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J Biol Chem, Vol. 274, Issue 44, 31305-31311, October 29, 1999
From the The low density lipoprotein receptor-related
protein (LRP) is a multifunctional endocytic cell-surface receptor that
binds and internalizes a diverse array of ligands. The receptor
contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we
studied the binding of these fragments to methylamine-activated The low density lipoprotein receptor-related protein
(LRP)1 is a 600-kDa membrane
glycoprotein that is a member of the low density lipoprotein (LDL)
receptor family of endocytic receptors (reviewed in Refs. 1 and 2). LRP
can bind and internalize a diverse spectrum of structurally unrelated
ligands in a calcium-dependent manner including
apolipoproteins, lipases, proteinases, proteinase-inhibitor complexes,
Kunitz-type inhibitors, matrix proteins, and other proteins such as
lactoferrin, Pseudomonas exotoxin A, and malaria circumsporozoite protein (1, 2). In addition, the blood coagulation
factor VIII was recently identified as a ligand of LRP (3). The broad
range of ligands suggests a role for the receptor in distinct
physiological and pathophysiological processes, ranging from
lipoprotein metabolism, cell growth and cell migration, fibrinolysis,
and thrombosis to atherosclerosis and Alzheimer's disease.
LRP is synthesized as a single polypeptide chain and is cleaved in the
trans-Golgi network by the endopeptidase furin into two
subunits, resulting in a 515-kDa fragment that contains the ligand
binding domains and an 85-kDa fragment comprising the transmembrane and
cytoplasmic domains. The subunits remain associated in a noncovalent fashion as they are routed to the cell surface (4, 5). LRP contains 31 class A cysteine-rich repeats, which are also present in the LDL
receptor and are therefore called LDL receptor class A (LDLRA) domains.
LRP has four clusters (denoted clusters I, II, III, and IV) with 2, 8, 10, and 11 LDLRA domains, respectively. Evidence is accumulating that
the clusters of LDLRA domains constitute the ligand binding domains of
the receptor (6-10). Except for receptor-associated protein (RAP)
binding to clusters III and IV (1, 7, 8), only cluster II was shown to
be involved in ligand binding (6-10). Specifically, it has been
demonstrated that Several studies have shown that RAP facilitates the proper folding and
subsequent trafficking of LRP within the early secretory pathway (8,
11-14). RAP inhibits the binding of all ligands to the receptor and is
thought to prevent premature binding of ligands to the receptor during
the trafficking to the cell surface (15-17). Some ligands for LRP
occupy distinct binding sites (9, 18) while other ligands compete with
each other for binding to LRP (19-22). Although RAP competes with all
known ligands, most ligands are not able to compete for RAP binding to
LRP. Examples of RAP competitors include lactoferrin and lipoprotein
lipase (23, 24). Other ligands, such as t-PA,
Materials--
Oligonucleotides were from Amersham Pharmacia
Biotech (Roosendaal, The Netherlands). Restriction enzymes and
DNA-modifying enzymes were from Life Technologies, Inc. (Breda, The
Netherlands). All other chemicals used were reagent grade from Sigma or
Merck (Darmstadt, Germany).
Proteins--
Purified human LRP and the anti-LRP monoclonal
antibody SPR Reagents and Instrumentation--
The Biacore®
2000 biosensor system and reagents, including an amine-coupling kit,
containing N-hydroxysuccinimide,
N-ethyl-N'-(3-diethylamino-propyl)carbodiimide, ethanolamine hydrochloride, and CM5 and SA5 sensor chips (research grade), were from Biacore AB (Uppsala, Sweden).
Construction of Expression Plasmids for Recombinant Receptor
Fragments--
The construction of plasmids, encoding recombinant LRP
cluster II fragments, was described previously (9). One of these plasmids, pZEM229R-II, was modified to facilitate the construction of
plasmids, encoding recombinant LRP cluster I, III, and IV fragments. The pZEM229R-II vector encodes the signal and pro-peptide sequence of
t-PA (amino acids Expression and Purification of Recombinant Receptor
Fragments--
The expression and purification of recombinant LRP
cluster II fragments was performed essentially as described (9).
