by secondary
and tertiary screening. Sequences of LSR
,
', and
forms can
be found in GenBankTM (accession numbers AF119667,
AF119668, and AF119669, respectively).
-actin cDNA that
was provided with the blot (CLONTECH). The
hybridization buffer used contained 5× SSPE, 10× Denhardt's solution, 0.5% SDS, 100 µg of salmon sperm DNA, and 50% deionized formamide. The filters were washed in 2× SSC, 0.5% SDS at room temperature and in 1× SSC, 0.1% SDS at 65 °C.
-D-thiogalactopyranoside. The cells were
collected by low speed centrifugation and lysed by repeated freeze-thaw
cycle and deoxyribonuclease I digestion. The cell membranes were
extracted by detergent, and the inclusion bodies were pelleted. After
three washes with 1% (w/v) sodium deoxycholate in PBS, the inclusion
bodies were solubilized in 6 M guanidine-HCl. The
renaturation of the recombinant protein was performed by a 100×
dilution in PBS. The renatured protein was purified and concentrated by
immobilized metal affinity chromatography with a nickel-ion affinity
column (Probond, Invitrogen); protein was eluted with 300 mM imidazol. The purity of the recombinant leptin was
determined by SDS-polyacrylamide gel electrophoresis to be >90%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of LSR elements by ligand and
Western blotting. Ligand blots were performed, as described
previously (10, 12), on protein solubilized from rat liver membranes
(400 µg/lane; lanes 1, 2, and
4) or the LSR 240-kDa band purified by preparative
electrophoresis (80 µg/lane; lane 3).
Nitrocellulose membranes were incubated with 40 µg/ml
125I-LDL in the absence (lane 1) or
presence (lanes 2 and 3) of 0.8 mM oleate or polyclonal anti-LSR antibodies prepared
against the 240-kDa band (lane 4), as described
under "Experimental Procedures."
View larger version (10K):
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Fig. 2.
Inhibiting effect of anti-LSR antibody on its
activity. A, oleate-induced binding of
125I-LDL (5 µg/ml) to rat liver plasma membranes was
measured as reported previously (12), except that after preincubation
with 0.8 mM oleate, membranes were washed six times and
incubated for 1 h at 4 °C with increasing concentrations of an
irrelevant IgG ( ) or anti-LSR IgG (
) before the addition of
125I-LDL. B, binding, uptake, and degradation of
125I-LDL (20 µg/ml) was measured (triplicate
determinations) in primary cultures of rat hepatocytes incubated 4 h at 37 °C in the presence or absence of 0.5 mM oleate
and 200 µg/ml irrelevant IgG (open bars) or
anti-LSR IgG (closed bars). In both A
and B, results shown as mean ± S.D. are expressed as
percentage of LSR activity (for A, the control value (100%)
was 0.45 µg of 125I-LDL bound per mg of membrane protein;
for B, oleate-induced binding, uptake, and degradation of
125I-LDL were 208 ± 24, 1215 ± 1.6, and
138 ± 38 ng/mg cell protein, respectively).
Lysates of cultured hepatocytes labeled for 2 h with
35S-Promix (methionine and cysteine) were obtained and
immunoprecipitated with the same antibodies. After SDS-polyacrylamide
gel electrophoresis under nonreduced conditions, three major bands
(240, 180, and 70 kDa) and two minor bands (115 and 90 kDa) were
identified (Fig. 3, lanes
2 and 3), while under stringent reducing
conditions, two main bands of 68 and 56 kDa were detected (Fig. 3,
lane 4). Analysis of gels run under nonreduced
conditions in the first dimension and reduced conditions in the second
dimension, indicated that the 240-kDa band resolved into a 68-kDa ()
and 56-kDa (
) band (data not shown). Occasionally the 68-kDa band
appeared as a doublet; a representative example is provided in Fig. 3,
lane 5. After 30 min of labeling followed by a
60-min chase to allow the expression of newly synthesized protein on
the cell surface, treatment of hepatocytes for 5 min with trypsin
degraded most of the 68- and 56-kDa subunits (data not shown). This
indicated that these proteins were primarily expressed on the cell
surface.
|
Anti-LSR IgG was next used to screen a GT11 expression library of
rat liver cDNA. Positive clones with a 1.8-kilobase insert were
obtained and found to have identical sequences. The full-length (2.1 kilobases) cDNA was obtained by 5'-rapid amplification of cDNA
ends PCR, cloned, and sequenced (Fig.
