From the Department of Plasma Proteins, Sanquin
Research at CLB, 1066 CX Amsterdam, The Netherlands and the
¶ Department of Pharmaceutics, Utrecht Institute for
Pharmaceutical Sciences, Utrecht University,
3584 CA Utrecht, The Netherlands
Received for publication, November 16, 2002
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ABSTRACT |
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Low-density lipoprotein receptor-related protein
(LRP) is an endocytic receptor that binds multiple distinct ligands,
including blood coagulation factor VIII (FVIII). FVIII is a
heterodimeric multidomain protein that consists of a heavy chain
(domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1,
and C2). Both chains contribute to high-affinity interaction with LRP.
One LRP-interactive region has previously been located in the C2
domain, but its affinity is low in comparison with that of the entire
FVIII light chain. We now have compared a variety of FVIII light chain
derivatives with the light chain of its homolog FVa for LRP binding. In
surface plasmon resonance studies employing LRP cluster II, the FVa and
FVIII light chains proved different in that only FVIII displayed
high-affinity binding. Because the FVIII a3-A3-C1 fragment was
effective in associating with LRP, this region was explored for
structural elements that are exposed but not conserved in FV.
Competition studies using synthetic peptides suggested that LRP binding
involves the FVIII-specific region Lys1804-Ala1834 in the A3 domain. In
line with this observation, LRP binding was inhibited by a recombinant
antibody fragment that specifically binds to the FVIII sequence
Glu1811-Lys1818. The role of this sequence in
LRP binding was further tested using a FVIII/FV chimera in which
sequence Glu1811-Lys1818 was replaced with the
corresponding sequence of FV. Although this chimera still displayed
residual binding to LRP cluster II, its affinity was reduced. This
suggests that multiple sites in FVIII contribute to high-affinity LRP
binding, one of which is the FVIII A3 domain region
Glu1811-Lys1818. This suggests that LRP
binding to the FVIII A3 domain involves the same structural elements
that also contribute to the assembly of FVIII with FIXa.
Coagulation factor VIII
(FVIII)1 serves its role in
the intrinsic coagulation pathway as a cofactor for factor IXa (FIXa)
in the proteolytic activation of factor X (for reviews, see Refs. 1 and
2). Functional absence of FVIII is associated with the bleeding
disorder hemophilia A. The cofactor is a 300-kDa glycoprotein that
comprises a discrete domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2) (2,
3). The A and C domains share 30-40% homology with the A and C
domains of the structurally related protein factor V (FV), whereas the
B domain and the short acidic regions a1, a2, and a3 are unique to
FVIII (4).
In plasma, FVIII circulates as a metal ion-linked heterodimer
consisting of a 90-220-kDa heavy chain (A1-a1-A2-a2-B) and an 80-kDa
light chain (a3-A3-C1-C2) (5, 6). The inactive protein is tightly
associated with its carrier protein, von Willebrand factor (7). Limited
proteolysis by either thrombin or factor Xa (FXa) converts the FVIII
precursor into its activated derivative (8, 9). The B domain and the
acidic region that borders the A3 domain are then removed from the
molecule (10), which leads to the loss of high-affinity binding to von
Willebrand factor (7). The resulting FVIIIa molecule consists of a
heterotrimer comprising the A2-a2 domain that is noncovalently
associated with the metal ion-linked A1-a1/A3-C1-C2 moiety (10).
Within the heavy and light chains of FVIII, several regions have been
identified as FIXa-interactive sites (11-13). A2 domain residues
Arg484-Phe509,
Ser558-Gln565, and
Arg698-Asp712 contribute to binding of the
heavy chain to FIXa (11, 12, 14). Within the FVIII light chain, the A3
domain region Glu1811-Lys1818 has been
identified as a FIXa-interactive site (13). In addition, FVIII regions
Arg484-Phe509 and
Lys1804-Lys1818 have also been identified as
target epitopes for antibodies that may occur in hemophilia A patients.
Such antibodies inhibit FVIII activity by interfering with the complex
assembly of FVIIIa and FIXa (15-17).
Recently, it has been demonstrated that FVIII interacts with the
multifunctional endocytic receptor low-density lipoprotein receptor-related protein (LRP) (18, 19). It is suggested that this
receptor plays a role in the clearance of FVIII from the circulation
(19, 20). LRP is a member of the low-density lipoprotein receptor
family, which also includes the low-density lipoprotein receptor, the
very low-density lipoprotein receptor, apoE receptor-2, and megalin
(for reviews, see Refs. 21 and 22). It is expressed in a variety of
tissues, including liver, lung, placenta, and brain (23). The receptor
consists of an extracellular 515-kDa Within FVIII, both the heavy and light chains contain LRP-interactive
sites. Both the A2 domain region Arg484-Phe509
and a so far unidentified region within the light chain are involved in
the high-affinity interaction with LRP (18, 19). Previously, we showed
that an anti-C2 domain antibody inhibits high-affinity binding of the
FVIII light chain to LRP, suggesting a major role for the C2 domain in
the interaction (18). However, the isolated recombinant C2 domain
demonstrates low-affinity binding to LRP compared with the intact FVIII
light chain (18). In the present study, we investigated the apparently
paradoxical role of the C2 domain in the interaction of the FVIII light
chain with LRP. To this end, the interaction between the FVIII light
chain and LRP is addressed using purified recombinant FVIII light chain fragments, synthetic peptides, recombinant antibody fragments, and a
chimeric FVIII light chain variant. This approach allowed us to
identify the FVIII light chain region
Glu1811-Lys1818 as a sequence that
contributes to the interaction with LRP.
Materials--
CNBr-Sepharose 4B was from Amersham Biosciences
(Uppsala, Sweden). Microtiter plates (Maxisorp), cell culture flasks,
Opti-MEM I medium, penicillin, and streptomycin were from Invitrogen
(Breda, The Netherlands). Grace's insect medium, Insect-XPRESS
medium, and fetal calf serum were purchased from BioWhittaker (Alkmaar, The Netherlands).
