Jan K Jensen

Anders Malmendal

Birgit Schiøtt

Sune Skeldal

Katrine E Pedersen

Leyla Celik

Niels Chr Nielsen

Peter A Andreasen

Troels Wind
email: wind@mb.au.dk.

Inhibition of plasminogen activator inhibitor-1

Keywords: plasminogen activator inhibitor-1, NMR, peptide, mutagenesis, endocytosis, ligand docking cfu, colony forming unit, CMC, critical micelle concentration, HBS

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INTRODUCTION

The plasminogen activators tPA and uPA are serine proteases catalysing the conversion of the zymogen plasminogen to the active serine protease plasmin which can degrade fibrin and other extracellular proteins. The activities of tPA and uPA are regulated by plasminogen activator inhibitor-1 (PAI-1) (for reviews, see [START_REF] Vaughan | PAI-1 and atherothrombosis[END_REF][START_REF] Durand | Plasminogen activator inhibitor-I and tumour growth, invasion, and metastasis[END_REF]). In tissues, plasminogen activation is concentrated on cell surfaces where the GPI-anchored uPA receptor (uPAR) recruits uPA (for a review, see [START_REF] Behrendt | The urokinase receptor (uPAR) and the uPAR-associated protein (uPARAP/Endo180): membrane proteins engaged in matrix turnover during tissue remodeling[END_REF]). Plasminogen activation is implicated in the spread of cancers in which uPA-catalysed plasmin generation in tumours leads to degradation of basement membranes, thereby supporting the cancer cell invasiveness. It was therefore initially surprising that a high tumour level of PAI-1 is an even better marker for a poor prognosis. The functions of PAI-1 in the spread of malignant tumours are not completely known, but one important aspect seems to be a proangiogenic effect of PAI-1 (for a review, see [START_REF] Durand | Plasminogen activator inhibitor-I and tumour growth, invasion, and metastasis[END_REF]). PAI-1 belongs to the serpin family of protease inhibitors (for a review, see [START_REF] Gettins | Serpin structure, mechanism, and function[END_REF]). During reaction of a serpin with its target serine protease, the P1-P1' bond in the reactive centre loop (RCL) of the serpin is cleaved and the protease becomes attached to the serpin by an ester bond between the active site Ser and the P1 residue [START_REF] Egelund | An ester bond linking a fragment of a serine proteinase to its serpin inhibitor[END_REF]. At this point, the N-terminal part of the RCL is inserted as β-strand 4A (s4A) in the serpin (Figure 1). The protease is thereby pulled to the opposite pole of the serpin, its active site distorted, hydrolysis of the ester bond prevented, resulting in a trapped covalent serpin-protease complex [START_REF] Dementiev | Active Site Distortion Is Sufficient for Proteinase Inhibition by Serpins: STRUCTURE OF THE COVALENT COMPLEX OF α 1 -PROTEINASE INHIBITOR WITH PORCINE PANCREATIC ELASTASE[END_REF][START_REF] Huntington | Structure of a serpin-protease complex shows inhibition by deformation[END_REF]. Insertion of the RCL changes the serpin from a metastable, stressed (S) conformation to a more stable, relaxed (R) conformation and the released energy is believed to fuel protease distortion [START_REF] Huber | Implications of the three-dimensional structure of α 1 -antitrypsin for structure and function of serpins[END_REF][START_REF] Gettins | The F-helix of serpins plays an essential, active role in the proteinase inhibition mechanism[END_REF].

For some serpins, including PAI-1, the N-terminal part of the RCL can be inserted without P1-P1' bond cleavage, resulting in formation of the latent state [10]. The S to R transition includes conformational changes around α-helix D (hD) and hE which serve as a flexible joint region when the small and the large serpin fragments slide apart to allow RCL insertion [START_REF] Stein | Serpin tertiary structure transformation[END_REF] (Figure 1). In PAI-1, a hydrophobic cavity in this region serves as the binding site for multiple PAI-1-neutralising compounds and is rearranged during the S to R transition [START_REF] Björquist | Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site-directed mutagenesis[END_REF][13][START_REF] Elliott | Topography of a 2.0 Å structure of α 1antitrypsin reveals targets for rational drug design to prevent conformational disease[END_REF]. Also, a high-affinty binding site for vitronectin exists in the flexible joint region of the S-form of PAI-1, but not in the R-form [START_REF] Jensen | The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand[END_REF]16].

PAI-1's complexes with its target proteases bind to and are endocytosed by certain receptors of the low density lipoprotein receptor family, including low density lipoprotein EXPERIMENTAL Cell culture U937 cells (ATCC CR1 1593) were cultured in RPMI 1640 medium. HEK 293T cells were cultured in Dulbecco Modified Eagle's Medium. Both media were supplemented with 10% foetal bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin. The medium for HEK293T additionally contained 1% nonessential amino acids. Cells were maintained in a humidified atmosphere with 5% CO 2 at 37 °C.