4A). Analysis of this sequence
indicated that it contained an open reading frame starting at base 218 within a Kozak consensus sequence (Fig. 4A) (28). The
predicted protein sequence showed no homology with that of the LDL
receptor or any of its related proteins (Fig. 4B) (2, 3).
Nevertheless, the different putative domains of the candidate cDNA
were compatible with a function as plasma membrane receptor (Fig.
4C). Indeed, a single cluster of hydrophobic residues of a
length sufficient to constitute a potential transmembrane-spanning domain and several putative cellular routing motifs were present. Among
those are a phosphorylation site that also corresponds to a partial
clathrin binding site (29) and a dileucine-lysosomal targeting signal
(30-32). All of these motifs were located toward the
NH2-terminal end, consistent with this domain being
intracellular. A cysteine-rich domain that corresponded to a TNF-
receptor signature was found near the transmembrane-spanning domain.
Such a motif is present in multiple copies on the extracellular domain
of various cytokine receptors but was present only as a single copy on
the putative LSR gene (33). Distal to this motif was a cluster of alternatively positively and negatively charged residues that provided
a potential apolipoprotein-binding site.
|
Multiple-tissue Northern blot analysis pointed toward the liver as the
primary site of LSR candidate gene expression (Fig. 5A); more detailed examination
of blots obtained with different liver RNA extracts revealed two bands
of ~2.1 and 1.9 kilobases, respectively (data not shown). We
therefore sought by reverse transcription-PCR for other mRNA with
overlapping sequences (Fig. 5B). A single band was obtained
for all set of primers except for one (bc'), which gave three different
products. Analysis of the sequence of the three products was consistent
with a mechanism of alternate splicing from a single precursor.
|
The three mRNAs were designated after their base number as LSR
2097, 2040, and 1893. The predicted molecular mass of LSR 2097 translation product is 65.8 kDa and matched that of the subunit (68 kDa) identified after immunoprecipitation (Fig. 3).
LSR 2040 encodes a 574 (63.8-kDa) amino acid ' subunit that is
identical to the
except that it lacks the dileucine repeat and its
second leucine-isoleucine is no longer properly positioned (Fig.
4C). Hence, the intracellular routing of the
' could
markedly differ from the
(30-32).
LSR 1893 encodes a 525-amino acid protein with a predicted molecular
mass of 58.3 kDa, which corresponded to LSR (56 kDa). This protein
lacks the putative lysosomal targeting signals, the transmembrane-spanning domain, and the cysteine-rich domain, but contains the putative FFA and ligand binding domains (Fig.
4C). We therefore speculate that the
or
' subunit
provides the transmembrane spanning domain and that the
subunits
are either located extra- or intracellularly. We next performed
semiquantitative analysis of immunoprecipitation data, taking into
account both the difference in molecular masses and the cysteine and
methionine content of the
,
', and
subunits. This analysis
showed that the
:
ratio was 2.9 ± 1 (n = 29). The histogram plot of the
:
ratios indicated that a 2:1
ratio was observed in 41%, a 3:1 ratio in 31%, and a 4:1 or 5:1 ratio
in 20% of all experiments. The predicted molecular masses of the
complexes were 185, 241, 297, and 353 kDa for the
1
2,
1
3,
1
4, and
1
5, respectively.
