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J. Biol. Chem., Vol. 276, Issue 35, 33241-33248, August 31, 2001
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From the
Received for publication, January 12, 2001, and in revised form, June 7, 2001
The reaction of lecithin:cholesterol
acyltransferase (LCAT) with high density lipoproteins (HDL) is of
critical importance in reverse cholesterol transport, but the
structural and functional pathways involved in the regulation of LCAT
have not been established. We present evidence for the direct binding
of LCAT to Human lecithin:cholesterol acyltransferase
(LCAT)1 is a 416-amino acid
glycoprotein circulating in plasma associated with lipids and
apolipoproteins in the high density lipoprotein (HDL) fraction (1).
LCAT plays a key role in cholesterol homeostasis by mediating the
production of most of the cholesteryl esters in human plasma. It has
been suggested (2, 3) that LCAT plays an important role in reverse
cholesterol transport (RCT) by creating a concentration gradient for
the efflux of free cholesterol from peripheral cells to HDL particles
and its conversion to cholesteryl esters. The cholesteryl esters are
internalized within the lipoprotein core for ultimate transport to the
liver for clearance or recycling. Thus, factors affecting the
structure, activity, or concentration of LCAT are likely to affect the
homeostasis of plasma cholesterol and the RCT process, one of several
proposed mechanisms (4) by which HDL may protect against atherosclerosis.
Recent investigations (5) suggest that LCAT can act as an antioxidant
and prevent the accumulation of oxidized lipid in plasma lipoproteins.
In human plasma, LCAT is almost entirely associated with lipoproteins
containing apoA-I, its principal physiological activator (6). Francone
et al. (1) have shown the presence of LCAT, cholesteryl
ester transfer protein, apoD, and a small amount of apoA-I in a complex
termed pre- We have recently found (7) an association between apoE and
The binding affinities of Structural and functional evidence for several binding domains in
different Materials--
Anti-human Blood Sampling--
Blood samples were obtained after an
overnight fast from male subjects with an apoE3/3 phenotype. Blood was
drawn from an arm vein into evacuated tubes containing
ethylenediamine-tetraacetate (EDTA, final concentration 1.5 mg/ml).
Blood collection tubes were immediately placed in ice. Plasma was
obtained by centrifugation (3,000 rpm, 15 min) and was kept in ice
until electrophoretic separation, which was routinely carried out
within 30 min of plasma isolation, or was frozen at Gel Electrophoresis--
Plasma samples were separated by
two-dimensional, non-denaturing gradient gel electrophoresis as
described previously (20, 21). In some experiments, only the
Immunoprecipitation Procedures--
LCAT associated with
In Vitro Binding Studies--
Preparation of Measurement of LCAT Activity and LCAT Mass--
LCAT activity
was measured as relative cholesterol esterification achieved during a
5-h incubation of plasma at 37 °C as described previously (21). The
LCAT activity of the LCAT· Preparation and Purification of
125I-rLCAT· Cell Culture--
HepG2 cells were cultured under standard
conditions. Briefly, HepG2 cells (ATCC HB 8065) were grown in Eagle's
minimum essential medium with nonessential amino acids, sodium
pyruvate, and 10% fetal bovine serum. After a 24-hour incubation,
serum-free medium from HepG2 (3 ml) were collected in the presence of 1 mmol/L phenylmethylsulfonyl fluoride, concentrated by using Centricon
filters (Amicon) to a final volume of 200 µl and separated by
two-dimensional gel electrophoresis.
Cell Association and Degradation Assays--
Mouse embryonic
fibroblasts (MEF1) that express LRP and mouse embryonic fibroblasts
genetically deficient in LRP (MEF2) were used. Cells were incubated
with 125I- Lipid and Lipoprotein Assays--
Cholesterol and triglyceride
concentrations were determined enzymatically on an autoanalyzer (Cobas
Mira, Roche Molecular Biochemicals). HDL-cholesterol concentration was
determined by measuring cholesterol in the supernatant after
heparin-manganese precipitation of apoB-containing lipoproteins in the
d > 1.006 g/ml fraction of plasma prepared by
ultracentrifugation where d is density. Plasma apoB and
apoA-I concentrations were measured by nephelometry (Behring
Nephelometer 100 Analyzer). ApoE phenotypes were determined by
immunoblotting of plasma separated by minigel electrophoresis (28). Low
density lipoprotein (LDL) (1.019 < d < 1.063)
and HDL3 (1.125 < d < 1.21 g/ml)
were isolated from normolipidemic plasma by sequential
ultracentrifugation using a Beckman ultracentrifuge. The total
HDL fraction was isolated from plasma by automated gel filtration
chromatography (FPLC).
