| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 38, 29324-29330, September 22, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
§¶
,,,, and
From the Department of Biochemistry, School of
Medicine, Medical College of Pennsylvania Hahnemann University,
Philadelphia, Pennsylvania 19129, the § Department of
Anatomy and Cell Biology and Pediatrics, SUNY Health Science Center at
Brooklyn, Brooklyn, New York 11203, the
Department of Pathology, Wake Forest
University School of Medicine, Winston-Salem, North Carolina
27157, and the ** Department of Medicine, Columbia University,
College of Physicians and Surgeons, New York, New York 10032
Received for publication, June 19, 2000
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Lipoprotein lipase (LPL) physically
associates with lipoproteins and hydrolyzes triglycerides. To
characterize the binding of LPL to lipoproteins, we studied the binding
of low density lipoproteins (LDL), apolipoprotein (apo) B17, and
various apoB-FLAG (DYKDDDDK octapeptide) chimeras to purified LPL. LDL
bound to LPL with high affinity (Kd values of
10 Lipoprotein lipase
(LPL)1 is a dynamic molecule
that performs several functions during the catabolism of
triglyceride-rich chylomicrons and very low density lipoproteins.
First, it is essential for the hydrolysis of triglycerides present in
the core of these lipoproteins. The hydrolysis results in the delivery
of free fatty acids to peripheral tissues. Defects in the enzyme
activity leads to Type 1 hyperlipidemia characterized by accumulation
of triglyceride-rich lipoproteins, defective chylomicron removal, and
the development of pancreatitis (1, 2). Second, LPL anchors
lipoproteins to the cell surface and matrix proteoglycans and increases
their retention by subendothelial matrix and uptake by cells. Third, it
acts as a ligand and binds to various members of the LDL receptor family and may play a role in the cellular endocytosis of lipoproteins (3, 4). LPL performs these various functions by physically interacting
with lipoproteins, proteoglycans, and receptors. LPL favors binding to
apoB-containing lipoproteins compared with apoA-I-containing lipoproteins. The molecular and biochemical basis for the binding of
LPL to apoB-containing lipoproteins are not completely understood.
It has been shown that protein-protein interactions between LPL and LDL
involve the N-terminal 17% of apoB (5-7). However, the LPL-binding
site(s) in the N-terminal 17% of apoB and apoB-binding site(s) in LPL
have not been identified. The N-terminal 17% of apoB has been
postulated to exist as an Materials--
Bovine milk LPL was purified using heparin
affinity chromatography as described before (14). For some experiments,
LPL was purified further by Biogel-P60 column chromatography. LPL
purified from heparin affinity column was applied to a 15-ml BioGel-P60 column equilibrated with five bed volumes of 10 mM Tris, pH
7.4, containing 1.5 M NaCl, and the enzyme was eluted in
the same buffer. All the assays were performed with the purified
immobilized homodimeric LPL. Antibodies used in ELISA for the detection
of apoB have been described (12, 15, 16).
Cells--
McA-RH7777 cells stably transfected with different
C-terminally truncated forms of apoB have been described (15, 17, 18). COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in Dulbecco's minimal essential medium containing 10% fetal bovine serum and 1%
antibiotic-antimycotic (Life Technologies, Inc.).
Binding of LDL to the Immobilized LPL--
The binding of LDL to
the immobilized LPL was studied as described earlier (5, 19). ELISA
plates were coated with 0.5 µg of purified, homodimeric bovine LPL in
100 µl of 0.05 M sodium borate, pH 9.5, in triplicate
wells by incubating at 4 °C for 18 h. Wells were washed with
PBS containing 0.3% bovine serum albumin (BSA), and incubated for 30 min at 37 °C to block all the protein-binding sites in the wells.
Wells were then incubated with indicated amounts of LDL diluted in PBS
and 0.3% BSA for 2 h at 37 °C. Various amounts of NaCl, Triton
X-102, tetrahydrolipstatin, heparin, or monoclonal antibodies were
added during these incubations. Microtiter wells were washed three
times with PBS and 0.3% BSA. A sandwich ELISA (15, 16) quantitated
apoB bound to immobilized LPL. In parallel, a standard curve for the
apoB was generated by coating wells with a monoclonal antibody, 1D1,
and incubating with different concentrations of LDL (0-14 ng/well) as
described before (15, 16).
Binding of Different apoB-FLAG Chimeras to LPL--
COS-7 cells
were transiently transfected with various FLAG chimeras (1 µg DNA/ml)
containing C-terminally truncated apoB cDNAs using Fugene-6
transfection reagent (Roche Molecular Biochemicals) and conditioned
media (72 h) were used to study the binding of different
secreted apoB polypeptides to immobilized LPL as described above (12).
Microtiter wells were coated with either LPL or M2, a monoclonal
antibody that recognizes FLAG octapeptide. In some experiments, wells
were blocked and washed with PBS and 0.05% Tween-20 instead of PBS and
0.3% BSA. The wells were then incubated with conditioned media
obtained from transfected COS cells. The amount of chimeras bound to
LPL or M2 were quantified using anti-apoB polyclonal antibodies (12).
The binding to M2 provides a measure for the amounts of chimeras
present in the conditioned media.
