Full-length and truncated forms of vitronectin provide insight into effects of proteolytic processing on function

Biochimica et Biophysica Acta 1545 (2001) 289^304 www.elsevier.com/locate/bba

Full-length and truncated forms of vitronectin provide insight into e¡ects of proteolytic processing on function Angelia D. Gibson 1 , Cynthia B. Peterson * M407 Walters Life Sciences Building, Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA Received 11 April 2000; received in revised form 5 December 2000; accepted 5 December 2000

Abstract A genetic polymorphism in the vitronectin allele directs the production of two distinct forms of the 459 amino acid glycoprotein. A methionine present at position 381 favors production of the single-chain form of vitronectin, while threonine at this position increases the susceptibility of vitronectin to cleavage just beyond its heparin-binding domain at residue 379. This reaction gives rise to a disulfide-bonded, two-chain form of vitronectin. In order to investigate the functional significance of the vitronectin polymorphism, the baculovirus system has been used to express recombinant full-length vitronectin and a truncated form of the molecule that represents the 62-kDa fragment of two-chain vitronectin. Both forms of vitronectin bind and neutralize heparin anticoagulant activity. The proteins also bind PAI-1 and stabilize its active conformation. These experiments suggest that the C-terminal 80 amino acids do not confer a functional difference in the two allelic variants. Immunoassays and gel filtration experiments indicate that both full-length and truncated recombinant forms of vitronectin are multimeric. Together with other reports from this laboratory, these results provide information regarding the primary binding sites for two vitronectin ligands and further define regions that may be involved in multimerization of the protein. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Vitronectin; Heparin; Serpin; Proteolytic processing

1. Introduction

Abbreviations: PAI-1, plasminogen activator inhibitor 1; SDS^PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; h.p.i., hours post infection ; BSB, blue Sepharose bu¡er; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-bu¡ered saline ; BSA, bovine serum albumin; FPLC, fast performance liquid chromatography; PBS, phosphate-bu¡ered saline; HRP, horseradish peroxidase * Corresponding author. Fax: +1-865-974-6306; E-mail: [email protected] 1 Present address: Physicians' Education Resource, 3535 Worth Street, Dallas, TX 75246, USA.

Vitronectin is a human plasma protein that binds to a number of structurally dissimilar molecules. A notable interaction is the binding of vitronectin to heparin, inhibiting its characteristic rate enhancement on the thrombin/antithrombin reaction [1]. The glycoprotein binds plasminogen activator inhibitor 1 (PAI-1), stabilizing its inhibitory activity against serine proteases [2,3]. Also, association with vitronectin alters the shape and solubility of the terminal complement complex, preventing its insertion into cell membranes [4]. Furthermore, vitronectin acts as a molecular tether; it facilitates the adhesion

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of cells to the extracellular matrix, mediates interactions between the thrombin/antithrombin complex and epithelial cells, and promotes the adsorption of bacteria onto endothelial cells [5]. The structure of vitronectin is organized into several discrete domains, which provide the necessarily broad repertoire of binding epitopes for target ligands. The N-terminal somatomedin B domain is likely the key binding site for integrins [6], PAI-1 [7^10], and the urokinase/plasminogen activator receptor [11], and a central hemopexin domain is thought to mediate binding to some bacteria [12,13]. A highly charged sequence near the C-terminus mediates binding to heparin [14^16] and collagen [17,18]. The C-terminal domain containing the heparin-binding sequence may also serve as a binding site for plasminogen [19], complement factors [20], and perforin [20,21], and as a secondary binding site for PAI-1 [22^24] and/or the urokinase receptor [11]. Enumeration and localization of discrete binding sites for these and other ligands is an ongoing e¡ort in the work on vitronectin from a variety of laboratories. A feature of vitronectin that has received much attention over the years is its conformational lability. Although the majority of circulating vitronectin is monomeric, the protein also adopts an oligomeric conformation. Such higher ordered oligomeric forms of the glycoprotein have been detected in the extracellular matrix [25], platelet releasates [26], and within the K-granules of platelets [26]. As a result of its increased valency, the multimeric form of vitronectin represents a more potent molecule, binding to ligands like heparin [27,28], PAI-1 [29], collagen [17], and integrins [30] with higher `apparent' a¤nities than does native vitronectin. In vivo, vitronectin can also exist in a full-length or a two-chain cleaved form. Cleavage occurs by a yet unidenti¢ed trypsin-like protease that clips vitronectin at arginine 379, just beyond the heparin-binding domain. The presence of threonine rather than methionine at position 381 increases the likelihood that vitronectin will be cleaved at position 379 [31]. The resulting molecule is a two-chain form of vitronectin: a 62-kDa N-terminal heavy chain, containing the PAI-1 and heparin-binding domains, and a 10kDa C-terminal light chain. A single disul¢de bond holds the two chains together.

Early proteolysis studies, which were conducted to identify functional domains in vitronectin, revealed that the molecule is most susceptible to proteolytic cleavage just beyond the somatomedin B domain and within the heparin-binding sequence [14,22,32]. Cleavage within either of these areas by enzymes like plasmin or trypsin dramatically reduces a¤nity of vitronectin for many of its target ligands [22,33,34]. But how does cleavage at position 379, which is strategically located at the C-terminus of the major functional domain of vitronectin, a¡ect the function of the molecule? Does this cleavage regulate vitronectin function? Do the two forms of vitronectin vary in ligand-binding activity? Is either more prone to multimerization? The studies that are reported here were designed to investigate whether there are functional di¡erences in the full-length and a 62-kDa form of vitronectin. The baculovirus system has been utilized to express recombinant, full-length vitronectin as well as a truncated molecule, which corresponds to the heavy chain of the proteolysed two-chain vitronectin. A variety of immunological, chromatographic and kinetic assays were performed to characterize the conformation and functional status of the recombinant proteins. The results indicate that the oligomeric state, heparin-binding activity and PAI-1-binding activity of full-length and two-chain vitronectin are indistinguishable. 2. Materials and methods 2.1. Materials Plasma-derived vitronectin was puri¢ed with modi¢cations as described [35,36]. Multimeric vitronectin was generated according to the method of Zhuang et al. [37]. PAI-1 was puri¢ed as described by Lawrence et al. [38] and was the generous gift of Dr. Joseph Shore (Henry Ford Research Institute, Detroit, MI). Human thrombin and antithrombin were generously provided by Dr. Frank C. Church (Department of Hematology, University of North Carolina, Chapel Hill, NC). Monoclonal anti-vitronectin antibodies were obtained from Quidel. The conformation-speci¢c monoclonal antibodies mAb 1244, mAb 153 and mAb 8E6 were generously provided by Dr. Diet-