We used two-dimensional gel electrophoresis to separate plasma
apolipoprotein complexes in the HDL size range (Fig.
1A). LCAT protein co-migrated
in approximately equal proportions with To provide evidence for a direct association of LCAT with
Interaction of Lecithin:Cholesterol Acyltransferase
(LCAT)·
2-Macroglobulin Complex with Low Density
Lipoprotein Receptor-related Protein (LRP)
EVIDENCE FOR AN 
2-MACROGLOBULIN/LRP
RECEPTOR-MEDIATED SYSTEM PARTICIPATING IN LCAT CLEARANCE*
,,¶
Cardiovascular Genetics Laboratory, McGill
University Health Center/Royal Victoria Hospital, Montréal,
Québec H3A 1A1, Canada and the § Hyperlipidemia and
Atherosclerosis Research Group, Clinical Research Institute of
Montreal, Montréal, Québec H2W 1R7
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin (2M) in
human plasma to form a complex 18.5 nm in diameter. Forty percent of
plasma LCAT-HDL was associated with 2M; moreover, most
of the LCAT in cerebrospinal fluid and in the medium of cultured human
hepatoma cell line was associated with 2M. Purified
recombinant human LCAT (rLCAT) labeled with 125I bound to
native and methylamine-activated 2M
(2M-MA) in vitro in a time- and
concentration-dependent manner, and this binding did not
depend on the presence of lipid. rLCAT bound to 2M-MA with greater affinity than to 2M. Furthermore, rLCAT did
not activate 2M as phosphatidylcholine-specific
phospholipase C does. Reconstituted HDL particles (LpA-I)
inhibited the binding of rLCAT to 2M more efficiently
than native HDL3 did. LCAT associated with
2M was enzymatically inactive under both endogenous and exogenous assay conditions. Purified rLCAT alone did not bind to
low density lipoprotein receptor-related protein (LRP) as
lipoprotein lipase (LPL) does; however, when rLCAT was combined with
2M-MA to form a complex, binding, internalization, and
degradation of rLCAT took place in LRP-expressing cells (LRP
+/+) but not in cells deficient in LRP (LRP
/). It is concluded that the binding of LCAT to
2M inhibits its enzymatic activity. Furthermore, the
finding supports the possibility that the LRP receptor can act in
vivo to mediate clearance of the LCAT-2M complex
and may significantly influence the bioavailability of LCAT.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-LpA-I that is involved in the
esterification and transfer of cell-derived cholesterol.
2-macroglobulin (2M) in human plasma,
when we observed that LCAT and apoE migrate to the same position in
two-dimensional electrophoretic gels together with 2M,
in particles 18.5 nm in diameter. This raises the possibility that LCAT
circulates in plasma in association with 2M. Human
2M, the largest known proteinase inhibitor
(Mr = 720,000), is found at high concentrations
(2-5 µM) in plasma and in extravascular spaces (8, 9).
It plays a pivotal role in the clearance of proteinases from the
circulation and in regulating their activity in fibrinolysis,
coagulation, and complement activation (10, 11). 2M is
also a carrier of specific cytokines and various non-proteolytic
proteins that include the transforming growth factor TGF-, the
platelet-derived growth factor-BB (12), the -amyloid peptide
(13), and recently, apolipoprotein E (7).
