From the
The low density lipoprotein receptor-related
protein/
The
The structural basis underlying
the high affinity interaction of such a multitude of functionally
diverse ligands to the receptor is only poorly understood. Moestrup
et al.(21) demonstrated by ligand blot analysis that
rat
To understand the structure and function relations
of LRP, we aim to delineate the structural determinants that constitute
the ligand-binding sites of the receptor. The present study was
performed to obtain monoclonal antibodies specifically directed toward
ligand-binding domains of the receptor. The interaction of all known
ligands to LRP is dependent on the binding of Ca
Combinatorial immunoglobulin repertoire cloning is a
technique that allows the selection of specific Fab fragments from
large, phage-displayed Fab fragment libraries
(24) . We have
employed this technique and constructed a phage library displaying Fab
portions derived from the IgG repertoire of a mouse that had been
immunized with purified human LRP.
Here we report the isolation of a
Fab fragment (Fab A8) that mimics a LRP ligand. It binds in a
Ca
To obtain inhibitory antibodies to
LRP, we exploited the advantages of a technique based on phage display,
enabling us to select Fab fragments rapidly from large repertoires with
properties that directly depend on the selection assays employed. We
hypothesized that specific selection of antibodies binding to divalent
cation-dependent epitopes would increase the chance of isolating
antibodies directed toward the ligand-binding sites of the receptor.
Hence, we modified the phage-panning procedures in order to select such
Fab phages (EDTA elution).
Panning resulted in the enrichment of a
single type of Fab phage (Fab A8) that was dependent on the presence of
Ca
The inhibition of the binding of u-PA
The importance of the
PAI-1-binding site was shown in previous studies demonstrating that
t-PA
The observation that pro-u-PA degradation by COS-1 cells could not
be completely inhibited by Fab A8 should be ascribed to the activation
of pro-u-PA to u-PA and the subsequent complex formation with PAI-1
synthesized by the cells. As suggested by Nykjet al. (39), pro-u-PA is protected from binding to and uptake by LRP by
initial binding to the urokinase receptor. After activation, complex
formation with PAI-1 takes place, and rapid uptake of this complex by
the COS-1 cells occurs mediated by LRP. Experiments performed by
Kounnas et al.(5) , who incubated HepG2 cells with
either [
The
availability of inhibitory monoclonal anti-LRP Fab fragments like Fab
A8 may prove to be useful for the analysis of the molecular mechanisms
that mediate the regulation of ligand binding by the receptor. Our
future efforts will be directed toward the isolation of additional
inhibitory Fab fragments and the precise localization of the
ligand-binding sites that are associated with these
``antibodies.'' In addition to facilitating the description
of the structure of the receptor, these antibodies may help to reveal
the physiological roles of LRP-mediated endocytosis in tissue culture
and animal models.
-macroglobulin receptor (LRP) mediates
endocytosis of a number of structurally unrelated ligands, including
complexes of plasminogen activator inhibitor type 1 (PAI-1) and
tissue-type plasminogen activator (t-PA) or urokinase plasminogen
activator (u-PA), free t-PA, single-chain urokinase (pro-u-PA),
-macroglobulin-protease complexes, and lipoprotein
lipase. So far, all ligands have in common the fact that they bind to
the receptor in a Ca
-dependent way and the fact that
binding to the receptor can be inhibited by a 39-40-kDa protein,
termed the receptor-associated protein. To obtain inhibitory antibodies
for the analysis of the structure and function of the receptor we
applied the combinatorial immunoglobulin repertoire cloning technique
in order to specifically select monoclonal Fab fragments directed
against Ca
-dependent epitopes. In this report we
describe the isolation of a Fab fragment (Fab A8) showing a high
relative affinity for the receptor (0.5 nM). The binding of
this Fab fragment to purified LRP is inhibited in the presence of 5
mM EDTA, receptor-associated protein, and lipoprotein lipase
(IC
values of 1.4 and 31 nM, respectively). By
immunoblotting of CNBr-digested LRP it is shown that Fab A8 binds to a
fragment that harbors the second cluster of cysteine-rich
complement-type repeats flanked by epidermal growth factor repeats.
