© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Identification of Soluble Forms of Lectin-Like Oxidized LDL Receptor-1
From the Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University (N.K., H.K., M.M., T.K.), and the Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka (T.S., T.M.), Japan. T. Murase is now at Biological Science Laboratories, Kao Corp, Ichikaimachi, Tochigi, Japan.
Correspondence to Noriaki Kume, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail nkume{at}kuhp.kyoto-u.ac.jp
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Abstract—Lectin-like oxidized LDL receptor-1 (LOX-1) is a type II membrane protein belonging to the C-type lectin family molecules, which can act as a cell-surface endocytosis receptor for atherogenic oxidized LDL. In this study, we show that soluble forms of LOX-1 are present in conditioned media of cultured bovine aortic endothelial cells (BAECs) and CHO-K1 cells stably transfected with LOX-1 cDNA. Immunoblot analysis of conditioned media from TNF-
–activated
BAECs and CHO-K1 cells stably expressing LOX-1 revealed that soluble
LOX-1 has an approximate molecular mass of 35 kDa. In
TNF-–activated BAECs, cell-surface expression of LOX-1
precedes soluble LOX-1 production. Cell-surface biotinylation
followed by immunoprecipitation and immunoblotting
showed that soluble LOX-1 in cell-conditioned media is derived from
LOX-1 expressed on the cell surface. Production of soluble
LOX-1 was inhibited by PMSF, suggesting that PMSF-sensitive proteases
may be involved in this process. Purification of soluble LOX-1 by
high-performance liquid chromatography and
N-terminal amino acid sequencing of soluble LOX-1 identified the 2
cleavage sites between Arg86-Ser87 and
Lys89-Ser90, which were located in the membrane
proximal extracellular domain of LOX-1. The data demonstrate that
cell-surface LOX-1 can be cleaved at 2 different sites and transformed
into soluble forms. Further studies may explore therapeutic and
diagnostic applications of soluble LOX-1 in
atherosclerotic diseases.
Key Words: LOX-1 • oxidized LDL • proteolysis • atherosclerosis • endothelial cells
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Introduction |
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Endothelial activation elicited by oxidized LDL (ox-LDL) and its lipid constituents has been implicated in the pathogenesis of atherosclerosis.1 2 We recently identified a novel receptor for atherogenic ox-LDL, designated lectin-like ox-LDL receptor-1 (LOX-1), in vascular endothelial cells.3 LOX-1 is a 50-kDa type II membrane protein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain, whose structure belongs to the C-type lectin family. Interestingly, LOX-1 does not share any homology with other receptors for ox-LDL, including class A and B scavenger receptors. Functional analysis revealed that LOX-1 supports binding, internalization, and proteolytic degradation of ox-LDL but not significant amounts of acetylated LDL.4 Our recent studies have shown that LOX-1 can also bind oxidized or aged red blood cells, as well as apoptotic cells, thus further suggesting its physiological roles in vivo.5 With regard to the regulation of expression, LOX-1 can be upregulated by inflammatory stimuli, such as tumor necrosis factor (TNF)-
and
phorbol ester,6 and by a mechanical stimulus, fluid shear
stress,7 in cultured bovine aortic
endothelial cells (BAECs). More importantly, expression
of LOX-1 is highly upregulated in atherosclerotic lesions of
humans.8 These results suggest that LOX-1 may be expressed
locally and play important roles in atherogenesis and inflammatory
responses in vivo. In the past decade, a number of membrane proteins have been shown to be converted into soluble molecules by proteolytic cleavage at the membrane proximal site of the extracellular domain.9 10 11 12 Elevated levels of soluble forms of membrane proteins in circulating blood in humans may reflect increased expression of membrane proteins and disease activities.10 12 13 14 15 16 17 In addition, soluble forms of receptors may interact with its ligands, inhibit the binding of its ligands to the cell-surface receptor, and thus modulate the pathophysiology.10 18 19 20
In the present study, therefore, we explored the possibility that LOX-1, a novel membrane receptor for ox-LDL, may also be proteolytically cleaved and released as soluble forms. We have identified soluble LOX-1 in conditioned media of cultured cells that express LOX-1 on their cell surface and determined the cleavage sites by protein purification and N-terminal amino acid sequencing.
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Methods |
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Reagents
DMEM and Ham’s F12 medium were obtained from Nissui. FCS was purchased from Sanko Junyaku. TNF-
was obtained from
Boehringer Mannheim, and protease inhibitors were
from Wako Pure Chemicals.