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mar Sei¡ert (Scripps, La Jolla, CA). Polyclonal antivitronectin antibodies were produced at Rockland Laboratories. Antibodies speci¢c for bovine albumin were obtained from Sigma Chemical Company. Goat anti-rabbit IgG and horse anti-mouse IgG, both covalently linked with peroxidase, were obtained from Vector Laboratories. An immunoa¤nity column was made by coupling the monoclonal antibody from Quidel to a protein G column using the Immunopure Protein IgG Orientation Kit (Pierce) [39]. 2.2. Cell culture Cultures of Spodoptera frugiperda (Sf9) or Trichoplusia ni (Hi 5) were grown routinely as monolayers or in suspension in side-arm spinner £asks with gentle stirring (100 rpm) at room temperature. Sf9 cells were typically maintained as monolayer cultures in Corning T-75 cm2 tissue culture £asks in Ex-Cell 400 (J.R.H. Biosciences) or Ex-Cell 401 (J.R.H. Biosciences) containing 10.0% Graces Insect Cell Medium (Gibco BRL) and 1.0% fetal bovine serum (Gibco BRL). Hi 5 cells were grown in Ex-Cell 400 or ExCell 401 insect cell medium without serum supplement. All growth media was supplemented with L-glutamine (Gibco BRL) to a ¢nal concentration of 100 mM and 20 Wg/ml gentamicin (Gibco BRL). Additionally, penicillin (50 U/ml) and streptomycin (50 Wg/ml) were added to cultures as needed. 2.3. Virus generation A recombinant transfer plasmid (pBlueBac-VN) for generating the baculovirus encoding full-length, human vitronectin was obtained from Dr. David Sane (Winston-Salem, NC) [40]. Details regarding the construction of the transfer plasmid are found in Zhao and Sane [40]. Note that this vitronectin cDNA encodes Met-381, the allelic variant that is associated with the single-chain form of human plasma vitronectin. To generate recombinant virus, Sf9 cells were cotransfected with 1 Wg of linearized wild-type AcNPV viral DNA and 4 Wg pBlueBac-VN by the cationic liposome method. Two days after transfection, the medium from cells was serially diluted 1:10, 1:40 and 1:1000 and the dilutions were screened for recombinant virus by plaque assay [41]. Plaques from

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recombinant virus were identi¢ed, and the virus was then subjected to a second round of plaque puri¢cation before being ampli¢ed. The recombinant virus encoding the large N-terminal fragment of vitronectin, residues 1^380, was generated using the Fastbac system (Gibco BRL). Polymerase chain reaction (PCR) was used to amplify the gene sequence encoding the vitronectin signal sequence and amino acids 1^382 using the sense primer T7, 5P-TAA TAC GAC TCA CTA TAG GG-3P, and antisense primer PCR4, 5P-CCA CGT GGC GCG GGA CGG CCG GCG GGA-3P. The PCR4 oligonucleotide encodes Thr-381, the allelic variant of human vitronectin that is associated with processing to the two-chain (62-kDa+10-kDa) form of the protein. The PCR-ampli¢ed DNA was subcloned into the TA cloning vector pCRII (Invitrogen). The fragment corresponding to amino acids 1^382 was excised by cutting the plasmid with BamHI and EcoRI and cloned into the same sites in pVL1393. An EagI digestion was performed, excising a small fragment at the 3P end of the gene, to produce an in-frame stop codon that truncated the recombinant gene product at position 380. The vitronectin gene fragment was then excised using BamHI/PstI and was cloned into the same sites in pFastBac. The resulting clone, pFastbac594, was then transformed to the commercial Escherichia coli strain, DH10bac, which contains the entire baculovirus genome on a large circular plasmid. The transformed bacteria were plated on LB plates containing 10 Wg/ml tetracycline, 7 Wg/ml gentamicin, 50 Wg/ml kanamycin, 40 Wg/ml IPTG and 300 Wg/ml Bluo-gal, a substrate for L-galactosidase. A blue/white screen identi¢ed clones containing the recombinant bacmid, and bacmid DNA was then puri¢ed from white colonies grown in liquid cultures. Insect cells were transfected with the recombinant bacmid DNA using Cellfectin reagent (Gibco BRL) according to the manufacturer's recommendations. 2.4. Virus ampli¢cation Stocks of recombinant baculovirus encoding Bac594 or BBVN were ampli¢ed by infecting 30-ml cultures of Sf9 cells (1U106 cells/ml) with 2 ml of the appropriate virus stock. The spent medium containing ampli¢ed virus was harvested 72 h post infection

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(h.p.i.) by gentle centrifugation in a Beckman JA-17 rotor at 4500 rpm. The supernatants were removed aseptically and stored at 4³C in light-protective containers until use. Ampli¢ed virus stocks were screened for protein production by varying the volume of virus that was added to monolayer cultures of Hi 5 cells (0.3^ 1.0U106 cells/ml) in serum-free media. The cells and supernatants were separated by centrifugation 72 h.p.i. Cells were suspended to 1/10th the original culture volume with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS^PAGE) loading dye containing L-mercaptoethanol. Supernatants were mixed 1:1 (v:v) with SDS^PAGE loading dye. The samples were boiled for 5 min and analyzed by immunoblotting. The infection conditions that yielded the highest amount of undegraded protein in the media were replicated in large-scale suspension cultures to produce protein for puri¢cation. 2.5. Immunoblotting Protein samples were separated by PAGE on 10% polyacrylamide SDS gels [42]. Proteins were transferred to nitrocellulose ¢lters using a Semi-Dry Blotter (Bio-Rad) in transfer bu¡er (0.35 M glycine, 25 mM Tris, containing 20.0% methanol). The ¢lters were blocked as with 10% non-fat dry milk in phosphate-bu¡ered saline (PBS). The nitrocellulose membrane was washed three times after this and all incubation steps with PBS containing 0.05% Tween. To detect recombinant vitronectin the ¢lter was incubated in a 1:2000 dilution of polyclonal anti-vitronectin rabbit serum in a 2.0% solution of non-fat dry milk in PBS. The blot was then incubated with a 1:1333 dilution of horseradish peroxidase (HRP)linked goat anti-rabbit antibody in a 20 ml solution of 2.0% milk in PBS. Immunostained protein bands were visualized by developing in a freshly prepared solution of 30 ml PBS containing 50 mg/ml 4-chloronaphthol and a 1:3000 dilution of 30% H2 O2 . 2.6. Protein puri¢cation Large-scale suspension cultures of Hi 5 cells were infected with recombinant virus at a density of 1U106 cells/ml using the appropriate volume of ampli¢ed virus stock determined empirically as de-

