From the Tohoku University Gene Research Center,
Sendai 981-8555, Japan, the ¶ Department of Animal Science,
College of Agriculture, Chonnam National University, Kwangju 500-600, Korea, the
Graduate School of Human Life Science, Osaka City
University, Osaka 558-8585, Japan, the ** Department of
Pathology, Tohoku University Graduate School of Medicine, Sendai
980-8574, Japan, the
Third Department of
Internal Medicine, Fukui Medical University, Fukui 910-1193, Japan, and
the §§ Division of Nephrology, Endocrinology,
and Vascular Medicine, Department of Medicine, Tohoku University
Graduate School of Medicine, Sendai 980-8574 and Yanagisawa Orphan
Receptor Project, Exploratory Research for Advanced Technology (ERATO),
Japan Science and Technology Corporation, Tokyo 135-0064, Japan
Received for publication, November 22, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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By expression cloning using fluorescent-labeled
high density lipoprotein (HDL), we isolated two clones that conferred
the cell surface binding of HDL. Nucleotide sequence of the two clones revealed that one corresponds to scavenger receptor class B, type 1 (SRBI) and the other encoded a novel protein with 228 amino acids. The
primary structure of the newly identified HDL-binding protein resembles
GPI-anchored proteins consisting of an N-terminal signal sequence, an
acidic region with a cluster of aspartate and glutamate residues, an
Ly-6 motif highly conserved among the lymphocyte antigen family, and a
C-terminal hydrophobic region. This newly identified HDL-binding
protein designated GPI-anchored HDL-binding protein 1 (GPI-HBP1), was
susceptible to phosphatidylinositol-specific phospholipase C treatment
and binds HDL with high affinity (calculated Kd = 2-3 µg/ml). Similar to SRBI, GPI-HBP1 mediates selective lipid
uptake but not the protein component of HDL. Among various ligands for
SRBI, HDL was most preferentially bound to GPI-HBP1. In contrast to
SRBI, GPI-HBP1 lacked HDL-dependent cholesterol efflux. The
GPI-HBP1 transcripts were detected with the highest levels in heart
and, to a much lesser extent, in lung and liver. In situ
hybridization revealed the accumulation of GPI-HBP1 transcripts in
cardiac muscle cells, hepatic Kupffer cells and sinusoidal endothelium,
and bronchial epithelium and alveolar macrophages in the lung.
High density lipoprotein
(HDL)1 plays a key role in
the transportation of cholesterol to extrahepatic tissues including
steroidogenic tissues and in the reverse transportation of cholesterol
from extrahepatic tissues to the liver (1). Unlike the low density lipoprotein (LDL) receptor pathway, the delivery of cholesterol from
HDL to cells is mediated by selective lipid uptake from HDL particles
and is independent of internalization of HDL. Reverse cholesterol
transportation requires the extraction of cholesterol from extrahepatic
cells by HDL and the subsequent delivery of cholesterol to hepatocytes.
Several HDL-binding proteins have been identified including class B
type I scavenger receptor (SRBI) (2, 3), two candidate hepatic HDL
receptors designated HDL-binding proteins 1 and 2 (4, 5), 80- and 130-kDa GPI-anchored HDL-binding proteins expressed in human
macrophages (6), 110-kDa GPI-anchored HDL-binding protein expressed in
HepG2 cells (7), and recently characterized 95-kDa HDL-binding protein
(8). To date, only SRBI appears to be a physiological HDL receptor
based on the selective uptake of cholesterol esters into cells and the
efflux of cholesterol from cells to HDL mediated by SRBI (1).
Consistent with the postulated physiological role, SRBI is highly
expressed in tissues that selectively take up cholesterol esters from
HDL including liver, adrenal gland, testis, and ovary (3). Although
hepatic overexpression of SRBI mediated by an adenovirus encoding SRBI resulted in a dramatic reduction of plasma cholesterol (9), the
targeted disruption of the murine SRBI gene led to a
modest increase in plasma cholesterol (10). This finding may suggest the presence of other HDL receptors that cooperate with SRBI in the
metabolism of HDL.