Other Analyses--
Protein was determined using the Coomassie
Plus reagent (Pierce) with BSA as a standard (20). Optical
density in ELISA plates was measured using a Dynatech MRX microplate
reader (Dynatech Labs, Chantilly, VA). The data were plotted as the
means ± S.D., and the binding isotherms were analyzed using
Prism2 (Graphpad, San Diego, CA). The molecular masses used for
LDL, LPL, Triton X102, and heparin were 512, 120, 0.757, and 16.5 kDa, respectively.
Nature of Interactions between apoB and LPL--
The binding of
human plasma LDL (0-100 µg/well) to the immobilized LPL was
saturable and exhibited a rectangular hyperbola (Fig.
1A). The
Bmax and Kd values were
0.018 ± 0.0002 (average ± standard error) pmol and
0.057 ± 0.004 nM, respectively. The r2
value for the curve was 0.9991. The Kd values ranged between 60 and 160 pM in five independent experiments. In
previous studies, we had shown that LDL binds to MTP with
Kd values ranging between 10 and 30 nM
(21). Thus, LDL binds to LPL with severalfold higher affinity than MTP.
The differences in the binding affinities between LPL and MTP for LDL
were directly compared (Fig. 1B). In the concentration range
of LDL (0-32 µg/well) used, significant binding of LDL to LPL was
observed. In contrast, we could not detect the binding of LDL to MTP at
these concentrations. Higher concentrations of LDL are required to get
measurable binding to MTP (21). Next, we compared the binding of LDL to
LPL with its binding to 1D1, a monoclonal antibody that recognizes
amino acids 474-539 in apoB (22). LDL bound with similar affinity to
LPL and 1D1 (data not shown). These studies indicated that the LDL-LPL
binding involves high affinity interactions that are greater than that
found for MTP and are similar to the binding of LDL to its monoclonal
antibody.
To determine whether the interactions between LDL and LPL were ionic or
hydrophobic in nature, we studied the effect of different concentrations of NaCl or Triton X102 on the binding of LDL to immobilized LPL. For control, we studied the effect of these reagents on the binding of LDL to its monoclonal antibody, 1D1. Increasing salt
concentrations (0.5-2.0 M) inhibited binding of LDL to LPL (Fig. 2A). The binding of LDL
was inhibited 70% at 2 M NaCl, whereas the binding of LDL
to 1D1 was not significantly inhibited by salt. LDL-LPL binding was
also inhibited to 80% in the presence of 0.4 mM Triton
X102 (Fig. 2B). Again, LDL-1D1 binding was not significantly inhibited. Similar inhibition of LDL-LPL binding was observed using
Tween-20 (data not shown). Thus, detergents specifically inhibit
LDL-LPL binding and do not affect LDL-1D1. The inhibition of the
LPL-LDL binding by both salt and detergents suggests that the binding
between LDL and LPL may be complex and involves both hydrophobic and
hydrophilic interactions.
apoB17, an N-terminal fragment of apoB that does not bind lipids (17,
23, 24), has been shown to interact with LPL (19). Thus, it was of
interest to determine whether apoB17 also interacts with LPL by both
ionic and hydrophobic interactions. The binding of apoB17 to LPL was
more sensitive to salt concentrations (Fig. 3A) compared with the binding
of LDL to LPL (Fig. 2A). At the lowest concentrations (0.25 M) of NaCl used, apoB17-LPL binding was inhibited by 75%
(Fig. 3A), whereas LDL-LPL binding (Fig. 2A) was
not inhibited. At higher concentrations of salt (2.0 M), apoB17 LPL Binding Site in apoB--
We next attempted to identify the
amino acid sequences in apoB17 that interact with LPL. To identify the
LPL-binding site within the N-terminal 17% (amino acids 1-781) of
apoB, we expressed different apoB polypeptides as FLAG chimeras (25).
FLAG (DYKDDDDK) is an octapeptide that is commonly used as an epitope
tag. The plasmids were transiently transfected into COS-7 cells, and
the conditioned media were used to examine the secretion of
FLAG/apoB chimeras using an anti-FLAG monoclonal antibody, M2 (Table
I). Different chimeric proteins were secreted to different extents as
determined by the binding of these chimeras to M2. Next, we studied the
binding of secreted chimeras to immobilized LPL (Table I). The amounts of B:1-781 (B17F) bound
to M2 and LPL were similar, indicating that similar amounts of B:1-781
bound to immobilized M2 and LPL. FLAG/apoB chimeras containing B:1-300
and B:270-570 were secreted to a similar extent and are consistent
with our previous studies (12). Both of these peptides bound to similar extents. Because all the chimeras bound to LPL, consideration was given
to the possibility that the binding observed between LPL and FLAG/apoB
chimeras may be due to interactions between FLAG and LPL. Immobilized
M2 and LPL were incubated with same amounts of either B:270-570 (Fig.
4A) or B17F (Fig.