PAI-1

Natural, glycosylated wild-type human PAI-1 was purified from serum-free conditioned medium of dexamethasone-treated HT-1080 fibrosarcoma cells [START_REF] Munch | Interconversions between active, inert and substrate forms of denatured/refolded type-1 plasminogen activator inhibitor[END_REF]. Murine PAI-1 cDNA was a kind gift from Dr. Ann Gills, Katholieke Universiteit, Leuven, Belgium. A DNA fragment encoding His 6 -tagged murine PAI-1 was constructed by PCR and cloned for expression in E. coli as described previously for human PAI-1 [START_REF] Jensen | The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand[END_REF]22]. Site-directed mutagenesis, expression of His 6 -tagged PAI-1 variants in E. coli and purification by metal affinity chromatography and size exclusion chromatography was performed as described [START_REF] Jensen | The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand[END_REF]22]. The specific inhibitory activity of PAI-1, i.e. the fraction of PAI-1 capable of inhibiting the proteolytic activity of uPA, was determined in a chromogenic assay as described previously [22].

Peptides

The following peptides were purchased from Eurogentec, Seraing, Belgium, from Biopeptide, San Diego, CA, USA, or from Peptide Protein Research Ltd., Wickham, UK: NH 2 -DVPCFGWCQDA-COOH; biotin-DVPCFGWCQDA-CONH 2 ; biotin-DVPCFGWCQDAGAKK-CONH 2 , all with a disulphide bridge constrained conformation.

Linear forms with the two cysteines replaced by serines were obtained for control experiments. We predominantly used freshly prepared stock solutions of less than 500 µM biotin-paionin-1 in Hepes-buffered saline (HBS; 140 mM NaCl, 10 mM Hepes, pH 7.4). If storage was required, stock solutions were snap-frozen in liquid N 2 and kept at -80 °C.

Miscellaneous materials

Vitronectin in a multimerised form was purchased from BD Biosciences, chicken ovalbumin and heparin from porcine intestinal mucosa was purchased from Sigma-Aldrich, rat PAI-1 was purchased from Molecular Innovations, and uPA was purchased from Wakamoto Pharmaceutical Company. α 1 -proteinase inhibitor was a gift from Professor J.

Enghild (Department of Molecular Biology, University of Aarhus, Denmark). Antithrombin III was a gift from the late Professor S. Magnusson (Department of Molecular Biology, University of Aarhus, Denmark) Receptor-associated protein (RAP) was prepared as described previously [START_REF] Nykjaer | Purified α 2 -macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the α 2 -macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes[END_REF]. Antibodies were described previously as follows: Monoclonal anti-PAI-1 antibodies Mab-1, Mab-2, Mab-3, Mab-5, Mab-6, and Mab-7 [START_REF] Nielsen | Monoclonal antibodies to human 54,000 molecular weight plasminogen activator inhibitor from fibrosarcoma cells--inhibitor neutralization and one-step affinity purification[END_REF][25][START_REF] Bødker | Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor-1 against inactivating agents[END_REF]; polyclonal rabbit anti-PAI-1 antibody [START_REF] Offersen | The myofibroblast is the predominant plasminogen activator inhibitor-1expressing cell type in human breast carcinomas[END_REF]; and monoclonal anti-uPA Mab-2 [28]. Full length LRP-1A purified from human placentas was a kind gift from J. Gliemann [START_REF] Moestrup | Analysis of ligand recognition by the purified alpha 2macroglobulin receptor (low density lipoprotein receptor-related protein). Evidence that high affinity of alpha 2-macroglobulin-proteinase complex is achieved by binding to adjacent receptors[END_REF].

Receptors

Isolation of PAI-1-binding peptides from a phage-displayed peptide library

Phage displayed random peptide libraries in the formats X 7 , CX 7 C, CX 10 C and CX 3 CX 3 CX 3 C (C, cysteine; X, any residue) were obtained from Erkki Koivunen, Division of Biochemistry, University of Helsinki, Finland [START_REF] Koivunen | Peptides in cell adhesion research[END_REF]. Selection of PAI-1 binding peptides was performed using standard procedures [START_REF] Smith | Libraries of peptides and proteins displayed on filamentous phage[END_REF]. Phage and PAI-1 expressed from HT-1080 cells (100 nM) were incubated in MBS (140 mM NaCl, 10 mM MES, pH 6) supplemented with 1% bovine serum albumin (BSA) and 5% glycerol (MBS-BG) for 1 h and formed phage-PAI-1 complexes were captured on polystyrene-adsorbed monoclonal anti-PAI-1 antibody Mab-7

(1 st and 4 th round of panning), Mab-5 (2 nd and 5 th round of panning) or Mab-3 (3 rd round of panning). After washing with MBS, bound phage was eluted with 0.1 M glycine, pH 2.2. For all rounds of panning, enrichment of binders was monitored by determining the amount of input-and output-phage, measured as colony forming units (cfu).