Purification of clusters I and III from conditioned media was done by
affinity chromatography, using Sepharose-coupled monoclonal antibody
CLB-CAg 69. After binding, columns were washed with HEPES-buffered
saline (HBS, 20 mM HEPES (pH 7.4), 150 mM NaCl)
and eluted with HBS, containing 1 M NaCl. To further purify
the cluster I and III fragments, an FPLC Superose 12 gel filtration
column was used. The cluster IV fragment was purified by a single
affinity-purification step, using a RAP-Sepharose column. This column
was washed with HBS and eluted with HBS containing 10 mM
EDTA. All purified cluster preparations were concentrated in
HEPES-buffered 1 M NaCl in Centricon 10 or 30 concentrators
(Amicon, Beverley, MA) by successive rounds of centrifugation in a
Sorvall high speed centrifuge for 1 h at 4 °C at a speed of
7000 rpm. Finally, the preparations were dialyzed against filtered and
degassed, modified HBST buffer (containing 20 mM HEPES (pH
7.4), 150 mM NaCl, 2 mM CaCl2,
0.005% (v/v) Tween 80). All cluster fragment preparations were
analyzed by non-reducing (12.5% (w/v)) SDS-polyacrylamide gel
electrophoresis and subsequent silver staining. Typical yields ranged
from 100 to 800 µg/liter of conditioned media, depending on the
particular fragment and purification method.
Concentration Determination of Proteins--
All protein
concentrations were determined, using a microBCA protein assay reagent
kit with bovine serum albumin (BSA) as a standard (Pierce). In
addition, the concentrations of cluster II and IV fragments were
determined, using surface plasmon resonance (SPR) as described
previously (9).
Mapping of Ligand Binding to Soluble Recombinant Receptor
Fragments Using SPR--
To determine the ligand-binding
characteristics of the different recombinant receptor fragments, all
ligands were immobilized at high density on CM5 sensor chips. Whereas
LpL was biotinylated and bound to a streptavidin-coated SA5 sensor
chip, the anti-LRP monoclonal Fab fragment A8 and the anti-LRP
monoclonal Kinetic Determinations Using SPR--
Purified human LRP was
immobilized on a CM5 sensor chip by amine coupling at a low density of
approximately 16 fmol/mm2 to determine kinetics of ligand
binding to LRP. Interactions between recombinant receptor fragments and
ligands were measured as follows: pro-u-PA (18 fmol/mm2),
fVIII light chain (155 fmol/mm2), t-PA·PAI-1 (37 fmol/mm2), LpL (23 fmol/mm2), and RAP (8 fmol/mm2) were immobilized to the sensor chip and different
concentrations of receptor fragments were passed over the sensor chip.
The different ligands or receptor fragments were passed over three
separate channels with immobilized LRP or ligand, respectively, and one control (non-immobilized) channel at 25 °C at a flow rate of 20 µl/min, using modified HBST as running buffer. Each determination was
performed at least in duplicate at different concentrations (n = 5) in the appropriate concentration range (around
KD values). The BIAevaluation software
(Biacore AB, Uppsala, Sweden) was used for analysis of the association
and dissociation profiles of the sensorgrams. Interaction constants
were determined by performing non-linear fitting of data, corrected for
bulk refractive index changes, according to a one- or a two-site model
employing previously described equations (9). Data were fitted to a
two-site model if a one-site model proved inadequate as judged from
residual plots and statistical parameters (data not shown). The data
were validated by subjecting them to tests of self-consistency
(36).
Analysis of Cluster II or IV Binding to RAP Which is Bound to
LRP, Cluster II, or Cluster IV--
First, 100 ng of LRP, cluster II,
or cluster IV was immobilized for 16 h at 4 °C in microtiter
wells in 50 mM NaHCO3 (pH 8.6) in a volume of
50 µl. Second, wells were blocked for 1 h at 37 °C with 3%
(w/v) BSA in modified HBST buffer in a volume of 300 µl, washed with
modified HBST buffer, and incubated with 100 nM RAP in
modified HBST buffer. Next, wells were washed and incubated for 1 h at 37 °C in modified HBST buffer with a range of biotinylated cluster II or IV concentrations. Proteins were biotinylated using an
EZ-LinkTM sulfo-NHS-LC-biotinylation kit, following the
instructions of the supplier (Pierce). Bound proteins were detected
using streptavidin-horseradish peroxidase (Amersham Pharmacia Biotech,
Roosendaal, The Netherlands) as described (28). Experiments were
performed in duplicate. As controls, cluster II or IV binding to RAP
bound to BSA and direct cluster II or IV binding to immobilized BSA,
LRP, cluster II, or cluster IV was measured.