2M for different
non-proteolytic proteins depend on its conformation, but
2M in plasma is present almost entirely in the native
conformation. This form of 2M is fully functional as a
proteinase inhibitor but is not recognized by the cell surface
receptor, which is an 2M receptor/low density lipoprotein receptor-related protein (LRP) (8, 14). This receptor is
responsible for the rapid plasma clearance of conformationally transformed 2M following its reaction with proteinases
or small primary amines that modify the 2M thiol
ester bonds. Gonias and co-workers have documented that the binding of
TGF- isoforms to 2M neutralizes the activity of
TGF- toward various cells (12, 15, 16). Indeed, 2M
may be involved in controlling apoptosis (17), the immune system, and
atherogenesis (18, 19) via its regulating effects on TGF- activity.
2M forms led us to hypothesize that
2M modulates LCAT activity and concentration in plasma
and may be involved in LCAT clearance via an LRP-mediated endocytic
process. The physical association of LCAT and 2M has not
been previously reported. The present study aims at providing evidence
for the association of LCAT with 2M and to examine how
these interactions could be affected by native and activated forms of
2M and by apolipoproteins known to bind LCAT. Cellular
binding, internalization and degradation assays were used to evaluate
the role of activated 2M and LRP receptor in mediating
the clearance of LCAT by cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2M monoclonal antibody,
affinity-purified rabbit anti-goat IgG and affinity-purified goat
anti-mouse IgG antibody were purchased from Biodesign International
(Kennebunk, ME). Methylamine hydrochloride,
phosphatidylcholine-specific phospholipase C and sphingomyelinase were
purchased from Sigma. Partially purified, anti-human
2M polyclonal antibody and anti-human IgG polyclonal antibody were purchased from ICN Pharmaceuticals Inc. (Aurora, OH).
HepG2 cells (HB-8065) were purchased from American Type Culture Collection (ATCC). Anti-LCAT goat polyclonal antibody and purified human recombinant LCAT (rLCAT) were a gift from Dr. Henry Pownall, Baylor College of Medecine,s Houston, TX. C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase (hrLCATH6) was a
gift from Dr John S. Parks, Wake Forest University School of Medecine,
Winston-Salem, NC. Anti-LCAT monoclonal antibody 2H11 was generously
provided by Dr. Ross Milne, University of Ottawa Heart Institute. Mouse
embryonic fibroblasts (MEF1) that express LRP, and mouse
embryonic fibroblasts genetically deficient in LRP (MEF2) were
generously provided by Dr. Joachim Herz, Department of Molecular
Genetics, University of Texas Southwestern Medical Center at Dallas.
70 °C until
analysis of lipids and apolipoproteins.
2M-containing pre-2-migrating segment
(~2 cm) of agarose gels was separated in the second dimension.
2M was isolated from human plasma by
immunoprecipitation. Plasma (120-500 µl) was incubated overnight at
4 °C with 50-200 µl of anti-human 2M antibody or
with control anti-human IgG antibody. The immunoprecipitates were
centrifuged at 10,000 rpm for 6 min and washed three times with buffer
(20 mM HEPES, pH 7.5, 0.15 M NaCl, 0.1%
Triton, and 10% glycerol). They were analyzed by 4-22.5%
SDS-polyacrylamide gel electrophoresis, together with molecular weight
standards (Amersham Pharmacia Biotech). The presence of LCAT and
2M in immunoprecipitates was detected by immunoblotting.
The efficiency of 2M immunoprecipitation was assessed as
>95%, based on the absence of 2M in the supernatant.
2M-MA and LPL were
iodinated using IODO-GEN® iodination reagent
(1,3,4,6-tetrachloro-3-6-diphenylglycouracil, Pierce) (22). rLCAT
and hrLCATH6 were iodinated as described by Bolin and Jonas (23). Free
iodine was removed by PD10 column chromatography. Iodinated proteins
were dialyzed extensively at 4 °C against phosphate-buffered saline,
pH 7.4. These 125I-rLCAT preparations retained ~85% of
control acyltransferase activity with rHDL substrates. LCAT activity
decreased slightly over time. No difference in the binding of
125I-rLCAT to 2M was observed between
125I-rLCAT preparations that retained 85% or 30% of
control acyltransferase activity with rHDL substrates.
