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J. Biol. Chem., Vol. 275, Issue 49, 38206-38212, December 8, 2000

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Mapping of a Minimal Apolipoprotein(a) Interaction Motif Conserved in Fibrin(ogen) - and -Chains^*

Regina Klose, Friedrich Fresser, Silvano Köchl^§, Walther Parson^§, Andreas Kapetanopoulos, Jamila Fruchart-Najib^¶, Gottfried Baier, and Gerd Utermann

From the  Institute for Medical Biology and Human Genetics, Universität Innsbruck, 6020 Innsbruck, Austria, the ^§ Institute for Legal Medicine, Universität Innsbruck, 6020 Innsbruck, Austria, and the ^¶ U325 INSERM, Departement d'Athérosclérose, Institut Pasteur de Lille, 59019 Lille Cedex, France

Received for publication, April 28, 2000, and in revised form, September 8, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen - and -chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen  and  modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lipoprotein(a) (Lp(a))¹ from human plasma is composed of a low density lipoprotein core and the highly polymorphic apo(a), covalently linked to apo B-100 by a single disulfide bridge (1, 2). apo(a) contains 10 distinct tandem repeats, named kringle IV types 1-10, closely resembling plg kringle IV followed by single plg kringle V-like and protease-like domains (3). The homology at the cDNA level between plg and apo(a) modules is 75-85% for the kringle IV domains and 94% for the protease domain (3). As a result of a size polymorphism in the apo(a) gene, more than 30 different apo(a) isoforms have been found in human plasma, differing in the number of the kringle IV type 2 repeat (4-6).

Several epidemiological studies indicate that elevated Lp(a) levels are an independent risk factor for coronary heart disease (7). Lp(a) accumulates in atherosclerotic lesions of coronary bypass patients and can be cross-linked to the fibrin thrombus (8-10). Binding of Lp(a) to fibrin(ogen) has been hypothesized to underlie a postulated role of Lp(a) in wound healing. Bound Lp(a) may protect the thrombus from premature digestion by plasmin and serve as an important source of phospholipids and cholesterol for membrane biogenesis and cell proliferation at the site of injury (11-13). Whereas the physiological role of Lp(a) remains unknown, several hypotheses have been proposed to account for the pathogenicity of Lp(a) (14). The high degree of homology between apo(a) and plg has been suggested to form the basis for the pathogenicity of Lp(a) as a modulator of fibrinolysis (15-18). Lp(a) effectively competes with plg for binding sites on fibrin and fibrinogen and reduces the generation of active plasmin (18-21). It has been demonstrated that Lp(a) increases smooth muscle cell migration and proliferation by inhibition of transforming growth factor- activation by plasmin (16, 17, 22). In addition, Lp(a) competes with plg for binding to receptors present on endothelial cells and monocytes (23, 24).

The aim of the study was to identify critical motifs in fibrin(ogen) interacting with Lp(a)/apo(a) for the understanding of the functions and pathophysiological properties of Lp(a). Here we present fibrin(ogen) - and -chain sequences interacting with apo(a) in the yeast two-hybrid system and the identification of a conserved 30-amino acid fibrin(ogen) minimal peptide motif that is sufficient for binding to apo(a)/Lp(a) in vitro.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast Strains and Media-- The genotype of the Saccharomyces cerevisiae reporter strain HF7c used for the two-hybrid screening, is MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3:: (GAL4 17-mers)₃-CYC1-lacZ. The strain CG1945, used for the -galactosidase assays, is characterized by an additional cycloheximide resistance. The genotype of SFY526, used to test interactions between GAL4 DNA binding domain and GAL4 transcription activation domain fusions, is MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, can^r, gal4-542, gal80-538, URA3::GAL1-lacZ (CLONTECH Laboratories, Inc, Palo Alto, CA). Strains were grown under standard conditions in rich or synthetic medium with appropriate supplements at 30 °C.