Proteins--
Plasma-derived FVIII light chain and its
FXa-cleaved derivative were prepared as described previously (28, 29).
Anti-FVIII monoclonal antibodies CLB-CAgA, CLB-CAg117, and CLB-CAg12
have been described previously (28, 30). Single-chain variable domain
antibody fragments (scFv fragments) directed against the light chain of
FVIII were expressed in Escherichia coli strain TG1 and
purified by metal chelate chromatography (QIAGEN, Hilden, Germany) as
described previously (31, 32), with the exception that scFv fragments
KM36 and KM41 were eluted in 150 mM NaCl, 100 mM imidazole, and 20 mM Hepes (pH 7.4). The
anti-FVa light chain monoclonal antibody CLB-FV5 was obtained by
standard hybridoma techniques and will be described in detail
elsewhere.2 Synthetic
peptides encompassing human FVIII regions
Trp1707-Arg1721 (WDYGMSSSPHVLRNR),
Lys1804-Lys1818 (KNFVKPNETKTYFWK),
Tyr1815-Ala1834 (YFWKVQHHMAPTKDEFDCKA),
His1822-Ala1834 (HMAPTKDEFDCKA),
Thr1892-Ala1901 (TENMERNCRA),
Glu1908-His1919 (EDPTFKENYRFH),
Thr1964-Lys1972 (TVRKKEEYK),
Lys2049-Gly2057 (KLARLHYSG), and
Asp2108-Gly2117 (DGKKWQTYRG) were synthesized
by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry following
the manual "T-bag" method (33) or employing an Applied Biosystems
Model 430A instrument (Amersham Biosciences, Roosendaal, The
Netherlands; Medprobe AS, Oslo, Norway). Peptides were >95% pure as
determined by high-pressure liquid chromatography analysis, and their
identity was confirmed by mass spectrometry. Purified placenta-derived
LRP (34) was a generous gift from Dr. S. K. Moestrup (University
of Aarhus, Aarhus, Denmark). The bacterial vector encoding glutathione
S-transferase-fused receptor-associated protein was kindly
provided by Dr. J. Kuiper (Leiden University, Leiden, The Netherlands).
Glutathione S-transferase-fused receptor-associated protein
was expressed in E. coli strain DH5 Recombinant Proteins--
The plasmid pCLB-BPVdB695, encoding
the FVIII B domain deletion variant FVIII- Purification of the FVa Light Chain--
Human FV was obtained
from human plasma provided by our institute (Sanquin Plasma Products).
Full-length FV was purified by immunoaffinity
chromatography.2 The FVa light chain was prepared by
incubating FV (10 µM) with thrombin (2 µM)
in buffer containing 100 mM NaCl, 5 mM
CaCl2, 5% (v/v) glycerol, and 50 mM Tris (pH
7.4) for 2 h at 37 °C. Thrombin was inactivated by hirudin
(Sigma), and the FVa light chain was purified by immunoaffinity
chromatography on CNBr-Sepharose 4B coupled to the anti-FV light chain
monoclonal antibody CLB-FV5 (3 mg/ml). The immunoaffinity matrix was
washed with 100 mM NaCl, 50 mM EDTA, and 50 mM Tris (pH 7.4) and eluted with 100 mM NaCl, 5 mM CaCl2, 55% (v/v) ethylene glycol, and 50 mM Tris (pH 7.4). Purified FVa light chain was dialyzed
against 150 mM NaCl, 5 mM CaCl2,
50% (v/v) glycerol, and 50 mM Tris (pH 7.4) and stored at
Expression and Purification of Recombinant LRP
Fragments--
Recombinant LRP clusters II and IV were expressed in
baby hamster kidney cells using Opti-MEM I medium supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin (27). After harvesting of the medium, CaCl2 was added to a final concentration of
10 mM. Purification of LRP clusters II and IV from the
conditioned medium was performed by a single purification step using
glutathione S-transferase-fused receptor-associated protein
coupled to CNBr-Sepharose 4B as an affinity matrix. The matrix was
collected in a column; washed with buffer containing 150 mM
NaCl, 5 mM CaCl2, and 50 mM Hepes
(pH 7.4); and eluted with 150 mM NaCl, 20 mM
EDTA, and 50 mM Hepes (pH 7.4). Subsequently, purified LRP
cluster preparations were concentrated in Centricon 10 concentrators
(Millipore Corp., Bedford, MA) by successive rounds of centrifugation
at 4000 × g for 1 h at 4 °C. Finally, the
preparations were dialyzed against 150 mM NaCl, 2 mM CaCl2, and 20 mM Hepes (pH 7.4)
and stored at 4 °C.
Solid-phase Binding Assays--
Recombinant LRP cluster II or IV
(1 pmol/well) was adsorbed onto microtiter wells in 50 mM
NaHCO3 (pH 9.8) in a volume of 50 µl for 16 h at
4 °C. Wells were blocked with 2% (w/v) HSA, 150 mM
NaCl, 5 mM CaCl2, and 50 mM Tris
(pH 7.4) in a volume of 200 µl for 1 h at 37 °C.