2M-MA, 2M, and binding
experiments were performed as previously described (7). The effect of
various apolipoproteins and reconstituted HDL particles on the binding
of rLCAT to 2M was determined by adding these substances
(as specified for each experiment) to reaction tubes prior to the
addition of 125I-rLCAT. In certain experiments, samples
were treated with phospholipases or trypsin before addition of
125I-rLCAT. Bound and unbound 125I-rLCAT was
separated by non-denaturing gradient gel electrophoresis 2.5-18% at
60 V (16 h, 15 °C). No dissociation of 125I-rLCAT bound
to 2M was detected with this electrophoretic system as
assessed by reseparating electroeluted 125I-labeled
rLCAT·2M or 125I-labeled
rLCAT·2M-MA complexes. In experiments using
125I-rLCAT, gels were dried and exposed at 70 °C to
XAR-2 Kodak film for 4-72 h. Exposed films were used as a template to
identify the position of appropriate bands for excision and counting.
2M complex was assayed as
described previously (24) but with minor modifications. The
[3H]cholesterol-labeled r(LpA-I) substrate was prepared
as previously described (25) and was diluted in buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM NaN3, and 0.01% EDTA) to give final apoA-I concentrations ranging from 107 to 106
M. Each reaction mixture contained 50 µl of 30 mg/ml
bovine serum albumin and 50 µl of 100 mM
-mercaptoethanol in a final volume of 300 µl. Incubation was for
3 h at 37 °C. LCAT activity was determined as the amount of
esterified cholesterol that was incorporated into apoA-I-containing
proteoliposomes. LCAT mass was measured by the method of Qu et
al. (26), with purified LCAT as standard.
2M-MA
Complex--
125I-rLCAT was incubated with
2M-MA at 2:1 molar ratio for 24 h at 37 °C.
Radiolabeled rLCAT·2M-MA complex was then separated from free 125I-rLCAT by 500-kDa exclusion filters and
isolated from preparative non-denaturing gradient gels by
electroelution. The integrity of the complex was evaluated by
electrophoresis under non-denaturing conditions on 3-15% gradient
gels followed by autoradiography.
2M-MA, 125I-rLCAT,
125I-LPL and 125I-rLCAT·2M-MA
complex at the concentrations indicated in the figure legends.
Cell-specific binding, association, and degradation were measured after
5-h incubations at 4 °C or 37 °C. Aliquots of culture medium were
taken for degradation of radiolabeled ligand by measurement of
non-iodide 125I (soluble in 10% trichloroacetic
acid) as previously described (27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HDL and 2M
plasma protein. LCAT was consistently found to co-migrate with plasma
2M protein, having a molecular diameter of 18.5 nm. (Fig. 1A, right panel), detected with
125I-labeled anti-2M monoclonal antibody.
LCAT migrated in the same position as 2M (Fig.
1A, left panel). LCAT associated with
2M was also detected in human cerebrospinal fluid (CSF)
and in the medium of cultured HepG2 (Fig. 1B). Most of the
LCAT in CSF and secreted by HepG2 cells was associated with
2M and had an apparent diameter of 18.5 nm, as was the
case for LCAT·2M in the plasma. -LCAT species
present in CSF and the medium of cultured HepG2 were smaller than
-LCAT in human plasma. The control sample in this experiment (Fig.
1B, right panel) was purified plasma
2M, which contained bound LCAT.
View larger version (62K):
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Fig. 1.
Separation of human plasma, CSF, and medium
of cultured human hepatoma cells (HepG2) by two-dimensional gradient
gel electrophoresis showing LCAT co-migrating with plasma
2M. Upper panels, plasma (100 µl) from a
normolipidemic subject was separated according to charge in the first
dimension (from left to right) by agarose gel
electrophoresis and then according to size in the second dimension
(top to bottom) by polyacrylamide gradient
(3-20%) gel electrophoresis. LCAT was detected with anti-LCAT
monoclonal antibody (2H11) and then with goat anti-mouse
125I-labeled IgG. 2M was detected by
immunoblotting with an 125I-labeled anti-2M
antibody. Lower panels, LCAT co-migrating with CSF, medium
of cultured HepG2, and purified native plasma 2M are
indicated by vertical arrows.