Phage was prepared from 16 individual clones eluted from each of the panning rounds 2 to 5 and tested for PAI-1 binding in ELISA format 1 (see below). PAI-1-binding peptides were identified by sequencing the corresponding phage [25]. Concentrated phage (~10 12 cfu/mL) was prepared by precipitation with NaCl and PEG 6000 [START_REF] Smith | Libraries of peptides and proteins displayed on filamentous phage[END_REF], followed by resuspension in MBS supplemented with 10% glycerol and 0.02% NaN 3 .

The binding of the phage to PAI-1 from HT-1080 cells was compared by incubating ~10 9 cfu/mL phage with PAI-1 (300 to 0. S1). A total of 1000 structures were calculated using the "Crystallographic and NMR system" (CNS) program package version 1.1 [40] using a total of 51 NOE derived distance constraints (Table S2). The obtained structures were visualised using the program MOLMOL [START_REF] Koradi | MOLMOL: a program for display and analysis of macromolecular structures[END_REF]. Based on the orientations of the aromatic Phe 5 and Trp 7 side chains, 88 of the 100 structures with the lowest energy could be divided into 9 distinct conformational groups.

Molecular modelling

The molecular modelling studies were carried out using the Cerius2 suite of programmes (version 4.8, Accelrys Software Inc.) and the pdb-files 1DVM (active PAI-1 carrying the stabilising mutations N152H/K156T/Q321L/M356I, a.k.a. PAI-1 14-1B [42,[START_REF] Stout | Structures of active and latent PAI-1: a possible stabilizing role for chloride ions[END_REF]) and 1LJ5 (latent PAI-1, Stein, P.E. and Baek, K., unpublished work). All crystal waters were deleted and hydrogen atoms were added in Cerius2 using standard protonation states. Cavity search was carried out with a 1.4 Å probe on a 0.5 Å grid, saving only sites with an opening of less than 8.0 Å and a volume of more than 62.5 Å 3 . Docking of the paionin-1 peptide with charged termini was done using the lowest energy structures from each of the 9 conformations identified in the NMR study and docking these was done using the LigandFit module of Cerius2. Flexible docking with the cff1.01 force field was performed by allowing Monte Carlo conformational search of the peptide structure inside the protein (10,000 MC-trials, max. 10 conformations saved). Soft potentials were included to model some plasticity into the protein. A rigid body minimization was included for optimal placement of the peptide inside the cavity (10 iterations, final minimization with 100 iterations). The generated peptide conformations were clustered using the complete linkage option and 3 ligand-protein structures for each of the 9 original paionin-1 conformations were saved. In these poses, the peptide structure was fully minimized (max. 10,000 iterations using the Cerius2 smart minimizer). The poses were selected based on the scoring functions calculated in Cerius2 and visual inspection ensuring that both termini of paionin-1 were protruding from the surface of PAI-1. Remaining strain introduced during the molecular docking was relieved by partial minimisation using NAMD [START_REF] Kalé | NAMD2: Greater scalability for parallel molecular dynamics[END_REF] and the CHARMM27 force field [START_REF] Mackerell | All-atom empirical potential for molecular modeling and dynamics studies of proteins[END_REF]. Protein residues within 3.0 Å of docked paionin-1 in the structure of latent PAI-1 as well as paionin-1 itself were allowed to move during the final minimization.

Determination of the critical micelle concentration

The conductivity at each peptide concentration was measured on a standard conductivity-meter by titrating it into pure water adjusted to pH 7.4 with NaOH. As a reference, a profile for titrating the same volumes of buffer without peptide was determined and subtracted from the peptide profile. The change in hydrodynamic size as a determinant of critical micelle concentration was determined by photon correlation spectroscopy using a Zetasizer Nano ZS (Malvern Instruments). The analysis was performed at 25 °C in HBS while titrating in biotin-paionin-1. Sampling time and analysis was set to automatic.

Analysis of paionin-1 binding by ELISA

In the four ELISA formats described below, all incubations were performed with 100 µL per well in MaxiSorp 96-well plates (Nunc) coated with 250 ng antibody in 0.1 M Na 2 CO 3 , pH 9.6 (formats 1, 2 and 4), or in Ni-NTA HisSorb Strips (Qiagen) (format 3).

Unless stated otherwise, PBS supplemented with 2% BSA was used for blocking the wells after coating, PBS supplemented with 0.2% BSA was used as the standard buffer for all incubations, and PBS supplemented with 0.05% Tween-20 was used for washing. Phage was detected with a HRP-conjugated monoclonal anti-M13 antibody (Amersham Biosciences, diluted 5,000-fold). Wells were developed with 0.5 mg/mL ortho-phenylenediamine (OPD, In ELISA format 1, PBS was replaced with MBS and BSA was omitted from the reaction buffer in experiments involving XR-5118 or bis-ANS. PAI-1 from HT-1080 cells in the indicated concentrations was captured on monoclonal anti-PAI-1 antibody Mab-5. Phage (~10 9 cfu/mL) was allowed to bind in the presence of various additions as indicated for each experiment and detected.