scribed above. The spent medium from the cells was harvested 72 h.p.i. by centrifugation at 4500 rpm in a Beckman JA-14 rotor for 20 min at 4³C. A cocktail of protease inhibitors was added to the ¢nal concentrations described: 100 Wg/ml phenylmethylsulfonyl £uoride, 0.5 Wg/ml leupeptin, 1.0 Wg/ml aprotinin, 1 Wg/ml pepstatin and 1.0 mM EDTA. For long-term storage, 0.05% NaN3 was added to prevent the growth of microorganisms. The medium was ¢ltered through a 0.45-Wm ¢lter and mixed with 15 ml Blue-Sepharose CL-B (Pharmacia) equilibrated in blue Sepharose bu¡er (BSB; 50 mM Tris, 0.15 M NaCl, 0.1 mM EDTA, pH 7.4). The Blue-Sepharose slurry was stirred gently at 4³C for 12^16 h. The resin was then poured into a 3U 10-cm column and washed with 100 ml BSB at a £ow rate of 1 ml/min. Bound protein was eluted from the Blue-Sepharose by washing the column with BSB containing 2.0 M NaCl. Fractions containing protein were identi¢ed by immunoblotting. Those fractions were pooled and dialyzed to low-salt BSB, and the protein was passed over an immunoa¤nity column. The column was washed extensively with BSB. Bound protein was eluted using Gentle Ab/Ag Elution Bu¡er (Pierce) in 2.0-ml fractions. Fractions were analyzed by SDS^PAGE followed by immunoblotting. The fractions were immediately pooled and dialyzed to BSB. After dialysis, the immunopure protein was pooled, concentrated to at least 0.5 mg/ml, and stored at 4³C. The protease inhibitor cocktail was added to all bu¡ers used for chromatography, dialysis or storage. Purity was assessed by the presence of a single band on a Coomassie-stained reducing SDS polyacrylamide gel and by comparing the concentration of vitronectin determined in a quantitative enzymelinked immunosorbent assay (ELISA) to the total protein concentration determined by the bicinchoninic assay method [43] using the Pierce BCA Protein Assay Reagent Kit. As has been our practice in published work on multimeric vitronectin derived from human plasma, vitronectin concentrations are always expressed as protomer concentrations so that assay results can be compared directly. The oligomeric state of the puri¢ed recombinant proteins was assessed by sedimentation equilibrium in a Beckman XL-A ultracentrifuge using our previously published method [37].

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2.7. Vitronectin immunoassays

2.8. Solid-phase binding assays

Recombinant vitronectin was quanti¢ed using a competitive ELISA. Microtiter plates were coated with 50 Wl of a 1.0 Wg/ml solution of native human vitronectin at 4³C for 16 h. After washing the plates three times with Tris-bu¡ered saline (TBS; 20 mM Tris, 0.15 M NaCl, pH 7.4), the wells were blocked at 37³C for 2 h with 200 Wl of 2.0% (w/v) bovine serum albumin (BSA) in TBS containing 0.01% Tween 20. The wells were washed three times with TBS after this and all subsequent incubation steps. Serial dilutions of 20.0^0.04 Wg/ml vitronectin in TBS containing 0.2% BSA and 0.01% Tween were added to the wells and mixed with an equal volume of monoclonal anti-vitronectin IgG (Quidel) to a ¢nal antibody dilution of 1:25 000. HRP-linked horse anti-mouse IgG diluted 1:1000 in TBS/Tween/BSA was used to detect the bound monoclonal antibodies. The plates were then developed with a 0.2 mg/ml solution of 2,2P-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in 50 mM sodium citrate, pH 5.5, containing a 1:2000 dilution of 30% hydrogen peroxide. The extent of antibody binding was determined by measuring the absorbance at 405 nm in a Microtek microplate reader. Several absorbance readings were performed over time following the addition of the substrate to ensure that data were in the linear range of the assay. Recombinant vitronectin was tested for reactivity with conformation-speci¢c antibodies in a competitive ELISA, essentially as described [44]. Vitronectin was immobilized on the surface of microtiter plates. Constant amounts (50 ng/ml) of the mAbs 153 and 1244 were added along with serial dilutions of native vitronectin, multimeric vitronectin, medium containing recombinant r72-kDa vitronectin or medium containing recombinant r62-kDa vitronectin. A 1:50 000 dilution of 8E6 was used for these assays. Bound monoclonal IgG was detected using biotin-conjugated goat anti-mouse IgG, followed by streptavidin alkaline phosphatase conjugate and the chromogenic substrate pNPP. Experiments were performed in triplicate. The absorbance of each sample was measured at 405 nm in a microplate reader.

A direct assay was used to measure vitronectin binding to heparin-coated microtiter plates. For coating, a 100 Wl solution of heparin (1.0 mg/ml in 50 mM sodium carbonate, pH 9.6) was incubated in microtiter wells for 16 h at 4³C. The plates were washed three times with PBS/casein/Tween (PBS containing 0.1% (w/v) casein and 0.1% (v/v) Tween 20) after this and all other incubation steps. Nonspeci¢c binding was prevented using a blocking solution of PBS containing 0.03% casein (w/v) and 0.05% (v/v) Tween 20. After blocking, the plates were incubated with serial dilutions of puri¢ed plasma-derived or recombinant vitronectin. The plates were probed for vitronectin binding using polyclonal anti-vitronectin (1:5000) followed by goat anti-rabbit IgG conjugated to HRP (1:1000). All dilutions were made in PBS/casein/Tween. The plates were developed as described above using the chromogenic substrate ABTS, and the absorbance was measured at 405 nm. Controls to con¢rm the speci¢c binding of the various forms of vitronectin to heparin in the assay were conducted using non-coated microtiter plates, with identical blocking and incubation conditions. In all cases, the background non-speci¢c binding to these plates without heparin was insigni¢cant at the vitronectin concentration ranges used in the assay. This assay has been successfully used in our laboratory previously to measure speci¢c binding of a recombinant C-terminal vitronectin domain to heparin [10]. Interactions between recombinant vitronectin and PAI-1 were measured using a slight modi¢cation of the competitive binding assay described by Sei¡ert and Loskuto¡ [7]. Brie£y, microtiter plates were coated with 50 Wl of a 1 Wg/ml solution of native human vitronectin at 4³C for 16 h. After washing the plates three times with PBS, the wells were blocked with 200 Wl of 3.0% (w/v) BSA in PBS at 37³C for 1 h. The wells were washed three times with PBS/Tween/BSA (PBS containing 0.1% (w/v) BSA and 0.1% (v/v) Tween 20) after this and all subsequent incubation steps. Serial dilutions of plasmaderived vitronectin (3^0.0003 Wg/ml) or medium containing recombinant vitronectin were added to the plates and mixed with 0.4 nM PAI-1 to give a ¢nal PAI-1 concentration of 0.2 nM in 100 Wl. Polyclonal