4B) in the presence of increasing concentrations of FLAG
peptide (0 to 400 µg/ml). Increasing concentrations of FLAG inhibited
(50-80%) the binding of B:270-570 and B17F to M2 but had no effect
on their binding to LPL (Fig. 4). Thus, the binding observed between
FLAG/apoB chimeras and LPL is not due to FLAG/LPL interactions. Taken
together, these studies indicated that LPL binds both B:1-300 and
B:270-570 and that apoB17 (amino acids 1-791) contains multiple
binding sites for LPL.
apoB-binding Site in LPL--
It is not known whether the enzyme
activity and the active site of LPL are required for its binding to
LDL. To study the importance of lipase activity in apoB binding, we
used tetrahydrolipstatin; this compound will completely inhibit LPL
activity at 0.1 mM most likely by binding to the active
site of the enzyme (26-28). We specifically considered the possibility
that the lipase activity was important for LDL, but not apoB17,
binding. Immobilized LPL was incubated with either LDL or apoB17 in the
presence of different indicated concentrations of tetrahydrolipstatin
(Fig. 5). The binding of LDL and apoB17
to LPL was not inhibited by tetrahydrolipstatin. These studies indicate
that the apoB-LPL binding does not require enzyme activity.
Next, attempts were made to identify a region in LPL that might be
involved in apoB binding (Fig. 6).
Monoclonal antibody, 5D2, is known to inhibit LPL activity and has been
shown to recognize an epitope between amino acids 380-410 in LPL (29).
At apoB and Proteoglycans May Bind to a Similar Site in LPL--
It
is known that apoB contains several binding sites for heparin (31) and
that LPL binds to GAGs present on the endothelial cells. For this
reason we tested whether the high affinity LDL-LPL binding might be due
to the presence of small amounts of heparin co-eluted with LPL during
heparin affinity chromatography. This hypothesis was ruled out using
two independent approaches. First, LPL purified by heparin affinity
chromatography was subjected to Biogel P60 size exclusion
chromatography in 1.5 M NaCl buffer to remove traces of
heparin. The enzyme purified by Biogel chromatography was then used to
study LDL binding. The Biogel P60 purified LPL bound better than the
LPL purified from heparin affinity chromatography (Fig.
7A). Nonlinear regression
analysis revealed that LDL bound to two different preparations of LPL
with similar affinity. The Kd values for the binding
of LDL to heparin and Biogel purified LPL were 0.14 ± 0.06 and
0.19 ± 0.07 nM (averages ± S.E.), respectively.
In contrast, the maximum binding of LDL to Biogel purified LPL was 68%
higher than its binding to heparin purified LPL. The
Bmax values for the binding of LDL to Biogel
purified and heparin purified LPL were 0.034 ± 0.004 and
0.057 ± 0.06 pmol (averages ± S.E.), respectively. Second,
LPL purified by heparin affinity chromatography and Biogel
chromatography was immobilized and subjected to treatment with
heparinase and heparitinase (Fig. 7B). The binding of LDL to
Biogel purified LPL was not altered by the treatment of the enzyme with
heparinase and heparitinase. In contrast, treatment of heparin purified
LPL by these enzymes resulted in ~50% increased binding of LDL.
These studies indicate that the removal of heparin from LPL
preparations increases the number of binding sites for LDL and suggests
that heparin might inhibit LPL-LDL binding. This was directly
determined by studying the inhibition of the binding of LDL to
immobilized LPL by heparin (Fig. 8).
Heparin (100 µM) inhibited 50% of the LDL-LPL binding. Higher concentrations of heparin did not increase the inhibition. These
studies suggested that heparin inhibits 50% of the binding of LDL to
LPL. The other 50% of binding is resistant to heparin probably because
it involves hydrophobic interactions. Taken together, we conclude that
LDL- and heparin-binding sites in LPL may be juxtaposed and compete for
each other.
Interactions between apoB and LPL--
The interactions between
apoB and LPL were of high affinity with Kd values in
the 10
The high affinity binding between LDL and LPL is due to both
hydrophobic and hydrophilic interactions between these molecules. The
evidence for hydrophobic interactions was derived from studies involving the effect of detergents. In a previous study, LPL binding to
LDL immobilized on microtiter plate assays failed to show hydrophobic interactions (5). Thus, the geometry of the interactions or the
exposure of apoB versus lipids may differ if the LDL, rather than LPL, is in solution. In the current study, the LDL-LPL binding was
very sensitive to detergents; it was completely inhibited by 0.4 mM Triton X102. It should be pointed out that 0.1% Triton (1.5 mM) is usually used for immunoprecipitation and
solubilization studies (33). In addition, 0.01% of Tween-20 inhibited
The hydrophobic interactions between lipoproteins and LPL may be
physiologically significant in the hydrolysis of lipoproteins. Triton
WR1339 is generally used in metabolic studies to determine the
production rates of lipoproteins (34) because it inhibits the
hydrolysis of lipoproteins during circulation. The mechanism of
inhibition of hydrolysis by Triton WR1339 is unknown, but it is
generally believed that the detergent covers the lipoprotein surface
and prevents lipolysis by LPL. Our studies may provide a biochemical
explanation for the inhibition of lipolysis by Triton WR1339. We
propose that Triton inhibits the hydrophobic interactions between
lipoproteins and LPL and inhibits hydrolysis of lipids in various
lipoproteins. It is expected that the hydrophobic interactions between
lipoproteins and LPL would be stronger with larger lipoproteins and may
also provide an explanation for greater rates of hydrolysis of larger,
triglyceride-rich lipoproteins by LPL.
Evidence for ionic interactions between LDL and LPL binding comes from
studies involving the use of salt (Figs. 2A and
3A), heparin (Fig. 8), and suramin (data not shown). The
inhibition of the binding of LDL to LPL was less sensitive to salt than
its binding to MTP. The amount of salt required for the 50% inhibition of the binding of LDL to LPL and MTP are 1.0 M (Fig.