Cell Culture
BAECs were cultured in DMEM containing 10%
heat-inactivated FCS in an atmosphere of 95% air, 5%
CO2 at 37°C. Wild-type CHO-K1 cells were
maintained in F12/10% FCS. CHO-K1 cells stably expressing bovine LOX-1
(BLOX-1-CHO) were maintained in F12/10% FCS supplemented with 10
µg/mL of blasticidin S as previously described.3 6
Immunoblot Analysis
Cells were lysed with 62.5 mmol/L Tris/HCl (pH 7.4), 2%
SDS, and 10% glycerol. Cell-conditioned media were concentrated by
Centricon 10 (Amicon). Equal protein concentrations of the whole-cell
lysates and cell-conditioned media were subjected to
SDS-polyacrylamide (12%) gel electrophoresis (SDS-PAGE) under
reducing conditions, followed by electroblotting onto an Immobilon
polyvinylidene difluoride (PVDF) transfer membrane
(Millipore). Membranes were then incubated with a monoclonal antibody
(mAb) directed to bovine LOX-13 6 7 and horseradish
peroxidase (HRP)–labeled anti-mouse immunoglobulin (Amersham) as the
second antibody. Bands were visualized by chemiluminescence reagents
(ECL Western blotting detection reagents, Amersham).
Cell-Surface Biotinylation and Immunoprecipitation
Cells were washed twice with PBS and incubated with 0.5 mg/mL of
sulfo-NHS-LC-biotin (Pierce) at room temperature for 15 to 30
minutes. After being washed with PBS, cells were cultured in F12 medium
with 5% FCS for 30 minutes at 37°C. The culture media were then
replaced with serum-free F12 medium, and the cells were incubated at
37°C for 24 hours. Cell-conditioned media were collected and filtered
through a 0.22-µm filter, then incubated overnight at 4°C with
anti-biotin agarose (Sigma), which was followed by
centrifugation and extensive washing.
Immunoprecipitated samples were denatured at 98°C in SDS sample
buffer (62.5 mmol/L Tris/HCl [pH 6.8], 2% SDS, 10% glycerol,
5% 2-mercaptoethanol, 1% bromophenol blue) and subjected to
immunoblotting with anti–LOX-1 mAb or streptavidin-HRP
(Amersham).
Purification of Soluble LOX-1
Cell-conditioned media from BLOX-1-CHO cells cultured in
serum-free F-12 media for 2 days were filtered through a 0.22-µm
filter and subjected to ammonium sulfate precipitation. Precipitates in
80% saturated ammonium sulfate were then centrifuged at
20 000g for 20 minutes, after which the pellet was
dissolved in PBS and dialyzed against 66.7 mmol/L phosphate buffer
(pH 7.4). After centrifugation at 15 000g
for 15 minutes, the supernatant was applied to a Q Sepharose
(Pharmacia) column equilibrated with buffer A (20 mmol/L HEPES,
0.1% CHAPS, pH 7.2). The column was washed with 5 column volumes of
buffer A and eluted with buffer A containing 250 mmol/L NaCl.
Eluted proteins were concentrated by Centricon 10, diluted with buffer
A, and then subjected to a Mono Q 5/5 high-performance liquid
chromatography (HPLC) ion exchange column (Pharmacia).
The column was washed with buffer A and eluted with a linear
concentration gradient (0 to 500 mmol/L) of NaCl in buffer A.
Aliquots of the fractions (0.5 mL) were subjected to
immunoblotting with an anti–LOX-1
mAb.3 Fractions containing soluble LOX-1 were diluted
with buffer C (20 mmol/L MES, 0.1% CHAPS, pH 6.5) and subjected
to a Mono S 5/5 HPLC ion exchange column (Pharmacia). The column was
eluted with a linear NaCl gradient (0 to 500 mmol/L NaCl) in
buffer C. Aliquots of the fractions (0.5 mL) were subjected to
immunoblotting with an anti–LOX-1 mAb. Fractions
containing soluble LOX-1 were pooled, concentrated with Centricon 10,
diluted with buffer A, and applied to a heparin Sepharose CL-6B column
(Pharmacia). The column was washed with 5-fold column volumes of buffer
A and 80 mmol/L NaCl/buffer A, followed by elution with 160
mmol/L NaCl/buffer A. Aliquots of the fractions (1.5 mL) were subjected
to immunoblotting with an anti–LOX-1 mAb and silver
staining. Fractions containing soluble LOX-1 were then applied to a
blue Sepharose 6 FF (Pharmacia) column equilibrated with buffer A.