Subsequently, the FVIII light chain was incubated at various
concentrations in a volume of 50 µl of buffer containing 150 mM NaCl, 5 mM CaCl2, 1% (w/v) HSA,
0.1% (v/v) Tween 20, and 50 mM Tris (pH 7.4) for 2 h
at 37 °C. After three rapid washes (<5 s each) with 150 mM NaCl, 5 mM CaCl2, 0.1% (v/v)
Tween 20, and 50 mM Tris (pH 7.4), bound ligand was
detected by incubation with peroxidase-conjugated monoclonal antibody
CLB-CAg12 in the same buffer for 15 min at 37 °C. During this latter
incubation period, one would expect that the FVIII light chain would
completely dissociate from the immobilized LRP clusters. However,
subsequent rebinding of the FVIII light chain to the LRP clusters
allows the formation of new FVIII·LRP cluster complexes, which can be detected by this sensitive method. In surface plasmon resonance (SPR),
this is prevented by a continuous buffer flow. Because of these
differences in experimental approach, the solid-phase binding assay is
compared only with SPR analysis in a qualitative manner. Antibody
CLB-CAg12 did not interfere with binding of FVIII fragments to LRP or
its clusters (data not shown). In competition experiments, the FVIII
light chain (25 nM) was incubated with wells containing
immobilized LRP clusters either in the presence or absence of serial
dilutions of competitor in a volume of 50 µl for 2 h at
37 °C. Residual FVIII binding was detected as described above. Data
were corrected for binding to empty microtiter wells, which was <5%
relative to binding to wells containing immobilized LRP clusters.
Surface Plasmon Resonance--
The kinetics of protein
interactions was determined by SPR analysis using a
BIAcoreTM 2000 biosensor system (BIAcore AB, Uppsala). LRP
(16 fmol/mm2), the FVIII light chain (71 fmol/mm2), the a3-A3-C1 fragment (67 fmol/mm2),
the FVa light chain (76 fmol/mm2), or scFv EL14 (67 fmol/mm2) was covalently coupled to the dextran surface of
an activated CM5 sensor chip via primary amino groups using the amine
coupling kit (BIAcore AB, Uppsala, Sweden) as recommended by the
supplier. One control flow channel was routinely activated and blocked
in the absence of protein. Association of analyte was assessed in 150 mM NaCl, 2 mM CaCl2, 0.005% (v/v)
Tween 20, and 20 mM Hepes (pH 7.4) for 2 min at a flow rate
of 20 µl/min at 25 °C. Dissociation was allowed for 2 min in the
same buffer flow. Sensor chips were regenerated by several pulses of
either 100 mM H3PO4 or 20 mM EDTA, 1 M NaCl, and 50 mM Hepes
(pH 7.4) at a flow rate of 20 µl/min. The association
(kon) and dissociation
(koff) rate constants were determined using
BIAevaluation Version 3.1 software (BIAcore AB). Data were corrected
for bulk refractive index changes and fitted by nonlinear regression
analysis according to a one- or two-site binding model. Equilibrium
dissociation constants (Kd) were calculated from the
ratio koff/kon. The
Kd value for low-affinity interactions was estimated
by steady-state affinity analysis using BIAevaluation software. In
competition experiments, the FVIII light chain (50 nM) was
incubated with immobilized LRP (16 fmol/mm2) either in the
presence or absence of serial dilutions of competitor for 2 min at a
flow rate of 20 µl/min at 25 °C.
Binding of the FVIII-(1811-1818)/FV Light Chain to LRP Cluster
II--
The recombinant FVIII-(1811-1818)/FV or recombinant wild-type
FVIII light chain was coupled to immobilized scFv EL14 to a density of
20 fmol/mm2 in buffer containing 150 mM NaCl
and 50 mM Tris (pH 7.4). scFv KM36 (100 nM),
scFv KM41 (40 nM), or LRP cluster II (25-125
nM) was passed over separate channels with the immobilized
FVIII-(1811-1818)/FV or recombinant wild-type FVIII light chain,
respectively, and one control channel (scFv EL14-coated) in 150 mM NaCl, 2 mM CaCl2, 0.005% (v/v)
Tween 20, and 20 mM Hepes (pH 7.4) for 2 min at a flow rate
of 20 µl/min at 25 °C. scFv EL14 was previously isolated from a
hemophilia A patient with inhibitory antibodies against FVIII (32). It
is directed against the FVIII C2 domain and competes with monoclonal
antibody CLB-CAg117 for FVIII binding (32, 40). scFv EL14 inhibits
neither FVIII procoagulant activity nor FVIII light chain binding to
LRP. This is in agreement with the observation that the affinity of LRP
cluster II for the directly immobilized FVIII light chain
(Kd = 56 ± 12 nM) or the scFv
EL14-captured FVIII light chain (Kd = 58 ± 7 nM) was similar upon SPR analysis.
Interaction between LRP and FVIII Light Chain
Fragments--
We have previously shown that the isolated
FVIII C2 domain (i.e.
Ser2173-Tyr2332) associates with LRP less
effectively than the intact FVIII light chain (18). In this study, we
explored the possibility that additional sites in the FVIII light chain
contribute to LRP binding. To this end, we monitored the interaction of
four FVIII derivatives with immobilized LRP by SPR analysis. These
derivatives included the FVIII light chain, the a3-A3-C1 moiety
(i.e. Glu1649-Asn2172), the
C-terminal C2 domain, and a FVIII light chain fragment with the
N-terminal acidic region deleted by cleavage at Arg1721 by
FXa (i.e. Ala1722-Tyr2332).
As shown in Fig. 1, all FVIII fragments
displayed time-dependent association with immobilized LRP,
followed by dissociation, which appeared to be
dose-dependent, as the highest response was observed at the
highest LRP density (data not shown). The data showed complex binding
behavior in which multiple components were involved. This is the result
of not only the inherent biological properties of both LRP
(i.e. clusters II and IV) (27) and FVIII, but also the
immobilization procedure of LRP. The latter may result in partial
blocking of FVIII interaction sites. Therefore, a heterogeneous two-site binding model was required to approximate the binding behavior
of the FVIII light chain, the FXa-cleaved light chain, and the a3-A3-C1
fragment with immobilized LRP. The calculated association
(kon) and dissociation
(koff) rate constants that followed from this
model were in the same order of magnitude for these fragments (Table
I). This resulted in comparable
Kd values describing a high-affinity and a slightly
lower affinity interaction with immobilized LRP, viz. 18 and
59 nM for the FVIII light chain, 22 and 60 nM
for the FXa-cleaved light chain, and 26 and 74 nM for the
a3-A3-C1 derivative (Table I). In contrast, no LRP binding could be
observed by SPR analysis employing C2 domain concentrations <250
nM, indicating that the affinity for this interaction is
low. This is in agreement with our previous study, in which we also
demonstrated inefficient binding of the isolated C2 domain to LRP (18).