Expression and Purification of Soluble Recombinant Receptor
Fragments--
To investigate the ligand-binding properties of all
four putative ligand binding domains of LRP, we expressed soluble
recombinant receptor fragments, comprising clusters I, II, III, and IV
(Fig. 1), in transfected baby hamster
kidney cells. Based on the findings that clusters II and IV fragments
strongly bind to RAP (6-8), these fragments were purified by one-step
affinity chromatography with RAP coupled to Sepharose. Affinity
chromatography with Sepharose coupled to the monoclonal antibody
CLB-CAg 69, directed against a tag was used to purify the cluster I and
III fragments containing this particular tag. Most of the latter
fragments consists of aggregated, SDS-resistant, high molecular weight
material. To remove high molecular weight protein from the monomeric
receptor fragments, gel filtration was performed using an FPLC Superose 12 column. The purified, monomeric preparations of clusters I, II, III,
and IV were analyzed by non-reducing SDS gel electrophoresis (Fig.
2). Heterogeneity of these monomeric
preparations is due to a different degree of N-linked
glycosylation, as we have reported before (9). Furthermore, the
mobility of the fragments is somewhat slower than would be expected on
the basis of the calculated molecular masses (approximately 12.1, 44.2, 48.7, and 52.6 kDa for clusters I, II, III, and IV, respectively),
consistent with variable N-linked glycosylation.
Mapping of Ligand Binding to Soluble Recombinant Receptor Fragments
Using SPR--
To examine the ligand-binding characteristics of the
four receptor fragments, we tested the binding of the following
ligands: t-PA, PAI-1, t-PA·PAI-1 complexes, pro-u-PA,
Kinetics of Ligand Binding to LRP, Cluster II, and Cluster
IV--
To further characterize the ligand-binding properties of
clusters II and IV, we determined the rate constants for the binding of
pro-u-PA, t-PA·PAI-1 complexes, LpL, RAP and fVIII light chain to
LRP, clusters II and IV (Table II). LRP
was immobilized at low density on a sensor chip to determine the
kinetics of ligand binding to the native LRP molecule. The resulting
binding curves were fitted according to a two-site model if a
single-site model did not appropriately describe the interaction (Table
II). The kinetics of LpL binding to LRP could not be accurately
determined due to a high degree of nonspecific binding of LpL to the
sensor chip. Pro-u-PA binds to LRP according to a two-site model with similar mediate affinities of 63.8 and 54.3 nM,
respectively. The interaction of t-PA·PAI-1 complexes could be
accurately described by a single-site model, yielding a
KD value of 9.2 nM. RAP binds to
high and mediate affinity sites on LRP with KD
values of 2.2 and 34.9 nM, respectively, and fVIII light
chain binds to LRP with mediate and low affinities of 52.1 and 130.3 nM, respectively. To determine the kinetics of ligand
binding to clusters II and IV, ligands were immobilized at low density
on sensor chips. As shown in Table II, pro-u-PA displays a slightly
higher affinity for cluster II (33.3 nM) than for cluster
IV (83.3 nM). This is also concluded for t-PA·PAI-1
complexes that bind to clusters II and IV with
KD values of 16.7 and 48.8 nM,
respectively. LpL binds with comparable affinities to both clusters
(cluster II; 32.6 nM, cluster IV; 21.9 nM). A
similar conclusion can be drawn for RAP (cluster II; 12.7 nM, cluster IV; 18.0 nM). The light chain of
fVIII binds to clusters II and IV with KD values
of 121.4 and 87.8 nM, respectively. From the quantitative
data presented in Table II, it can be concluded that clusters II and IV
are highly similar with respect to their ligand-binding properties,
further substantiating the concept of a major functional duplication in LRP.
Mapping of Ligand Binding to Subfragments of Cluster II--
To
investigate whether the ligands occupy distinct binding sites or
whether they bind to the same segment of cluster II, we determined the
binding of RAP Can Bind to Two Receptor Fragments Simultaneously--
The
findings presented in the previous paragraph suggest that RAP inhibits
ligand binding by direct competition or steric hindrance of each of the
LRP ligands. However, previous results (9) show that it is unlikely
that RAP regulates ligand binding solely by a mechanism involving
mutually exclusive binding to overlapping binding sites. Instead, those
results favor a model in which one molecule of RAP would induce a
conformational change in the receptor by interacting with multiple
receptor domains simultaneously. This conformational change would
render the receptor incapable of ligand binding. This model would imply
that one RAP molecule should be capable of binding multiple clusters
simultaneously. To test this hypothesis, we used an enzyme-linked
immunosorbent assay to measure the binding of increasing concentrations
of either cluster II or cluster IV to RAP, which, on its turn, was
bound to either LRP, cluster II, or cluster IV (Fig.