2M, we used a purified anti-2M IgG
fraction to immunoprecipitate 2M from the plasma of
three normolipidemic subjects. The amount of LCAT associated with
2M immunoprecipitate was assessed by immunodetection of
LCAT and 2M (Fig.
2A, upper panel)
after separation by SDS-polyacrylamide gel electrophoresis. In
lane a the nonspecific IgG immunoprecipitation revealed one
IgG band detected by the second antibody used in the LCAT detection.
Lanes b, c, and d demonstrate, in addition, an
LCAT band with molecular mass of ~66 kDA. The absence and
presence of 2M in respective samples are shown in the
(Fig. 2A, lower panel). Removal of
2M from plasma by affinity chromatography reduced most
of the LCAT associated with 2M but not -migrating
LCAT (data not shown). The amount of plasma LCAT associated with
2M was estimated to be ~40% of total plasma LCAT in
the HDL size range (see later, Fig. 6A).
View larger version (48K):
[in a new window]
Fig. 2.
Association of plasma LCAT with
2M isolated by immunoprecipitation (A) and
association of LCAT with commercially available 2M
(B and C). A, plasma from three
normolipidemic subjects (200 µl) was immunoprecipitated with
anti-human-2M (lanes b, c and d)
or with nonspecific anti-human-IgG antibodies (lane a, one
subject only). Immunoprecipitates were separated by SDS-polyacrylamide
gradient-gel electrophoresis. LCAT and 2M were detected
by immunoblotting with polyclonal goat anti-human LCAT antibodies and
then with 125I-labeled rabbit anti-goat IgG antibody
(A, upper panel). 2M was detected by
125I-labeled anti-2M antibody (lower
panel). B, immunodetection of LCAT in native and
methylamine-treated commercial 2M (100 µg) separated
by nondenaturing gel electrophoresis (lanes a and
b, respectively). C, commercial 2M separated
by SDS polyacylamide gradient gel electrophoresis under nonreducing
conditions and in the presence of a reducing agent
(-mercaptoethanol) (lanes a and b,
respectively). Purified rLCAT (5 µg) (lane c) was used as
control. 125I-labeled molecular mass standards are also
shown in panels A, B, and C.
LCAT was also present in commercial preparations of 2M,
purified by metal chelate chromatography. Lane a of the
left panel of Fig. 2B shows that a significant
amount of LCAT was immunodetectable in commercial 2M
separated, in this case, by non-denaturing gradient gel
electrophoresis; the complex had an apparent diameter of 18.5 nm. The
specificity of anti-LCAT antibody was confirmed by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by
immunodetection of LCAT in commercial preparations of
2M.
LCAT was present with molecular masses of 66 kDa, as shown in
lanes a and b of Fig. 2C. In this
experiment purified rLCAT was used as control, lane c. The
difference in apparent molecular mass between plasma LCAT dissociated
from native purified 2M and rLCAT (Fig. 2C)
was because of different degrees of glycosylation as previously
reported (29).
Methylamine activation of commercial 2M did not
dissociate LCAT from 2M, as shown by the similar amount
of LCAT associated with 2M under non-denaturing
conditions (Fig. 2B, lanes a and b,
respectively). Under non-reducing conditions, SDS gel electrophoresis caused a part of LCAT to dissociate from commercial 2M,
as shown in lane a of Fig. 2C.
LCAT·2M complex was resistant to boiling in the
presence of SDS. In addition, the in vitro formation of 125I-rLCAT·2M was not inhibited by
iodoacetic acid, indicating that free sulfhydryl groups are not
required for complex formation, providing evidence for the non-covalent
association of LCAT with 2M (data not shown).
The binding of LCAT to activated (2M-MA) and native
2M was investigated in vitro by incubation of
125I-rLCAT at 37 °C with different concentrations of
2M for various times. Bound and unbound
125I-rLCAT were separated by nondenaturing gel
electrophoresis, and 125I-rLCAT was quantified by direct
scintillation counting. LCAT binding was found to occur in a time- and
concentration-dependent manner. Maximum binding was reached
after 6 h for both 2M and 2M-MA and
remained constant for the remaining 18 h of the experiment (data
not shown). 2M-MA had a 2-fold greater capacity to bind LCAT relative to 2M (Fig.