In this study, we identified a novel HDL-binding protein by expression
cloning from a murine hepatic cDNA library. This newly identified
HDL-binding protein designated GPI-anchored HDL-binding protein 1 (GPI-HBP1) belongs to the GPI-anchored lymphocyte differentiation antigen Ly-6 family, binds HDL with high affinity on the cell surface,
and mediates selective lipid uptake from HDL particles. We also
describe the ligand specificity and tissue expression of GPI-HBP1.
Materials--
Human HDL, LDL, acetylated LDL and oxidized LDL,
and newborn calf lipoprotein-deficient sera were prepared essentially
as described previously (2, 11). 125I-Labeled HDL was
prepared according to the procedure as described previously (12).
Oxidized LDL was prepared by dialyzing 1 ml of LDL (4-10 mg/ml)
(prepared without Standard Procedures--
Standard molecular biology techniques
were performed essentially as described by Sambrook and Russell (15).
cDNA clones were subcloned into pBluescript vectors and sequenced
by the dideoxy chain termination method with a BigDye Terminator Cycle
Sequencing Ready Reaction kit (PE Biosystems) and a DNA sequencer
(model 310, PE Biosystems). To analyze RNA in murine tissues,
commercially available Northern blots (Clontech
laboratories) were used for Northern blot analysis.
32P-Labeled probes were prepared by priming with random hexanucleotides.
Expression Cloning--
A cDNA library was constructed from
poly(A) RNA isolated from the livers of LDL receptor-deficient male
mice (16) in the pZeoSV2 vector (Invitrogen) using a NotI
unidirectional primer. The cDNA library consisted with ~3 × 105 clones, and these clones were divided into
small pools (300 clones/pool). Plasmid DNA from each pool was prepared
using the QIAprep 96 Turbo Miniprep kit (Qiagen). On day 0, LDL
receptor-lacking Chinese ovary cells, ldlA7 (17), were plated into
96-well plates (5 × 104/well) in minimum essential
medium Cell Culture and Transfection--
The entire coding regions of
murine GPI-HBP1 and SRBI were subcloned into the pRC/CMV vector
(Invitrogen) for stable transfection. CHO ldlA7 cells were transfected
with the expression plasmid using the LipofectAMINE reagent
(Invitrogen) according to the manufacturer's instructions. Stably
transfected cells were selected in Ham's F-12 medium containing 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mM
glutamine (medium B) supplemented with 5% fetal bovine serum and 250 µg/ml G418 for 2 weeks. For 125I-HDL and DiI-HDL
binding studies and [3H]cholesterol efflux assays (see
below), cells were plated in 6-well (250,000 cells/well) dishes in
medium B supplemented with 5% newborn calf lipoprotein-deficient serum
and cultured for 48 h.
Phosphatidylinositol-specific Phospholipase C (PIPLC)
Treatment--
GPI-HBP1-expressing cells were incubated for 1 h
in medium B supplemented with 5% newborn calf lipoprotein-deficient
serum with or without 1 unit/ml PIPLC. Cells were then washed and
incubated for the measurement of DiI-HDL uptake.
125I-HDL-binding and Association Assays--
Cells
were washed once with medium B and then refed with medium B containing
0.5% (w/v) fatty acid-free BSA and the indicated concentrations of
125I-HDL. After a 2-h incubation at 37 °C, cells were
washed once with 50 mM Tris-HCl, pH 7.4, and 0.15 M NaCl (buffer A) containing 2 mg/ml BSA followed by two
quick washes with buffer A without BSA. Cells were then solubilized
with 0.1 N NaOH for 30 min at room temperature on a shaker,
and we determined the amounts of cell-associated radioactivity using a
Fluorimetric Assay of DiI-HDL Uptake--
DiI-HDL was used to
measure the cellular association and uptake of fluorescence by cells
stably expressing GPI-HBP1 or SRBI according to the procedure as
described by Acton et al. (3). Cells were washed once with
medium B and then refed with medium B containing 0.5% (w/v) fatty
acid-free BSA and the indicated concentrations of DiI-HDL. After
incubation at 37 °C for 2 h, cells were washed twice with PBS
containing 5 mM CaCl2 and 5 mM MgCl2 (5 min/wash). Cell-associated DiI was then
solubilized in 0.5 ml of Me2SO at room temperature for
2 h, and the fluorescence was measured using a spectrofluorimeter.