We next raised polyclonal antibodies against a 15-amino acid synthetic
peptide with a sequence identical to that found within the highly
charged putative apolipoprotein-binding domain i.e. on LSR
between residues 556 and 570 and on LSR
between residues 488 and 502 (peptide 170; see Fig. 4C). Western blotting with these anti-LSR peptide antibodies indicated that they recognized primarily two proteins with apparent molecular masses of 66 and 58 kDa,
respectively (Fig. 6A). The
signal is 2-3-fold more intense with the
band than with the
,
consistent with the results of the stoichiometry analysis after
immunoprecipitation. Interestingly, this antisynthetic peptide antibody
also identified a low abundance band with an apparent molecular mass of
~75 kDa. The origin and function of this band are currently unclear.
The antipeptide antibody significantly inhibited LDL binding to LSR in
plasma membranes isolated from rat liver by ~40% and in primary
cultures of rat hepatocytes by ~80% (Fig. 6, B and
C). Similar to what was observed with polyclonal
anti-240-kDa band (Fig. 2), the inhibitory effect was more pronounced
in the binding assay that used intact cells than in that which relied
on isolated plasma membranes. These data provided the first direct
evidence that the products of the newly identified gene were
responsible for LSR activity. Transfection experiments were then
performed to further support this conclusion.
|
Transient transfection of LSR into CHO-K1 cells increased the
binding of 125I-LDL after incubation with oleate (Fig.
7A, open
squares) while leaving virtually unchanged LDL binding
measured after incubations without oleate (Fig. 7A,
open circles). Cotransfection of both the
and
plasmid consistently increased the oleate-induced binding of LDL
(oleate (closed squares) versus no
oleate (closed circles)). Besides the
representative experiment shown in Fig. 7A, transient
transfections with the
plasmid alone increased oleate-induced
binding by 146 ± 37% in three independent experiments. In the
same experiment, cotransfections with the
and
plasmids increased binding by 560 ± 406% (data not shown). Uptake of LDL followed a pattern similar to that of binding (Fig. 7B). In
nontransfected CHO cells, we were unable to detect the presence of
oleate-induced LDL degradation products in the incubation medium.
Transfection of the
plasmid alone failed to increase LDL
degradation (Fig. 7C) despite causing a significant increase
in oleate-induced LDL binding and uptake (Fig. 7, A and
B). An increase in oleate-induced degradation of LDL was
detected only after cotransfection with
and
plasmids (Fig.
7C, closed squares). The increase in
oleate-induced binding of 125I-LDL to CHO-K1 cotransfected
with
and
was inhibited by more than 80% by polyclonal
antibodies directed against the 240-kDa band (data not shown). The
results of five different transient transfection experiments with
alone showed no detectable oleate-induced degradation products.
However, significant amounts of LDL degradation products were present
after cotransfection of
and
in three out of five experiments.
Transient transfections with
or with
and
did not modify the
binding, uptake, or degradation of LDL in absence of oleate. Thus,
transient transfection of LSR
and
reproducibly increased LSR
activity in CHO-K1 cells. However, the divergence in the dose-response
curves between binding/uptake and degradation after transient
transfections is consistent with other variables affecting the
functioning of LSR. We are therefore in need of a much deeper
understanding of the biology of this gene and of its products and
possibly of other genes that contribute to the regulation of this new
pathway.
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Competition experiments were performed next to test the ligand
specificity of the LSR expressed in CHO-K1 cells cotransfected with and
plasmid. As shown in Fig. 7D, chylomicrons were the most efficient ligand, while LDL and VLDL demonstrated similar affinity, with a slight but reproducible advantage to the latter. High
density lipoproteins were 5-8-fold less efficient competitors than LDL
and had an affinity similar to that of Pronase-treated LDL. However, in
keeping with our previous observations, cyclohexanedione modifications
of apoB arginine and lysine residues only slightly reduced LDL affinity
for LSR (11). These data suggest that the apoB acidic residues
interacting with positively charged residues of the LSR putative ligand
binding domain are sufficient to allow the binding of the particles to
the LSR. Together, these data indicated that the ligand specificity of
the receptor expressed in transiently cotransfected CHO cells closely
resembled that of native LSR present in rat liver plasma membranes and
in normal human fibroblasts (10-12).