Plasmids-- apo(a)KIV-2 and apo(a)KIV-6 were cloned as described in Ref. 25. apo(a)KIV-5, -7, -8, -9, and -10 were cloned likewise using polymerase chain reaction and recombinant polymerase chain reaction primers (apo(a)KIV-5: 5'-CCAAGCGAATTCGGTGGCGGTGGATCCGCACTGACTGAGGAAACCCCC-3' and 5'-GCTTTGGTCGACTCATCATTCTTCAGAAGAAGCCTCTGTGCTTGGAT-3'; apo(a)KIV-7: 5'-CCAAGCGAATTCGGTGGCGGTGGATCCGCACCAACGGAGCAAAGCCCCA-3' and 5'-GCTTTGGTCGACTCATCATTCTTCAGAAGGAAGCTCTGTGCTTGGAACT-3'; apo(a)KIV-8: 5'-CCAAGCGAATTCGGTGGCGGTGGATCCGCACCAACTGAAAACAGCA-3' and 5'-GCTTTGGTCGACTCATCATTGTTCAGAAGGAGCCTCTGTGCT-3'; apo(a)KIV-9: 5'-CCAAGCGAATTCGGTGGCGGTGGATCCGCACCACCTGAGAAAAGCCCCTGT-3' and 5'-GCTTTGGTCGACTCATCATGCTTCAGAATGAGCCTCC-3'; and apo(a)KIVtype-10: 5'-CCAAGCGAATTCGGTGGCGGTGGATCCGCACCAACTGAGCAAAC-3' and 5'-ATTCCCGTCGACTCATCATTGTTCAGAAGGAGGCCCTAG-3'). Plasmids pCMV-A18 (A18 wt), pCMV-A18VP (KV-P), pCMV-A18_4174Arg (A18-Arg), and pCMV-A1832-35 (KIV 5-8) are described in Ref. 26. Plasmid pCMV-A18-AS (KIV 8-P) is described in Ref. 27.

Two-hybrid Screening and cDNA Isolation-- For the yeast two-hybrid screening, apo(a)KIV-6 was cotransformed with the human liver cDNA Matchmaker library in the pGAD10 vector (CLONTECH Laboratories, Inc., Palo Alto, CA) into the HF7c yeast strain, as described by the manufacturer, and the transformants were plated to synthetic dropout medium lacking leucine, histidine, and tryptophan but containing 5 mM 3-amino-1,2,4-triazole. The plates were incubated at 30 °C for up to 7 days.

-Galactosidase Reporter Activity-- His⁺ colonies were assayed for -galactosidase activity by transferring individual colonies on filters placed on selection medium. The plates were incubated for 2 days at 30 °C, and the filters lifted and immersed in liquid nitrogen for 10 s. After thawing at room temperature, the filters were placed on filter circles saturated with 0.2 mg/ml 5-bromo-4-chloro-3-indolyl--D-galactopyranoside in Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 30 mM -mercaptoethanol) in a Petri dish (permeabilized cells up) and incubated overnight as indicated. To verify the significance of the interaction a liquid culture assay using o-nitrophenyl -D-galactopyranoside as substrate was performed as described by the manufacturer (CLONTECH Laboratories, Inc., Palo Alto, CA). At least six individual cotransformants were assayed in the background of two different yeast strains. Only interactions of cotransformants in both yeast strains were asumed to be significant. The results are presented as the means ± S.D.

Peptide Synthesis and Characterization-- Biotinylated peptides FibG_207-235 and FibKA_207-235 were purchased at Neosystem (Strasbourg, France). Biotinylated peptides FibG_190-235 and FibKA_190-235 were prepared on an automated synthesizer ABI 421A (Applied Biosystems Inc.) using standard Boc/Bzl strategy. Biotin was coupled to the peptidyl-resin via N-hydroxy-succinimidyl-6-(biotinamido)-hexanoate in dimethyl formamide for 16 h at room temperature (28). The biotinylated peptidyl-resin was then washed twice with ter-amylalcohol, acetic acid, ter-amylalcohol, and diethylether. The peptides were cleaved from the vacuum-dried resins and simultaneously deprotected according to high hydrogen fluoride procedure (29). The peptides were purified and characterized by reversed phase high pressure liquid chromatography, capillary electrophoresis, and amino acid analysis. The biotinylated control peptide derived from protein kinase C (RFARKGSLRQKNVY) was from Genosys (Cambridge, UK).

Immunoblotting of Gal4 Binding Domain Fusion Baits in Yeast Extracts-- A total of 5 ml of transformed yeast cells grown overnight in selective medium lacking tryptophan were used to inoculate 15 ml of yeast extract peptone dextrose medium. At an A₆₀₀ of 0.5, the cells were pelleted, washed, resuspended at 5 × 10⁸ cells/ml in ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 µg/ml aprotinin/leupeptin, 1 mM PMSF) and frozen at 20 °C. Samples were analyzed by SDS-PAGE (10%) and transferred to polyvinylidene fluoride membrane (Millipore, Vienna, Austria), and fusion proteins were detected using a GAL4 DNA binding domain specific antibody (Santa Cruz Inc, Santa Cruz, CA), followed by a rabbit antimouse IgG-peroxidase conjugate and a chemiluminescence detection kit (ECL reagent; Amersham Pharmacia Biotech).