1A) and 0.05 M (21), respectively. This may be
because LPL binds to LDL involving both hydrophilic and hydrophobic
interactions, whereas LDL-MTP binding involves hydrophilic interactions only.
There are significant differences with respect to the salt sensitivity
of LDL-LPL and apoB17-LPL interactions. However, such differences for
the effect of salt on the binding of LDL and apoB17 to MTP have not
been observed (21). The apoB17-LPL binding was more sensitive to salt
than the LDL-LPL binding (compare Figs. 2 and 3), whereas the salt
sensitivity of apoB17-LPL was the same as that observed for either
apoB17-MTP or LDL-MTP binding (21). Different amounts of salt required
for the inhibition of the binding of LDL and apoB17 to LPL supports the
idea that LDL binds to LPL via both ionic and hydrophobic interactions,
whereas apoB17-LPL binding is ionic. Because hydrophobic interactions
are not inhibited by salt, higher amounts of salt are required to
inhibit the binding of LDL to LPL. Although salt is known to alter LPL
dimeric structure (35), in the current study LPL was immobilized on
microtiter plates and is not expected to monomerize. Furthermore, the
differences between the binding of LPL to LDL and apoB17 (Figs. 2 and
3) indicate that the effect of salt observed is not related to changes
in the LPL structure. Thus, we conclude that although the tertiary structure of LPL or apoB could have been altered either by the salt or
the detergent, both protein-protein and protein-lipid interactions are
involved in LPL LPL-binding Sites in apoB--
To determine the LPL-binding site
in apoB17, we used apoB-FLAG chimeras that have been successfully used
in the identification of apoB-binding site in MTP (12). LPL bound to
B17F, B:1-300, and B:270-570. The binding of these chimeras was not
due to FLAG-LPL binding (Fig. 4). Thus, we conclude that LPL binds to
apoB at multiple sites. Different binding sites for LPL on apoB may
result in the anchoring of several LPL molecules on lipoproteins and facilitate rapid and simultaneous lipid hydrolysis at different sites.
apoB-binding Site in LPL--
The binding of LDL to LPL does not
require enzymatic activity. Tetrahydrolipstatin is a potent inhibitor
of LPL and is known to completely inhibit the enzyme activity. It did
not inhibit LDL-LPL binding (Fig. 5). This is rather surprising because
of the observations that LDL-LPL binding involves hydrophobic
interactions and most likely involves the binding of LPL to lipids
present in the LDL. These studies indicate that the lipid-binding site in LPL may be independent of its lipid hydrolyzing activity. Thus, it
is conceivable that LPL binds to LDL apolipoproteins and lipids and
subsequently accesses triglyceride molecules for hydrolysis.
The binding of LDL was inhibited by a monoclonal antibody 5D2,
indicating that amino acids 380-410 or a region close to these residues might be involved in LDL binding. It is quite possible that
the inhibition of LDL-LPL binding by antibodies may be due to steric
exclusion of LDL by the antibodies and may not be due to competition
for the LDL-binding site. Thus, the suggestion that amino acids
380-410 are involved in apoB binding should be confirmed by
independent methods such as site-directed mutagenesis. Nonetheless, it
is interesting to note that a similar region in the C terminus of LPL
has been implicated for its binding to the LDL receptor-related protein
and Sortilin/neurotensin receptor-3 (4, 36, 37). Nielsen et
al. (36) have shown that amino acids 308-384 and 404-430 in LPL
bind to the LDL receptor-related protein. This region is characterized
by the presence of high density of positive charges and is probably
exposed on the surface of the molecule (4). It is also interesting to
note that the binding of LPL to LDL receptor-related protein,
neurotensin receptor-3, and LDL is inhibited by heparin. Thus, it is
conceivable that the binding sites for 5D2 monoclonal antibody, apoB,
heparin, LDL-receptor related protein, and the Sortilin/neurotensin
receptor-3 in LPL may be the same or juxtaposed, and this site may be
critical for the binding of the enzyme to different biologically
important molecules.
Modulation of LDL-LPL Binding by Glycosaminoglycans--
LPL is
well known to bind GAGs. apoB has also been suggested to bind to GAGs
but does so weakly in physiologic salt solutions (38). In the present
study, we show that the protein-protein interactions between LDL-LPL
involving ionic residues are inhibited by heparin (Fig. 8). Thus, GAGs
can modulate catabolism of lipoproteins by LPL in several ways. First,
by binding to both LPL and lipoproteins, GAGs may help bring substrate
and enzyme together for efficient hydrolysis on the endothelium.
Second, by inhibiting the binding of LPL to lipoproteins they can bring
enzyme and substrate apart and modulate the extent of hydrolysis.
Third, they can dislodge both lipoproteins and LPL from the endothelial
cell surface for removal from vascular system. Once LPL leaves its site
of physiologic action on the lumenal endothelial surfaces, it
circulates in the bloodstream attached to LDL, prior to its removal by
the liver. If LDL occupies the GAG-binding site of LPL, then it would
prevent LPL from reassociating with the endothelial surface, thus
insuring its removal.