3, upper and left
panels). The affinity of LCAT binding to both forms of
2M was assessed from a double-reciprocal plot of the
concentration-dependent binding data (right
panel). Dissociation constants (KD) calculated
from the slope of the regression lines were 3.93 and 1.9 µM for the nonactivated and activated forms of
2M, respectively, indicating that LCAT had a greater
affinity to bind to 2M-MA than to 2M.
|
We have previously reported that phosphatidylcholine-specific
phospholipase C (PC-PLC) activate native 2M (7). To
verify that LCAT does not bind to 2M through its well
characterized proteinase-trapping mechanism, we determined whether the
conformation of native 2M could be affected by LCAT
treatment. Native 2M isolated by metal chelate
chromatography (having detectable quantities of bound LCAT (Fig.
2B, lane a)) was incubated for 12 h at
37 °C with sphingomyelinase (SM-ase), PC-PLC, or rLCAT.
2M-MA and trypsin-2M
(2M-T) were used as activated forms of 2M
in this experiment. PC-PLC (but not SM-ase or rLCAT) caused
2M to become "activated" (as evidenced by the
smaller size and increased mobility of 2M separated by
nondenaturing gradient gel electrophoresis) as shown (Fig.
4).
|
To verify that the association of LCAT does not depend on the presence
of lipid, we investigated whether the binding of 125I-rLCAT
could be prevented by prior treatment of 2M with
phospholipases (sphingomyelinase and phosphatidylcholine-specific
phospholipase C) or delipidating solvents. No decrease was observed in
the binding of 125I-rLCAT to native 2M (data
not shown).
We next investigated whether HDL particles would affect the association
of LCAT with native 2M. Native 2M (50 µg) was incubated with increasing amounts of reconstituted HDL,
r(LpA-I), or native HDL3 (Fig.
5). 50 µg of r(LpA-I) or
HDL3 inhibited the association of LCAT with native
2M by 50 and 30% respectively. At all concentrations used in this experiment, r(LpA-I) particles were more efficient inhibitors than native HDL3.
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We measured the ability of LCAT·2M complex to esterify
cholesterol in vitro by examining the effect of removing the
LCAT·2M complex from plasma by immunoprecipitation of
2M using a purified anti-2M IgG fraction
and nonspecific anti-human IgG. The fractional esterification rate of
cholesterol in plasma was the same in 2M-depleted plasma
samples as in IgG-depleted plasma samples (Fig.
6B). As ~40% of the plasma
LCAT was associated with 2M (Fig. 6A), this result shows that LCAT associated with 2M in plasma is
inactive in the esterification of cholesterol. To investigate this
observation further, we assayed for LCAT activity using a
proteoliposome substrate. The same amount of LCAT (1 µg) present in
commercial 2M preparation (native 2M and
2M-MA), purified plasma 2M (isolated by
ultracentrifugation (d > 1.25) and separated from
others plasma proteins by FPLC, thus reducing the possibility of
inactivating LCAT by the purification process) and the IgG-depleted HDL
fraction (isolated by FPLC) was tested for LCAT activity. Under these
conditions, there was no significant LCAT activity in either
conformation of commercial and purified plasma 2M in
contrast to the activity associated to HDL fraction (Fig.
6C).
|
-radiation spectrometer. Values given are means ± S.D. of
five different experiments. Radioactivity in the To evaluate the ability of 125I-rLCAT·2M
complex to interact with the LRP receptor, we combined
125I-rLCAT with unlabeled 2M-MA, and the
resulting complex was purified. The inset of Fig. 8, shows
gel electrophoretic separation of free 125I-rLCAT
(lane a) and 125I-rLCAT·2M-MA
complex (lane b). Equivalent amounts of
125I-2M-MA,
125I-rLCAT·2M-MA, 125I-rLCAT,
and 125I-LPL were incubated with LRP-expressing cells LRP
(+/+) and control cells that did not express LRP (/) at 4 °C for
5 h, and the levels of specific binding were measured. As shown in
Fig. 7B, incubation with cells
expressing the LRP receptor resulted in the binding of
125I-rLCAT·2M-MA complex, but cells
deficient in this receptor (MEF2, LRP /) were unable to bind
significant amounts of 125I-rLCAT·2M-MA
complex. In separate experiments (Fig. 7A) we have confirmed
the absence of LRP from MEF2 (LRP /) cells by measuring the
specific binding of 125I-2M-MA.