Because of the low affinity, the Kd value for this
interaction was obtained by two calculation methods: from
kon and koff and from
steady-state affinity analysis using the response calculated at
equilibrium. The affinities that followed from this analysis were
similar, viz. 3.6 ± 1.7 µM (Table I) and
3.4 ± 0.2 µM, respectively. Collectively, these results show that there is a high-affinity LRP-binding site in the
A3-C1 region (i.e. Ala1722-Asn2172)
and a low-affinity site in the C2 domain.
Binding of the FVIII Light Chain to Immobilized LRP Clusters II and
IV--
A previous study showed that LRP ligand-binding clusters II
and IV mediate the interaction with the FVIII light chain (27). In the
present study, we used a solid-phase binding assay to address the
question of whether or not LRP clusters II and IV can compete for
binding to the FVIII light chain. As demonstrated in Fig. 2 (inset), the FVIII light
chain was able to bind immobilized LRP cluster II in a
dose-dependent manner. This observation is in agreement
with a previous study in which SPR analysis was used to monitor the
interaction between LRP cluster II and the immobilized FVIII light
chain (27). Competition studies revealed that LRP clusters II and IV
competed for binding to the FVIII light chain (Fig. 2). LRP cluster II
displayed a dose-dependent inhibition of FVIII light chain
binding to immobilized LRP cluster IV. These data imply that LRP
clusters II and IV share a similar binding region within the FVIII
light chain.
Binding of LRP Cluster II to the Immobilized FVa Light
Chain--
In view of the known homology between FVIII and FV (4), the
question may arise as to whether or not the light chains of FVIII and
FVa share LRP cluster II-binding properties. To this end, serial
dilutions of LRP cluster II were incubated with immobilized FVIII and
FVa light chains upon SPR analysis. As shown in Fig. 3, the light chains of FVa and FVIII
proved different in that only FVIII displayed high-affinity binding to
LRP cluster II. These observations indicate that the a3-A3-C1 domains
of the FVIII light chain contain a high-affinity LRP cluster
II-interactive region that is not conserved in the FVa light chain.
Effect of Synthetic Peptides on FVIII Light Chain Binding to LRP
Cluster II--
We constructed a panel of synthetic peptides that
mimic the surface loops of the a3-A3-C1 domains. The observation that
the FVa light chain does not efficiently associate with LRP cluster II
was used as a selection criterion for construction of synthetic peptides that are unique to FVIII. The solvent accessibility of these
loops was verified by hydropathy analysis (13) and by studying the
three-dimensional model of the intact FVIII heterodimer (41). The
synthetic peptides comprised Trp1707-Arg1721,
Lys1804-Lys1818,
Tyr1815-Ala1834,
His1822-Ala1834,
Thr1892-Ala1901,
Glu1908-His1919,
Thr1964-Lys1972,
Lys2049-Gly2057, and
Asp2108-Gly2117 (Table
II). Subsequently, these peptides were
tested for their ability to interfere with the interaction between the
FVIII light chain and immobilized LRP cluster II. As shown in Table II,
the Lys1804-Lys1818 and
Tyr1815-Ala1834 synthetic peptides efficiently
inhibited the interaction of the FVIII light chain and immobilized LRP
cluster II. Half-maximum inhibition (IC50) was reached at
peptide concentrations of ~1.9 and 16.8 µM,
respectively. The other synthetic peptides did not show such an
inhibitory effect. These observations suggest that sequence
Lys1804-Ala1834 within the A3 domain of FVIII
contains important residues involved in the interaction with LRP.
Effect of scFv Fragments on FVIII Light Chain Binding to LRP or LRP
Cluster II--
We previously employed phage display to isolate
recombinant scFv fragments from a patient with inhibitory antibodies
directed against residues within the A3 domain region
Gln1778-Asp1840 (31). These scFv fragments
were evaluated for their ability to interfere with the interaction
between the FVIII light chain and LRP or LRP cluster II. The first
scFv, referred to as scFv KM36, is directed against a region within
Gln1778-Asp1840, but does not require
Arg1803-Lys1818 for FVIII binding (31). The
second scFv, designated as scFv KM41, is directed against region
Arg1803-Lys1818 and inhibits FVIII
procoagulant activity (31). As shown in Fig.
4A, scFv KM36 did not affect
the interaction between the FVIII light chain and immobilized LRP
cluster II. Accordingly, scFv KM36 did not interfere with binding of
the FVIII light chain to full-length LRP upon SPR analysis (Fig.
4B, inset). scFv KM36 slightly increased the
response of FVIII light chain binding to LRP, which was due to a small
increase in mass as the result of FVIII light chain (80 kDa)·scFv
KM36 (30 kDa) complex formation. In contrast, the presence of scFv KM41
inhibited the binding of the FVIII light chain to LRP cluster II (Fig.
4A). The effect of scFv KM41 on the interaction between the
FVIII light chain and LRP was further studied by SPR analysis. As shown
in Fig. 4B, association of the FVIII light chain with
immobilized LRP was inhibited in the presence of scFv KM41. These data
suggest a role for the FVIII A3 domain region
Arg1803-Lys1818 in the interaction with
LRP.