Binding studies using
I-labeled ligands and immobilized
receptor show that Fab A8 partially inhibits the binding of
[
I]u-PA
PAI-1 complexes (IC
= 1.1 nM) and completely inhibits the binding of
[
I]pro-u-PA to the receptor (IC
= 2.2 nM). No inhibition was observed for the
binding of
I-labeled methylamine-activated
-macroglobulin or
[
I]t-PA
PAI-1 to LRP. Degradation of
[
I]u-PA
PAI-1 complexes by COS-1 cells was
also partially (43%) inhibited by Fab A8. Our results provide evidence
for the presence of an interaction site for pro-u-PA localized in the
second cluster of cysteine-rich repeats that is unrelated to the
t-PA
PAI-1 or methylamine-activated
-macroglobulin interaction sites.
-macroglobulin receptor/low density
lipoprotein receptor-related protein (LRP)
(
)
is a
member of the low density lipoprotein receptor gene family and has been
shown to be a multiligand clearance receptor (reviewed in Refs. 1 and
2). The ligands for the receptor include complexes of urokinase-type
plasminogen activator (u-PA) and plasminogen activator inhibitor type 1
(PAI-1) or protease nexin, the single-chain form of u-PA (pro-u-PA),
tissue-type plasminogen activator (t-PA), and t-PA
PAI-1
complexes,
-macroglobulin-protease complexes, or
lipoproteins enriched in apoE or associated with lipoprotein lipase,
lactoferrin, and Pseudomonas exotoxin A (3-15).
Recently, the binding of minor group common cold virus to LRP was
described
(16) , and evidence has been presented that LRP
mediates the uptake of tissue factor pathway inhibitor
(17) .
Purification of LRP has revealed the existence of a receptor-associated
protein (RAP), capable of inhibiting the binding of all known ligands
to the receptor (14, 18-20).
-macroglobulin and u-PA
PAI-1 complexes bind
to specific distinct binding sites located in a CNBr-generated 75-kDa
fragment (amino acids 776-1399) containing the second cluster of
complement-type repeats (cluster II domain) flanked by one
amino-terminal and two carboxyl-terminal cysteine-rich epidermal growth
factor type repeats. RAP was also shown to bind to the fragment and to
inhibit the binding of both ligands to the receptor. Using a
recombinant DNA approach to express functionally restricted LRP
minireceptors, Willnow et al.(22) demonstrated that
RAP has a minor affinity for the fourth cluster of complement-type
repeats. The binding sites for other ligands on LRP have not been
identified yet.
to
the receptor, and it has been postulated that Ca
may
induce allosteric changes that are required to expose the
ligand-binding sites (23). Hence, we used a strategy for selection of
anti-LRP antibodies directed against Ca
-dependent
epitopes.
-dependent manner with high affinity to immobilized
purified LRP and competes partially for binding of u-PA
PAI-1
complexes and completely for binding of pro-u-PA to the receptor.
Proteins and Chemicals
Human LRP,
M
, and monoclonal antibodies
MR
-5 (IgM) and
MR
-1 (IgG)
were purified as
described
(19, 21, 23, 26) . Recombinant
RAP was provided by Dr. H. C. Th(University of Aarhus).
LpL was from Sigma, human two-chain u-PA was from Serono (Anbonne,
Switzerland), two-chain t-PA was from Biopool (Umea, Sweden), and
active PAI-1 was a gift from Dr. T. M. Reilly (Merck Laboratories).
Pro-u-PA was a gift from Dr. J. Henkin (Abbott). Proteins were
radiolabeled by using the IODO-GEN method (Pierce). Na
I
was from the Radiochemical Centre. Specific activities were 1-4
10
Bq/mol of ligand. Complexes between PAI-1 and
[
I]u-PA or [
I]t-PA were
prepared as described
(27) . All other chemicals used were
reagent grade (Sigma).
Construction of a Phage-displayed Fab Library
A
Balb/C mouse was immunized with purified human LRP. To that end, the
mouse was injected intraperitoneally three times during a period of 6
weeks with 10 µg of purified LRP diluted 1:1 in either complete or
incomplete Freund's adjuvant. After the third boost anti-LRP
immunoglobulin serum titers were checked by fluorescence-activated cell
sorter analysis of COS-1 cells derived from African green monkey kidney
(ATCC CRL 1650). After a final intravenous boost, the mouse was
sacrificed, and the spleen was isolated. Total RNA was isolated
according to the method of Chomczynski and Sacchi
(28) .