In ELISA format 2, wells were coated with a polyclonal rabbit-anti PAI-1 antibody.

Samples of mostly active PAI-1 variants (100 nM) were prepared and split into three aliquots which were incubated 16 h at 0 °C to preserve activity; incubated 16 h at 37 °C to obtain latent material; or reacted with a 2-fold excess of uPA for 30 min at room temperature and then stored at 0 °C for 16 h. Three-fold dilution series of the different forms of PAI-1 variants were incubated in the wells and after washing, paionin-1 phage clone B (~10 9 cfu/mL) was allowed to bind PAI-1. The wells were washed and bound phage detected.

In ELISA format 3, 50 nM His 6 -tagged PAI-1 variants were incubated 16 h at 37 °C to obtain latent material. These samples and samples of active PAI-1 were immobilised in HisSorb wells. After washing, paionin-1 phage clone B (10 9 cfu/mL) was allowed to bind PAI-1. The wells were washed and bound phage was detected as described above. To ensure that equal amounts of the individual PAI-1 variants were bound to the wells, a parallel ELISA was performed with 1 µg/mL polyclonal rabbit-anti-PAI-1 antibody instead of phage and with HRP-conjugated swine-anti-rabbit serum (DAKO, diluted 2,000-fold) instead of anti-M13 antibody.

In ELISA format 4, wells were coated with a polyclonal rabbit-anti PAI-1 antibody followed by incubation with recombinant PAI-1 variants (5 nM) and 0.3 µM biotin-paionin-1 peptide. After washing the wells, captured PAI-1-peptide complexes were detected with HRPconjugated avidin (DAKO, diluted 10,000-fold) and a peroxidase reaction as described above.

Equal loading of PAI-1 was assured by a parallel ELISA with 1 µg/mL monoclonal anti-PAI-1 Mab-1 antibody instead of peptide and with HRP-conjugated rabbit-anti-mouse serum (DAKO, diluted 2,000-fold) instead of HRP-conjugated avidin.

Analysis of paionin-1 binding by surface plasmon resonance (SPR)

The peptide biotin-DVPCFGWCQDAGAKK-CONH 2 was coupled to a CM5-chip (BIACORE) according to the manufacturer's amine-coupling protocol at pH 5.0 to a final immobilised ligand response of approximately 3000 RU (1.6 pmol). Detection of binding was carried out at 25 °C on a BIACORE X instrument at a flow rate of 10-20 μl/min, using 10 mM Hepes, 140 mM NaCl, 3 mM EDTA, 0.005% Tween-20, degassed and filtered (0.22 μm)

as running buffer. The concentration of the applied proteins was 0.7 µM. Signals were corrected for those obtained from a serially connected ethanolamine blocked flow cell on the same CM5 chip.

Solid phase receptor-binding assays

Purified receptors were coated in microtiter wells overnight in 50 mM NaHCO 3 , pH 9.6, with a receptor density adjusted to obtain 5-10% binding of 125 I-uPA-PAI-1 complex, corresponding to receptor concentrations in the coating buffer of around 0.5 µg/mL. The wells were blocked with 10% BSA in binding buffer (10 mM HEPES, pH 7.8, 140 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 ) for 2 h, washed, and incubated over night at 4 °C with 20-40 pM 125 I-ligand. Washing and incubation with ligand were done in binding buffer with 1% BSA.

After the incubation, the supernantants were removed for γ-counting and the wells washed.

Bound ligand was released from the wells with 10% SDS and taken for γ-counting. Ligand binding was expressed as bound divided by total ligand and corrected for non-specific binding, i.e. the radioactivity recovered from wells without receptor. These data were then corrected for small variations in the actual relative amount of complex, by the use of the binding of complex to parallel wells coated with 20 µg/mL anti-PAI-1 Mab-2 antibody.

Receptor-mediated ligand degradation U937 cells were washed 3 times with serum-free medium supplemented with 0.5% BSA and incubated for 45 min at 37 °C in 0.5 mL of the same buffer with 10 pM 125 I-labelled ligand and a cell density of 10 × 10 6 cells/mL. The cultures were then made 7% with respect to trichloroacetic acid and centrifuged. Supernatant and pellets were γ-counted separately.