Tissue Culture and Transient Transfection-- The human hepatocarcinoma cell line HepG2 (30) and CHO cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured as recommended by the American Type Culture Collection. Transient transfection of cells was achieved by liposome-mediated gene transfer with the LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's protocol. After overnight incubation the transfection mixture was replaced by 2 ml of growth medium. After 48 h, cell culture supernatants were harvested, treated with proteinase inhibitors (1 mM PMSF, 5 µg/ml of each aprotinin and leupeptin), and centrifuged for 10 min each at 300 and 4000 × g to remove cells and cell debris, respectively. The Lp(a) content was assayed as described elsewhere (31, 32)

Human Plasma Samples and Preparation of Lp(a)-- Venous blood samples from healthy donors were collected in EDTA tubes, treated with proteinase inhibitors (1 mM PMSF, 5 µg/ml of each aprotinin and leupeptin), and centrifuged for 10 min at 300 × g to remove cells. Following enzyme-linked immunosorbent assay determination of Lp(a) plasma levels and prior to diluting for the pull-down experiments, the integrity of plasma apo(a) was analyzed by SDS-PAGE and immunoblotting. The Lp(a) isoforms used consisted of 18 apo(a) kringle units for plasma pull-down experiments and 21 apo(a) kringle units for pull-downs of purified Lp(a). Preparation of Lp(a) was performed by density centrifugation as described in Ref. 6.

Peptide Pull-down-- Diluted Lp(a) preparations, diluted plasma samples and cell supernatants that had been diluted with HBS (50 mM HEPES, 200 mM NaCl, pH 7.5) with or without 0.2 M EACA were precleared with volume Pansorbin cells (Calbiochem, San Diego, CA) at 4 °C for 1 h. Precleared samples were incubated overnight at 4 °C with the indicated biotinylated peptide at 3 nM final peptide concentration followed by addition of 20 µl of Biobeads-streptavidin (Merck) for the last 30 min. Precipitates were collected on a magnetic rack and washed four times with HBS/0.05% Tween 20 buffer and protease inhibitors (1 mM PMSF, 5 µg/ml of each aprotinin and leupeptin). Analysis of experiments described was determined by densitometric quantification of nonsaturated bands of the resulting immunoblots. The linearity of density and Lp(a) concentration within the density range observed in the experiments was verified by analyzing a Western blot with serial Lp(a) dilutions (not shown). The amount of Lp(a) bound to magnetic beads alone (36% of the maximal signal obtained for FibG_190-235) or the signal obtained for pCMV vector control transfections (4% of the maximal signal obtained for A18 apo(a)/Lp(a)), respectively, was subtracted. Because of some gel to gel variability, statistical analysis of data are expressed as the means ± S.E. of at least two (cell culture supernatants) and five (purified Lp(a) and human plasma) independent experiments. Relative values are expressed as percentages of the maximal binding set at 100% for calculation purposes.