Single-stranded cDNA was prepared using RNase H reverse transcriptase (Superscript II, Life Technologies, Inc.).
The construction of phage-displayed Fab libraries has been described in
detail
(29, 30) . Briefly, specific amplification of
cDNAs encoding the F
portions of IgG
and
IgG
and
light chains was accomplished by polymerase
chain reaction using a set of specific primers. Polymerase chain
reaction products were subsequently cloned into phagemid pComb3. After
transformation of Escherichia coli strain XL1-Blue and
infection with VCSM13 helper phages (Stratagene), a phage-displayed Fab
library was obtained that consisted of 1.8
10
independent colonies.
DNA Sequence Analysis
Sequencing of DNA, encoding
the CDR3 regions of Fab heavy chains, was performed using the Sequenase
2.0 sequencing kit (U. S. Biochemical Corp.).
Panning of the Phage-displayed Fab
Library
Selection of LRP-binding Fab phages was performed in a
biopanning assay as described by Burton et al.(25) .
Briefly, 1 µg of LRP was coated for 16 h at 4 °C onto wells of
microtiter plates (enzyme immunoassay/radioimmunoassay plates, Costar),
and unbound protein was washed away using Tris-buffered saline (TBS)
(20 mM Tris, pH 7.4, 150 mM NaCl). Wells were blocked
with TBS, 3% (w/v) BSA for 1 h at 37 °C, and wells were washed once
with TBS. 10 Fab phages were incubated in TBS, 1% (w/v)
BSA at 37 °C for 2 h. After extensive washing with TBS, 0.05% (v/v)
Tween 20, bound Fab phages were eluted with TBS, 10 mM EDTA.
Suspensions of eluted phages were reamplified in E. coli.
Panning procedures were repeated 3 times.
Screening of the Phage-displayed Fab Library by Phage
ELISA
Small scale phage preparations (10 plaque-forming units/µl) obtained from single colonies of the
subsequent rounds of selection were analyzed for binding to immobilized
LRP by phage ELISA
(31) . Briefly, 10
phages were
incubated for 2 h at 37 °C on coated LRP or BSA in a final volume
of 50 µl of TBS, 1% (w/v) BSA in the presence or absence of EDTA as
indicated. Bound Fab phages were detected by incubation with polyclonal
anti-M13 biotin conjugate (5 Prime
3 Prime, Inc.) followed by
incubation with horseradish peroxidase-coupled streptavidin (Amersham
Corp.) and staining with tetramethylbenzidine. Extinction was measured
at 450 nm.
Preparation of Soluble Fab
Preparation of soluble
Fab was performed as described
(30) . Briefly, the DNA coding for
the M13 gene III product was deleted by subcloning allowing for the
expression of soluble Fab in the periplasmic space of E. coli and subsequent purification from the culture supernatant. The
culture supernatant was centrifuged for 1 h at 4 °C at 10,000 rpm
and filtered using 0.22-µm filters (Schleicher & Schuell,
Inc.). Subsequently, the Fab fragment was purified from the supernatant
to homogeneity by affinity chromatography using a Sepharose-coupled rat
anti-mouse Ig light chain monoclonal antibody (CLB, Amsterdam,
The Netherlands). The Fab fragment preparation was essentially pure
when analyzed by SDS-polyacrylamide gel electrophoresis combined with
silver staining. Purification procedures yielded approximately 2 mg of
Fab protein/liter of E. coli culture supernatant, as
determined by measuring extinction at 280 nm.
Determination of Relative Affinities of Antibodies by
Competitive ELISA
The relative affinities of the Fab fragments
were determined as described previously
(32, 33) . 50 ng
of LRP was coated for 1 h at 37 °C onto 96-well ELISA plates
(NUNC), blocked with TBS, 3% (w/v) BSA, and incubated with serial
dilutions of the selected antibodies to establish the antibody
concentrations that result in half-maximal binding. These
concentrations were then used in a competitive ELISA in which the
binding of the antibody to immobilized LRP was competed with a range of
soluble LRP concentrations. After an incubation period of 1 h at 37
°C, plates were washed, and bound Fab fragments were detected as
described above using rat anti-mouse light chain antibodies
coupled to biotin. The relative affinities were estimated from the
concentrations of soluble LRP that give a half-maximal inhibition.