In summary, we show that LDL
12 M) similar to that observed for the
binding of LDL to its receptors and 1D1, a monoclonal antibody to LDL,
and was greater than its affinity for microsomal triglyceride transfer
protein. LDL-LPL binding was sensitive to both salt and
detergents, indicating the involvement of both hydrophobic and
hydrophilic interactions. In contrast, the N-terminal 17% of apoB
interacted with LPL mainly via ionic interactions. Binding of various
apoB fusion peptides suggested that LPL bound to apoB at multiple sites
within apoB17. Tetrahydrolipstatin, a potent enzyme activity inhibitor,
had no effect on apoB-LPL binding, indicating that the enzyme activity was not required for apoB binding. LDL-LPL binding was inhibited by
monoclonal antibodies that recognize amino acids 380-410 in the
C-terminal region of LPL, a region also shown to interact with heparin
and LDL receptor-related protein. The LDL-LPL binding was also
inhibited by glycosaminoglycans (GAGs); heparin inhibited the
interactions by ~50% and removal of trace amounts of heparin from
LPL preparations increased LDL binding. Thus, we conclude that the high
affinity binding between LPL and lipoproteins involves multiple ionic
and hydrophobic interactions, does not require enzyme activity and is
modulated by GAGs. It is proposed that LPL contains a surface exposed
positively charged amino acid cluster that may be important for various
physiological interactions of LPL with different biologically important
molecules. Moreover, we postulate that by binding to this cluster, GAGs
modulate the association between LDL and LPL and the in
vivo metabolism of LPL.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical domain (8-10). This region is
essential for the secretion of various C-terminal lipid-binding
sequences of apoB (11). Recently, this region has been shown to contain
elements necessary for MTP binding (12, 13). Although the N-terminal
region of apoB interacts with LPL, the physiological importance of this
is unclear. To better understand these interactions, we tested the
effects of salt, detergents, and glycosaminoglycans (GAGs) on LPL-apoB
binding. Furthermore, we attempted to identify the binding sites
responsible for LPL-apoB interactions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
[in a new window]
Fig. 1.
Binding of human LDL to immobilized LPL.
A, binding to LPL. Mictotiter wells were coated with 0.5 µg of purified LPL, washed, and incubated (2 h, 37° C) in
triplicate with 100 µl of different concentrations of LDL. The
amounts of apoB bound to LPL were measured by ELISA. The binding of LDL
was subjected to nonlinear regression analysis using one binding site
isotherm. The means ± S.D. are plotted as line graphs
and error bars, respectively. The data are representative of
five independent experiments. B, comparative binding of LDL
to LPL and MTP. Microtiter wells were coated with 0.5 µg of purified
bovine LPL or MTP and incubated in triplicate with indicated amounts of
human LDL for 2 h at 37 °C. The amount of apoB bound to the
immobilized proteins was measured by ELISA. Averages and standard
deviations are plotted as bar graphs and error
bars, respectively. The data are representative of three
independent experiments.
View larger version (13K):
[in a new window]
Fig. 2.
Effect of NaCl and Triton X102 on the binding
of LDL to LPL. Micotiter well plates were coated with LPL (0.5 µg/well) or 1D1 (1 µg/well), incubated in triplicate (2 h,
37 °C) with 100 µl of LDL (100 ng/ml) in the presence of various
indicated concentrations of NaCl (A) or Triton X102
(B), and apoB bound to the immobilized LPL was quantified by
ELISA. Wells incubated with LDL alone represented 100% binding,
whereas wells incubated with no LDL served as blank. Blank values were
subtracted from all wells. The average percentage inhibition values by
the presence of salt or detergent are plotted as line
graphs, whereas standard deviations are plotted as error
bars. The data for salt and detergent effects are representative
of seven and ten experiments, respectively.
LPL binding was inhibited to ~85%, but apoB17-1D1 binding was not inhibited (Fig. 3A). These studies indicated that
apoB17-LPL binding involves ionic interactions. Next, we studied the
effect of Triton X102 to determine whether apoB17-LPL interactions also involve hydrophobic interactions (Fig. 3B). apoB17-LPL
binding was not significantly inhibited by 400 µM Triton
X102. Note that at this concentration of Triton X102, LDL-LPL binding
was inhibited by ~80% (Fig. 2B). Taken together, these
studies indicate that the binding of LPL to LDL involves both ionic and
hydrophobic interactions, whereas the binding of LPL to the N-terminal
17% of apoB involves mainly ionic interactions.
View larger version (12K):
[in a new window]
Fig. 3.
Effect of NaCl and Triton X102 on the binding
of apoB17 to LPL. Micotiter well plates were coated with LPL (0.5 µg/well) or 1D1 (1 µg/well) incubated in triplicate (2 h, 37 °C)
with 100 µl of conditioned media obtained from McA-RH7777
cells stably transfected with plasmids expressing human recombinant
apoB17 in the presence of various indicated concentrations of either
NaCl (A) or Triton X102 (B). The apoB17 bound to
LPL was quantified by ELISA. Wells incubated with the conditioned
media alone represented 100% binding of apoB17, whereas wells
incubated with nonconditioned media served as blank. Blank
values were subtracted from all wells and the inhibition because of the
presence of either salt or detergent was calculated. The average
percentage inhibition because of the presence of salt or detergent is
plotted as a line graph, whereas standard deviations are
plotted as error bars. The data are representative of two
independent experiments.