Immunoprecipitation and Immunoblotting-- Cell culture supernatants adjusted to end concentration of 0.6% SDS and 1% Triton X-100 in HBS were treated with proteinase inhibitors (1 mM PMSF, 5 µg/ml of each aprotinin and leupeptin) and subsequently precleared with 100 µl of Pansorbin cells (Calbiochem) at 4 °C for 30 min. After centrifugation the supernatants were immunoprecipitated overnight with a monospecific polyclonal rabbit anti-apo(a) antibody (Behringwerke AG, Marburg, Germany) at 5 µg/ml final antibody concentration, followed by addition of 40 µl of protein A-Sepharose (Amersham Pharmacia Biotech) for the last 2 h. Immunoprecipitates were collected by centrifugation for 2 min at 10000 g at 4 °C and washed four times with 1 ml of washing buffer (0.2% SDS, 1.25% Triton X-100, 1 mM PMSF, 5 µg/ml of each aprotinin and leupeptin in HBS). The final pellet was resuspended in 15 µl of SDS-PAGE sample buffer and subjected to reducing SDS-PAGE on commercially available on 4-12% Bis-Tris- or 3-9% Tris-acetate gels (Novex, San Diego, CA). Immunoblot analysis was performed using apo(a)-specific monoclonal antibody 1A2 (33) as described (1).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of Fibrinogen Clones Interacting with apo(a) Kringle IV Type 6 in the Yeast Two-hybrid System-- In this study, we have attempted to identify novel apo(a)/Lp(a) binding proteins by the GAL4 two-hybrid interaction trap (34) approach screening a human liver cDNA library with the unique apo(a) kringle IV type 6 as a bait. This kringle IV subtype was used for the library screening because of its low self-activation rate compared with other kringle IV subtypes (not shown). To identify cDNA clones encoding proteins that interact with apo(a) kringle IV type 6, we transformed the yeast host strain HF7c, carrying a GAL4-HIS3 selection and GAL4--galactosidase reporter gene with the kringle IV type 6-expression plasmid as a bait and a human liver cDNA library with the cDNA fused to the GAL4 activation domain. A total of 2.4 × 10⁶ transformants were subjected to positive genetic growth selection on HisLeuTrp plates, containing 5 mM 3-amino-1,2,4-triazole. 25 colonies stained positive for -galactosidase activity. To determine whether activation of the GAL4-dependent reporter genes reflects a specific interaction of the encoded proteins with the kringle IV type 6 bait, each cDNA clone was rescued from yeast colonies and retransformed into the same yeast strain in the absence or presence of the GAL4 kringle IV type 6 bait expression plasmid. Additionally, we transformed each of the putative ligand expression plasmids with an expression plasmid with the GAL4 DNA binding domain fused to a nonspecific protein (p53), which is not expected to interact with proteins that bind to apo(a). Four of the 25 clones showed specific interaction, activating -galactosidase expression exclusively in the presence of the GAL4 kringle IV type 6 bait. Sequence analysis of these positive clones using primers in the pGAD10-flanking sequence revealed two cDNA sequences of so far unknown identity and two cDNA sequences showing a 100% match to fragments of the human fibrinogen - and -chain, respectively. The fibrinogen -chain clone (K6/) extended from residues 1-310, and the fibrinogen -chain clone (K6/) extended from residues 189-295 (Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Interaction of fibrinogen clones with apo(a) kringle IV type 6 bait in the two-hybrid screening. Position of the positive clones K6/ and K6/ (arrows) obtained on screening of a human liver cDNA library are shown next to the corresponding fibrin(ogen) chain. The consensus binding domains for apo(a) kringle IV type 6 defined by the -chain clone K6/1 (arrow) are shaded. Also included is the amino acid sequence alignment and consensus sequence of this conserved - and -chain region. Peptide sequences of FibG190-235 (bold) and FibG207-235 (underlined) are marked. Lysine to alanine exchanges in the peptide sequences are indicated underneath the -chain sequence. Residue numbering corresponds to native human fibrinogen, in which the -chain runs from Gln1 to Gln461 and the -chain runs from Tyr1 to Val411. Scissors mark the thrombin cleavage site on the fibrinogen -chain.

Further Characterization of the Binding Site-- The matched sequences of both clones are located within the carboxyl-terminal globular domain of fibrinogen. These carboxyl-terminal sections of the fibrinogen - and -chains are characterized by high sequence and structural homology (35). A carboxyl-terminal fragment of K6/ containing the sequence overlapping with K6/ was cloned in fusion with the GAL4 activation domain, and the resulting plasmid K6/1 (Fig. 1) was assayed in yeast cells for interaction with the GAL4 DNA binding domain-apo(a) kringle IV type 6 fusion protein to localize the region of the fibrinogen -chain clone that mediated binding to apo(a) kringle IV type 6. The positive result in the two-hybrid assay showed that the 64 carboxyl-terminal residues of fibrinogen -chain that correspond by sequence alignment to amino acids 189-246 of fibrinogen -chain are sufficient for binding to apo(a) kringle IV type 6.

Moreover, we performed a two-hybrid assay with the fibrinogen clones K6/1 and K6/ using the apo(a) kringle IV types 2, 5, 6, 7, 8, 9, and 10 as baits. Kringle subtypes 5, 7, and 10 showed a high rate of self-activation. Therefore, the interaction could not be evaluated (not shown). As expected, apo(a) kringle IV type 6 significantly interacted with the fibrinogen - and -chain in both strains, despite some quantitative differences observed. However, kringle IV types 2, 8, and 9 did not interact (Fig. 2). The expression of the bait and prey proteins has been tested by analyzing the extracted yeast proteins after SDS-PAGE and Western blot by GAL4 fusion domain-specific antibody. Expressed proteins of the expected molecular weights have been detected in the corresponding yeast protein extracts (not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Interaction of fibrinogen clones with apo(a) kringle IV types 2, 6, 8, and 9 baits in the two hybrid -galactosidase assay. The fibrinogen -chain subclone K6/1 and the -chain clone K6/ were assayed for interaction with four different kringle baits in yeast strains CG1945 and HF7c. The assay was performed as described under "Experimental Procedures."