|
To determine whether 125I-rLCAT alone might interact with
LRP-receptor, we added equivalents amount of 125I-rLCAT to
cultured MEF1 (LRP+/+) and control MEF2 (LRP/), and the level of
specific binding was measured. As shown in Fig. 7C, minimal
LCAT binding to LRP +/+ and LRP / cells is observed. The control
protein in this experiments was purified LPL, which bind to LRP +/+ but
not significantly to LRP / (Fig. 7D).
To evaluate the ability of
125I-rLCAT·2M-MA complex to interact with
the LRP receptor, equivalent amounts of
125I-rLCAT·2M-MA were incubated with LRP
(+/+) and control LRP (/) cells, and the level of cell association
and degradation of this complex was measured. As shown in Fig.
8, incubation with cells expressing the
LRP receptor resulted in the internalization and degradation of the
complex, but cells deficient in this receptor (LRP /) were
unable to internalize or degrade significant amounts of
125I-rLCAT· 2M-MA complex.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
We have shown that LCAT, co-migrates with 2M
in human plasma in an intermediate-sized complex (18.5 nm in diameter)
between LDL and HDL lipoproteins (Fig. 1A). Plasma LCAT
associated with 2M could be specifically
immunoprecipitated using antibodies directed against 2M.
Another experiment demonstrated that purified native 2M
(isolated by metal-chelate chromatography) consistently contained
detectable amounts of LCAT (Fig. 2B). LCAT became bound to
2M in a concentration-dependent manner (Fig.
3). Such binding does not depend on the presence of lipid molecules
and, for LCAT, is apparently noncovalent (Fig. 2C,
right panel, lane a). LCAT may be recognizing a
protein motif, such as an amphipathic -helix, that is common to
apolipoproteins and 2M and is independent of lipid
content. The association of LCAT with apolipoprotein complexes may thus
be due to direct protein-protein interaction, suggesting that LCAT
binding and activation by apolipoprotein are independent events. It was
documented that the association of LCAT to lipoprotein surfaces
essentially was independent of their composition (31).
We have previously shown (7) that the binding of apoE to
2M depends on the apoE phenotype. Charge or
conformational differences in LCAT mutants might similarly affect their
binding to 2M species in plasma. Adimoolam et
al. (32) have shown that two naturally occurring mutants of
human LCAT, T123I and N228K, expressed in COS-1 cells, bind to a
lipoprotein particle (rHDL) with about half the affinity of wild-type
LCAT. Again, activated 2M-MA binds two times as
much LCAT as native 2M (Fig. 3), and various cytokines and growth factors (e.g. TGF-1, NGF-, PDGF, bFGF, and
TNF-) also bind to 2M-MA with greater affinity than
to native 2M (12).
LCAT shares several sequence regions with other lipases (33).
Phosphatidylcholine-specific phospholipase C activates
2M (7), but rLCAT does not (Fig. 4). This suggests that
LCAT does not bind to 2M through its well characterized
proteinase-trapping mechanism (34).
The binding studies shown in Fig. 3 predict that LCAT bound with low
affinity to native 2M may dissociate from the complex in
the presence of LpA-I particles. We postulate that native discoidal HDL
particles affect the availability of active LCAT, which is crucial for
their maturation, by modulating its dynamic distribution between
-migrating HDL and 2M. When 125I-labeled
rLCAT was incubated with 2M in the presence of a 4-fold excess of r(LpA-I) over 2M, the association of rLCAT
with 2M was almost completely inhibited. Actually,
r(LpA-I) inhibited the binding of 125I-rLCAT with
2M more efficiently than did native HDL3
(Fig. 5).