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anti-PAI-1 antibodies diluted 1:10 000 in PBS/ Tween/BSA, followed by HRP-linked goat anti-rabbit IgG (1:1000) were used to detect bound PAI-1. Control samples with conditioned medium demonstrated no e¡ect on the PAI-1-binding results, demonstrating that results were directly attributable to the vitronectin samples assayed. 2.9. Kinetic analysis The rate of inactivation of thrombin by the protease inhibitor antithrombin in the presence of heparin was measured continuously using the chromogenic thrombin substrate Chromozym TH [45]. Reactions were performed in a 1-ml reaction volume in acrylic cuvettes (Sarstedt) at 23³C in a bu¡er of 40 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl and 0.1% PEG-8000. Heparin with an average molecular weight of 6000 was used in the reactions at a ¢nal concentration of 66.0 nM. The ¢nal concentrations of Chromozym TH, thrombin and antithrombin were held constant at 0.19 mM, 2.0 nM and 60.0 nM, respectively. The concentration of plasma-derived or recombinant vitronectin was varied in the assay reaction. Pseudo-¢rst-order rates of reaction were determined by non-linear least-squares analysis using the IGOR software package (Wavemetrics, Oswego, OR). 2.10. PAI-1 stabilization A 2.0 WM stock of PAI-1 was incubated in 25 mM HEPES, pH 7.4, containing 15 mM NaCl, 0.1% BSA and 1.3 mM EDTA at 37³C, along with 0 or 2 WM vitronectin (plasma-derived or recombinant). At various time intervals (0^8 h), the PAI-1 mixture was diluted in reaction bu¡er (0.1 M HEPES, 0.1 M NaCl, pH 7.4) and mixed with equal volumes of 100 nM trypsin (Sigma) to give a ¢nal concentration of 60 nM PAI-1. The PAI-1/trypsin mixture was incubated at room temperature for 10 min to allow complete inhibition of the protease. A small volume (10 Wl) of the protease/inhibitor sample was then added to 0.99 ml of reaction bu¡er, which contained 0.1 mM S-2222, a chromogenic substrate. Final concentrations of trypsin and PAI-1 in the reaction were 0.5 nM and 0.6 nM, respectively. The reaction proceeded for 5 min at 37³C in an acrylic cuvette (Sar-

stedt), and the absorbance at 405 nm was measured using a spectrophotometer interfaced with a computer. The rate of residual trypsin activity was determined from the slope of the A405 vs. time plot for each sample. The exact concentration of active PAI-1 (P) was determined for each sample by extrapolation to a standard curve. Data were collected for the standard curve prior to the experiment by varying the concentration of freshly diluted active PAI-1 incubated with trypsin. The rate of residual trypsin activity was plotted as a function of PAI-1 concentration to generate the standard curve. The PAI-1 activity was determined as a percent of the original active PAI-1 present for each sample using the equation below: 100…Px =P0 †; where Px is the concentration of active PAI-1 at time x determined by extrapolation to the standard curve, and P0 is the original concentration of active PAI-1 in the sample. Finally, the data were plotted for as percent PAI-1 vs. time in hours. 3. Results 3.1. Generating recombinant full-length and 62-kDa vitronectin in the baculovirus expression system Expression of recombinant forms of vitronectin was pursued using the baculovirus expression system. The recombinant plasmids pBlueBac-VN and pFastbac594 were used to transfer the recombinant vitronectin genes into the baculovirus genome. The resulting recombinant virus stocks, BBVN and Bac594VN, were then used to direct expression of full-length (r72-kDa) and truncated (r62-kDa) vitronectin in insect cells. Fig. 1 demonstrates that immunoreactive bands migrating near the 70 000 molecular weight marker could be detected in the spent media and in lysates from cells infected with BBVN, but not in cells infected with wild-type baculovirus or uninfected cells. Cells infected with the Bac594-VN virus stock also express and secrete a novel protein, which reacts with anti-vitronectin antibodies and migrates faster on SDS^PAGE, consistent with its smaller size. Non-reduced SDS^PAGE analysis on the

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8E6 [44,46] and 1244 [8] map to the ¢rst hemopexin repeat, whereas 153 recognized the somatomedin B domain [8]. All three antibodies avidly bind immobilized denatured vitronectin. This interaction can be inhibited in the presence of denatured or multimeric vitronectin, but not native vitronectin. For this reason, they have been useful to detect the `altered' or tissue-associated conformation of vitronectin in biological samples [8]. The recombinant proteins inhib-

Fig. 1. SDS^PAGE and Western blot analysis of vitronectin expression in the baculovirus system. Cultures of Sf9 cells were infected with the BBVN or Bac594-VN stocks. After 72 h, the cells and culture media were harvested, separated by centrifugation, mixed with SDS^PAGE loading dye containing L-mercaptoethanol, and boiled for 5 min. The proteins in each sample were separated on 10% polyacrylamide SDS gel and transferred to nitrocellulose for Western blotting. The blot was probed with polyclonal anti-vitronectin antibodies. Lanes 1 and 4 represent samples from cells infected with the wild-type baculovirus. Lanes 2 and 5 are samples from cultures infected with Bac594. Lanes 3 and 6 are samples from cells infected with BBVN.