Confirmation of apo(a)/Lp(a)-fibrin(ogen) Interaction Using Peptide-based Pull-down Assays-- The crystal structure of the fibrinogen module encompassing residues 144-411 (35) and the crystal structure of fibrinogen fragment D (36) revealed that the minimal kringle IV type 6 binding sequences of the - and -chains are located in a region of similar overall structure consisting of two helices interrupted by the solvent exposed B1-loop (35). We tested the biotinylated peptides comprising 190-235 (FibG_190-235) and 207-235 (FibG_207-235), respectively, as well as the corresponding lysine exchange mutants FibKA_190-235 and FibKA_207-235 for in vitro interaction with apo(a)/Lp(a). All four fibrinogen peptides were able to specifically pull down Lp(a) from human plasma, whereas the control peptide was not (Fig. 3). There was a reduced binding for the Lys/Ala peptide exchange mutant FibKA_207-235 in relation to wt fibrinogen peptides and FibKA_190-235, indicating that lysines affect binding of the shorter fibrinogen peptides (207-235) to Lp(a). However, and to our surprise, lysine residues appear not to be essential for this interaction.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Pull-down of Lp(a) with fibrinogen peptides from human plasma. A, evaluation of a representative experiment was determined by densitometric quantification. The maximal binding was set at 100% for calculation purposes. Statistical evaluation of five pull-down experiments expressed as the means ± S.E. is presented below. Control, 15.4% ± 1.7%; FibG190-235, 80.7% ± 21.2%; FibKA190-235, 97.7% ± 31.9%; FibG207-235, 100% ± 33.0%; FibKA207-235, 32.0% ± 4.2%. B, interacting complexes were formed in plasma by using four different fibrinogen peptides and control as indicated. Immunodetection for the presence of apo(a) was performed using anti-apo(a) mAb 1A2. A representative experiment is shown.

Next, employing purified Lp(a) instead of human plasma samples, again all fibrin(ogen) peptides bound Lp(a), whereas the control peptide did not (Fig. 4). Here we observed reduced binding efficiencies of the lysine exchange mutants FibKA_190-235 and FibKA_207-235 compared with the corresponding wt sequences FibG_190-235 and FibG_207-235.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Pull-down of Lp(a) purified from human plasma with fibrinogen peptides and control. A, statistical evaluation of the experiment was determined by densitometric quantification. The maximal binding was set at 100% for calculation purposes, and data are expressed as the means ± S.E. (n = 5). B, interacting complexes were formed in a solution of purified human Lp(a) by using four different fibrinogen peptides and a control peptide as indicated. The double band represents a PAGE artifact. Immunodetection for the presence of apo(a) was performed using anti-apo(a) mAb 1A2.

Interaction of FibG_207-235 with apo(a)/Lp(a) from Supernatants of Transfected HepG2 Cells-- HepG2 cell supernatants containing distinct apo(a)/Lp(a) derivatives were obtained after transient transfection of HepG2 cells with apo(a) expression vector constructs A18 wt, KV-P, and KIV 8-P representing wt apo(a) with 18 kringle units and two 3' deletions of different lenght (as outlined in Fig. 5). Equal amounts of apo(a)/Lp(a) (as measured by enzyme-linked immunosorbent assay) were used for pull-down experiments with FibG_207-235 and immunoprecipitation. As a result, A18 wt Lp(a) as well as KV-P Lp(a) showed a strong interaction with FibG_207-235. In contrast, only marginal binding of KIV 8-P apo(a) to FibG_207-235 was observed (Fig. 6, A and B). The three distinct forms of apo(a)/Lp(a) were expressed equally as verified by immunoprecipitation (Fig. 6C).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Schematic representation of wt and mutant apo(a) encoded by cDNA expression plasmids. Kringle IV types are numbered from 1 to 10. The W4174R mutation in pCMV A18-Arg and the premature stop codon in pCMV KIV 5-8 are indicated. SP, signal peptide; V, kringle V; PD, protease domain. The cDNA is flanked at the 5' end by the cytomegalovirus enhancer/promotor and a heterologous intron and at the 3' end by a SV40 polyadenylation site (1, 26).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Pull-down of apo(a)/Lp(a) with FibG207-235 and a control peptide from supernatants of HepG2 cells ectopically expressing different apo(a) derivatives. A, statistical evaluation of the experiment was determined by densitometric quantification. The maximal binding was set at 100% for calculation purposes, and data are expressed as the means ± S.E. (n = 3). B, interacting complexes were formed in cell supernatants containing equal amounts of apo(a)/Lp(a). Immunodetection for the presence of apo(a) was performed using anti-apo(a) mAb 1A2. C, immunoprecipitation of apo(a)/Lp(a) derivatives from HepG2 cell supernatants. Immunoprecipitates were formed using a monospecific polyclonal antibody raised against apo(a). Immunodetection for the presence of apo(a) was performed using anti-apo(a) mAb 1A2.