The FVIII Light Chain Sequence
Glu1811-Lys1818 Contains a Binding Site for
LRP--
The Lys1804-Lys1818 and
Tyr1815-Ala1834 synthetic peptides and scFv
KM41 are effective inhibitors of FVIII procoagulant activity by
interfering with the assembly of the FVIIIa·FIXa complex (13, 31). As the FVIII A3 domain region Glu1811-Lys1818
contributes to the interaction with FIXa (13), this particular FVIII
light chain region was investigated with respect to its role in the
interaction with LRP. As the FVa light chain did not interact with LRP
cluster II, a FVIII chimera was constructed in which residues
Glu1811-Lys1818 were replaced with the
corresponding residues of FV (i.e.
1704SSYTYVWH1711). This chimera is referred to
as FVIII-(1811-1818)/FV. The purified chimera was previously evaluated
for its ability to mediate the FIXa-dependent activation of
factor X. At saturating concentrations of FIXa, replacement of
the FVIII region Glu1811-Lys1818 with the
corresponding residues of FV abolished >80% of FVIII activity (42).
This indicates that the FVIII A3 domain region Glu1811-Lys1818 is indeed indispensable for
FVIII cofactor function.
The light chain of the FVIII-(1811-1818)/FV chimera was isolated
and first tested for binding to scFv fragments KM36 and KM41 by SPR
analysis. As shown in Fig. 5A,
scFv KM36 displayed similar binding to both the FVIII-(1811-1818)/FV
and wild-type FVIII light chains, suggesting that the chimera does not
suffer from significant structural defects. In contrast, scFv KM41 did
not recognize the immobilized FVIII-(1811-1818)/FV light chain,
whereas it readily reacted with the immobilized recombinant wild-type
FVIII light chain (Fig. 5B). These observations indicate
that the FVIII A3 domain region
Glu1811-Lys1818 contains residues critical for
binding to scFv KM41.
To further address the role of the FVIII light chain region
Glu1811-Lys1818 in the interaction with LRP,
we studied the interaction between the FVIII-(1811-1818)/FV light
chain and LRP cluster II by SPR analysis. To this end, the chimera was
compared with the recombinant wild-type FVIII light chain in the
interaction with LRP cluster II in terms of affinity. Although some
residual binding could be observed, LRP cluster II displayed a reduced
binding signal to the immobilized FVIII-(1811-1818)/FV light chain
(Fig. 6B) compared with the
intact FVIII light chain (Fig. 6A). This suggests that a
substantial part of the LRP-binding site was lost as a result of the
mutagenesis. To investigate the importance of the A3 domain region
Glu1811-Lys1818 in LRP binding in more detail,
the kinetic parameters that describe both interactions were addressed.
LRP cluster II bound the wild-type FVIII light chain with an affinity
of 58 ± 7 nM (koff = (1.9 ± 0.2) × 10 In this study, we have demonstrated that the A3 domain region
Glu1811-Lys1818 of the FVIII light chain
contributes to the high-affinity interaction with LRP. Several lines of
evidence support this conclusion. First, the A3 domain-derived
synthetic peptides Lys1804-Lys1818 and
Tyr1815-Ala1834 affected the interaction
between the FVIII light chain and LRP cluster II (Table II). Second, a
recombinant scFv directed against region
Glu1811-Lys1818 inhibited binding of the FVIII
light chain to LRP or its cluster II fragment (Fig. 4). Third, a
chimeric FVIII light chain variant in which residues
Glu1811-Lys1818 were replaced with the
corresponding residues of FV displayed a reduction in affinity for LRP
cluster II compared with the wild-type FVIII light chain (Fig. 6).
To date, no clear consensus sequence that mediates the interaction with
LRP has been identified. However, for a number of LRP ligands,
including receptor-associated protein, lipoprotein lipase, and
To date, two amino acid regions within the FVIII light chain that
contribute to the assembly of the FVIII light chain·LRP complex have
been identified. Besides a role for the A3 domain region
Glu1811-Lys1818 found in this study, also the
C-terminal C2 domain is known to contribute to the interaction with LRP
(18). The LRP-interactive site in the A3 domain seems more predominant
than the one in the C2 domain, as the isolated C2 domain exhibited a
low-affinity interaction with LRP (Kd Besides the high-affinity LRP-interactive site within the FVIII light
chain, the A2 domain region Arg484-Phe509
within the FVIII heavy chain is also thought to contain residues critical for the interaction with LRP (19). Interestingly, an antibody
that is directed against region Arg484-Phe509
completely inhibits the interaction between intact FVIII and LRP (19).
This observation suggests a minor role of the light chain in the
interaction between the intact FVIII heterodimer and LRP. However,
similar to the inhibitory mechanism of ESH4, steric interference cannot
be excluded. Both LRP-interactive regions within the FVIII A2 and A3
domains are positioned at the same flank of the FVIII molecule, as
judged from the three-dimensional model of the intact FVIII heterodimer
(41). As such, both regions might cooperate in the high-affinity
interaction with LRP. Therefore, it seems conceivable that full-length
antibodies directed against this flank of the FVIII protein are able to
completely inhibit FVIII binding to LRP. Similar inhibitory mechanisms
have been reported for the assembly of the FVIIIa·FIXa complex
(15-17, 31). Further studies, preferably employing site-directed
mutagenesis, are required to establish the relative importance of both
FVIII subunits in the assembly of the FVIII·LRP complex.
The complementary FVIII-interactive regions on the LRP molecule remain
to be elucidated. It is well established that the ligand-binding regions within LRP are located within its clusters (27). The FVIII
light chain is known to be equally effective in its association with
recombinant LRP clusters II and IV, whereas no interaction was observed
using LRP cluster I or III (27). Moreover, both LRP clusters II and IV
were demonstrated to compete for binding to the FVIII light chain (Fig.