secreted samples indicates that the recombinant vitronectins, assayed both from cell extracts and secreted into conditioned media, are present as highmolecular-weight multimers (data not shown). Quantitative ELISAs indicated that recombinant fulllength vitronectin was expressed at 10^50 Wg/ml, while cells infected with Bac594-VN expressed r62kDa vitronectin at 100^150 Wg/ml. These expression levels are typical for the baculovirus system, and they allow for production of biochemical quantities of material from large cultures. 3.2. Analyzing the conformation and oligomeric state of the recombinant proteins To investigate the conformation of the proteins, medium samples containing recombinant proteins were subjected to analysis in competitive ELISAs using a panel of conformation-speci¢c monoclonal antibodies. The recombinant proteins were tested for the ability to inhibit the interaction between immobilized denatured vitronectin and three monoclonal antibodies, 8E6, 153 and 1244. The antibodies

Fig. 2. Reactivity of recombinant vitronectins with conformationally sensitive monoclonal antibodies. Recombinant vitronectin was compared with human native and multimeric vitronectin for reactivity with monoclonal antibodies 8E6 (A), 1244 (B), and 153 (C). Antibody binding to immobilized urea-denatured human vitronectin was measured in the presence of increasing concentrations of native human vitronectin (hatched bars), human multimeric vitronectin (gray), medium containing r72-kDa vitronectin (white), or medium containing r62-kDa vitronectin (black). The data are expressed in terms of percent inhibition of antibody binding to the immobilized vitronectin.

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ited the binding of antibody to the immobilized vitronectin at concentrations comparable to that of multimeric vitronectin generated in vitro (Fig. 2). Native vitronectin did not react with the antibodies, and therefore did not diminish their binding to the immobilized vitronectin. These results clearly indicate that both recombinant proteins are in the `altered' conformation that is prone to multimerization [35,37,47]. To further test the oligomeric state of the recombinant proteins, a gel ¢ltration analysis was performed. The proteins in spent media from infected insect cells were precipitated with ammonium sulfate and dia-

lyzed against bu¡er before application to a Superose-12 gel ¢ltration column connected to a Pharmacia fast performance liquid chromatography (FPLC) system. Several protein peaks were observed in the FPLC pro¢le (Fig. 3). Western blots using polyclonal anti-vitronectin antibodies indicated that peak 1 contained vitronectin. The other predominant peak in the elution pro¢le was shown by Western blotting with anti-albumin antibodies to contain albumin; indeed, this peak coincided with the elution volume of a BSA standard with a molecular weight of 67 000. The recombinant vitronectin behaved like multimeric vitronectin generated in vitro by denaturation/renaturation and eluted close to the void volume of the column. Native plasma-derived vitronectin, with a molecular weight of 72 000 elutes in fraction 13, at a position indistinguishable from that of the albumin standard. 3.3. Purifying the recombinant protein

Fig. 3. Gel ¢ltration pro¢le for recombinant vitronectin. Gel ¢ltration pro¢les of cultures producing full-length (solid line) or truncated (dashed line) vitronectin are shown. Proteins were precipitated from spent media of insect cultures infected with BBVN or Bac594-VN by adding ammonium sulfate. The precipitated proteins were resuspended in a small volume of 0.1 M sodium phosphate, pH 7.5, containing 0.15 M NaCl and 1 mM EDTA, and the sample was dialyzed against the same bu¡er. After dialysis, the concentrated samples were applied to a Superose-12 gel ¢ltration column (1U30 cm; bed volume 23.7 ml) with a £ow rate of 0.5 ml/min using a Pharmacia FPLC system. The absorbance of the fractions was monitored at 280 nm, and the fractions were analyzed for the presence of vitronectin or albumin by immunoblotting with polyclonal anti-vitronectin or anti-albumin antibodies, respectively. The peak that corresponds to recombinant vitronectin is indicated. The elution position of an albumin standard is also marked. The exclusion limit of the column is 2U106 kDa. Native and multimeric human vitronectin elute from the same column at 13 ml and 9 ml volumes, respectively.

Zhao and Sane [40] reported that a single Mono-Q step was su¤cient to purify recombinant vitronectin from the serum-free media of infected insect cells, but Mono-Q chromatography failed to separate the protein adequately in the current studies. Due to heterogeneous charge distribution on the protein and its relatively low concentration, the recombinant vitronectin eluted from the anion exchange column over a broad range, which overlapped with the elution pro¢le of many other proteins. Hence, an alternative puri¢cation strategy was employed. Medium that was harvested from baculovirus-infected cells was applied to Blue-Sepharose resin as described in Section 2. The bound proteins were eluted from the column with 2.0 M NaCl. Fractions containing recombinant vitronectin were pooled and dialyzed to BSB. These samples were then applied to an immunoa¤nity column which had been generated by directionally crosslinking a monoclonal anti-vitronectin antibody to a protein G resin to permit optimal exposure of the antigen-binding Fab regions. After extensive washing, puri¢ed bound vitronectin was eluted with Gentle Elution Bu¡er2 as described. The purity of the protein was assessed qualitatively by SDS^PAGE analysis. Fig. 4 shows Coomassiestained SDS^PAGE gels and Western blots of puri¢ed recombinant 72-kDa and 62-kDa vitronectin.

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formed on the pre-puri¢cation samples shown in Fig. 3. 3.4. Testing the heparin-binding activity

Fig. 4. SDS^PAGE analysis of puri¢ed full-length and truncated vitronectin. Recombinant full-length and truncated vitronectin were puri¢ed from the spent media of Hi 5 cells infected with recombinant BBVN or Bac594-VN, respectively. Puri¢ed proteins were mixed with non-reducing (IAA) or reducing (LME) loading dye, boiled for 5 min, and separated on 10% polyacrylamide SDS gels. (A) Coomassie-stained gel. (B) Western blot probed with polyclonal anti-vitronectin antibodies. Recombinant 72-kDa vitronectin was loaded in lanes 1 and 3 of both gels. Recombinant 62-kDa vitronectin was loaded in lanes 2 and 4. The positions of the molecular weight markers are indicated.