Influence of Lysines or LBS on the Interaction of Fibrinogen Peptides with apo(a)/Lp(a) from Supernatants of Transfected HepG2 Cells-- HepG2 cells were transfected with the apo(a) expression vector constructs A18 wt and the mutant A18-Arg (Fig. 5). The latter is A18 apo(a) with a Trp-4174 to Arg substitution in the LBS of kringle IV type 10, which renders A18-Arg Lp(a) unable to bind to lysine-Sepharose (26). Equal amounts of apo(a)/Lp(a) (as measured by enzyme-linked immunosorbent assay) from the resulting supernatants were used for pull-down experiments with FibG_207-235 and the mutant peptide FibKA_207-235. The influence of the LBS on the interaction with the fibrinogen peptides was further evaluated by adding the lysine analogue EACA to the binding buffer. Consistent with pull-down experiments from plasma and with purified Lp(a), pull-down of A18 wt Lp(a) from HepG2 supernatants with the mutant peptide FibKA_207-235 resulted in decreased binding efficiency compared with the wt FibG_207-235 peptide. Binding of FibG_207-235 to A18 wt Lp(a) was significantly reduced after preventing lysine-dependent interactions by addition of EACA. Binding of both fibrinogen peptides to the A18-Arg mutant Lp(a) was greatly reduced when compared with the binding to A18 wt Lp(a). Again further reduction in binding of both peptides was observed in the presence of EACA (Fig. 7).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Pull-down of apo(a)/Lp(a) with fibrinogen peptides and a control peptide from supernatants of HepG2 cells ectopically expressing different apo(a) derivatives in the presence and absence of EACA. A, statistical evaluation of the experiment was determined by densitometric quantification. The maximal binding was set at 100% for calculation purposes, and data are expressed as the means ± S.E. (n = 2). B, interacting complexes were formed using fibrinogen peptides FibG207-235 (FibG) and FibKA207-235 (FibKA) and a control peptide in cell supernatants containing equal amounts of apo(a)/Lp(a). The reaction was performed with (+) or without () 0.2 M EACA in incubation buffer. Immunodetection for the presence of apo(a) was performed using anti-apo(a) mAb 1A2. Note that the right panel showing A18-Arg Lp(a) was exposed three times longer than the left panels showing A18 wt Lp(a) and control. C, immunoprecipitation of apo(a)/Lp(a) derivatives from HepG2 cell supernatants. Immunoprecipitation and immunodetection of apo(a) was performed as described for Fig. 6C.

Interaction of FibG_207-235 with apo(a) from Supernatants of Transfected CHO Cells-- To address the question whether the low to absent binding of FibG_207-235 to KIV 8-P apo(a) is due to the presence of this mutant form as free apo(a) exclusively, we transfected apoB100 negative CHO cells with the apo(a) expression vector constructs and performed pull-down experiments with equal amounts of apo(a) from the cell supernatants. We observed strong binding of A18 wt apo(a) and reduced binding of KV-P apo(a) to FibG_207-235 (Fig. 8, A and B). Binding to KIV 8-P was below the value obtained by the control peptide. Equal expression of the three apo(a) forms was controlled by immunoprecipitation (Fig. 8C).

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Pull-down of apo(a) with FibG207-235 and a control peptide from supernatants of CHO cells ectopically expressing different apo(a) derivatives. A, statistical evaluation of the experiment was determined by densitometric quantification. The maximal binding was set at 100% for calculation purposes, and data are expressed as the means ± S.E. (n = 3). B, interacting complexes were formed in cell supernatants containing equal amounts of apo(a). Immunodetection for the presence of apo(a) was performed using anti-apo(a) mAb 1A2. C, immunoprecipitation of apo(a) derivatives from CHO cell supernatants. Immunoprecipitation and immunodetection of apo(a) was performed as described for Fig. 6C.