Binding of different apoB sequences to the immobilized LPL
View larger version (17K):
[in a new window]
Fig. 4.
Effect of different concentrations of FLAG on
the binding of apoB/FLAG chimeras to M2 and LPL. Anti-FLAG
monoclonal antibody, M2 (2 µg/well), and LPL (0.5 µg/well) were
immobilized and incubated with conditioned media (72 h
post-transfection) obtained from COS-7 cells transfected with plasmids
expressing either B:270-570 (A) or B17F (B) in
the presence of increasing concentrations of FLAG peptide. The amount
of apoB bound to LPL and M2 was determined by ELISA. Averages and
standard deviations are plotted as line graphs and
errors bars, respectively. The data are representative of
two independent experiments.
View larger version (16K):
[in a new window]
Fig. 5.
Effect of tetrahydrolipstatin on the binding
of either LDL or apoB17 to LPL. Immobilized LPL was incubated in
triplicate with LDL or conditioned media from McA-RH7777 cells
expressing apoB17 in the presence or absence of indicated
concentrations of tetrahydrolipstatin for 2 h as described in the
legends to Figs. 2 and 3. ELISA determined the amounts of apoB bound.
Wells incubated with LDL or apoB17 in the absence of
tetrahydrolipstatin served as control (100%). The average percentages
of control and standard deviations are plotted as line
graphs and error bars, respectively.
4 µmg/ml, this antibody inhibited LDL-LPL binding by about
70-90% (Fig. 6). Another monoclonal antibody, Mab, whose
binding epitope has not yet been determined and that inhibits LPL
activity and is known to block LPL binding to very low density
lipoproteins (30), was less effective. These studies indicate that
amino acids 380-410 of LPL, other amino acids proximal to these
residues, or these amino acids with other proximal residues in LPL
might constitute a site for LDL binding.
View larger version (18K):
[in a new window]
Fig. 6.
Effect of two different anti-LPL monoclonal
antibodies on LDL-LPL binding. Immobilized LPL was incubated in
triplicate with LDL in the presence or absence of indicated
concentrations of two different monoclonal antibodies developed against
LPL. Mab represents a monoclonal antibody described by
Goldberg et al. (30), whereas 5D2 represents
monoclonal antibody described by Brunzell and co-workers (29). After
2 h at 37 °C wells were washed, and apoB was determined by
ELISA. The averages and standard deviations are plotted as line
graphs and error bars, respectively. The data are
representative of three independent experiments.
View larger version (21K):
[in a new window]
Fig. 7.
Effect of removal of heparin on the binding
of LPL to LDL. A, bovine milk LPL purified by heparin
affinity chromatography was subjected to Biogel P60 column
chromatography in the presence of high salt (1.5 M) as
described under "Experimental Procedures." Both LPL preparations
were immobilized (0.5 µg/well), and the binding of different
concentrations of LDL was studied as described in Fig. 1. The binding
isotherms were subjected to nonlinear regression analysis, and averages
and standard deviations are plotted as line graphs and
error bars, respectively. B, two different
preparations of LPL were immobilized and blocked with 0.3% BSA in
phosphate-buffered saline. Wells were washed three times with 1 mM Tris, 30 mM sodium acetate, 10 mM EDTA, pH 8.0, and incubated with equal amounts of both
the enzymes as indicated for 1 h at 37 °C. The enzyme solutions
were removed, and plates were washed with PBS and 0.3%BSA.
Subsequently, wells were incubated with LDL (10 ng/ml) for 2 h at
37 °C, washed, and then exposed to sheep anti-human apoB and
alkaline phosphatase-labeled rabbit anti-sheep IgG, and the amount of
apoB was quantified. The data are representative of two independent
experiments.
View larger version (13K):
[in a new window]
Fig. 8.
Effect of heparin on LDL-LPL binding.
Immobilized LPL (0.5 µg/well) was incubated in triplicate with human
plasma LDL (10 ng/well) and different indicated concentrations of
heparin for 2 h at 37 °C. The apoB bound was quantified by
ELISA. The average percentages of inhibition and standard deviations
are plotted as line graphs and error bars,
respectively. The data are representative of two independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12 M range (Fig. 1). Interestingly,
these interactions were found to be much stronger than those observed
for the binding of LDL to MTP (21) and were as strong as those observed
between antigen-antibody (LDL-1D1) binding (data not shown) and the
binding of LDL to its receptors in fibroblasts (32). The high affinity
interactions between LDL and LPL imply that they are physiologically
significant. These interactions may be crucial in ensuring the
hydrolysis of circulating lipoproteins before their recognition by
receptors for removal.
50% of the LDLLPL binding (data not shown). Usually, 0.05% of
Tween-20 is used in ELISA assays. Thus, LDL-LPL binding is inhibited by detergent concentrations that are lower than those used in various biochemical procedures of protein characterization. Furthermore, detergents did not inhibit apoB17-LPL binding (Fig. 3). Therefore, we
conclude that the effect of detergents were specific to LDL-LPL binding. These studies indicate that detergents were probably inhibiting hydrophobic interactions between LDL and LPL independent of
structural modification of proteins.
LDL binding. Furthermore, we conclude that
interactions between LPL and apoB17 are protein-protein interactions
involving ionic amino acid residues.