Ligand degradation was defined as non-trichloroacetic acid precipitable radioactivity divided by the total amount of added radioactivity. The degradation in incubations without cells were scored as nonspecific degradation and subtracted from all other data [START_REF] Kjøller | Differential regulation of urokinase-type-1 inhibitor complex endocytosis by phorbol esters in different cell lines is associated with differential regulation of alpha 2-macroglobulin receptor and urokinase receptor expression[END_REF][START_REF] Jensen | Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells[END_REF]. Under the conditions used degradation proceeded linearly with time. Copyright 2006 Biochemical Society

RESULTS AND DISCUSSION

Selection of PAI-1 binding peptides from phage-displayed peptide libraries

A mixture of four different phage-displayed random peptide repertoires was panned against PAI-1 in order to isolate specific PAI-1-binders. As bait, we used human glycosylated PAI-1 from HT-1080 fibrosarcoma cells. To avoid denaturation of PAI-1 by its immobilisation on plastic [25,[START_REF] Deng | The PAI-1/vitronectin interaction: two cats in a bag?[END_REF], phage was allowed to bind PAI-1 in solution before capture of phage-PAI-1 complexes on immobilised monoclonal anti-PAI-1 antibody.

Enrichment of antibody-binding peptides was avoided by using alternating antibodies, i.e.

Mab-7, -5 or -3, for capture. The output:input ratio of phage increased 100-fold from round 1 to round 5 (data not shown). Individual phage from selection rounds 2 to 5 were tested for PAI-1-binding in a sandwich ELISA and sequencing of positives identified a short disulphideconstrained loop CFG(W/Y/F)C as a well-defined consensus-motif (Table 1). To compare the isolated phage, their EC 50 for PAI-1 binding was determined and revealed no gross variation among the different peptides, advocating that the consensus-residues are the major determinants of binding (Table 1). Importantly, these EC 50 values do not reflect the actual affinity between the displayed peptide and PAI-1 since each phage carry more than one copy of the peptide thus causing avidity effects, i.e. an apparently tighter phage-PAI-1 binding than warranted by the affinity in the 1:1 peptide-PAI-1 complex. In the following, a peptide with the sequence DVPCFGWCQDA will be referred to as paionin-1. This sequence is a chimera of clones A and B devoid of the flanking cysteines (Table 1). That this second disulfide was dispensable for binding could be inferred from the isolation of clone G (ILCFGMC, see Table 1).

The paionin-1 peptide inhibited the PAI-1-binding of phage clone B with an IC 50 of 1.8 ± 0.8 µM (n=3) while a linear version with the two cysteines replaced by serines had no effect on binding (Figure 2A). This advocates that the constraint imposed by the disulphide locks the CFGWC loop in a conformation required for binding and/or that the cystine contributes to PAI-1-binding by engaging in protein contacts. The binding of paionin-1 phage was further evaluated through competition experiments with monoclonal anti-PAI-1 antibodies with known epitopes and with vitronectin: Mab-1 binds the hI/s5A-loop and hC [START_REF] Bødker | Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor-1 against inactivating agents[END_REF] and both Mab-2 and Mab-6 binds hF and the hF/s3A-loop [25]. The vitronectin binding site is located between s1A, s2A, hE and hF [START_REF] Jensen | The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand[END_REF]16]. Mab-2, Mab-6, and vitronectin competed with paionin-1 phage for binding, Mab-1 slightly augmented binding, and an irrelevant antibody (anti-uPA Mab-2) had no effect (Figure 2B). Considering the epitopes for the employed antibodies and vitronectin, these data are consistent with a binding site for paionin-1close to hF of PAI-1. PAI-1-binding of phage was also inhibited by the organochemical PAI-1 neutralizers XR-5118 and bis-ANS, which are known to bind hydrophobic surfaces in the flexible joint region of PAI-1 [13, 48, 49], and by heparin which binds basic residues in and around hD [START_REF] Ehrlich | Elucidation of structural requirements on plasminogen activator inhibitor 1 for binding to heparin[END_REF] (Figure 2A). In summary, these competition experiments suggested a hydrophobic binding site for the disulphide-constrained CFGWC loop of paionin-1 in the flexible joint region of PAI-1.

Solution structure of paionin-1

Expecting that the main chain and to some extent the side chains of a five residue ring such as the CFGWC motif of paionin-1 would be stabilised by inherent strain we analysed paionin-1 by liquid state NMR spectroscopy. The peptide proton signals were well resolved in the resulting spectra and all except for those of the N-terminal amide were assigned (Table S1). A total of 51 NOEs could be identified and used in the structure calculations (Table S2).

In the 100 calculated structures with the lowest energy, the main-chain of the CFGWC ring was well conserved with a low RMSD (1.08 ± 0.51 Å). The structure of the residues flanking the CFGWC ring is less well-defined as the only forces directing their conformation seem to be steric repulsion by other residues which leads to the predominantly orthogonal orientation of both termini relative to the planar CFGWC ring (Figure 3A). As expected from the primary structure, paionin-1 is polarised with respect to hydrophobicity since the two hydrophobic side chains of the Phe 5 and Trp 7 points in the opposite direction of the more polar segments flanking the ring.