3). From the data, we can conclude that
RAP is able to bind simultaneously to clusters II and IV. Furthermore,
RAP is also able to bind to two clusters II or two clusters IV and both
clusters II and IV can bind to RAP bound to LRP, suggesting that RAP is
capable of forming an intermolecular bridge between LRP molecules. In
addition, half-maximal saturation of cluster II binding to RAP, which
is bound to LRP, cluster II, or cluster IV, is obtained at virtually
identical concentrations, namely at 1.4, 1.7, or 1.4 nM,
respectively. Finally, it can be deduced that also nearly identical
values are found for cluster IV binding to RAP, bound to LRP, cluster
II, or cluster IV, namely 1.8, 2.5, or 4.3 nM,
respectively. Apparently, one RAP molecule can bind two clusters
simultaneously with comparable affinities and might even be able to
bind to multiple LRP molecules.
We have generated a set of recombinant LRP fragments, comprising
the four putative ligand-binding domains, generally referred to as
clusters I, II, III, and IV, to study the structure and function of
this universal clearance receptor. Until now, a systematic investigation on the properties of the individual clusters is lacking,
since predominantly reports on ligand binding to cluster II have been
published (6-10). We demonstrate that each of the ligands tested binds
with similar affinity to both cluster II and cluster IV. Furthermore,
in both cases binding of these ligands is calcium-dependent
(data not shown). These observations suggest that LRP contains two
duplicated domains that have fully retained their functional
properties. In a study by Willnow et al. (7), using clusters
II and IV LRP-minireceptors, binding of t-PA·PAI-1 and u-PA·PAI-1
complexes to cluster II could be detected, but not to cluster IV.
Moreover, in contrast to our findings, no binding of Although we observe in this study that t-PA·PAI-1 complexes can bind
both to the separate cluster II and IV, the interactions of these
complexes with LRP could be most accurately described by a single-site
model. Consequently, a KD value for LRP is
derived that corresponds to a higher affinity than those of the
separate clusters II and IV. In the case of RAP, the
KD values for the ligand interactions with the
fragments are not in accordance with those for the intact LRP molecule.
This may be explained by the fact that the data for RAP binding to LRP
were fitted according to a two-site model, whereas we observed that RAP
binds, next to cluster II and IV, also weakly to cluster III,
suggesting a three-site model. Furthermore, according to the RAP
inhibition model in Fig. 4, the kinetics
of RAP binding to LRP is even more complex than a three-site model.
Nevertheless, the interactions can be faithfully fitted by a two-site
model, apparently since the algorithm employed contains sufficient
parameters to describe these interactions.
To further delineate the ligand-binding sites on cluster II, we mapped
the binding of ligands to subdomains of cluster II. Clearly, each of
the ligands tested binds to the same region of cluster II, spanning
epidermal growth factor repeat E4 and LDLRA domains C3-C7. RAP binds
to a fragment comprising the LDLRA domains C5-C7 and the anti-LRP
monoclonal Fab fragment A8 to the domains C8-C10 (9). Recently, Vash
et al. (10) reported on the binding of RAP, lactoferrin, and
PAI-1 to soluble LRP subfragments of cluster II. These investigators
showed binding of these ligands to LDLRA repeats C5-C7 and concluded
that the lactoferrin-binding site extends beyond C5-C7. With respect
to RAP, our results agree well with those of Vash et al.,
since in both studies C5-C7 has been identified as the binding site.
However, we did not observe PAI-1 binding to C5-C7 alone, but
concluded that the PAI-1-binding site only partially overlaps the
RAP-binding site on cluster II and extends toward the N terminus.