Moreover, we tested binding of the mutant apo(a) KIV 5-8 to FibG_207-235. This mutant which lacks the kringle IV types 5-8 and does not form Lp(a) particles (26) also showed interaction with the fibrinogen peptide FibG_207-235 at low concentrations (not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fibrinolysis is a surface-controlled process leading to the plasmin-catalyzed proteolysis of fibrin. The adsorption of plg to fibrin and the surface dynamics of fibrin and plg transformation during this process have been well characterized (37). The plg paralogue apo(a) also binds to fibrinogen. However so far there is little information about the apo(a) fibrinogen interaction at the molecular level. We have confirmed direct physical interaction between apo(a) and fibrinogen by the yeast two-hybrid system. With the apo(a) unique kringle IV type 6 as bait, we have isolated two of the three fibrinogen subunits (fibrinogen - and -chain) from a human liver cDNA library, and we were able to localize the apo(a) binding sites within these two individual fibrinogen subunits. The strongest similarities between the fibrinogen - and -chain subunits are located in their carboxyl termini with stretches of about 250 amino acids sharing marked homology. The two fibrinogen sequences isolated from the positive two-hybrid clones revealed 64 overlapping amino acids that have been shown to be sufficient for interaction with kringle IV type 6 by the two-hybrid -galactosidase filter assay. In cross-linked fibrin this interacting amino acid sequence is a constituent of fragment D. It is noteworthy that also the plg binding site of fibrin has been localized to the carboxyl-terminal fragment D (38). In the intact fibrin polymer the two proposed interacting carboxyl-terminal domains are located in close proximity within the two distal globular domains of the ()-fibrinogen dimer (36). These globular domains at the surface of fibrin seem to be easily accessible for large molecules as Lp(a), and it has indeed been demonstrated that Lp(a) blocks specifically carboxyl-terminal lysine residues on the surface of fibrin (19). Moreover, binding leads to a large conformational change that may prevent other molecules from interacting with fibrin(ogen) (39).

Because of the high sequence and structural homology in the binding region of the - and -chain of fibrinogen defined by two-hybrid interaction with apo(a) kringle IV type 6, we restricted the further investigation of the binding site in a more physiological environment to the -chain that does not contain the insertion and extra disulfide bridge found in the corresponding -chain B1-loop region (35). The shorter peptide FibG_207-235 contains the two helices flanking the loop, whereas FibG_190-235 contains an additional third helix preceeding the loop. We reproducibly demonstrated interaction of wt fibrinogen peptides (FibG) and corresponding mutated peptides (FibKA) with Lp(a) from different sources indicating a specific interaction. The minor quantitative differences observed between pull-downs (as in Figs. 3 and 4) are most likely due to different experimental setups e.g. buffer composition or state of native versus purified Lp(a)/apo(a). Taken together the four fibrinogen peptides significantly interacted with Lp(a)/apo(a).

However, our study does not exclude additional binding sites for apo(a)/Lp(a) in the globular domain of fibrin(ogen). The occurrence of more than one binding site has been demonstrated for other fibrin ligands. FibG_190-235 overlaps with part of the MAC-1 binding site characterized by Tang et al. (40). A subsequent report described a second binding motif for MAC-1 on a distant -sheet of the carboxyl-terminal fibrinogen domain (41). This is due to the close proximity of some distant antiparallel -sheets in the folded domain (35).

So far studies investigating the interaction of apo(a)/Lp(a) and fibrin(ogen) report that binding of Lp(a) or apo(a) to fibrin(ogen) is inhibited by the lysine analogue EACA, indicating that LBSs in apo(a) are predominantly involved in fibrin(ogen) binding (42, 43). A sequence comparison between human and rhesus monkey Lp(a), which differ in their lysine binding activities, led to the suggestion that kringle IV type 10 of apo(a) plays a dominant role in the binding of Lp(a) to lysine (44). Transgenic mice with a mutated kringle IV type 10 LBS show reduced lesion development (45), indicating a key role for this LBS in the pathogenicity of Lp(a). A recent report by Lou et al. (46) implicated fibrin(ogen) as one of the major sites of apo(a) accumulation in the vessel wall of apo(a) transgenic mice preceeding atherosclerosis. Weak LBS have also been identified in apo(a) kringle IV types 5-8 (26, 47-49). By comparison of the binding behavior of wt apo(a)/Lp(a) and apo(a)/Lp(a) with a mutated LBS in kringle IV type 10, a second LBS outside kringle IV type 10 was suggested to be responsible for fibrin binding (50). The LBS in kringle IV type 10 is accessible both in free apo(a) and in apo(a) covalently linked to low density lipoprotein, whereas the so called minor binding site located on kringle IV types 5-8 is masked in Lp(a) and becomes only accessible by the use of detergents (26) or on release of low density lipoprotein from the particle by reducing agents (50).