By visual inspection we found that in 88 of the 100 structures the Phe 5 and Trp 7 side chains adapted one of nine different conformations relative to the CFGWC ring, one example depicted in Figure 3A. Accordingly, these 88 structures were divided into nine conformational groups based on the orientation of these two side chains (Table 2 and Figure 3B). Since there was no correlation between the calculated energy and the grouping of the 88 structures we conclude that from an energetic perspective each of the conformations is equally likely to occur.

Molecular modelling of the paionin-1 interaction with PAI-1

The X-ray crystal structures of highest resolution of active PAI-1 carrying the stabilising mutations N152H/K156T/Q321L/M356I [START_REF] Stout | Structures of active and latent PAI-1: a possible stabilizing role for chloride ions[END_REF] and latent PAI-1 (Stein, P.E. and Baek, K., unpublished work) were searched for cavities capable of accommodating paionin-1.

In both structures, the calculations revealed only one cavity with a total volume compatible with paionin-1 binding. The cavity was positioned in the flexible joint region between hD, hE and s2A, thus overlapping with that identified by Elliot et al. (cavity 2 in [START_REF] Elliott | Topography of a 2.0 Å structure of α 1antitrypsin reveals targets for rational drug design to prevent conformational disease[END_REF]). The total volume of the cavity was almost conserved between active PAI-1 14-1B (~350 Å 3 ) and latent PAI-1 (~290 Å 3 ), but the cavity in active PAI-1 14-1B consists of numerous narrow extensions from the centre of the cavity, whereas the cavity in the latent form consists almost entirely of one single moiety.

We next used in silico docking procedures to evaluate whether paionin-1 would fit into the identified cavities in active PAI-1 14-1B and latent PAI-1. The lowest energy structure of paionin-1 from each of the 9 conformational groups was used as the ligand for docking and three converged solutions were obtained from each of the peptide structures. That the peptides containing the CFGWC motif listed in Table 1 can bind PAI-1 in the context of a phage, in which the displayed peptide sequence is preceded by Ala-Asp-Gly-Ala and followed by the phage coat protein [START_REF] Smith | Libraries of peptides and proteins displayed on filamentous phage[END_REF], allowed us to disqualify docking solutions in which the termini of paionin-1 do not protrude from the surface of PAI-1. Accordingly, only three solutions were acceptable (one solution shown in Figure 4A), all with the entire CFGWC ring of paioinin-1 buried deep into the cavity of latent PAI-1. Importantly, since the ligand docking algorithm mimics structural flexibility through the use of soft potentials, the docked structures contained atomic overlaps between paionin-1 and PAI-1. Nonetheless, the cavity search and subsequent docking routines unequivocally pointed to the flexible joint region as the binding site for paionin-1 and suggested a preferential binding to the latent conformation of PAI-1.

Solubility properties of biotin-paionin-1

Handling of the paionin-1 peptide demanded the addition of DMSO to solubilise the peptide and 20% DMSO was needed to keep the peptide in solution for NMR. For direct binding studies, we wished to employ a biotin-labelled peptide. Expecting that removal of the charge at the C-terminus by amidation and introduction of the apolar biotin at the N-terminus would reduce the solubility even further and obtaining inconsistent results with peptide concentrations above 50 µM, we decided to test if the biotinylated peptide forms aggregates at high concentrations. The conductivity profile obtained when titrating biotin-paionin-1 into pure water demonstrated a clear break in the linear response at a concentration of ~100 µM, suggesting that micelles are formed in water with a critical micelle concentration (CMC) close to this value (Figure 5), and since the CMC decreases with increasing saline concentration, the CMC of biotin-paionin-1 will be lower in HBS. However, the high dielectric constant of HBS made it impossible to determine the CMC of biotin-paionin-1 in this buffer by conductivity measurements. As an alternative approach we used photon correlation spectroscopy to estimate the CMC for biotin-paionin-1 in HBS and found it to be between 30 and 40 µM (data not shown). In light of these observations, it seemed reasonable to assume that biotin-paionin-1 has a CMC near 40 µM at physiological ionic strength. Hence, any data obtained at biotin-paionin-1 concentrations above this threshold may be flawed by nonspecific biochemical and cell biological effects due to the formation of aggregates, presumably micelles.

Mapping of the paionin-1 binding site by site-directed mutagenesis

In order to map the paionin-1 binding site, we studied its binding to PAI-1 variants in ELISAs and by SPR. First, a preferential binding of paionin-1 phage to latent PAI-1 and to the uPA-PAI-1 complex, as compared to active PAI-1, was shown (Figure 6A). Second, compared to the binding observed with latent human PAI-1, paionin-1 showed impaired binding to latent murine PAI-1 (Figure 6B). Third, when the binding of the paionin-1 peptide with an N-terminal biotin and a C-terminal amide (biotin-paionin-1) to recombinant PAI-1 variants was tested, a clear preference for latent PAI-1 over active PAI-1 or PAI-1 14-1B was observed (Figure 6C). Furthermore, we found that biotin-paionin-1 showed decreased binding to latent forms of the variants PAI-1(R78E), PAI-1(R117E/R120E), and PAI-1(K71A/R78A/Y81A/K82A). In contrast, biotin-paionin-1 binding to latent PAI-1 was not affected by the triple mutation R103A/M112A/Q125A, which reduces the apparent affinity for vitronectin more than 3,000-fold [START_REF] Jensen | Construction of a plasminogen activator inhibitor-1 variant without measurable affinity to vitronectin but otherwise normal[END_REF] (Figure 6C). The binding of paionin-1 to active and uPA-complexed PAI-1(R78E), PAI-1(R117E/R120E), and PAI-1(K71A/R78A/Y81A/K82A) was tested in an ELISA similar to the one depicted in Figure 6A and no binding was detected (data not shown).