The amount of DiI in each sample expressed as equivalent amounts of
DiI-HDL (micrograms of protein) was calculated by comparing the
fluorescence intensity of the sample to that from a standard curve
generated by dissolving DiI-HDL in Me2SO. Specific DiI
uptake was determined by subtracting the values obtained with parental
vector transfected cells from those obtained with a given expression plasmid.
[3H]Cholesterol Efflux from Stably Transfected CHO
ldlA7 Cells--
HDL-dependent cholesterol efflux study
was performed according to the procedure described by Gu et
al. (19). Cells (~70% confluent) were incubated for 48 h
with 0.2 µCi/ml [1,2-3H]cholesterol (40-60 Ci/mmol,
Amersham Biosciences). After washing five times with PBS
containing 1% fatty acid-free BSA, radiolabeled cells were incubated
overnight in Ham's F12 containing 1% fatty acid-free BSA to allow for
the equilibration of cellular cholesterol pools. Cells were then washed
and incubated for the indicated times in efflux medium (Ham's
F12, 0.5% fatty acid-free BSA) with or without 40 µg/ml HDL. The
efflux medium was collected and clarified by centrifugation for 1 min
with a Microcentrifuge, and the radioactivity of each
supernatant was determined by liquid scintillation counting. Cells were
solubilized with 1% Triton X-100 in PBS for 30 min at room
temperature, and the amount of [3H]cholesterol in each
lysate was determined. Total cellular [3H]cholesterol was
calculated as the sum of the radioactivity in the efflux medium plus
the radioactivity in the cells and was used to calculate the
[3H]cholesterol efflux (percent of total
[3H]cholesterol released into the medium).
In Situ Hybridization--
Digoxigenin-11-UTP-labeled
single-stranded RNA probes were prepared with a digoxin/RNA-labeling
mixture and the corresponding T3 or T7 RNA polymerase (Roche Molecular
Biochemicals) according to the manufacturer's
instructions. The entire coding region of murine GPI-HBP1 cDNA was
subcloned into the pBluescript II vector (Stratagene) and used to
prepare single-stranded RNA probes. Tissues from a C57BL/6J male mouse
(10-weeks old) were fixed in PBS containing 4% paraformaldehyde at
4 °C for 12 h, dehydrated, and embedded in paraffin using a
standard procedure. In situ hybridization was performed
using 6-µm thick sections mounted on silane-coated glass slides.
After digestion with 10 mg/ml proteinase K at 37 °C for 20 min, the
tissue sections were hybridized with antisense or sense RNA probes at
50 °C for 16 h. For the reaction with antidigoxigenin antibodies, slides were washed with 100 mM Tris-HCl, pH 7.5 and 150 mM NaCl (buffer B), treated with 0.5% blocking
reagent (Roche Molecular Biochemicals) in buffer B, and then incubated
with alkaline phosphatase-coupled anti-digoxigenin antibodies (diluted
1:750 in buffer B, Roche Molecular Biochemicals) for 1 h.
Expression Cloning of HDL-binding Proteins--
We screened a
cDNA library from murine liver for cDNAs that facilitate the
binding of HDL when transiently expressed in LDL receptor-lacking ldlA7
cells. After screening a total of 960 pools of ~300 cDNA each, we
obtained two pools of cDNAs that stimulated HDL binding to levels
that were significantly higher than background. We then plated a total
of 960 colonies from two positive pools into individual wells of
96-well plates. We prepared cDNAs from the pooled rows and columns
of the plates. Four of these pools gave positive results. We then
assayed individual clones from the wells at the intersections of the
positive rows and columns and obtained two positive clones (Fig.