Although we identified the fibrin(ogen) peptide motif binding to apo(a)/Lp(a) through its interaction with kringle IV type 6 in the yeast two-hybrid system, the pull-down experiments indicate that kringle IV type 6 is not a major fibrin(ogen) binding site in intact apo(a)/Lp(a). Rather the interactions of FibG_207-235 with the apo(a) deletion mutants indicate that this peptide interacts with some other kringle IV motifs in apo(a) e.g. kringle IV type 10. An interaction of kringle IV type 10 (and some of the other kringle IV types) with the fibrin(ogen) clones could not be demonstrated in the two-hybrid system because of the strong self-activation of this kringle subtype (not shown). The ability of the mutant KIV 5-8 apo(a) to interact with FibG_207-235 although the minor LBS has been deleted also shows that the interaction of kringle IV types 5-8 with FibG_207-235 is not essential. Moreover, this mutant protein interacts with FibG_207-235 even at low concentrations (not shown). Of the four tested apo(a) mutants only proteins containing kringle IV types 1-4, 9, and 10 were able to bind to the fibrin(ogen) peptide. Deletion of the carboxyl-terminal kringle V and protease domain considerably weakened the interaction with FibG_207-235 (Fig. 6). The binding may also be context-dependent and influenced by the overall conformation of apo(a) in the particle. This might be a reason for the marginal binding of the splice site mutant KIV 8-P apo(a) to FibG_207-235. This mutant apo(a) contains kringle types 1-7 and only 34 amino acids of kringle IV type 8 that are likely to be misfolded, probably influencing the conformation of the mutant molecule.

Importantly, however, we observed only minimal residual binding of FibG_207-235 to the mutant A18-Arg Lp(a), suggesting a major role of the LBS in apo(a) kringle IV type 10 for the FibG_207-235 and apo(a)/Lp(a) interaction (Fig. 7). Mutations of lysine residues in the fibrinogen peptide FibG_207-235, the W4174R mutation in the apo(a) KIV-10 LBS and the presence of EACA all resulted in a reduced interaction of the fibrinogen peptide with apo(a)/Lp(a). This clearly demonstrates the LBSs and particularly the LBS in apo(a) kringle IV type 10 is involved in the interaction. However, EACA alone did not totally block the interaction of wt FibG_207-235 with wt apo(a)/Lp(a). On the other hand EACA reduced binding even when both the LBS in apo(a) kringle IV type 10 and the lysine residues in the fibrinogen peptide were mutated. This suggests a more complex interaction, which in addition to the LBS in kringle IV type 10 involves other sites. There is one publication reporting a lysine-insensitive component of the interaction of isolated apo(a) kringle IV type 10 and plasmin modified fibrinogen (51). It further suggests an effect of EACA on apo(a)/Lp(a) beyond the inhibition of lysine binding. This might be due to changes in Lp(a) conformation promoted by EACA as demonstrated by Fless et al. (52). Taken together, our data for the first time clearly identify an apo(a)/Lp(a) binding site in fibrin(ogen). They also confirm the LBS in apo(a) kringle IV type 10 as one fibrin(ogen) binding site in apo(a)/Lp(a).

The interaction of Lp(a) and fibrin(ogen) is well established, but so far no binding site in fibrin(ogen) had been identified. Here we present the FibG_207-235 sequence as a novel and sufficient binding site for Lp(a)/apo(a) in vitro. Our finding may have practical consequences because the FibG_207-235 sequence may represent a pharmaceutical target site to interfere with the pathological interaction of Lp(a)/apo(a) and fibrinogen. However, more work has to be done to elucidate the full functional relevance of this interaction in vivo.

	ACKNOWLEDGEMENT

We are grateful to Dr. J. Müller (Roche, Penzberg, Germany) for providing recombinant apo(a) constructs.

	FOOTNOTES

^* This work was supported in part by grants from the EC-Biomed 2 shared cost project P95-0898, the Austrian "Fond zur Förderung der wissenschaftlichen Forschung" (P11695-MED and P12819-GEN), and the "Legerlotz-Stiftung" and Austrian Federal Bank Grant 7665/1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Inst. for Medical Biology and Human Genetics, Schoepfstr. 41, A-6020 Innsbruck, Austria. E-mail: Gottfried.Baier@uibk.ac.at.

Published, JBC Papers in Press, September 8, 2000, DOI 10.1074/jbc.M003640200

	ABBREVIATIONS

The abbreviations used are: Lp(a), lipoprotein(a); apo(a), apolipoprotein(a); LBS, lysine binding site; plg, plasminogen; PMSF, phenyl-methyl-sulfonyl-fluoride; CHO, Chinese hamster ovary; EACA, -aminocaproic acid; HBS, HEPES-buffered saline; wt, wild type; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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