We finally tested the recombinant variants in their latent form for biotin-paionin-1

binding by SPR analysis (Figure 6D). For these experiments, biotin-paionin-1 with a Gly-Ala-Lys-Lys extension on the C-terminus was immobilised on the sensor chip by amide coupling and used in SPR binding experiments. The obtained sensorgrams were in agreement with the ELISA data as an impaired binding of PAI-1(R78E), PAI-1(R117E/R120E), and PAI-1(K71A/R78A/Y81A/K82A) was evident (Figure 6D). Using the same SPR setup, no binding between biotin-paionin-1 and the serpins antithrombin III and α 1 -proteinase inhibitor could be detected. In agreement with this, no binding site for paionin-1 in the flexible joint region of these serpins could be identified by an in silico cavity search (data not shown).

The charge-reversing mutations in PAI-1(R78E/R117E/R120E), located in hD (Arg78), hE (Arg117) and the hE/s1A loop (Arg120), were previously reported to impair binding to the PAI-1-neutralizing compounds AR-H029953XX and bis-ANS [START_REF] Björquist | Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site-directed mutagenesis[END_REF]13]. XR-5118 was found to have an overlapping, but not identical binding site [13,[START_REF] Einholm | Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1[END_REF]. Thus, the impaired binding of paionin-1 to variants carrying one or more of these mutations is consistent with paionin-1 binding to this hydrophobic surface area in the flexible joint and in agreement with the competition studies with bis-ANS and XR-5118 (Figure 2A). Likewise, the decreased binding of paionin-1 to PAI-1(K71A/R78A/Y81A/K82A) and the ability of heparin to compete with paionin-1 for binding (Figure 2A) is consistent with Lys71, Arg78 and Lys82 in hD being important for heparin binding [START_REF] Ehrlich | Elucidation of structural requirements on plasminogen activator inhibitor 1 for binding to heparin[END_REF]. The competition of paionin-1 binding by vitronectin (Figure 2B) is readily explainable by steric hindrance from the nearby vitronectin binding site [START_REF] Jensen | The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand[END_REF]16]. The impaired binding to murine PAI-1 may result from the replacement of Arg120 with Gln in this ortholog.

In summary, these ELISA and SPR data consistently advocate a binding site for paionin-1 in the flexible joint region of human PAI-1 that becomes more accessible upon the S to R transition. As depicted in Figure 4B, the residues found to be important for paionin-1 binding surround the entrance to the binding cavity proposed from the ligand docking algorithms thus lending support to this analysis.

Paionin-1 and the anti-proteolytic activity of PAI-1

We tested if biotin-paionin-1 was able to affect the activity of PAI-1 towards uPA or to change the rate of latency transition of PAI-1. To this end we employed a chromogenic assay which monitored the activity of uPA in the absence or presence of PAI-1 which had been treated with different concentrations of biotin-paionin-1 for various periods of time. Biotinpaionin-1 did not prevent formation of the uPA-PAI-1 complex but increased the rate of PAI-1 latency transition ~2-fold at concentrations higher than 50 µM, i.e. above the determined CMC (data not shown). This effect is most likely not related to specific peptide-binding but rather a non-specific effect of biotin-paionin-1 aggregates or micelles, analogues to the acceleration of latency transition caused by >100 µM Triton X-100, which has a CMC of 200-300 µM [START_REF] Gils | Modulation of plasminogen activator inhibitor 1 by Triton X-100-identification of two consecutive conformational transitions[END_REF]. That biotin-paionin-1 does not impair the activity of PAI-1 though it binds the same area as the PAI-1 neutralisers bis-ANS and XR-5118 exemplifies that the effect of targeting this area depends on the properties of the employed compound. For instance, negatively charged neutralisers including bis-ANS induce PAI-1 polymerisation whereas XR-5118, which carries a positive charge, does not [13,48,[START_REF] Einholm | Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1[END_REF].