LPL binding involves high affinity
interactions involving hydrophilic and hydrophobic forces. The
N-terminal region of apoB contains multiple binding sites for ionic
interactions with LPL. The enzymatic activity of LPL is not required
for LDL binding. The LDL-binding region of LPL also binds to monoclonal
antibodies, heparin, and LDL receptor-related protein. We speculate
that LPL may contain a surface exposed positively charged amino acid
cluster that may be important for various physiological interactions of
LPL with different biological molecules.
| |
ACKNOWLEDGEMENT |
|---|
We gratefully appreciate the technical assistance of Neeru Nayak.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grants DK-46900, HL62301, and HL-64272) and funds from the American Heart Association (National Center).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.
¶ Established investigator of the American Heart Association.
To whom correspondence should be addressed: Dept. of Anatomy
and Cell Biology and Pediatrics, SUNY Health Science Center at Brooklyn, 450 Clarkson Ave., Box 5, Brooklyn, New York, NY 11203. Tel.:
718-270-4790; Fax: 718-270-3732; E-mail:
mahmoodhussain@netmail.hscbklyn.edu.
Published, JBC Papers in Press, July 5, 2000, DOI 10.1074/jbc.M005317200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: LPL, lipoprotein lipase; apo, apolipoprotein; BSA, bovine serum albumin; ELISA, enzyme linked immunoassay; GAG, glycosaminoglycans; LDL, low density lipoprotein(s); MTP, microsomal triglyceride transfer protein; PBS, phosphate buffered saline.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Brunzell, J. D. (1995) in The Metabolic and Molecular Bases of Inherited Disorders (Scriver, C. R. , Beaudet, A. L. , Sly, W. S. , and Valle, D., eds) , pp. 1913-1932, McGraw-Hill, Inc., New York |
| 2. | Bhatnagar, A. (1999) in Lipoproteins in Health and Disease (Betteridge, D. , Illingworth, D. R. , and Shepherd, J., eds) , pp. 737-752, Arnold, London |
| 3. | Goldberg, I. J. (1996) J. Lipid Res. 37, 693-707 |
| 4. | Hussain, M. M., Strickland, D. K., and Bakillah, A. (1999) Annu. Rev. Nutr. 19, 141-172 |
| 5. | Choi, S. Y., Sivaram, P., Walker, D. E., Curtiss, L. K., Gretch, D. G., Sturley, S. L., Attie, A. D., Deckelbaum, R. J., and Goldberg, I. J. (1995) J. Biol. Chem. 270, 8081-8086 |
| 6. | Pang, L., Sivaram, P., and Goldberg, I. J. (1996) J. Biol. Chem. 271, 19518-19523 |
| 7. | Choi, S. Y., Pang, L., Kern, P. A., Kayden, H. J., Curtiss, L. K., Vanni-Reyes, T. M., and Goldberg, I. J. (1997) J. Lipid Res. 38, 77-85 |
| 8. | Segrest, J. P., Jones, M. K., Mishra, V. K., Anantharamaiah, G. M., and Garber, D. W. (1994) Arterioscler. Thromb. 14, 1674-1685 |
| 9. | Segrest, J. P., Jones, M. K., Mishra, V. K., Pierotti, V., Young, S. H., Borén, J., Innerarity, T. L., and Dashti, N. (1998) J. Lipid Res. 39, 85-102 |
| 10. | Segrest, J. P., Jones, M. K., and Dashti, N. (1999) J. Lipid Res. 40, 1401-1416 |
| 11. | Gretch, D. G., Sturley, S. L., Wang, L., Lipton, B. A., Dunning, A., Grunwald, K. A. A., Wetterau, J. R., Yao, Z., Talmud, P., and Attie, A. D. (1996) J. Biol. Chem. 271, 8682-8691 |
| 12. | Hussain, M. M., Bakillah, A., Nayak, N., and Shelness, G. S. (1998) J. Biol. Chem. 273, 25612-25615 |
| 13. | Bradbury, P., Mann, C. J., Köchl, S., Anderson, T. A., Chester, S. A., Hancock, J. M., Ritchie, P. J., Amey, J., Harrison, G. B., Levitt, D. G., Banaszak, L. J., Scott, J., and Shoulders, C. C. (1999) J. Biol. Chem. 274, 3159-3164 |
| 14. | Obunike, J. C., Edwards, I. J., Rumsey, S. C., Curtiss, L. K., Wagner, W. D., Deckelbaum, R. J., and Goldberg, I. J. (1994) J. Biol. Chem. 269, 13129-13135 |
| 15. | Hussain, M. M., Zhao, Y., Kancha, R. K., Blackhart, B. D., and Yao, Z. (1995) Arterioscler. Thromb. Vasc. Biol. 15, 485-494 |
| 16. | Bakillah, A., Zhou, Z., Luchoomun, J., and Hussain, M. M. (1997) Lipids 32, 1113-1118 |
| 17. | Yao, Z., Blackhart, B. D., Linton, M. F., Taylor, S. M., Young, S. G., and McCarthy, B. J. (1991) J. Biol. Chem. 266, 3300-3308 |
| 18. | Wang, H., Yao, Z., and Fisher, E. A. (1994) J. Biol. Chem. 269, 18514-18520 |
| 19. | Sivaram, P., Choi, S. Y., Curtiss, L. K., and Goldberg, I. J. (1994) J. Biol. Chem. 269, 9409-9412 |
| 20. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 |