Possible reasons for this discrepancy might be due to differences in
immobilization, tag position, and the degree of glycosylation. It is of
note to mention that Orlando et al. (37) showed that the
multi-ligand endocytic receptor megalin (gp330), which is closely
related to LRP, also contains a binding site for apoE- Two models have been proposed for the inhibition of ligand binding to
LRP by RAP. The first model assumes a close spatial association of a
RAP-binding site to each of the independent LRP ligand-binding sites
(38). Consequently, RAP would inhibit ligand binding by direct
competition or steric hindrance of each of the LRP ligands. This
concept is supported by findings reported here, notably that the
RAP-binding site on cluster II is adjacent to and partially overlaps
with an important ligand binding domain. On the other hand, we have
shown that RAP and the anti-LRP Fab A8 each have their own distinct
binding site on cluster II, consistent with the observation that
cross-competition on the isolated cluster II domain could not be shown
for these molecules (9). However, RAP efficiently competes for binding
of Fab A8 to intact LRP, whereas the reverse could not be demonstrated
(9), despite the fact that the affinity of RAP and A8 for LRP is
similar. Collectively, these observations render the option unlikely
that RAP regulates ligand binding solely by a mechanism that involves
mutually exclusive binding to overlapping binding sites. Instead, these
findings would favor a RAP inhibition model that requires LRP moieties that are located outside the cluster II of the receptor. Indeed, an
alternative model for the regulation of ligand binding by RAP proposes
a RAP-induced conformational change in the LRP molecule (9, 18, 39). It
was demonstrated that both LRP and RAP harbor multiple interaction
sites for their mutual interaction (7, 8, 13). Based on analysis of the
primary structure, it was revealed that RAP contains an internal
triplication of structural autonomous domains comprising residues
1-100 (D1), 101-200 (D2), and 201-323 (D3) (13, 40). When expressed
individually, each of the single domains of human RAP maintains its
functional integrity and binds to LRP (13, 40). D1 binds to cluster II, D2 to cluster IV, and D3 to all three RAP binding cluster domains (II,
III, and IV) (13). Based on these reported multiple binding sites on
RAP and LRP, one may envision a model in which one molecule of RAP can
induce a conformational change in the receptor by interacting simultaneously with multiple receptor domains (Fig. 4B).
This conformational change would render the receptor incapable of
ligand binding. We show in this study that a single RAP protein can
simultaneously bind to clusters II and IV with comparable affinities,
an observation that is in accordance with the latter model. It should
be noted, however, that these experiments were performed with separate
cluster fragments and do not strictly prove that these events occur
within an intact LRP molecule. Superficially, the adaptation of a
different shape of LRP has been suggested by electron microscopic
experiments, indicating that LRP may display considerable structural
variability (41). In aggregate, our data support aspects of both models and therefore it cannot be excluded that RAP inhibition of ligand binding involves both competition or sterical hindrance and a conformational change.
In conclusion, we have shown that LRP contains ligand-binding sites on
cluster II and IV that are very similar with regard to ligand-binding
properties, suggesting a functional duplication within the receptor.
Finally, we have provided data on the mechanism of the complex
interactions between ligands and the intracellular chaperone RAP and
their receptor LRP.
*
This work was supported by Grant 902-26-175 from the
Netherlands Organization for Scientific Research, by Swedish Medical Research Council Grant 31X-12 203-03A, and by Concerted Action BIOMED
Program Contract BMH4-98-3324 from the European Commission.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. Tel.: 31-20-5665344;
Fax: 31-20-6915519; E-mail: j.g.neels@amc.uva.nl.
The abbreviations used are:
LRP, low density
lipoprotein receptor-related protein;
C, complement-type repeat;
PAI-1, plasminogen activator inhibitor type-1;
RAP, receptor-associated
protein;
SPR, surface plasmon resonance;
t-PA, tissue-type plasminogen
activator;
HBS, HEPES-buffered saline;
HBST, HBS plus Tween 20;
The Second and Fourth Cluster of Class A Cysteine-rich
Repeats of the Low Density Lipoprotein Receptor-related Protein
Share Ligand-binding Properties*
§,,, and
Department of Biochemistry, Academic Medical
Center, University of Amsterdam, Meibergdreef 15, 1105 AZ
Amsterdam, The Netherlands and the ¶ Department of Medical
Biochemistry and Biophysics, Umeå University,
S-901 87 Umeå, Sweden
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin, pro-urokinase-type plasminogen
activator, tissue-type plasminogen activator (t-PA), plasminogen
activator inhibitor-1, t-PA·plasminogen activator inhibitor-1
complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway
inhibitor, lactoferrin, the light chain of blood coagulation factor
VIII, and the intracellular chaperone receptor-associated protein
(RAP). No binding of the cluster I fragment to any of the tested
ligands was observed. The cluster III fragment only bound to the
anti-LRP monoclonal antibody 2MR3 and weakly to RAP.