1). Nucleotide sequencing of these clones
and subsequent BLAST search revealed that one corresponds to murine
SRBI cDNA (3) and the other (designated pHRC7) encoded a protein of
228 amino acids (Fig. 2).
Structure of a GPI-anchored HDL-binding Protein--
A hydropathy
plot of the deduced amino acid sequence of the cDNA shows the
presence of two hydrophobic regions (Fig. 2), one at the N terminus and
the other at the C terminus. The former corresponds to a classical
signal sequence of probable 19 amino acids in length, whereas the
latter strongly resembles the hydrophobic region of GPI-anchored cell
surface proteins. Excluding the N-terminal 19 amino acids, the mature
protein would then consist of 209 amino acids with a calculated
Mr of 22,603. This value is greatly smaller than
those values of the previously characterized candidate receptors for
HDL (4-8).
The predicted amino acid sequence revealed the presence of a region
highly enriched with acidic amino acids (aspartate and glutamate) and a
region similar to a highly conserved domain termed Ly-6 motif, which
occurs singly in the GPI-anchored lymphoid differentiation antigen Ly-6
family, and is repeated 3-fold in urokinase-type plasminogen activator
receptor. These results predicted that the cloned protein consists of
an N-terminal signal sequence, an acidic region with a cluster of
aspartate and glutamate residues, an Ly-6 motif, and a C-terminal
hydrophobic region.
Based on the structural similarity with the GPI-anchored Ly-6 family
proteins, we analyzed the effects of phosphatidylinositol-specific PIPLC treatment on the HDL binding. As shown in Fig.
3, the PIPLC treatment almost completely
abolished the uptake of DiI-HDL, suggesting that the cloned protein is
indeed GPI-anchored. Therefore, we designate the cloned HDL-binding
protein as GPI-HBP1.
EST data base search identified an isoform lacking the C-terminal
half of GPI-HBP1. This isoform (designated sHBP1) shares the N-terminal
signal sequence, the acidic region, and part of the Ly-6 motif with
GPI-HBP1 but lacks the C-terminal hydrophobic region, suggesting that
the isoform is a secreted form generated by alternative splicing.
Consistent with the deduced structural feature, ldlA7 cells
transiently transfected with sHBP1 failed to bind DiI-HDL on the cell
surface (Fig. 1C).
BLAST searches of the GenBankTM databases revealed that
GPI-HBP1 is structurally related to Ly-6 molecules, a set of
GPI-anchored cell surface proteins belonging within a superfamily that
includes the urokinase-type plasminogen activator receptor, the
complement inhibitor CD59, the sperm antigen SP10, and more distantly,
the snake venom neurotoxin family. The Ly-6 motif consists of ~90 amino acids with ten highly conserved cysteine residues (20). A
comparison of the sequence of the Ly-6 motif in GPI-HBP1 with those in
the Ly-6 family members revealed that these cysteine residues are
completely conserved in the Ly-6 motif of GPI-HBP1 (data not shown).
The phylogenetic tree of the Ly-6 family proteins of various origins
indicates that GPI-HBP1 is most closely related to Ly-6 molecules (data
not shown).
HDL Binding--
To characterize lipoprotein-binding
properties, cDNAs encoding GPI-HBP1 and SRBI were stably expressed
in ldlA7 cells. The binding of 125I (125I-HDL)
or fluorescent (DiI-HDL) labeled HDL was measured following incubation
of the cells. As shown in Fig. 4,
A and B, the saturation binding of
125I-HDL was observed in both GPI-HBP1- and SRBI-expressing
cells at 4 and 37 °C. Although the maximal binding of
125I-HDL in GPI-HBP1-expressing cells was 3-fold lower than
that of SRBI-expressing cells, the calculated Kd
values of the two proteins were within the same range (2-3 µg/ml).
The relative maximal binding activity between SRBI and GPI-HBP1 could
not be determined because the expression levels of these two proteins in ldlA7 cells were unknown. Similar saturation binding was observed when cells were incubated with DiI-HDL at 4 °C (Fig. 4C).
In contrast, when GPI-HBP1-expressing cells were incubated with DiI-HDL
at 37 °C, the amounts of DiI-HDL binding by the cells were ~3-fold higher than those at 4 °C and unsaturable (Fig. 4D).