The purity was determined quantitatively by comparing the total protein concentration measured using the bicinchoninic acid method to the vitronectin concentration, which was measured using a quantitative vitronectin-speci¢c ELISA. Analytical ultracentrifugation analysis on the puri¢ed recombinant samples indicated that the proteins were high-molecularweight oligomers, in agreement with the analyses per-

To test whether recombinant vitronectin represented a functional model for human multimeric vitronectin, both r72-kDa and r62-kDa vitronectin were analyzed for heparin-binding activity. Preliminary studies using heparin Sepharose chromatography had indicated that the two forms of the protein bind heparin. Like plasma-derived multimeric vitronectin, the recombinant proteins bound avidly to heparin Sepharose and could only be eluted using a relatively high salt concentration (0.5 M). Native (monomeric) vitronectin elutes from heparin columns at much lower salt concentrations (0.12 M). Both proteins also bound to immobilized heparin in direct solid-phase immunoassays, as shown in Fig. 5. This ¢gure also shows that native vitronectin binds much more weakly to the immobilized heparin under these assay conditions. The di¡erence in heparin reactivity between the native and multimeric forms of vitronectin in solid-phase assays has been previously reported [25,35] and is indicative of speci¢c binding to heparin that is in£uenced by the multivalent nature of the interaction with the multimer [27,28]. In all cases, the levels of binding are signi¢cantly elevated com-

2

Gentle Elution Bu¡er was tested for its e¡ects on the oligomeric state of plasma-derived vitronectin, to con¢rm that it would not alter the conformation of the recombinant proteins. Plasma-derived vitronectin was diluted in the bu¡er. After a 1-h incubation at room temperature, the chaotropic bu¡er was removed by dialysis and the conformation of the protein was determined by gel ¢ltration chromatography. After this treatment, vitronectin maintained its native conformation. Hence, the chaotropic composition of this bu¡er is not expected to signi¢cantly alter the conformation of the recombinant proteins.

Fig. 5. Solid-phase heparin-binding assays. Puri¢ed samples of recombinant 72 kDa vitronectin (R), recombinant 62-kDa vitronectin (F), native human vitronectin (a), and multimeric vitronectin (b) were tested for the ability to bind immobilized heparin. The heparin was immobilized in the wells of microtiter plates as described in Section 2. After blocking with casein, vitronectin was added to the wells in increasing concentrations. Bound vitronectin was detected using polyclonal anti-vitronectin antibodies.

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arin activity by 50% is 0.05 WM for either form of recombinant vitronectin. This is in close agreement with the 0.1 WM value observed with multimeric plasma-derived vitronectin [28]. These data indicate that both forms of recombinant vitronectin produced in the baculovirus system bind and neutralize heparin in a concentration-dependent manner that is quite similar to that of plasma-derived multimeric vitronectin. 3.5. Analyzing PAI-1 binding and stabilization

Fig. 6. Heparin neutralization assays for recombinant vitronectin. Heparin activity was measured by an increase in the reaction rate of antithrombin inactivation of thrombin. The concentration of active thrombin was monitored continuously over time by hydrolysis of the Chromozym TH substrate, as described in Section 2. Kapp values were determined by ¢tting the data using least-squares analysis and the IGOR pro software package. The e¡ects of recombinant vitronectin on Kapp were measured at di¡ering concentrations and are standardized to the rate of the heparin-catalyzed thrombin inhibition in the absence of added vitronectin. The data points for multimeric human vitronectin (b), r72-kDa vitronectin (R), and r62-kDa vitronectin (O) are indicated. The solid lines do not represent mathematical ¢ts to the data but are presented to show the overall trend in the data.

pared to any non-speci¢c binding that occurs in the ELISA. Clearly, both recombinants mimic the highvalency interactions with heparin that are characteristic of the multimeric form of human vitronectin [28]. Also, solution-phase kinetic heparin-binding assays were performed to quantitatively compare the heparin-binding activity of the full-length and truncated forms of recombinant vitronectin. These assays measure thrombin inhibition by antithrombin in the presence of heparin, which serves as a catalyst and increases the rate of reaction. Molecules that compete with antithrombin and thrombin for heparin binding reduce the rate of interaction of thrombin with antithrombin (Kapp ). The e¡ects of various concentrations of recombinant vitronectin on the heparin-catalyzed Kapp for thrombin antithrombin are shown in Fig. 6. Both molecules compete for heparin binding. The concentration needed to neutralize hep-

In order to determine whether recombinant vitronectin binds PAI-1, an immunoassay was performed. Solid-phase immunoassays have been used routinely to analyze the interaction between plasma-derived vitronectin and PAI-1 [7,23,29]. In this assay, plasma-derived vitronectin was immobilized to a microtiter plate. Medium containing recombinant vitronectin was serially diluted and incubated with pure, active PAI-1. PAI-1 binding to the immobilized vitronectin was then detected using polyclonal antibodies directed against the serpin. Both recombinant forms of vitronectin competed with the immobilized

Fig. 7. Competitive PAI-1-binding assays. Samples of multimeric human vitronectin (b), or media containing r72-kDa (R) or r62-kDa (O) vitronectin were tested for PAI-1 binding in a competitive assay in which native human vitronectin was immobilized on the wells of a microtiter plate. PAI-1 was added in the presence of increasing concentrations of competing vitronectin or medium. Bound PAI-1 was detected using polyclonal anti-PAI-1 antibody. Percent binding inhibition was calculated using the equation: 100(Ax 3A0 )/A0 , where A0 is the absorbance at 405 nm in the absence of added vitronectin and Ax is the absorbance at a given concentration of vitronectin.

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plasma-derived vitronectin to PAI-1 increased the t1=2 to 6 h. Both forms of recombinant vitronectin puri¢ed from the spent media of insect cells resulted in stabilization of PAI-1 activity to the same extent of native vitronectin, with a t1=2 value of 6 h. These data are represented in Fig. 8. The increase in t1=2 of PAI-1 upon binding to vitronectin has been reported in the literature, with values that are highly variable (up to 24 h with multimeric vitronectin [50]) and dependent on assay conditions. 4. Discussion

Fig. 8. PAI-1 stabilization assays for recombinant vitronectin. Samples of puri¢ed r72-kDa vitronectin (R) or r62-kDa vitronectin (O) were tested for their ability to stabilize PAI-1. The assays were performed by incubating 2 WM PAI-1 alone (b) or with an equimolar concentration of recombinant vitronectin at 37³C. At various time points, PAI-1 was diluted in reaction bu¡er and mixed with trypsin. The PAI-1:trypsin mixture was allowed to incubate at room temperature for 10 min to allow complete interaction. After this incubation period, residual trypsin activity was measured using the chromogenic substrate S-2222, as described in Section 2. The trypsin rates were extrapolated to a standard curve to calculate the actual concentration of active PAI-1 remaining in the sample. This value was compared to the known concentration of active PAI-1 at the beginning of the time course to obtain percent PAI-1 activity.