Competition for Fab Binding to Purified LRP by RAP or
LpL
Conditions were essentially as described for the competitive
ELISA. Competition for Fab binding to LRP was investigated by adding
increasing amounts of RAP or LpL. Residual Fab binding was measured as
described above.
Immunoblotting of CNBr-digested LRP
LRP digested
with CNBr was separated on a SDS-polyacrylamide gel (4-16%;
nonreducing conditions) and electroblotted onto Immobilon filters as
described (21). Immunostaining was performed using IgM
MR
-5, polyclonal rabbit anti-human LRP serum, and
Fab fragment Fab A8. As secondary antibodies, rat polyclonal anti-mouse
light chain antibodies coupled to biotin and polyclonal goat
anti-rabbit antibodies coupled to alkaline phosphatase (Promega) were
used. Staining was performed using 5-bromo-4-chloro-3-indolyl phosphate
and nitro blue tetrazolium chloride (Promega).
Ligand Binding to Purified LRP
Assays were
essentially as described
(3) . Incubations were performed with
I-labeled methylamine-activated
-macroglobulin (
M
),
t-PA
PAI-1 complexes, u-PA
PAI-1 complexes, or pro-u-PA for
16 h at 4 °C in Hepes buffer (140 mM NaCl, 10 mM
Hepes, 2 mM CaCl
, 1 mM MgCl
,
2% (w/v) BSA, pH 7.8) in the presence of a range of Fab A8
concentrations. After washing, bound radioactivity was removed with 10%
(w/v) SDS and counted. Values were corrected for binding to BSA-coated
wells.
Inhibition of Ligand Degradation by COS-1
Cells
COS-1 cells were seeded in 6-well plates (NUNC) and grown
to confluency in Iscove's modified medium containing 5% (v/v)
fetal calf serum (Life Technologies, Inc.), penicillin, streptomycin,
and Fungizone for at least 24 h prior to incubations. Ligand
degradation experiments were performed as follows. Cells were washed
with phosphate-buffered saline and binding medium (Iscove's
medium supplemented with 0.01% (v/v) Tween 20, 1 mM
CaCl, 1 mM MgCl
, 50 mM
-aminocaproic acid, 0.02% (w/v) sodium azide) and incubated for 2
h at 37 °C with 0.4 nM
[
I]u-PA
PAI-1 complexes or 3 nM
[
I]t-PA
PAI-1 complexes in binding medium,
in either the presence or the absence of 1 µM Fab fragment
or 1 µM RAP. After incubation, proteins present in the
cell culture supernatant were precipitated using 10% (w/v)
trichloroacetic acid/phosphate-buffered saline, supplemented with 0.5%
(w/v) KI. Nonprecipitable radioactivity was determined in a
counter.
RESULTS
Selection of Anti-LRP Fab Phages Directed toward
Ca
LRP-binding Fab
phages were selected from a phage-displayed Fab library by a panning
assay using immobilized LRP and elution of bound Fab phages with EDTA.
After four rounds of panning, individual phage stocks were prepared and
screened in a phage ELISA (Fig. 1). Fab phage isolates from the
first and second round of panning did not show any specific binding to
LRP. One Fab phage out of 10 isolated from the third round of panning
specifically bound to LRP, and binding was completely abolished in the
presence of 5 mM EDTA. This Fab phage did not show any binding
to coated BSA in either the presence or the absence of 5 mM
EDTA. All Fab phage isolates from the fourth round of panning
specifically bound to LRP, whereas no binding was observed either to
coated LRP in the presence of 5 mM EDTA or to coated BSA in
both the presence and the absence of EDTA. These results show that we
selected Fab phages that bind to LRP exclusively when divalent cations
(Ca-dependent Epitopes
) are present.
Figure 1:
Selection
of Ca-dependent anti-LRP Fab phages. Microtiter wells
were coated with 100 ng of LRP or 3% (w/v) BSA and incubated with
10
phages derived from single phage isolates from four
consecutive rounds of panning (A-D). Incubations were
performed in the presence or absence of 5 mM EDTA. Bound
phages were detected using sheep anti-M13 polyclonal antibodies
conjugated to biotin. Black, hatched,
double-hatched, and dotted bars indicate the binding
of anti-LRP Fab phages to LRP, BSA, LRP in the presence of 5
mM EDTA, and BSA in the presence of 5 mM EDTA,
respectively.