2). It is therefore unclear which of the two LRP clusters is involved
in the interaction with the FVIII light chain. However, as judged from
the three-dimensional model of the membrane-bound FVIII heterodimer,
region Glu1811-Lys1818 is positioned in close
proximity to the membrane surface (41). It is therefore conceivable
that LRP cluster IV, being located closest to the membrane surface, is
more important in the interaction with FVIII compared with LRP cluster
II. The preferential binding of the FVIII A2 domain to individual LRP
clusters remains unknown. This leaves the possibility that one or more
LRP clusters accommodate the binding to intact FVIII. The latter model
is compatible with the recent observation that two separate LRP
clusters cooperate to mediate high-affinity binding to
Intriguingly, both LRP-interactive sites within the FVIII A3 and A2
domains were previously identified as FIXa-interactive regions (13, 14,
19, 49). This suggests that LRP binding to these domains involves the
same structural elements that also contribute to the interaction
between FVIII and FIXa. Therefore, one interesting question is whether
LRP plays a regulatory role in the assembly of the FVIIIa·FIXa
complex (50). Such a modulating role for LRP is further suggested by
the observation that LRP also recognizes FIXa as a ligand (51). Whether
or not LRP is capable of binding both FVIIIa and FIXa simultaneously
and to what extent the FVIIIa·FIXa complex is dissociated by LRP
remain speculative and need further study.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain that is noncovalently
linked to a transmembrane 85-kDa
-chain (24). The
-chain contains
four clusters of a varying number of complement-type repeats that
mediate the binding of many structurally and functionally unrelated
ligands (25-27). The FVIII light chain has been demonstrated to
interact with recombinant LRP clusters II and IV, whereas no binding
was observed to LRP clusters I and III (27).
EXPERIMENTAL PROCEDURES
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DISCUSSION
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and purified on
glutathione-Sepharose as described (35). Baby hamster kidney cells
expressing recombinant LRP ligand-binding clusters II and IV have been
described previously (27) and were kindly provided by Dr. H. Pannekoek
(Academic Medical Center, University of Amsterdam, Amsterdam, The
Netherlands). Human serum albumin (HSA) was from the Division of
Products of CLB. Protein was quantified by the method of
Bradford (36) using HSA as a standard.
(868-1562), has been
described previously (37) and was used as a template to construct the
plasmid coding for the FVIII-(1811-1818)/FV chimera.
Oligonucleotide primers derived from the FVIII light chain sequence
containing the FVIII/FV codon replacements (see Table II) were employed
to construct the plasmids using the overlap extension PCR mutagenesis
method (38). Sequence analysis was performed to verify the presence of
the mutations in the plasmid. Transfection of FVIII-encoding plasmids
into murine fibroblasts (C127) cells was performed as described
previously (37). Stable cell lines expressing wild-type FVIII or the
FVIII-(1811-1818)/FV chimera were maintained in cell factories in RPMI
1640 medium supplemented with 5% fetal calf serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, 1 µg/ml amphotericin B, and 0.8 µg/ml deoxycholate. FVIII-containing medium was harvested three
times/week. The medium was subsequently filtered to remove cell debris
and concentrated ~10-fold employing a hollow fiber cartridge
(Hemoflow F5, Fresenius, Bad Homburg, Germany). Benzamidine was added
to a final concentration of 10 mM, and concentrates were
stored at
20 °C. FVIII was purified from the concentrated medium
by immunoaffinity chromatography employing antibody CLB-CAg117 and
Q-Sepharose chromatography according to an established procedure (37).
FVIII light chains were prepared by incubating the purified
FVIII-(1811-1818)/FV chimera and wild-type FVIII in buffer containing
40 mM EDTA, 100 mM NaCl, and 50 mM
Tris (pH 7.4) for 4 h at 25 °C. Subsequently, the
FVIII-(1811-1818)/FV and wild-type FVIII light chains were purified by
Q-Sepharose chromatography. Recombinant proteins were eluted in buffer
containing 1 M NaCl and 50 mM Tris (pH 7.4), dialyzed against 150 mM NaCl and 50 mM Tris (pH
7.4), and stored at 4 °C. The construction of the plasmid encoding
the recombinant C2 domain (i.e.
Ser2173-Tyr2332) has been described previously
(16). The plasmid pACgp67b-His-a3-A3-C1, encoding the FVIII a3-A3-C1
fragment (i.e. Glu1649-Asn2172),
was constructed by PCR employing the oligonucleotide primers 5'-TTACTCGAGGAAATAACTCGTACTACTC-3' (sense) and
5'-AATGCGGCCGCTTCAATTTAAATCACAGCCCAT-3' (antisense) using pCLB-BPVdB695
as a template (37). The amplified DNA fragment was purified, digested
with XhoI and NotI, and ligated into pBluescript.
The resulting construct was verified by sequencing. Subsequently,
pBluescript-a3-A3-C1 was digested with EspI and NotI, and the obtained fragment was purified and ligated
into the EspI/NotI-digested pACgp67b-80K plasmid
(39). A DNA fragment encoding a polyhistidine tag
(5'-ATTGGATCCGGCCATCATCATCATCATCATGGCGGCAGCCCCCGCAGCTTTCAAAAGCCCGGGGCCATGGGA-3') was digested with BamHI and NcoI and cloned into
the BamHI/NcoI-digested pACgp67b-a3-A3-C1
plasmid. Using the baculovirus expression system, recombinant a3-A3-C1
and C2 fragments were obtained by infection of insect cells as
described (16). The a3-A3-C1 fragment was purified from Insect-XPRESS
medium by immunoaffinity chromatography using the anti-A3 domain
antibody CLB-CAgA coupled to CNBr-Sepharose 4B as an affinity matrix.