Except for t-PA, we found that each of the ligands tested binds both to
cluster II and to cluster IV. The affinity rate constants of ligand
binding to clusters II and IV and to LRP were measured, showing that
clusters II and IV display only minor differences in ligand-binding
kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of
cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we
show that one RAP molecule can bind to different clusters
simultaneously, supporting a model in which RAP binding to LRP induces
a conformational change in the receptor that is incompatible with
ligand binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin-light chain, complexes
between urokinase-type plasminogen activator (u-PA) and its inhibitor
plasminogen activator inhibitor-1 (PAI-1), lactoferrin, an anti-LRP Fab
fragment (denoted Fab A8), and RAP bind to cluster II. In addition,
complexes between tissue-type plasminogen activator (t-PA) and PAI-1
were shown to bind to cluster II, while no binding of t-PA·PAI-1
complex to cluster IV could be detected (7, 9). The present study was
performed to systematically examine the binding of a large number of
structurally and functionally distinct ligands to each of the clusters
I to IV to obtain more insight into the molecular elements that
contribute to the remarkable ligand binding capacity of LRP. We
demonstrate that, although there are small differences concerning the
kinetics of the interactions, clusters II and IV are highly similar in
their ligand-binding properties, revealing a major functional
duplication in the receptor.
2-macroglobulin (2M), and very low
density lipoprotein, do not block the binding of RAP to LRP (23). It is
as yet unclear how RAP is capable of antagonizing all ligand binding to
LRP. In a previous study we have shown that, even though the affinity
of RAP for cluster II and LRP is similar, RAP is a poor inhibitor of
ligand binding to the isolated cluster II compared with its inhibition
of ligand binding to the intact LRP (9). This suggests that RAP does not inhibit ligand binding solely in a competitive or sterical manner.
The current study provides further evidence for a model of inhibition
of ligand binding by RAP, involving a RAP-induced conformational change
in the LRP molecule.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2MR3 were kindly provided by Dr. S. K. Moestrup (Institute of Medical Biochemistry, University of Aarhus,
Aarhus, Denmark). Two-chain t-PA was from Biopool (Umeå, Sweden).
Procedures to purify active PAI-1 were essentially as described (25,
26). t-PA·PAI-1 complexes were prepared as described (9). Human
tissue factor pathway inhibitor was a generous gift from Dr. L. Aarden
(Central Laboratory of Blood Transfusion, Amsterdam, The Netherlands).
Human apoE, human lactoferrin and recombinant glutathione
S-transferase-fused RAP (GST-RAP) were kindly provided by
Dr. J. Kuiper (Sylvius Laboratory, University of Leiden, Leiden, The
Netherlands). Since the GST tag does not interfere with binding
properties of RAP (27), GST-RAP was used throughout the present study
and is referred to as RAP. Isolation of the monoclonal Fab fragment Fab
A8 as well as the isolation of the monoclonal antibody CLB-CAg 69, directed against a peptide derived from the human coagulation factor
VIII (fVIII), were essentially as described (28, 29). Mouse pro-u-PA was a gift from Dr. J. Henkin (Abbot Laboratories, Abbot Park, IL). Bovine LpL was purified from milk and biotinylated as described (30, 31). fVIII light chain was kindly provided by Dr. P. Lenting
(Central Laboratory of Blood Transfusion, Amsterdam, The Netherlands).
Native human 2-macroglobulin was a kind gift from Dr. W. Boers (Academic Medical Center, Amsterdam, The Netherlands) and was
activated by incubating with 200 mM methylamine in 50 mM Tris-HCl (pH 7.8), 220 mM NaCl for 4 h
at room temperature. Unreacted methylamine was removed by dialysis at
4 °C against 20 mM HEPES (pH 7.4), 150 mM
NaCl, 2 mM CaCl2, 0.005% (v/v) Tween 80.