Studies by Adimoolam et al. have documented that normal LCAT
dissociates from rHDL after one catalytic cycle (32). It is possible
that the fraction of LCAT bound preferentially to 2M reflects a catabolic compartment. We may say that an important proportion of HDL-sized LCAT (40%) is sequestered into in an inactive pool by 2M, which does not react with cholesterol of
lipoprotein origin (Fig. 6) and leads to the concept that plasma
2M inhibits the enzymatic activity of LCAT. It is not
possible to use a water-soluble substrate such as
p-nitrophenylvalerate to provide evidence that the LCAT
associated with 2M is catalytically active; the latter being a substrate for serine proteases and esterases, it might also
interact with chymotrypsine (30), which binds to native purified
2M.
LCAT with low activity has been demonstrated in CSF (35). LCAT may be
synthesized in the brain (36). Recently, Collet et al. (37)
reported that cultured nerve cells secreted a functional LCAT protein.
The present study shows that most of the LCAT in CSF was associated
with 2M (Fig. 1B). Although the functional significance of LCAT binding to 2M in CSF remains to be
determined, this complex may be involved in mediating the clearance of
LCAT by the LRP receptor in the brain.
One can also speculate that native 2M could be involved
in the stabilization, the decreased plasma clearance, and the
resistance of plasma LCAT to proteinase cleavage. Indeed, we have
observed that significant amounts of rLCAT resisted trypsin
digestion when bound to 2M, whereas unbound rLCAT was
readily degraded (data not shown).
The results presented in this study provide direct evidence that LRP is
a receptor for the LCAT·2M-MA complex (Figs. 7 and 8),
thus indicating that it could mediate LCAT·2M-MA
binding, endocytosis, and degradation in any tissue that expresses LRP. This work provides evidence for the first time that the LRP receptor may play an important role in the catabolism of LCAT.
Further study of the interaction between LCAT and 2M may
provide new insights into plasma factors affecting HDL particles remodeling, the mechanism of reverse cholesterol transport, and the
clearance of LCAT via an 2M/LRP receptor that is
dependent on the conformation of 2M in
vivo.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Denise Dubreuil, Head Nurse at the Clinical Research Institute of Montreal Lipid Clinic, Nancy Doyle and Lucie Boulet for their technical support for this project, and Joanne Griffith for editorial assistance. hLCATH6 was generously provided by Dr. John S. Parks. Anti-LCAT monoclonal antibody was generously provided by Dr. Ross Milne and goat antiserum to human LCAT and purified LCAT were generously provided by Dr. Henry Pownall. MEF was a gift from Dr. Joachim Herz. LPL was generously provided by Dr Ira Goldberg. CSF was generously provided by Dr Jude Poirier. The helpful advice of Drs. Jonathan Lamarre, Laurence Mabile, Steven L. Gonias, Arnold von Eckardstein, and the late Peter J. Dolphin is gratefully acknowledged.
| |
FOOTNOTES |
|---|
* This work was supported by a grant from Pfizer within the framework of university/industry (Canadian Institutes of Health research program DOP40845 and CIHR grant MOP15042) and by La Succession J. A. De Sèves.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: McGill University Health Center/Royal Victoria Hospital, Cardiology Division M4.76, 687 Pine Ave. West, Montréal, Québec, Canada H3A 1A1. Tel.: 514-842-1231 ext. 4642; Fax: 514-843-2843; E-mail: jacques.genest@muhc.mcgill.ca.
Published, JBC Papers in Press, July 2, 2001, DOI 10.1074/jbc.M100326200
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ABBREVIATIONS |
|---|
The abbreviations used are:
LCAT, human
lecithin:cholesterol acyltransferase;
HDL, high density lipoprotein;
LDL, low density lipoprotein;
RCT, reverse cholesterol transport;
2M, 2-macroglobulin;
LRP, lipoprotein
receptor-related protein;
LPL, lipoprotein lipase;
rLCAT, recombinant
LCAT;
hr, human recombinant;
MEF, mouse embryonic fibroblasts;
MA, methylamine-activated;
FPLC, fast protein liquid chromatography;
CSF, cerebrospinal fluid;
PC-PLC, phosphatidylcholine-specific phospholipase
C.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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