Similarly, the amounts of DiI-HDL binding at 37 °C by
SRBI-expressing cells were ~6-fold higher than those at 4 °C.
Furthermore, compared with 125I-HDL binding, the
degradation of 125I-HDL (trichloroacetic
acid-soluble 125I) at 37 °C was almost negligible
in both cells (data not shown). These data indicate that GPI-HBP1,
similar to SRBI (3), mediates selective lipid uptake but not the
protein component of HDL.
Effects of SRBI Ligands--
We next analyzed the effects of
various ligands for SRBI (2, 14) on the binding of 125I-HDL
to GPI-HBP1-expressing cells. As shown in Fig.
5, in the presence of excess unlabeled
HDL, the binding of 125I-HDL to GPI-HBP1- and
SRBI-expressing cells was strongly reduced. Compared with the
strong inhibition of 125I-HDL binding to SRBI-expressing
cells by human apoAI (free form), phosphatidylserine, and acetylated
LDL (inhibited by ~75%), the inhibitory effects by these compounds
were relatively lower in GPI-HBP1-expressing cells (reduced by
~50%). Oxidized LDL, native LDL, and human apoAII had relatively
weak inhibitory effects on 125I-HDL binding to GPI-HBP1-
and SRBI-expressing cells. These data show that HDL is bound to
GPI-HBP1 most preferentially among various SRBI ligands including HDL,
phosphatidylserine, and acetylated LDL.
Cholesterol Efflux--
In addition to selective lipid
uptake from HDL particles, SRBI mediates HDL-dependent
cholesterol efflux. To test whether GPI-HBP1 also mediates cholesterol
efflux, the cells were labeled with [3H]cholesterol,
allowed for the equilibration of cellular cholesterol pools, and then
incubated with or without HDL. As shown in Fig. 6, SRBI-expressing cells exhibited
HDL-dependent cholesterol efflux with
time-dependent manner, whereas almost no cholesterol efflux was seen in GPI-HBP1-expressing cells in the absence or the presence of
HDL. The lack of cholesterol efflux in GPI-HBP1 cells indicates that
GPI-HBP1 mediates selective lipid uptake only, whereas SRBI mediates
both influx and efflux of cholesterol.
Expression of GPI-HBP1 Transcripts--
Northern blot analysis of
RNA from various murine tissues revealed hybridization of the HBP1
probe to a major transcript of 0.8 kilobases with the highest
expression in heart and, to a much lesser extent, in lung and liver
(Fig. 7). Apparently, no transcripts were
detected in other tissues including brain, kidney, skeletal muscle,
spleen, and testis.
To locate cells expressing GPI-HBP1 transcripts, in situ
hybridization was performed using tissue sections from murine liver, heart, and lung. In the liver, the accumulation of hybridization signals for GPI-HBP1 transcripts appearing dark blue were detected most
intensely in the Kupffer cells and sinusoidal endothelium, but no
significant accumulation was detected in the parenchymal cells (Fig.
8, panel A). In the heart, the
intense hybridization signal was detected in cardiac muscle cells (Fig.
8, panel C). The intense hybridization signals were also
detected in bronchial epithelium and in alveolar macrophages in the
lung (Fig. 8, panel E).
In contrast to the abundant expression of SRBI in steroidogenic tissues
(3, 21), GPI-HBP1 transcripts were highly accumulated in the Kupffer
cells as well as in alveolar macrophages of the lung. Based on the
abundant expression in these scavenger cells and the lack of
cholesterol efflux, it is suggested that GPI-HBP1 plays a role in the
initial entry of HDL cholesterol into these scavenger cells for further
transportation of cholesterol. To elucidate the precise biological role
of GPI-HBP1 and to determine any disorders caused by the absence of the
protein, the generation of mice lacking GPI-HBP1 is currently underway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-hydroxytoluene) against saline solution
containing 5 µM Cu2SO4 (2 × 500 ml) for 24-48 h at 4 °C.