CLB-CAgA-Sepharose was incubated with medium containing the a3-A3-C1
fragment for 16 h at 4 °C. After binding, the immunoaffinity
matrix was collected; washed with buffer containing 1 M
NaCl and 50 mM Tris (pH 7.4); and eluted with 150 mM NaCl, 55% (v/v) ethylene glycol, and 50 mM
lysine (pH 11). Elution fractions were immediately neutralized with 1 M imidazole (pH 6); dialyzed against 150 mM
NaCl, 50% (v/v) glycerol, and 50 mM Tris (pH 7.4); and
stored at
20 °C. The recombinant C2 domain was purified employing
the same immunoaffinity chromatography technique, except that the
anti-C2 domain antibody CLB-CAg117 was used instead of CLB-CAgA.
20 °C.
RESULTS
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View larger version (11K):
[in a new window]
Fig. 1.
Binding of FVIII light chain fragments to
immobilized LRP. LRP immobilized on a CM5 sensor chip at 16 fmol/mm2 was incubated with the FVIII light chain (150 nM; solid line) and the FXa-cleaved light chain
(150 nM; dashed line) (A) and with
the a3-A3-C1 fragment (150 nM; solid line) and
the isolated C2 domain (750 nM; dashed line)
(B). Incubations were performed in 150 mM NaCl,
2 mM CaCl2, 0.005% (v/v) Tween 20, and 20 mM Hepes (pH 7.4) at a flow rate of 20 µl/min for 2 min
at 25 °C. Dissociation was initiated upon replacement of the ligand
solution with buffer. Response is indicated as resonance units
(RU) and is corrected for nonspecific binding, which was
<5% relative to LRP-coated channels.
Kinetic parameters for binding of the FVIII light chain and its
derivatives to immobilized LRP
View larger version (21K):
[in a new window]
Fig. 2.
Binding of the FVIII light chain to
recombinant LRP fragments. The FVIII light chain (LCh;
25 nM) was incubated with immobilized LRP cluster IV (1 pmol/well) in a volume of 50 µl of 150 mM NaCl, 5 mM CaCl2, 1% (w/v) HSA, 0.1% Tween 20, and 50 mM Tris (pH 7.4) in the presence or absence of various
concentrations of recombinant LRP cluster II (0-600 nM)
for 2 h at 37 °C. After washing with the same buffer, bound
FVIII light chain was quantified by incubation with
peroxidase-conjugated anti-FVIII antibody CLB-CAg12 for 15 min at
37 °C. Residual binding is expressed as the percentage of binding in
the absence of competitor and is corrected for nonspecific binding
(<5% relative to binding to LRP cluster IV-immobilized wells).
Inset, serial dilutions of the FVIII light chain were
incubated with immobilized LRP cluster II (1 pmol/well) in a volume of
50 µl of 150 mM NaCl, 5 mM CaCl2,
1% (w/v) HSA, 0.1% Tween 20, and 50 mM Tris (pH 7.4) for
2 h at 37 °C. After washing with the same buffer, bound FVIII
light chain was quantified as described above. Data represent the
means ± S.D. of three experiments.
View larger version (13K):
[in a new window]
Fig. 3.
Binding of LRP cluster II to the immobilized
FVa light chain. The FVIII light chain (71 fmol/mm2;
curve I) or the FVa light chain (76 fmol/mm2;
curve II) on a CM5 sensor chip were incubated with LRP
cluster II (100 nM) in 150 mM NaCl, 2 mM CaCl2, 0.005% (v/v) Tween 20, and 20 mM Hepes (pH 7.4) at a flow rate of 20 µl/min for 2 min
at 25 °C. Dissociation was initiated upon replacement of the ligand
solution with buffer. Response is indicated as resonance units
(RU) and is corrected for nonspecific binding, which was
<5% relative to coated channels.
Effect of FVIII a3-A3-C1 fragment-derived synthetic peptides on the
interaction between the FVIII light chain and LRP cluster II
View larger version (18K):
[in a new window]
Fig. 4.
Effect of scFv fragments on the interaction
between the FVIII light chain and LRP. A, the FVIII
light chain (LCh; 25 nM) was incubated with
immobilized LRP cluster II (1 pmol/well) in a volume of 50 µl of 150 mM NaCl, 5 mM CaCl2, 1% (w/v) HSA,
0.1% Tween 20, and 50 mM Tris (pH 7.4) in the presence of
various concentrations (0-100 nM) of scFv KM41 ( ) or
scFv KM36 (
) for 2 h at 37 °C. After washing with the same
buffer, bound FVIII light chain was quantified by incubation with
peroxidase-conjugated anti-FVIII antibody CLB-CAg12 for 15 min at
37 °C. Residual binding is expressed as the percentage of binding in
the absence of competitor and is corrected for nonspecific binding
(<5% relative to binding to LRP cluster II-immobilized wells). Data
represent the means ± S.D. of three experiments. B,
the FVIII light chain (50 nM) was incubated with
immobilized LRP (16 fmol/mm2) as described in the legend of
Fig. 1. Binding was assessed in the absence (curve 1) or
presence of increasing concentrations of scFv KM41 (20, 60, 300, and
500 nM (curves 2-5, respectively)). Complexes
were allowed to form for 30 min before SPR analysis. Inset,
the FVIII light chain (50 nM) was incubated with
immobilized LRP (16 fmol/mm2) as described above. Binding
was assessed in the absence (solid line) or presence
(dashed line) of scFv KM36 (500 nM).
RU, resonance units.
View larger version (13K):
[in a new window]
Fig. 5.
Binding of scFv KM41 to the
FVIII-(1811-1818)/FV light chain. scFv EL14 (67 fmol/mm2) on a CM5 sensor chip was incubated with either
the recombinant wild-type FVIII or recombinant FVIII-(1811-1818)/FV
light chain to a density of 20 fmol/mm2 in 150 mM NaCl and 50 mM Tris (pH 7.4). scFv KM36 (100 nM) (A) and scFv KM41 (40 nM)
(B) were passed over two separate channels with the
immobilized wild-type FVIII (solid lines) and
FVIII-(1811-1818)/FV (dashed lines) light chains,
respectively, in 150 mM NaCl, 2 mM
CaCl2, 0.005% (v/v) Tween 20, and 20 mM Hepes
(pH 7.4) for 2 min at a flow rate of 20 µl/min at 25 °C. Response
is indicated as resonance units (RU) and is corrected for
nonspecific binding.