The phagemid DNA-encoding CDR3
regions of heavy chains of all LRP-binding Fab phages were sequenced
and were found to be identical, indicating the selection of a single
type of Fab phage, denoted Fab A8. Upon comparison, no apparent
homology was observed between the Fab A8 heavy chain CDR3 region
(YPFWPYH) and the primary structure of LRP ligands apoE, LpL, PAI-1,
pro-u-PA, and RAP.
Determination of Relative Affinities of Fab
Fragments
To further characterize the properties of the selected
Fab A8, the Fab A8 phagemid DNA was modified for expression of soluble
Fab fragment, and Fab A8 was purified from E. coli supernatant
by affinity chromatography. Relative affinities of Fab fragments were
determined by performing a competitive ELISA
(32) . In these
experiments, Fab A8 was compared with a Fab fragment (Fab A2) that
recognizes LRP in a Ca-independent way. Fab A2 (CDR3
region YDFNTSTGYY) was taken from a panel of anti-LRP Fab fragments
obtained by a panning procedure in which bound Fab phages were eluted
using glycine HCl (pH 2.2). A competitive ELISA was performed using a
constant amount of coated LRP (50 ng) and a range of soluble LRP
(10
-10
M)
(Fig. 2). Relative affinities (corresponding to IC
values) were 2.8 and 0.62 nM for Fab A2 and A8,
respectively. These values were comparable with the relative affinity
of an anti-LRP monoclonal antibody obtained by classical hybridoma
technology (
MR
-1, 5.6 nM, data not
shown).
Figure 2:
Determination of relative affinities of
Fab A2 and A8 by competitive ELISA. Microtiter wells were coated with
50 ng of LRP and incubated with half-maximal saturating amounts of Fab
A2 or A8. Incubations with a range of soluble LRP concentrations were
performed to determine 50% inhibition values, representing relative
affinities of the Fab fragments. Detection was performed using a rat
anti-mouse light chain monoclonal antibody coupled to biotin.
, Fab A2;
, Fab A8. Experiments were performed in
triplicate.
Binding of Fab A8 to LRP Is Inhibited by RAP and
LpL
Because binding of all ligands to LRP is
Ca-dependent and can be prevented by RAP, the binding
of Fab A8 to LRP was tested for inhibition by RAP (Fig. 3). LpL
has been found to compete for the binding of RAP to LRP
(34) .
Therefore we tested the inhibition of Fab fragment binding by LpL.
Whereas binding of Fab A2 is not prevented by RAP and LpL over a
concentration range of 10
-10
M, the binding of Fab A8 is completely inhibited upon
addition of RAP (IC
= 1.4 nM) or LpL
(IC
= 31 nM). In a control experiment, no
direct binding of Fab A8 to immobilized RAP or LpL was observed.
Figure 3:
Effects of RAP and LpL on the binding of
Fab A2 or A8 to immobilized LRP. The binding of Fab A2 and Fab A8 to 50
ng of immobilized LRP was determined in an ELISA as described under
``Materials and Methods'' in the presence of increasing
amounts of RAP or LpL as indicated. and
, Fab A2 in the
presence of RAP and LpL, respectively;
and
, Fab A8 in the
presence of RAP and LpL, respectively. Data were corrected for binding
to BSA-coated wells. Experiments were performed in
triplicate.
Fab A8 Binds to a CNBr Fragment of LRP Containing the
Second Cluster of Complement-type Repeats
In a previous study,
binding sites on LRP for M
,u-PA
PAI-1
complexes and RAP were localized by ligand-blotting experiments using
CNBr-derived fragments of purified LRP
(21) . We have used the
same approach to localize the epitope of Fab A8 on LRP. As can be seen
in Fig. 4, no bands were stained upon incubation without primary
antibodies (lane 1), whereas incubation with polyclonal rabbit
anti-LRP serum visualized a subset of the CNBr-derived LRP fragments
(lane 2). The monoclonal IgM antibody
MR
-5 has been shown to bind to an epitope located
between residues 1165 and 1246 of the receptor
(21) . This region
is contained within a CNBr-derived fragment of 624 residues (amino
acids 776-1399) that spans the second cluster of complement-type
repeats. Lane 3 shows the immunostaining of this fragment
(indicated by the arrow) by monoclonal antibody
MR
-5. The higher molecular mass immunoreactive
band (±80 kDa) is due to incomplete cleavage at the
Met
-Ser
bond
(21) . Fab A8
recognizes the same pattern of bands as
MR
-5
(lane 4). These results demonstrate that Fab A8 also binds to
an epitope associated with the second cluster of cysteine-rich repeats
in LRP.