Proteins bound to the column were eluted in a stepwise manner with
0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, and 0.45 mol/L NaCl.
N-Terminal Amino Acid Sequence Analysis of Soluble
LOX-1
Sequence analysis of purified soluble LOX-1 was
performed with automated Edman degradation on a Hewlett Packard G1005A
protein sequencing system.
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Results |
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Production of Soluble LOX-1 in TNF-
–Activated BAECs
and BLOX-1-CHOTo examine whether BAECs produce soluble forms of LOX-1, cell-conditioned media and whole-cell lysates from BAECs cultured in the presence or absence of TNF-
were subjected to
immunoblotting with a mouse mAb directed to the
extracellular domain of LOX-1.3 Figure 1
demonstrates that both expression of
membrane-anchored LOX-1 and production of soluble LOX-1 were
induced by TNF-. Soluble LOX-1 has an approximate molecular mass of
35 kDa, as shown by immunoblotting of the
cell-conditioned media. In untreated BAECs, in contrast, neither the
membrane-bound nor the soluble form of LOX-1 was detectable by
immunoblotting. Soluble LOX-1 was detectable in
conditioned media of BAECs treated for 24 hours with 1 to 5 ng/mL of
TNF- (Figure 2). In response to 5
ng/mL of TNF-, soluble LOX-1 was detectable as early as 8 hours
after the treatment and kept increased for 
24 hours (Figure 3). Conversely, cell-surface expression
of LOX-1 was detectable within 4 hours after TNF- treatment and
remained elevated for 36 hours, as previously reported (Figure 3).6 These results indicate that expression of
membrane-bound LOX-1 appeared to precede release of soluble LOX-1 in
BAECs.
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We also examined whether soluble LOX-1 was produced in CHO-K1 cells
transfected with bovine LOX-1 cDNA (BLOX-1-CHO). As shown in Figure 4, large amounts of soluble LOX-1 with an
approximate molecular mass of 35 kDa were detectable in conditioned
media from BLOX-1-CHO but not in those from untransfected CHO-K1
cells.
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Soluble LOX-1 in Cell-Conditioned Media Derives From
Cell-Surface LOX-1
To determine whether soluble LOX-1 in cell-conditioned media
results from proteolytic cleavage of membrane-anchored LOX-1 expressed
on the cell surface, we carried out immunoprecipitation of
cell-conditioned media after cell-surface biotinylation. After
cell-surface biotinylation of BLOX-1-CHO, cell-conditioned media were
harvested and immunoprecipitated with anti-biotin agarose.
Immunoprecipitates were subsequently analyzed by Western
blotting with anti–LOX-1 mAb or streptavidin-HRP. As shown in Figure 5, biotinylated 35-kDa bands, which
appear to correspond to soluble LOX-1, were detectable in conditioned
media of BLOX-1-CHO. This clearly demonstrates that soluble LOX-1 in
cell-conditioned media is derived from LOX-1 expressed on the cell
surface.
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Cleavage of the Membrane-Anchored LOX-1 Requires Protease
Activities Sensitive to PMSF
To gain insights into mechanisms responsible for the release of
soluble LOX-1 from the cell surface, we tested the effects of various
protease inhibitors on soluble LOX-1 release from
TNF-–activated BAECs and BLOX-1-CHO cells. Protease
inhibitors were used at concentrations that are known to
specifically inhibit the protease activities without exhibiting
cytotoxicity.20 As shown in Figure 6A, PMSF, a potent serine protease
inhibitor, markedly inhibited soluble LOX-1 release.
Leupeptin, TLCK, and other serine protease inhibitors
partially blocked the soluble LOX-1 release. Conversely, neither E64
(cysteine protease inhibitor), aprotinin (serine protease
inhibitor), pepstatin (acid protease
inhibitor), nor calpain inhibitor II affected
soluble LOX-1 release. In TNF-–activated BAECs, PMSF
similarly inhibited the soluble LOX-1 release as shown in BLOX-1-CHO
(Figure 6B). None of these protease inhibitors
caused morphological changes in BLOX-1-CHO or
TNF-–activated BAECs (data not shown). Taken together,
soluble LOX-1 is produced and released by proteolytic cleavage of
cell-surface LOX-1 by proteases sensitive to PMSF.