vitronectin for binding PAI-1 (Fig. 7). The e¡ective concentration range needed to inhibit PAI-1 binding to the immobilized vitronectin was consistent with that of puri¢ed plasma-derived vitronectin. Another important test of vitronectin function addressed its stabilization of PAI-1. PAI-1 is extremely labile, undergoing a conformational change that leads to inactivity [48]. The half-life (t1=2 ) is estimated to be 1.5^3 h depending on assay conditions [3,49]. Vitronectin binds PAI-1 and stabilizes the active conformation, increasing the t1=2 of its conversion to latency two- to four-fold. The stabilizing e¡ect of recombinant vitronectin was measured with the PAI-1 stabilization assay described in Section 2. PAI-1 incubated at 37³C without added vitronectin lost the ability to inhibit trypsin within 4 h. The t1=2 was 1.5 h, consistent with reported values. Adding

Most of the vitronectin that is used for functional studies is puri¢ed from pools of human plasma. Because of the genetic polymorphism at position 381 in vitronectin, only about 50% of vitronectin in any preparation is single-chain, 72-kDa vitronectin. Addressing di¡erences in the two forms of vitronectin has been a burdensome task because the proteins are not easily separated in plasma pools. Furthermore, the protease responsible for this cleavage has not been identi¢ed, and even limited proteolysis with other trypsin-like proteases generally results in cleavage within the heparin-binding sequence rather than at arginine 379. Contradictory results have been reported on whether the single-chain and two-chain forms di¡er in ligand binding. Nevertheless, the studies with the mixture of forms isolated from plasma have proceeded on the tacit assumption that functional di¡erences are insigni¢cant. It is important to establish the validity of this argument. Heterologous expression systems are useful tools for exploring the results of substitutions, insertions and truncations on protein function. Bacterial systems have been used for expressing small domains within vitronectin, including the somatomedin B domain [8,9], the central hemopexin domain [51], and the C-terminal heparin-binding domain of the protein [52]. However, these prokaryotic expression systems have not been useful for generating full-length vitronectin. This is likely due in part to its numerous post-translational modi¢cations and intricate disul¢de arrangement. Eukaryotic expression systems o¡er appealing alternatives for generating recombinant vitronectin, but such strategies have enjoyed only moderate

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success. Cherny et al. [6] reported expression and secretion of full-length wild-type and mutated vitronectin in transfected BHK cells. Under the expression conditions, vitronectin was cleaved at position 379 such that almost half of the protein existed in the two-chain form. Vitronectin was puri¢ed from the spent media of transfected cells according to the Yatohgo method [53], which involves denaturation and reduction. Recombinant vitronectin promoted integrin-dependent cell adhesion, whereas the cell-binding activity was markedly reduced for point mutants in which the integrin-binding RGD sequence was changed to RGE or RAD. No other functions were characterized in these studies. In 1993, Zhao and Sane [40,54] reported expression and secretion of vitronectin in baculovirus-infected Sf9 cells. The recombinant vitronectin was sulfated, phosphorylated and glycosylated. It promoted integrin-speci¢c cell binding and bound to immobilized PAI-1. In a separate report, Zhao and Sane [55] described the expression of wild-type vitronectin and two deletion mutants of the protein lacking the somatomedin B domain (1^44) and the heparin-binding domain (343^376). However, the structure and function of the protein was not further characterized. Realizing the potential of the baculovirus system for advancing the understanding of vitronectin biochemistry, this project extends the work of Zhao and Sane by characterizing the structure and function of recombinant vitronectin produced in the baculovirus system. The structure and function of a truncated vitronectin that corresponds to the large fragment of two-chain vitronectin has also been evaluated to investigate the functional di¡erences in the two genetic variants of vitronectin. 4.1. Recombinant vitronectin is similar in structure and function to the puri¢ed oligomeric human protein A necessary objective for this work was to compare the recombinant proteins to vitronectin that is obtained from human plasma. A technical goal in our research on these various forms of vitronectin was the development of sensitive assay methods for stocks of recombinant vitronectin that will be useful in screening larger numbers of mutants for functional perturbations. For this reason, it was imperative that

we evaluate methods that are useful on small quantities of unpuri¢ed recombinant proteins. Also, it was important to ensure that our puri¢cation strategies did not alter function by comparing results from similar assays on vitronectin in baculovirus culture medium and in a puri¢ed form. Vitronectin was expressed and secreted from baculovirus-infected Hi 5 cells grown in serum-free medium, and the oligomeric state of recombinant vitronectin was assessed before puri¢cation. In gel ¢ltration chromatography experiments, both recombinant proteins eluted from the column in the same fractions as plasma-derived vitronectin that had been rendered multimeric by chemical denaturation. The conformation of the recombinant proteins was investigated further using conformation-speci¢c monoclonal antibodies, which bind preferentially to the multimeric form of vitronectin that is generated by refolding in vitro [44,56,57]. These antibodies also recognize a minor component of vitronectin in plasma and the major form of vitronectin in the extracellular matrix, but the antibodies do not bind the monomeric form of vitronectin, which is the predominant form of vitronectin in plasma. Furthermore, mAbs 153, 1244, and 8E6 react with vitronectin/ thrombin/antithrombin complexes and vitronectin/ PAI-1 complexes [56^58], which are thought to contain a multivalent form of vitronectin. Both recombinant proteins reacted strongly with the conformation-speci¢c antibodies. The multivalent form of vitronectin that is produced by denaturation has commonly been used as a model for the multivalent form of vitronectin that is found in vivo. The mechanisms that lead to multimerization in vivo are not well established but they clearly do not involve high temperature or chaotropic agents. The common assumption at this point is that binding of vitronectin to the thrombin/antithrombin complex, complement, or PAI-1 results in vitronectin self-association. Indeed, there is strong evidence that vitronectin associates with PAI-1 to form high-molecular-weight complexes [59] that promote formation of stable vitronectin multimers ([58], K. Minor, M.N. Blackburn, C.B. Peterson, unpublished results). Also, the proportion of high-molecular-weight vitronectin is increased after coagulation [60]. Most of the multimeric vitronectin thus formed