1,1'-Dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI)
was obtained from Molecular Probes (Eugene, OR) and was used to prepare
fluorescent-labeled HDL as described previously (13). Human apoAI and
AII, phosphatidylinositol-specific phospholipase C (Bacillus
cereus, specific activity of 7.1 units/mg), egg yolk
phosphatidylcholine, and brain phosphatidylserine were from Sigma.
Phospholipid liposomes were prepared by extrusion through polycarbonate
membranes as described previously (14): phosphatidylcholine liposomes
(phosphatidylcholine/free-cholesterol, molar ratio 2:1) and
phosphatidylserine liposomes
(phosphatidylcholine/phosphatidylserine/free-cholesterol, molar ratio
1:1:1).
supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (medium A). On day 1, the cells in each plate were transfected with 0.1 µg of a cDNA
pool using Effectene transfection reagent (Qiagen) according to the
manufacturer's protocol. After incubation for 16 h, the
transfection mixture was replaced with medium A. On day 3, the
monolayers were refed with minimum essential medium
containing 2 µg/ml protein of DiI-HDL and 5% lipoprotein-deficient serum. After a
2-h incubation at 37 °C, the plates were washed twice with PBS
containing 10 mg/ml BSA and then twice with PBS, and the cells were
fixed with 3% formaldehyde in PBS for 15 min at room temperature. The
presence of DiI in the fixed cell was detected by visual inspection
under fluorescent microscopy. Positive pools were serially subdivided
and retested to obtain positive cDNA clones.
-counter. The protein content was determined using the method of
Lowry et al. (18). For 4 °C binding studies, the protocol
was identical to that at 37 °C with the exception that the cells
were prechilled on ice for 15 min and incubated with
125I-HDL at 4 °C for 2 h. Specific cell association
or binding was determined by subtracting the values obtained with
parental vector (pRC/CMV) transfected cells from those obtained with a
given expression plasmid.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Visualization of receptor mediated uptake of
fluorescent HDL by CHO cells expressing murine GPI-HBP1.
Transiently expressing cells were used in this study. Murine GPI-HBP1
cDNA (pHRC7) (A), murine SRBI cDNA (B),
murine sHBP1 cDNA (C), and parental vector (pZeoSV2)
(D) were independently introduced into LDL
receptor-deficient ldlA7 cells as described under "Experimental
Procedures." After a 2-h incubation with DiI-HDL at 37 °C, the
cells were washed and fixed, and the presence of fluorescent DiI in the
fixed cell was detected under fluorescent microscopy.
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Fig. 2.
Amino acid sequence of murine GPI-HBP1.
The amino acid sequence of murine GPI-HBP1 deduced from the cDNA is
compared with that of murine sHBP1. Identical amino acids are indicated
by asterisks. The N- and C-terminal hydrophobic amino acids
were boxed. A cluster of negatively charged amino acids is
underlined. The Ly-6 motif is indicated by a dotted
underline.
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Fig. 3.
Effect of PIPLC treatment on DiI-HDL
uptake. A, murine GPI-HBP1 was transiently expressed in
ldlA7 cells as described under "Experimental Procedures." After a
1-h incubation with or without PIPLC at 37 °C, cells were washed and
incubated for the measurement of DiI-HDL uptake. B, DiI-HDL
uptake. The amount of cell-associated DiI was determined as described
under "Experimental Procedures." Values are the average of
triplicate determinations, and error bars represent the
range of the three measurements.
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Fig. 4.
Concentration dependence of HDL
binding to and HDL lipid uptake by GPI-HBP1 and SRBI-expressing
cells. CHO ldlA7 cells were stably transfected with expression
vectors for murine GPI-HBP1, murine SRBI, or the control
("parental") vector (pRC/CMV) as described under "Experimental
Procedures." Cells were plated in six-well dishes and incubated with
the indicated amounts of either 125I-HDL (A and
B) or DiI-HDL (C and D) at either
4 °C (A and C) or 37 °C for 2 h. The
amounts of specific 125I-HDL binding or DiI uptake
(spectrofluorimetry) were determined by subtracting the values obtained
with parental vector (pRC/CMV) transfected cells from those obtained
with a given expression plasmid as described under "Experimental
Procedures." Similar results have been observed in multiple
independent experiments, and the data shown are representative.