35 to 1) (32), followed by a 16-amino acid
"tag" (KKEDFDIYDEDENQSP) that contains the antigenic determinant of
an anti-fVIII monoclonal antibody CLB-CAg 69 (33), which is followed by
the cluster II coding sequence. pZEM229R-II DNA was partially digested
with XhoI, blunt-ended using T4 DNA polymerase, and ligated
to remove the original XhoI site of pZEM229R (34). The
resulting vector, containing only one XhoI site between the tag and cluster II coding sequence, was digested with XhoI
and BamHI to remove the cluster II sequence. Subsequently,
the (partially overlapping) phosphorylated oligonucleotides
adZEM1 (5'-TCGAGACGTACGACTAGTTAGTGAG-3') and adZEM2
(5'-GATCCTCACTAACTAGTCGTACGTC-3') were annealed and ligated into the
XhoI/BamHI-digested pZEM229R-II vector to
introduce a SpeI site and translation termination codons.
The resulting plasmid is referred to as pZEN. DNA fragments, encoding
cluster I (amino acids 6-91 (numbering according to Herz et
al. (35))), cluster III (amino acids 2503-2922), and cluster IV
(amino acids 3313-3759), were obtained by polymerase chain reaction
using a plasmid DNA, containing the full-length human LRP cDNA (a
gift from Dr. J. Herz, University of Texas Southwestern Medical
Center, Dallas, tx) as a template and primers LRPIF
(5'-GGACTCGAGAAGACTTGCAGCCCCAAGC-3') and LRPIR
(5'-AACTAGTCTCTCGGCAGTGGGGCCCCT-3'); LRPIIIF
(5'-GGACTCGAGTCCTCTTGCCGAGCACAA-3') and LRP3RB
(5'-AACTAGTGATGTGGCAGCCACGCTCG-3'); and LRPIVF
(5'-GGACTGGAGTCCAACTGCACGGCTAGC-3') and LRPIVR
(5'-AACTAGTGATGCTGCAGTCCTCCTC-3'), respectively. Polymerase chain
reaction products were digested with XhoI and
SpeI and, subsequently, ligated into the
XhoI/SpeI-digested plasmid pZEN. All constructs
were verified by DNA sequence analysis, using an ALF DNA sequencer
(Amersham Pharmacia Biotech).
2MR3 were bound to biotinylated rat
anti-mouse kappa light chain monoclonal antibody (CLB Products,
Amsterdam, The Netherlands), which was bound to a SA5 sensor chip.
Measurements were performed at 25 °C at a flow rate of 5 µl/min.
Concentrations of the different recombinant receptor fragments, which
were passed over the immobilized ligands, varied from nanomolar to
micromolar range.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
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Fig. 1.
Recombinant receptor fragments used in the
ligand mapping studies. Schematic overview of the domain structure
of LRP, containing clusters I, II, III, and IV. Enlargements of the
four clusters have been drawn separately. The symbols for the various
subdomains are indicated in a separate inset (top
right) as are the previously described (9) cluster II
subfragments (top left). The presence of the tag
sequence that facilitates detection and purification is indicated as
well as the epidermal growth factor repeat (E) and the LDLRA
repeats (C) within certain fragments.
View larger version (84K):
[in a new window]
Fig. 2.
Analysis of purified recombinant receptor
fragments. Silver staining of a nonreducing 12.5% (w/v)
SDS-polyacrylamide gel. Lane 1, cluster I;
lane 2, cluster II; lane 3,
cluster III; lane 4, cluster IV. The sizes of
molecular mass markers are indicated on the left
side.
2M, RAP, tissue factor pathway inhibitor, apoE,
lactoferrin, LpL, and fVIII light chain. In addition, we assayed the
binding of an anti-LRP Fab fragment and an anti-LRP monoclonal
antibody, denoted A8 and 2MR3, respectively. Each
ligand was immobilized at high density on a sensor chip, and receptor
fragments were passed over the sensor chip surface at concentrations
varying from nanomolar to micromolar range (Table
I). Clearly, we did not observe binding
of cluster I to any of the tested ligands. However, when the monoclonal
antibody CLB-CAg 69 was immobilized on a sensor chip, which is directed against the tag present in the recombinant cluster I fragment, binding
of cluster I was observed when the fragment was passed over the
antibody (not shown). This indicates that, although the cluster I
fragment is recognized by the tag-binding antibody, it does not contain
ligand-binding properties. Cluster III binds to the anti-LRP monoclonal
antibody 2MR3 and to RAP, provided the latter was
immobilized in very high density, conditions that are indicative for a
weak interaction. Surprisingly, cluster IV binds to an identical
repertoire of ligands as cluster II, except for the Fab fragment A8,
which is apparently specifically raised against cluster II.