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is associated with the ¢brin clot (C.B. Peterson, J.I. Weitz, T.J. Podor, submitted), and this ¢brin-bound vitronectin stabilizes PAI-1 in the vicinity of the clot [61]. Baculovirus-infected insect cells produce an oligomerized form of the vitronectin without chemical denaturation that is similar in conformation and epitope exposure to the denatured/renatured form of plasma vitronectin. The antibodies that recognize the recombinant form of vitronectin generated in this expression system are the same that recognize the matrix- and tissue-associated form of human vitronectin in other experiments [8,44,46]. Thus, the recombinant vitronectin likely resembles the form of human vitronectin that is produced in vivo and deposited in the extracellular matrix or in platelet Kgranules. More studies will be necessary to understand fully the folding process of recombinant vitronectin. Several experiments were performed to evaluate whether the recombinant vitronectin behaved similarly to plasma vitronectin with respect to heparin binding. A¤nity chromatography and ELISA assays indicated that both recombinant proteins bind heparin on a solid phase in a concentration range similar to multimeric vitronectin. Solution-phase kinetic assays provide more evidence that recombinant vitronectin binds heparin with an a¤nity that is comparable to multimeric vitronectin. Puri¢ed recombinant vitronectin neutralized heparin activity with a Ki of 0.05 WM. Ki values are about 10-fold higher for native vitronectin and are approximately 0.1 WM for multimeric vitronectin [28]. There are no signi¢cant di¡erences in the ability of recombinant or plasmaderived multimeric vitronectin to bind or neutralize heparin. The multivalent nature of the recombinant proteins is re£ected in the high salt concentrations required to elute recombinant vitronectin from heparin Sepharose and the low protein concentrations needed to neutralize heparin activity. PAI-1 binding has been tested by two separate methods. First, an ELISA assay demonstrates that recombinant vitronectin associates with the serpin. Zhao and Sane [40] had shown that vitronectin from the baculovirus system binds to immobilized PAI-1, but immobilized PAI-1 is reported to be in the latent conformation [62]. The current studies utilize a competitive ELISA, which monitors the ability

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of vitronectin in solution to compete with soluble PAI-1 for binding to immobilized vitronectin. While the conformation of vitronectin that is immobilized to the plate is altered [63], it is unlikely that the assay a¡ects the conformation of either PAI-1 or the competing vitronectin in solution. A solution-phase kinetic assay was also used to study the interaction between PAI-1 and recombinant vitronectin. Plasma vitronectin binds PAI-1, preventing the molecule from adopting its latent conformation. Vitronectin prolongs the t1=2 of active PAI-1 two- to four-fold. Both of the recombinant vitronectin molecules stabilize PAI-1 activity, increasing the t1=2 from 1.5 to 6 h and are therefore functional with respect to their e¡ect on PAI-1. 4.2. The C-terminal 80 amino acids are not necessary for multimerization One way to regulate vitronectin function is to interfere with its ability to form multimeric, high-valency species. Stockman et al. [25] have suggested that plasmin-mediated cleavage of vitronectin within the heparin-binding domain prevents vitronectin selfassociation. Are the C-terminal amino acids of vitronectin required for multimerization? The present study reveals that both recombinant forms of vitronectin are multimeric, and both react with conformation-speci¢c monoclonal antibodies. The studies indicate that the C-terminal 80 amino acids are not necessary either for multimerization or for adopting the proper fold at the N-terminus that confers reactivity with the monoclonal antibodies 8E6, 153 or 1244. Thus, endogenous cleavage of plasma vitronectin at position 379 is not expected to compromise the ability to adopt the multivalent conformation that is associated with more e¡ective ligand binding. Although it was previously suggested that the heparin-binding sequence is involved in multimerization [25], this has since been disproved [47]. Apparently, sequences upstream of the heparin-binding region beginning at residue 345 are involved in self-association of vitronectin. One possibility is that the hemopexin-like regions in the central domain mediate this function in a manner analogous to the association of hemopexin domains recently documented by X-ray crystallography [64].

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4.3. The recombinant model for the heavy chain (62-kDa fragment) of vitronectin suggests that proteolytic cleavage at position 379 does not alter vitronectin binding to heparin or PAI-1 Plasmin, thrombin, and elastase cleave within or near the heparin-binding region (345^379) of vitronectin. Findings regarding functional e¡ects of proteolytic processing by the various enzymes in this region have been inconsistent. Numerous reports document attenuated heparin and PAI-1 binding to the clipped form of vitronectin, and many suggest that this may be a feedback mechanism for controlling vitronectin prothrombotic activity [15,25,32]. Alternatively, Sane et al. [65] have suggested that the 62-kDa chain of two-chain cleaved vitronectin actually binds heparin more avidly, a deduction that was based on a preferential binding of biotinylated heparin to the 62-kDa chain in ligand blotting assays. The current studies indicate that there is no di¡erence in the heparin-binding activity of full-length and truncated vitronectin, as both bind with comparable a¤nity to heparin. This provides clear evidence that the heparin-binding elements are not present in the C-terminal 80 amino acids of vitronectin, and that the 80 amino acids at the C-terminus of full-length vitronectin do not diminish function of the heparinbinding sequence. This result is consistent with the surface orientation of the heparin-binding sequence that has been demonstrated for vitronectin [28,52]. There was no di¡erence in either of the recombinant forms for binding PAI-1 or for stabilizing the protein. Consistent with domain mapping studies, which localize the PAI-1-binding site to the somatomedin B domain or the heparin-binding domain, the C-terminal 80 amino acids are not required for PAI1 binding. Additionally, they do not appear to confer folding information to the upstream PAI-1- and heparin-binding sites. In conclusion, the baculovirus system has been employed to produce and purify recombinant forms of vitronectin that are functional and multimeric. Indeed, sensitive methods have been developed to test heparin- and PAI-1-binding functions of recombinant vitronectin in culture media and in its puri¢ed form. The expression system has provided pure

stocks of full-length and the 62-kDa vitronectin. In the absence of the C-terminal 80 amino acids, vitronectin retains structural information that directs multimerization, PAI-1 binding, and heparin binding. These studies clearly indicate that the truncated form of vitronectin can adopt the appropriate conformation to self-associate and acquire multivalency that e¡ectively strengthens the interaction with heparin. Thus, single-chain and two-chain vitronectin, with the C-terminal 80 amino acids attached via a disul¢de bond, are expected to function similarly in reactions that control thrombosis and ¢brinolysis. Acknowledgements We wish to thank Dr. Frank Church at the University of North Carolina, Chapel Hill, NC, for guidance in establishing the baculovirus expression system for use in our laboratory. Also, we are grateful to Dr. Dietmar Sei¡ert for assaying the recombinant vitronectins using the conformation-speci¢c monoclonal antibodies. This work was supported by Grant HL50676 from the Heart, Lung and Blood Institute of the National Institutes of Health and by an Established Investigator Award from the American Heart Association. Support was also received from a Professional Development Award to C.B.P. from the University of Tennessee, Knoxville, TN.

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