Because in transfection experiments the proteolytic degradation of LDL
did not systematically follow the uptake of the particles, we sought to
ascertain that LDL were indeed internalized. The fluorescent pattern of
CHO-K1 cells incubated with DiI-LDL treated with cyclohexanedione to
prevent LDL receptor-mediated binding and uptake were examined.
Virtually no uptake of cyclohexanedione-LDL was detectable after
incubation without oleate (Fig. 8,
left panel). A faint but detectable pattern of
fluorescence was found in nontransfected cells incubated with 0.5 mM oleate (Fig. 8, middle panel).
This corresponded most likely to endogenous LSR activity.
Cotransfection of LSR and
plasmids dramatically increased the
fluorescent pattern observed after incubations with oleate (Fig. 8,
right panel) while leaving it unchanged after
incubations without oleate (data not shown).
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DISCUSSION |
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Since 1994, ligand blotting in the presence of oleate has led to identification of candidate LSR proteins. In fibroblasts isolated from a patient with familial hypercholesterolemia, we initially described two bands of 85 and 115 kDa (10). When solubilized rat liver membrane proteins were used (12), three bands of 90, 115, and 240 kDa were observed. Optimization of the experimental conditions to improve the efficiency of transfer of large molecular weight complexes and minimization of the storage of the sample to avoid proteolytic degradation indicated that the ~240-kDa band accounted for most of LSR activity.
Partial purification of the unstable LSR complexes allowed the
production of polyclonal antibodies, which 1) established a link
between the candidate proteins identified by ligand blotting and LSR
function and 2) demonstrated immunological cross-reactivity between the
240-, 115-, and 90-kDa bands. These antibodies were then used to
characterize LSR proteins endogenously labeled with [35S]methionine and cysteine. Immunoprecipitation studies
under nonreduced and reduced conditions revealed that the LSR complex
was constituted of two main subunits designated (68 kDa) and
(56 kDa). Occasionally, the
band appeared as a doublet, consistent
with PCR analysis of LSR mRNA, which indicated that at least three
mRNAs are produced. However, the probes currently available do not
allow us to distinguish between the
and
'. Stoichiometry
analysis was therefore performed with the assumption that the
subunit is primarily expressed. This analysis indicated that the most
abundant (41%) LSR complexes were
1
2
with predicted molecular masses of 180 kDa and
1
3 with predicted molecular masses of 236 kDa (31%). Therefore, the results of immunoprecipitation experiments
appeared to match those of ligand blotting with respect to both the
apparent molecular mass of the LSR complexes and the relative abundance
of the
1
2 (46 versus 41%) and
1
3 (31 versus 31%) complexes.
Clearly, the LSR
subunit is more abundant than the
or
'.
Indeed, the
band was more pronounced in Western blots using
antibodies directed against a synthetic peptide derived from a sequence
common to
,
', and
subunits. Thus, three independent sets of
experiments indicated that the LSR is a multimeric receptor consisting
of two main subunits, organized either as a heterotrimer or tetramer and assembled through disulfide bridges. We hypothesize that it is
because of the instability of these complexes that the breakdown products of 90 and 115 kDa were those initially identified.
Screening of expression libraries using polyclonal antibodies led to the cloning of a candidate gene. This gene was selected for further analysis because 1) the predicted molecular mass of its products matched that of the two main LSR subunits; 2) the sequence predicted by the open reading frame was compatible with the function of a receptor; and 3) the gene is primarily expressed in the liver, i.e. the tissue in which most LSR activity has been found. We initially relied upon the production of antisynthetic peptide antibodies to establish the link between the newly identified gene and the LSR function. The inhibitory effect of these antibodies was pronounced (40-80%), detected in two independent types of assays, and followed a pattern similar to that of polyclonal anti-240-kDa band antibodies. However, the inhibitory effect was less pronounced with the antisynthetic peptide. We cannot therefore completely rule out the possibility that another gene accounts for part of LSR activity.