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Purification of Soluble LOX-1 From Conditioned Media of
BLOX-1-CHO
Because BLOX-1-CHO produces larger amounts of soluble LOX-1 with
the same molecular mass on SDS-PAGE than TNF-–activated
BAECs, we used BLOX-1-CHO to isolate and purify soluble LOX-1. Proteins
in cell-conditioned media were concentrated by ammonium sulfate
precipitation, applied to a Q Sepharose anion exchange column, and
subsequently subjected to a Mono Q 5/5 anion-exchange HPLC column.
Proteins bound to the column were eluted with a linear concentration
gradient of NaCl. Soluble LOX-1 was eluted between 0.08 and 0.15 mol/L
of NaCl, as detected by immunoblotting (Figure 7A). Fractions containing soluble LOX-1
were then applied to a Mono S cation exchange HPLC column and eluted
with a linear concentration gradient of NaCl. Soluble LOX-1 was eluted
between 0.16 and 0.27 mol/L of NaCl (Figure 7B). After
application to a heparin column chromatograph, fractions
containing soluble LOX-1 were applied to a blue Sepharose column.
Soluble LOX-1 was eluted at 0.2 mol/L NaCl when the column was eluted
with a stepwise concentration gradient of NaCl. SDS-PAGE and silver
staining of the purified protein showed that 1 major protein, which
corresponds to soluble LOX-1, was present in the purified fraction
(Figure 7C).
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N-Terminal Amino Acid Sequencing of Purified Soluble LOX-1 and
Identification of the Cleavage Sites
To determine the cleavage site of soluble LOX-1, the N-terminal
amino acid sequence of purified soluble LOX-1 was analyzed with
an automated protein sequencer after SDS-PAGE and transfer onto a PVDF
membrane. Two different N-terminal amino acid sequences, SAQES and
SEKSA, were found. These amino acid sequences correspond to the amino
acid numbers between 87 and 91 and between 90 and 94 of LOX-1,
respectively. These results thus demonstrate that purified 35-kDa
soluble LOX-1 consists of 2 forms of the proteins resulting from
proteolytic cleavage of the
Arg86-Ser87 and
Lys89-Ser90 bonds of LOX-1
(Figure 8), although SDS-PAGE
analysis of the purified soluble LOX-1 was not able to
distinguish these 2 proteins with similar molecular weights (Figure 7C
).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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LOX-1 has been identified as a cell-surface receptor for ox-LDL in endothelial cells.3 Our recent study indicated that LOX-1 can also support binding and phagocytosis of oxidized or aged red blood cells and apoptotic cells, suggesting its physiological roles as a "scavenger" receptor.5 Expression of LOX-1 is rapidly and dramatically induced by treatment with TNF-
6 and
fluid shear stress,7 indicating that this receptor may
play more important roles in inflammatory diseases. LOX-1 structurally
belongs to the C-type lectin family, which consists of an extracellular
lectin-like domain, a single transmembrane domain, and a short
cytoplasmic tail.3 Because a variety of membrane proteins
with a single transmembrane domain, including growth factor receptors
and cell adhesion molecules, can be cleaved and transformed into
soluble molecules,9 10 11 12 we tested the hypothesis that
LOX-1 may also be converted into soluble molecules. In the present
study, we provide evidence that soluble forms of LOX-1 are present
in cell-conditioned media of TNF-–activated BAECs, as well
as CHO-K1 cells transfected with LOX-1 cDNA. We also showed that
soluble LOX-1 results from proteolytic cleavage of cell-surface LOX-1
by proteases sensitive to PMSF and further identified the cleavage
sites. Among the scavenger receptor family molecules, LOX-1 is the
first that has been shown to be converted into soluble forms.
Soluble receptors, in general, can be generated by 2 different
mechanisms: alternative splicing out of the exon encoding the
transmembrane regions and proteolytic cleavage of the full-length
membrane-bound receptor.10 11 12 For instance, soluble forms
of cell-surface molecules, such as platelet and
endothelial cell adhesion molecule-1,21
P-selectin,22 and interleukin (IL)-4
receptor,23 are generated by alternative splicing of their
mRNA. Conversely, soluble forms of L-selectin,24 vascular
cell adhesion molecule-1,25 E-selectin,13 and
platelet-derived growth factor receptor26 are produced
by proteolytic processing of full-length membrane-bound molecules.