Error bars represent the range of variations in the
triplicate determinations.
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Fig. 5.
Inhibition of 125I-HDL binding to
GPI-HBP1 expressing cells by various compounds. GPI-HBP1 or SRBI
expressing cells were incubated with 10 µg/ml 125I-HDL at
4 °C for 2 h in the presence of 200 µg/ml of the indicated
compound. PC, phosphatidylcholine; PS,
phosphatidylserine; Ox-LDL, oxidized LDL; Ac-LDL,
acetylated LDL. The 100% control values for the binding
125I-HDL (in the absence of inhibitors) of GPI-HBP1- and
SRBI-expressing cells were 48.9 and 190 ng/mg protein, respectively.
Error bars represent the range of variations in the
triplicate determinations.
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Fig. 6.
Lack of HDL-dependent cholesterol
efflux in GPI-HBP1-expressing cells. ldlA7 cells stably expressing
GPI-HBP1 or SRBI were prelabeled with [3H]cholesterol as
described under "Experimental Procedures." Cells were incubated
for the indicated times in medium containing 0.5% BSA with or without
40 µg/ml HDL. Results are expressed as percentages of radioactivities
released in the culture media of the total radioactivities in media and
cells. Values are the average of triplicate determinations. The
error bars are too small to show.
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Fig. 7.
Tissue expression of GPI-HBP
transcripts. 2 µg of poly(A) RNA from the indicated murine
tissues was probed with 32P-labeled murine GPI-HBP1
(upper panel). The filters were exposed to Kodak X-Omat AR
film with an intensifying screen at 80 °C for 48 h. The same
samples were subsequently hybridized with a control probe for mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(lower panel) and exposed to Kodak X-Omat AR with an
intensifying screen at
80 °C for 6 h.
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Fig. 8.
In situ hybridization analysis of
GPI-HBP1 transcripts in mouse. Sections A,
C, and E were hybridized with an antisense probe
to murine GPI-HBP1. Sections B, D, and
F are negative controls with a sense probe. Hybridization
signals were visualized in blue. Tissue sections prepared
from liver (A and B), heart (C and
D), and lung (E and F) of a normal
male mouse were analyzed by in situ hybridization as
described under "Experimental Procedures." GPI-HBP1 transcripts
are localized in the Kupffer cells (indicated by arrows in
A) and sinusoidal endothelium (arrowheads in
A), but no significant accumulation is detected in the
parenchymal cells. In the heart, the hybridization signal is detected
in cardiac muscle cells (C). The intense hybridization
signals are seen in bronchial epithelium (arrow in
E) and alveolar macrophages (arrowhead in
E) in the lung. Bars, 50 µm
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ACKNOWLEDGEMENT |
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We thank Dr. M. Krieger for providing CHO ldlA7 cells.
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FOOTNOTES |
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* This work was supported in part by Grant RFTF97L00803 from the Japan Society for the Promotion of Science.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB095543.
§ Both authors contributed equally to this work.
¶¶ To whom correspondence should be addressed: Tohoku University Gene Research Center, 1-1 Tsutsumidori-Amamiya, Aoba, Sendai 981-8555, Japan. Tel.: 81-22-717-8875; Fax: 81-22-717-8877; E-mail: tfujino@biochem.tohoku.ac.jp.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211932200
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ABBREVIATIONS |
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The abbreviations used are: HDL, high density lipoprotein; LDL, low density lipoprotein; BSA, bovine serum albumin; CHO, Chinese hamster ovary; DiI, 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate; GPI, glycosylphosphatidylinositol; HBP1, HDL-binding protein 1; Ly-6, lymphocyte antigen 6; PBS, phosphate-buffered saline; PIPLC, phosphatidylinositol-specific phospholipase C; SRBI, scavenger receptor class B, type I; sHBP1, soluble HDL-binding protein 1.
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REFERENCES |
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