Transient transfection of the LSR subunit increased the
oleate-induced binding and uptake of LDL but did not increase
proteolytic degradation of the particle. This was only seen after
cotransfection of
and
plasmids. Clearly, further studies using
stably transfected cell lines are needed to define the biology of LSR
and understand how the
subunit contributes to the regulation of its
cellular routing. At this stage, however, transient transfection data
reproducibly established that the products of the LSR gene functioned
as lipoprotein receptors only when the cells were incubated with oleate.
The reasons for interexperimental variation in the efficiency of transient transfections are not clearly understood. The cell density and the time of plating appeared to be critical factors with optimum values being obtained with plating cell densities of 2.5-2.75 × 105 cells/well (six-well plates). The possibility that the assembly of LSR multimeric complex is under the regulation of molecular chaperons must also be considered. We are currently pursuing the identification of such a protein that coprecipitates with LSR and is visible as a 33-kDa band in Fig. 3, lane 4. The identification of this LSR-associated protein has been achieved through NH2 terminus sequencing, and its role in the regulation of LSR assembly and function is currently being investigated.
The ligand specificity of the reconstituted receptor closely resembles that of the LSR expressed in fibroblasts and in hepatocytes; triglyceride-rich lipoproteins are those with optimal affinity for the LSR (10, 12). We nevertheless used LDL in most experiments mainly because of technical considerations. Indeed, LDL, unlike triglyceride-rich lipoprotein, contains a single nonexchangeable apolipoprotein (apoB), is easily prepared in large quantities, and is radiolabeled mostly on its protein moiety. Because of this, LDL binding studies provided a better signal:noise ratio than those using 125I-VLDL or 125I-chylomicrons (data not shown). We do not believe, however, that under normal conditions LDL is a physiological ligand of the LSR. Indeed, LDL contains mainly cholesterol and cannot, when acted upon by hepatic lipase, produce FFA in sufficient amounts to induce the conformational shift of the LSR complex that unmasks the lipoprotein binding site (12). It is possible, however, that a significant part of LDL are cleared through the LSR when the LDL receptor is defective and plasma LDL concentrations are therefore increased. Such a mechanism could explain that the bulk of LDL is cleared by hepatocytes, even in subjects lacking the LDL receptor (34).
LSR is capable of binding unmodified TG-rich chylomicrons, and its
affinity is greatest for these as well as the larger of the VLDL
particles (11). We therefore postulate that it provides a significant
pathway for the clearance of chylomicrons. One would therefore
anticipate that it binds apoE. Previous studies have shown that
triolein phosphatidylcholine emulsions supplemented with apoE bind to
LSR while the same emulsions not supplemented with apoE do not (10).
Furthermore, VLDL isolated from subjects with the apoE 2/2 phenotype
and with symptomatic type III hyperlipidemia does not bind to LSR (10).
In addition, studies of subjects with type III hyperlipidemia have
shown that this condition was markedly influenced not only by the
presence of a specific apoE isoform (apoE 2/2) but also by yet
unidentified nutritional factors (35). The characterization of the LSR
gene also shed new light on the results of previous studies of the apoE
receptor (36, 37). Indeed, the fraction of apoE binding proteins,
initially found to be partially contaminated with
F1-ATPase, contained an unidentified 59-kDa apoE-binding
protein (37). The molecular weight of this protein matches closely the
predicted 58 kDa of the most abundant LSR subunit. It is possible
that prior to the assembly of the LSR complex, the most abundant
subunit is localized within the cell, i.e. the location of
the previously identified putative apoE receptor (37).
In contrast with the previously described apoE receptor, the LSR complex also binds apoB. The apoB domain that binds to LSR appears to be different from that of the LDL receptor. Indeed, cyclohexanedione modification of apoB basic residues suppresses its ability to bind to the acidic residues of the LDL receptor, whereas apoB binding to LSR is slightly, but not markedly, decreased by cyclohexanedione. This suggests that apoB acidic residue, interacting with LSR positively charged residues, are sufficient to mediate the binding.