Soluble IL-6 receptor can be generated by both alternative splicing and
proteolytic cleavage.10 11 27 28 Our results in this
report demonstrated that expression of cell-surface membrane-bound
LOX-1 precedes production of soluble LOX-1 in
TNF-–activated BAECs (Figure 3). In addition, large
amounts of both membrane-bound and soluble LOX-1 were produced in
BLOX-1-CHO but not in untransfected CHO-K1 cells (Figure 4).
Finally, cell-surface biotinylation followed by immunoprecipitation
provided evidence that production of soluble LOX-1 appears to
result from proteolytic cleavage of LOX-1 expressed on the cell surface
(Figure 5). Studies with various protease inhibitors
indicated that PMSF-sensitive protease activities appear to be involved
in the proteolytic cleavage to generate soluble LOX-1 (Figure 6). Furthermore, series of reverse transcription–polymerase
chain reaction analyses in TNF-–activated BAECs did
not detect LOX-1 mRNA lacking the transmembrane domain (data not
shown). These results thus provide evidence that soluble LOX-1 is
produced mainly by proteolytic cleavage of LOX-1 expressed on the cell
surface.
Purification and N-terminal amino acid sequencing of soluble LOX-1
identified the 2 different cleavage sites
Lys89-Ser90 and
Arg86-Ser87 in the membrane
proximal extracellular domain (Figure 7C). One of the amino acid
sequences of LOX-1 cleavage sites,
Lys89-Ser90, appears to be
identical to the L-selectin cleavage site, Lys321
and Ser322. Recognition of LOX-1 cleavage sites
by the putative converting enzyme, however, may depend on the protein
conformation rather than amino acid sequence, because 2 distinct amino
acid sequences of LOX-1 can be cleaved by this enzyme. In fact,
conformation- or position-dependent recognition of membrane proteins by
proteases has been implicated in the shedding of IL-6
receptor29 and L-selectin.24 30
Alternatively, 2 different proteases may specifically shed LOX-1 at 2
different sites, both of which are sensitive to PMSF.
Recent studies have indicated that shedding of L-selectin, as well as
proteolytic cleavage of TNF-, appears to be supported by
metalloproteases.31 32 33 TNF-–converting enzyme, which
cleaves TNF- from its precursor, has been identified at the
molecular level.34 35 Because LOX-1 can be cleaved at 2
different sites, 2 distinct enzymes might be responsible for the
digestion at these 2 different sites. In addition, our data suggested
that enzymes involved in the release of soluble LOX-1 appear to be
PMSF-sensitive and may possibly be serine proteases (Figure 6);
however, further studies would be needed to identify the molecules
involved in this process. These enzymes may be constitutively active in
various cell types, because large amounts of soluble LOX-1 can be
produced in BLOX-1-CHO as well as TNF-–activated BAECs.
Soluble receptors are present in circulating blood of humans, and plasma concentrations of soluble receptors are correlated with the levels of receptor expression on the cell surface and appear to reflect a certain disease status in vivo.13 14 15 16 Because expression of LOX-1 is inducible by inflammatory stimuli in vitro6 and in atherosclerotic lesions in vivo,8 circulating levels of soluble LOX-1 may be elevated in patients with atherosclerotic diseases. Therefore, measurement of plasma levels of soluble LOX-1 might also reflect the disease status and thus may potentially be useful to predict atherosclerotic progression in humans. Although further studies might be necessary to clarify the pathophysiological roles of soluble LOX-1, our preliminary studies failed to show inhibitory effects of soluble LOX-1 in the ox-LDL binding to the LOX-1 expressed on the cell surface (data not shown). This might result from the lower affinity of soluble LOX-1 for the ox-LDL–binding and/or multiple binding sites for LOX-1 on the ox-LDL particle.
In summary, the present report provides the first evidence that LOX-1 can be cleaved at the 2 sites located in the membrane proximal extracellular domain and secreted as soluble forms. Further studies related to pathophysiological functions of soluble LOX-1 and molecular mechanisms of LOX-1 cleavage might elucidate novel therapeutic and diagnostic values of soluble LOX-1 in atherogenesis and vascular diseases.
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Acknowledgments |
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This work was supported by Grants-in-Aid for Scientific Research (08407026, 08670788, and 09044293), for Scientific Research on Priority Areas (09281103 and 09281104), and for Creative Basic Research (09NP0601) from the Japanese Ministry of Education, Science, Sports, and Culture, as well as Research Grants for Cardiovascular Diseases (A8-1) from the Japanese Ministry of Health and Welfare.
Received July 9, 1999; accepted October 6, 1999.
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