Figure 4:
Immunostaining of CNBr-digested LRP.
Immobilon filters with protein digests were blocked with TBS, 3% (w/v)
BSA and subsequently incubated either without antibodies (lane
1) or with polyclonal rabbit anti-LRP serum (lane 2), IgM
MR
-5 (lane 3), or Fab A8 (lane
4). The arrow indicates the mobility of a 75-kDa
CNBr-derived fragment (amino acids 776-1399) that harbors the second
cluster of complement-type repeats. Staining was performed using goat
anti-rabbit polyclonal antibodies conjugated to alkaline phosphatase or
using monoclonal rat anti-mouse
light chain biotin
conjugate.
Fab A8 Competes for the Binding of u-PA
The immunoblotting
experiments showed that binding of Fab A8 to LRP is mediated by the
fragment that carries ligand-binding sites for
PAI-1
Complexes and pro-u-PA to Immobilized LRP
M
, u-PA
PAI-1 complexes, and RAP. To
test whether Fab A8 might compete for the binding of these ligands to
the receptor, immobilized LRP was incubated with
I-labeled ligands. Fig. 5shows that Fab A8 has no
effect on the binding of
[
I]
M
and on the
binding of [
I]t-PA
PAI-1 complexes to
immobilized purified LRP, whereas it partially blocks the binding of
[
I]u-PA
PAI-1 complexes to the receptor
(IC
= 2.2 nM). The binding of
[
I]pro-u-PA is completely prevented by Fab A8
(IC
= 1.1 nM).
Figure 5:
Fab A8 inhibition of binding of
[I]u-PA
PAI-1 complexes and
[
I]pro-u-PA to LRP. Microtiter wells were
coated with 15 ng of purified LRP, blocked with BSA, and incubated with
different
I-labeled LRP-ligands in the presence of
increasing Fab A8 concentrations. The bound radioactivity was
determined in a
counter. The values are the mean of triplicates
representing the binding of
[
I]
M
(
),
[
I]t-PA
PAI-1 complexes (
),
[
I]u-PA
PAI-1 complexes (
), and
[
I]pro-u-PA
(
).
The inhibition of LRP
binding to coated RAP or LpL by Fab A8 was also investigated. No
inhibition could be detected at Fab A8 concentrations up to 500
nM (data not shown).
Fab A8 Inhibits the Degradation of u-PA
Internalization and degradation of
[PAI-1
Complexes by COS-1 Cells
I]u-PA
PAI-1 complexes by COS-1 cells
were shown to be mediated by LRP
(3) . To test whether Fab A8 is
also capable of inhibiting LRP-dependent degradation in this system,
COS-1 cells were incubated with
[
I]t-PA
PAI-1 complexes and
[
I]u-PA
PAI-1 complexes, in either the
presence or the absence of 1 µM Fab A8 or 1
µM RAP. Whereas no inhibition of
[
I]t-PA
PAI-1 complex degradation was
observed, [
I]u-PA
PAI-1 complex
degradation was inhibited by 43% by Fab A8. Concomitant incubations
with RAP inhibited the degradation of
[
I]t-PA
PAI-1 by 99% and the degradation
of [
I]u-PA
PAI-1 complexes by 84%
(Fig. 6). Experiments performed with radiolabeled pro-u-PA showed
that inhibition of binding by 1 µM Fab A8 was partial
(40%, data not shown); a possible explanation for this observation will
be presented below.
Figure 6:
Fab
A8 inhibition of [I]u-PA
PAI-1 complex
degradation by COS-1 cells. COS-1 cells were treated for 2 h at 37
°C with 3 nM [
I]t-PA
PAI-1
complexes or 0.4 nM [
I]u-PA
PAI-1
complexes in the presence or the absence of 1 µM Fab A8 or
1 µM RAP. Degradation was measured as the trichloroacetic
acid-soluble radioactivity in the medium. Black bars,
degradation of [
I]t-PA
PAI-1 complexes;
hatched bars, degradation of
[
I]u-PA
PAI-1
complexes.