Collectively, these observations strongly indicate that LRP consists of
duplicated domains, of which cluster II and IV have fully retained
their functional properties.
Ligand binding to cluster fragments of LRP
indicates no binding of
fragment could be detected. The abbreviation l.c. stands for light
chain. The anti-LRP Fab fragment and the anti-LRP monoclonal antibody
tested represent Fab A8 and monoclonal antibody 2MR3,
respectively. TFPI, tissue factor pathway inhibitor.
Kinetics of ligand binding to LRP, cluster II, and cluster IV
1s1, and koff
values are in s1. Data represent the means ± S.E. of
six experiments. The term ND means that no accurate rate constants
could be determined due to a high degree of nonspecific binding of LpL
to the sensorchip.
2M, pro-u-PA, LpL, and factor VIII light
chain to recombinant subfragments of cluster II (see Fig. 1) by SPR.
The data in Table III shows that only
binding of fragment CL-II-1/2 to the tested ligands could be detected,
indicating that these ligands bind to a fragment of cluster II spanning
the amino-terminal flanking epidermal growth factor repeat E4 and LDLRA
domains C3-C7. The same fragment has been reported to bind PAI-1 and
t-PA·PAI-1 complexes and contains a RAP-binding site on C5-C7 (9).
Only the anti-LRP monoclonal Fab A8 was shown to bind to a part of
cluster II (C8-C10) that is distinct from the RAP-binding site (9). In
conclusion, each of the tested ligands bind to a region on cluster II
that partially overlaps with a RAP-binding site, except for monoclonal
A8.
Mapping of ligand binding to subfragments of cluster II
indicates no binding of fragment could
be detected.
View larger version (17K):
[in a new window]
Fig. 3.
Cluster binding to RAP bound to LRP, cluster
II, or cluster IV. A, increasing concentrations of
cluster II were bound to RAP which, on its turn, was bound to LRP ( 
)
or to cluster II (
) or to cluster IV (
). Controls of cluster II
binding to RAP bound to BSA (
) and direct cluster II binding to BSA
(
), LRP (
), cluster II (
), or cluster IV (
), in the absence
of RAP, are also shown. B, same as in A but
instead of increasing cluster II concentrations, increasing cluster IV
concentrations were used. Experiments were performed in
duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2M
to either one of the minireceptors could be detected. These discrepancies might be due to a different composition of the constructs employed or to a different experimental design to measure ligand binding. Furthermore, it is relevant to mention that we do not observe
binding of t-PA to any of the LRP cluster fragments, despite the fact
that t-PA binds with a relatively low affinity to intact LRP. Since the
separate cluster fragments were designed to maintain the integrity of
the LDLRA repeats, and not that of other subdomains, we tentatively
conclude that the LDLRA repeats do not contribute to the t-PA-binding
site of LRP.
View larger version (15K):
[in a new window]
Fig. 4.
Proposed model for inhibition of Fab A8
binding to LRP by RAP. A, Fab A8 and RAP can bind to
the cluster II fragment of LRP without competing for each other.
B, in the context of the intact LRP molecule, Fab A8 binding
to LRP is inhibited by RAP due to a conformational change in LRP
induced by RAP. This model may not only apply to the inhibition of Fab
A8 binding to LRP by RAP, but may also explain the apparent
non-competitive antagonistic effects of RAP on ligand binding to LRP in
general.
-migrating
very low density lipoprotein, LpL, aprotinin, lactoferrin, and RAP
spanning the fourth and fifth LDLRA repeats of cluster II of megalin.
These repeats are homologous to the C6 and C7 subdomains within LRP. In
addition, and similar to LRP, megalin appears to have more than one
RAP-binding site. From the current and above mentioned studies, it can
be deduced that a limited number of LDLRA repeats is apparently
sufficient to provide specific binding of a diverse array of ligands to
LRP and to other members of the LDL receptor family.
FOOTNOTES
ABBREVIATIONS
2M, 2-macroglobulin;
GST, glutathione
S-transferase;
LpL, lipoprotein lipase;
u-PA, urokinase-type
plasminogen activator;
apoE, apolipoprotein E;
LDL, low density
lipoprotein;
LDLRA, LDL receptor class A;
BSA, bovine serum albumin;
fVIII, factor VIII;
FPLC, fast performance liquid chromatography.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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