2 s
1 and
kon = (3.3 ± 0.3) × 105
M
1 s
1). Strikingly, LRP cluster
II interacted with the FVIII-(1811-1818)/FV light chain with 4-5-fold
lower affinity compared with the wild-type FVIII light chain
(koff = (4.3 ± 0.4) × 10
2 s
1, kon = (1.6 ± 0.4) × 105 M
1
s
1, and Kd = 262 ± 62 nM). These data demonstrate that the A3 domain region
Glu1811-Lys1818 contributes to the assembly of
the FVIII light chain·LRP complex.
View larger version (14K):
[in a new window]
Fig. 6.
Binding of LRP cluster II to the
FVIII-(1811-1818)/FV light chain. scFv EL14 (67 fmol/mm2) on a CM5 sensor chip was incubated with either
the recombinant wild-type FVIII or recombinant FVIII-(1811-1818)/FV
light chain to a density of 20 fmol/mm2 in 150 mM NaCl and 50 mM Tris (pH 7.4). LRP cluster II
(25, 50, 75, 100, and 125 nM) was passed over two separate
channels with the immobilized wild-type FVIII (A) and
FVIII-(1811-1818)/FV (B) light chains, respectively, in 150 mM NaCl, 2 mM CaCl2, 0.005% (v/v)
Tween 20, and 20 mM Hepes (pH 7.4) for 2 min at a flow rate
of 20 µl/min at 25 °C. Response is indicated as resonance units
(RU) and is corrected for nonspecific binding.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin, it has been established that positively charged residues at the ligand surface are involved in the interaction with LRP (43-46). Interestingly, the FVIII A3 domain region
Glu1811-Lys1818 (i.e.
1811ETKTYFWK1818) also contains two exposed
positively charged lysine residues at positions 1813 and 1818. Based on
the sequence of the inhibitory peptides
Lys1804-Lys1818 and
Tyr1815-Ala1834 (Table II), a possible
involvement of the overlapping 1815YFWK1818
region in the interaction with LRP is further suggested. However, compared with the homolog part within the A3 domain of FV
(i.e. 1704SSYTYVWH1711), the lysine
residues appear to be unique to the FVIII A3 domain (4). Replacement of
FVIII residues Glu1811-Lys1818 with the
corresponding residues of FV resulted in impaired binding to LRP
cluster II (Fig. 6). These results suggest that positively charged
residues within the FVIII region
Glu1811-Lys1818 mediate an electrostatic
interaction with LRP.
3.6 µM) (Table I). This is in agreement with a previous study
in which the isolated C2 domain showed only modest association with LRP
(18). In addition, the affinity for FVIII light chain binding to LRP
was not affected upon deletion of the C2 domain (Table I). However, it
has been demonstrated that an anti-C2 domain monoclonal antibody (ESH4) completely inhibits the interaction between the FVIII light chain and
LRP (18). The mechanism by which antibody ESH4 inhibits LRP binding is
not yet elucidated. Because the anti-C2 antibody does not require
region Glu1811-Lys1818 for its interaction
with the FVIII light chain (47), it is unlikely that ESH4 competes with
LRP for binding to the same site in the A3 domain. Therefore, one of
the mechanisms that could contribute to the inhibition includes steric
interference. In contrast, scFv KM41 only partially inhibited the
interaction between the FVIII light chain and LRP (Fig. 4). This might
be due to the relative small size of a scFv (
30 kDa) compared with a
complete antibody (
150 kDa). These observations suggest that,
besides region Glu1811-Lys1818, other
surface-exposed structural elements within the A3-C1 domains (i.e. Ala1722-Asn2172) contribute
to the assembly of the FVIII light chain·LRP complex. This is in line
with the observation that the FVIII-(1811-1818)/FV light chain
demonstrated residual binding to LRP cluster II (Fig. 6). The fact that
replacement of FVIII residues Glu1811-Lys1818
with residues of FV affected the affinity by 4-5-fold and as such had
relatively limited impact on the binding energy of the FVIII light
chain-LRP interaction also suggests that LRP binding involves an
additional or more extended binding site. In this context, it should be
mentioned that region Glu1811-Lys1818 within
the A3 domain of the FVIII light chain is part of a larger segment that
is exposed to the protein surface (i.e.
Glu1804-Lys1818) (13). Besides the lysine
residues at positions 1813 and 1818, this region contains two
additional unique FVIII lysine residues at positions 1804 and 1808, which might play a role in the interaction with LRP.
2-macroglobulin (48).
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ACKNOWLEDGEMENTS |
---|
We express our gratitude to E. Turenhout for providing vectors encoding scFv fragments and FVIII light chain fragments. We also thank Dr. H. Pannekoek for providing baby hamster kidney cells expressing LRP clusters II and IV.
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FOOTNOTES |
---|
* 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.
§ Present address: Laboratory for Thrombosis and Haemostasis, Dept. of Haematology, University Medical Center, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
To whom correspondence should be addressed: Dept. of Plasma
Proteins, Sanquin Research at CLB, Sanquin Blood Supply Foundation, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. Tel.:
31-20-512-3120; Fax: 31-20-512-3680; E-mail:
k.mertens@sanquin.nl.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M212053200
2 M. H. A. Bos, D. W. E. Meijerman, C. van der Zwaan, and K. Mertens, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: FVIII, factor VIII; FIXa, factor IXa; FV, factor V; FXa, factor Xa; LRP, low-density lipoprotein receptor-related protein; scFv, single-chain variable domain antibody fragment; HSA, human serum albumin; SPR, surface plasmon resonance.
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