The finding of a second receptor that binds both apoB and apoE was
rather unexpected. It is, however, not the first characterization of a
receptor distinct from the LDL receptor but capable of binding unmodified LDL. Indeed, Hoeg et al. (38) have characterized hepatic membrane receptors capable of binding LDL and with apparent molecular masses of 270 and 320 kDa. These correspond reasonably well
with those of the 1
3 and
1
4 LSR complexes. These binding sites
were expressed in membranes from subjects homozygous for familial
hypercholesterolemia, which eliminated the possibility that the LDL
binding site corresponds to the di- or trimer of the LDL receptor
(Mr ~130 kDa). Finally, similar to LSR, the
LDL binding sites were independent of calcium. Thus, the apparent molecular mass of the most abundant LSR subunit matches that of the
previously identified intracellular apoE-binding protein. However,
because of the multimeric organization of the LSR complex, it may also
account for the previously described paradoxal LDL binding site.
FFA are thus far the only molecular tools that cause a direct activation of LSR. The relative physiological importance of such mechanism, however, remains to be determined. Indeed, the concentrations of FFA that are needed to achieve such an effect are greater than those circulating in plasma bound to albumin, and it is currently unclear whether the concentrations generated at the site of lipolysis are sufficient to activate LSR. The possibility that other as yet unknown mechanisms of acute activation intervene must also be considered. However, it is our current hypothesis that the activity of the lipolytic enzymes LPL and hepatic lipase, present in the space of Disse (39, 40), act upon TG-rich particles generating FFA that induce the conformational shift of LSR, leading to its activation and the internalization of the particles. A key issue is to determine whether the receptor or the enzymes represent the rate-limiting step. Because genetic defects in LPL or its activator, apoCII, lead to massive hypertriglyceridemia, it is widely accepted that LPL is responsible for the clearance of plasma TG. Lipoprotein receptors, on the other hand, account for the removal of small cholesterol-enriched and triglyceride-depleted chylomicron remnants. Indeed, this might very well be the function of LRP (7). It is, however, likely that LSR acts in concert with the lipase system and contributes to the clearance of a significant fraction of plasma TG. In support of this interpretation is the finding that the LSR binds with high affinity large TG-rich chylomicrons (10). Further, overexpression of receptor-associated protein up to concentrations that inhibit LSR activity caused hypertriglyceridemia (8, 9). Also, injections with the LSR inhibitor lactoferrin induce a massive postprandial hypertriglyceridemia (10).2 In addition, LSR apparent number in the liver correlates strongly and negatively with postprandial plasma TG but not with plasma cholesterol. It is likely that the LSR represents a rate-limiting step for the removal of TG. We therefore speculate that the relative activity of LPL in peripheral tissues versus that of LSR in the liver plays a key role in determining the partitioning of dietary lipid between these different tissues.
Identification of the LSR gene will allow the production of transgenic
mice overexpressing or deficient for this receptor. These animal models
can be used to define the precise role of the liver in determining the
ability of individuals to dispose of dietary lipid.
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ACKNOWLEDGEMENTS |
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We thank Christine Ory, Nathalie Denoual, and Arnaud Botrel for excellent technical support. Dr. Didier Thoraval performed the screening of the expression library, and Alain Kerihuel provided assistance in image analysis.
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FOOTNOTES |
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* This work was supported by grants from INSERM, the Région Bretagne, the European Community, and Genset SA.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Genset Corp., 875 Prospect St., Suite 206, La Jolla, CA 92037. Tel.: 619-551-3000; Fax: 619-551-3044; E-mail: bihainb{at}genxy.com.
2 M. Masson, F. T. Yen, and B. E. Bihain, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TG, triglyceride(s); apo, apolipoprotein; BSA, bovine serum albumin; CHO, Chinese hamster ovary; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate; DMEM, Dulbecco's modified Eagle's medium; FFA, free fatty acid(s); LSR, lipolysis-stimulated receptor; LPL, lipoprotein lipase; LDL, low density lipoprotein(s); LRP, low density lipoprotein receptor-related protein; PBS, phosphate-buffered saline; VLDL, very low density lipoprotein; PCR, polymerase chain reaction.
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REFERENCES |
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