DISCUSSION
In this report we describe the selection of an inhibitory Fab
fragment directed toward LRP by combinatorial immunoglobulin repertoire
cloning. In many studies designed to demonstrate the role of LRP in
endocytosis, RAP inhibition has been used to confirm the involvement of
LRP
(4, 16, 35, 36, 37) .
However, as has recently been pointed out by Battey et
al.(38) , RAP also interacts with and inhibits ligand
binding to other members of the low density lipoprotein receptor gene
family. The monospecificity of inhibitory monoclonal antibodies may
facilitate studies on the importance of LRP in vivo without
affecting the receptor activities of other low density lipoprotein
receptor gene family members.
for its binding to immobilized LRP. The results of
the competitive ELISA experiments, which demonstrated the inhibition of
Fab A8 binding to the receptor by nanomolar concentrations of RAP,
indicated that this Fab fragment mimics the characteristics of a LRP
ligand. The finding that LpL, like RAP, can inhibit Fab A8 binding to
LRP, whereas the reverse could not be shown, confirms the observation
(34) that the structurally related RAP and LpL molecules share
multiple binding sites on LRP. Our binding studies with purified LRP
show that Fab A8 partially inhibits the binding of
[
I]u-PA
PAI-1 complexes and completely
inhibits the binding of [
I]pro-u-PA to the
receptor. Immunoblotting experiments with CNBr-derived LRP fragments
showed that Fab A8 is directed against an epitope in the second cluster
of cysteine-rich repeats of LRP containing the cluster II domain. The
inhibition of pro-u-PA binding to the receptor by Fab A8 demonstrates
the presence of a single binding site for pro-u-PA in the cluster II
domain. This binding site appears to be unrelated to the binding
site(s) of
M
, PAI-1, and t-PA
PAI-1
complexes. Specificity of the Fab fragment for LRP was shown upon
incubation with coated rabbit GP330; binding of Fab A8 could not be
detected.
PAI-1 complexes
by Fab A8 supports previous reports that localize the binding of these
complexes to the cluster II domain
(21, 22) .
Interestingly, this inhibition is only partial (50%), suggesting that
binding of u-PA
PAI-1 complexes to LRP is mediated by both the
u-PA moiety and the PAI-1 moiety of the complex. This observation is in
agreement with a recent report by Nykjet al.(39) describing the involvement of multiple binding sites
present on the protease and inhibitor parts of the complex. The fact
that t-PA
PAI-1 complex binding to LRP is not affected by Fab A8
further strengthens the conclusion that the u-PA moiety of the
u-PA
PAI-1 complex interacts with LRP independently of the PAI-1
interaction and that the recognition site on the receptor for u-PA is
different from the t-PA recognition site.
PAI-1 complexes can block binding of u-PA
PAI-1
complexes to immobilized LRP by 76% (cleaved PAI-1 inhibited binding up
to 55%, whereas t-PA did not inhibit at all)
(3) and that
u-PA
PAI-1 complexes can inhibit degradation of t-PA
PAI-1
complexes in human vascular smooth muscle cells by 43%
(40) .
These data, however, seem to be in contrast with the findings of Camani
et al.(37) , who showed in rat hepatoma cells that
u-PA
PAI-1 complexes cannot compete for degradation of
t-PA
PAI-1 complexes, indicating that the protease recognition
site is the most important binding determinant in the rat system.
I]pro-u-PA or
[
I]u-PA
PAI-1 complexes, demonstrated that
[
I]pro-u-PA was taken up by the cells without
activation and subsequent complex formation. The observed differences
might be ascribed to the different cell systems used.
-macroglobulin
receptor;
M
, methylamine-activated
-macroglobulin; BSA, bovine serum albumin; ELISA,
enzyme-linked immunosorbent assay; LpL, lipoprotein lipase; PAI-1,
plasminogen activator inhibitor type 1; u-PA, urokinase-type
plasminogen activator or urokinase; pro-u-PA, single-chain u-PA; RAP,
receptor-associated protein; t-PA, tissue-type plasminogen activator;
TBS, Tris-buffered saline.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.