©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Analysis of the Binding of Pro-urokinase and Urokinase-Plasminogen Activator Inhibitor-1 Complex to the Low Density Lipoprotein Receptor-related Protein Using a Fab Fragment Selected from a Phage-displayed Fab Library (*)

Ivo R. Horn , SK. Moestrup (1), Birgit M. M. van den Berg , Hans Pannekoek , Morten S. Nielsen (1), Anton-Jan van Zonneveld (§)

From the (1) Department of Biochemistry, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands and the Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
REFERENCES

ABSTRACT

The low density lipoprotein receptor-related protein/-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-PAPAI-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-PAPAI-1 to LRP. Degradation of [I]u-PAPAI-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-PAPAI-1 or methylamine-activated -macroglobulin interaction sites.


INTRODUCTION

The -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-PAPAI-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).

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 -macroglobulin and u-PAPAI-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 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 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.

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-dependent manner with high affinity to immobilized purified LRP and competes partially for binding of u-PAPAI-1 complexes and completely for binding of pro-u-PA to the receptor.


MATERIALS AND METHODS

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). NaI 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-PAPAI-1 complexes, u-PAPAI-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-PAPAI-1 complexes or 3 nM [I]t-PAPAI-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-dependent Epitopes

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) 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 (10-10M) (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 10-10M, 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-PAPAI-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-PAPAI-1 Complexes and pro-u-PA to Immobilized LRP

The immunoblotting experiments showed that binding of Fab A8 to LRP is mediated by the fragment that carries ligand-binding sites for M, u-PAPAI-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-PAPAI-1 complexes to immobilized purified LRP, whereas it partially blocks the binding of [I]u-PAPAI-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-PAPAI-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-PAPAI-1 complexes (), [I]u-PAPAI-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-PAPAI-1 Complexes by COS-1 Cells

Internalization and degradation of [I]u-PAPAI-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-PAPAI-1 complexes and [I]u-PAPAI-1 complexes, in either the presence or the absence of 1 µM Fab A8 or 1 µM RAP. Whereas no inhibition of [I]t-PAPAI-1 complex degradation was observed, [I]u-PAPAI-1 complex degradation was inhibited by 43% by Fab A8. Concomitant incubations with RAP inhibited the degradation of [I]t-PAPAI-1 by 99% and the degradation of [I]u-PAPAI-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-PAPAI-1 complex degradation by COS-1 cells. COS-1 cells were treated for 2 h at 37 °C with 3 nM [I]t-PAPAI-1 complexes or 0.4 nM [I]u-PAPAI-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-PAPAI-1 complexes; hatched bars, degradation of [I]u-PAPAI-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.

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 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-PAPAI-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-PAPAI-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.

The inhibition of the binding of u-PAPAI-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-PAPAI-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-PAPAI-1 complex binding to LRP is not affected by Fab A8 further strengthens the conclusion that the u-PA moiety of the u-PAPAI-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.

The importance of the PAI-1-binding site was shown in previous studies demonstrating that t-PAPAI-1 complexes can block binding of u-PAPAI-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-PAPAI-1 complexes can inhibit degradation of t-PAPAI-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-PAPAI-1 complexes cannot compete for degradation of t-PAPAI-1 complexes, indicating that the protease recognition site is the most important binding determinant in the rat system.

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 [I]pro-u-PA or [I]u-PAPAI-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.

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.


FOOTNOTES

*
This work was supported by Grant 902-26-128 from the Netherlands Organization for Scientific Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Academic Medical Center, Dept. of Biochemistry (K1-161), Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Tel.: 31-20-5665129; Fax: 31-20-6915519.

The abbreviations used are: LRP, low density lipoprotein receptor-related protein/-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.


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