| 21. | Hussain, M. M., Bakillah, A., and Jamil, H. (1997) Biochemistry 36, 13060-13067 |
| 22. | Pease, R. J., Milne, R. W., Jessup, W. K., Law, A., Provost, P., Fruchart, J. C., Dean, R. T., Marcel, Y. L., and Scott, J. (1990) J. Biol. Chem. 265, 553-568 |
| 23. | Du, E. Z., Kurth, J., Wang, S.-L., Humiston, P., and Davis, R. A. (1994) J. Biol. Chem. 269, 24169-24176 |
| 24. | Herscovitz, H., Hadzopoulou-cladaras, M., Walsh, M. T., Cladaras, C., Zannis, V. I., and Small, D. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 88, 7313-7317 |
| 25. | Shelness, G. S., Morris-Rogers, K. C., and Ingram, M. F. (1994) J. Biol. Chem. 269, 9310-9318 |
| 26. | Lookene, A., Skottova, N., and Olivecrona, G. (1994) Eur. J. Biochem. 222, 395-403 |
| 27. | Hadvary, P., Sidler, W., Meister, W., Vetter, W., and Wolfer, H. (1991) J. Biol. Chem. 266, 2021-2027 |
| 28. | Yin, B., Loike, J. D., Kako, Y., Weinstock, P. H., Breslow, J. L., Silverstein, S. C., and Goldberg, I. J. (1997) J. Clin. Invest. 100, 649-657 |
| 29. | Chang, S. F., Reich, B., Brunzell, J. D., and Will, H. (1998) J. Lipid Res. 39, 2350-2359 |
| 30. | Goldberg, I. J., Paterniti, J. R., Jr., France, D. S., Martinelli, G., and Cornicelli, J. A. (1986) Biochim. Biophys. Acta 878, 168-176 |
| 31. | Weisgraber, K. H., and Rall, S. C., Jr. (1987) J. Biol. Chem. 262, 11097-11103 |
| 32. | Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzymol. 98, 241-260 |
| 33. | Helenius, A., and Simons, K. (1975) Biochim. Biophys. Acta 415, 29-79 |
| 34. | Tietge, U. J. F., Bakillah, A., Maugeais, C., Tsukamoto, K., Hussain, M. M., and Rader, D. J. (1999) J. Lipid Res. 40, 2134-2139 |
| 35. | Bengtsson, G., and Olivecrona, T. (1983) Biochim. Biophys. Acta 751, 254-259 |
| 36. | Nielsen, M. S., Brejning, J., Garcia, R., Zhang, H., Hayden, M. R., Vilaro, S., and Gliemann, J. (1997) J. Biol. Chem. 272, 5821-5827 |
| 37. | Nielsen, M. S., Jacobsen, C., Olivecrona, G., Gliemann, J., and Petersen, C. M. (1999) J. Biol. Chem. 274, 8832-8836 |
| 38. | Goldberg, I. J., Wagner, W. D., Pang, L., Paka, L., Curtiss, L. K., DeLozier, J. A., Shelness, G. S., Young, C. S. H., and Pillarisetti, S. (1998) J. Biol. Chem. 273, 35355-35361 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Loeffler, J. Heeren, M. Blaeser, H. Radner, D. Kayser, B. Aydin, and M. Merkel Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor J. Lipid Res., February 1, 2007; 48(2): 288 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Khalil, W. D. Wagner, and I. J. Goldberg Molecular Interactions Leading to Lipoprotein Retention and the Initiation of Atherosclerosis Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2211 - 2218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Iqbal and J. W. McCauley Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site-directed mutagenesis J. Gen. Virol., September 1, 2002; 83(9): 2153 - 2159. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. O. Pentikainen, R. Oksjoki, K. Oorni, and P. T. Kovanen Lipoprotein Lipase in the Arterial Wall: Linking LDL to the Arterial Extracellular Matrix and Much More Arterioscler. Thromb. Vasc. Biol., February 1, 2002; 22(2): 211 - 217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Zimmermann, U. Panzenbock, A. Wintersperger, S. Levak-Frank, W. Graier, O. Glatter, G. Fritz, G. M. Kostner, and R. Zechner Lipoprotein Lipase Mediates the Uptake of Glycated LDL in Fibroblasts, Endothelial Cells, and Macrophages Diabetes, July 1, 2001; 50(7): 1643 - 1653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Obunike, E. P. Lutz, Z. Li, L. Paka, T. Katopodis, D. K. Strickland, K. F. Kozarsky, S. Pillarisetti, and I. J. Goldberg Transcytosis of Lipoprotein Lipase across Cultured Endothelial Cells Requires Both Heparan Sulfate Proteoglycans and the Very Low Density Lipoprotein Receptor J. Biol. Chem., March 16, 2001; 276(12): 8934 - 8941. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Boren, A. Lookene, E. Makoveichuk, S. Xiang, M. Gustafsson, H. Liu, P. Talmud, and G. Olivecrona Binding of Low Density Lipoproteins to Lipoprotein Lipase Is Dependent on Lipids but Not on Apolipoprotein B J. Biol. Chem., July 13, 2001; 276(29): 26916 - 26922. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