Effects of paionin-1 on binding of uPA-PAI-1 complex to VLDLR and LRP-1A

The four alanine substitutions in PAI-1(K71A/R78A/Y81A/K82A), located in the hC/hD-loop (Lys71) and hD (Arg78, Tyr81, and Lys82), reduces the affinity of the uPA-PAI-1 complex for endocytosis receptors of the LDLR family (Skeldal, S., Larsen, J.V., Pedersen, K.E., Petersen, H.H., Egelund, R., Christensen, A., Jensen, J.K., Gliemann, J., Andreasen, P.A., unpublished work). This is in agreement with a reported binding site for these receptors involving hD and hE of PAI-1 [18-20] and the impaired paionin-1 binding to PAI-1(K71A/R78A/Y81A/K82A) suggests an overlap between the binding sites for the peptide and the receptors. In light of this, the effect of paionin-1 on uPA-PAI-1 complex binding to VLDLR and LRP-1A was tested. Receptor was immobilised on the solid phase and the binding of purified 125 I-uPA-PAI-1 complex was determined in the presence of varying concentrations of peptide. The results clearly showed that biotin-paionin-1 inhibited the binding of the complex to LRP-1A with an IC 50 value of ~2.5 μM (Figure 7A). Similar data were obtained with soluble VLDLR (IC 50 ~5 μM, not shown). As a control, the peptide was shown not to affect the binding of 125 I-RAP to the receptors except when concentrations close to the CMC for biotin-paionin-1 were applied (Figure 7A and not shown). Noteworthy, a high variability of the effect on RAP-binding was observed. Considering that the extent of micelle formation will be temperature dependent close to the CMC, and that the experiments summarised in Figure 7A were performed at ambient temperature, this variability agrees with a non-specific effect of micelles. In contrast, paionin-1 without a biotin on the N-terminus did not have an inhibiting effect even at high concentrations (50 µM). Biotin alone or attached to the linear version of paionin-1 was also shown not to have an effect (data not shown). Our interpretation of these data is that the combination of the intact paionin-1 structure and the biotin are equally important for the function of the peptide, i.e. the structure dictates binding specificity and biotin the antagonizing function. The observed effect of the biotinylated paionin-1 peptide on uPA-PAI-1 complex binding to endocytosis receptors was confirmed in a cell culture system with the human histiocytic lymphoma derived cell line U937. From this assay it was evident that biotin-paionin-1 significantly reduced VLDLR-mediated endocytosis and degradation of 125 I-uPA-PAI-1 complex with an IC 50 value of ~10 µM (Figure 7B). Again a concentration in the area up to 30-40 µM was required to observe an effect of biotinpaionin-1 on 125 I-RAP endocytosis, an effect probably explainable by the formation of micelles.

In conclusion, we have isolated a PAI-1 binding peptide sequence, paionin-1, from a phage-displayed peptide library. We determined the solution structure of the peptide by NMR and used it for in silico docking of the peptide into X-ray crystal structures of PAI-1. These analyses suggested a binding site for paionin-1 in the flexible joint region of PAI-1. The predicted binding site was supported by the ability of other molecules targeting this region to compete with paionin-1 for binding and by site-directed mutagenesis. We demonstrated that the peptide with an N-terminal biotin extension is able to inhibit the binding of the uPA-PAI-1 complex to endocytosis receptors. However, the peptide needs to be developed further before being valuable as a tool for investigations of the molecular interactions of PAI-1. In particular, an improvement of affinity and solubility is needed in order to increase the presently small window between specific and non-specific effects. By being able to inhibit uPA-PAI-1 endocytosis receptor binding, biotin-paionin-1 exemplifies compounds which may prevent the recycling of free uPAR after internalisation of the uPAR-uPA-PAI-1 complex. This would disrupt the signalling function of uPA-PAI-1 endocytosis receptor binding and downregulate the pericellular proteolytic capacity. [START_REF] Stout | Structures of active and latent PAI-1: a possible stabilizing role for chloride ions[END_REF]), latent PAI-1 (PDB ID 1DVN [START_REF] Stout | Structures of active and latent PAI-1: a possible stabilizing role for chloride ions[END_REF]), and the complex between the serpin α 1 -PI and the protease elastase (PDB-ID 2D26 [START_REF] Dementiev | Active Site Distortion Is Sufficient for Proteinase Inhibition by Serpins: STRUCTURE OF THE COVALENT COMPLEX OF α 1 -PROTEINASE INHIBITOR WITH PORCINE PANCREATIC ELASTASE[END_REF]). For the serpins, the RCL is in red, the large serpin fragment is in orange, the small serpin fragment including hF is in blue and hD and hE from the flexible joint region are in green. Elastase is shown in purple. Monoclonal anti-uPA Mab-2 was included as a control.

A

  stop codon succeeding the codon for amino acid 772 was introduced into the human VLDLR type-I cDNA [29]. This cDNA, encoding soluble VLDLR (residues 1-772) without the cytosolic domain and transmembrane region, was then cloned into the plasmid pcDNA3.1(-) (Invitrogen) and authenticated by DNA sequencing. E. coli DH5α cells (Invitrogen) were used for plasmid propagation and transient transfection of HEK 293T cells was carried out by standard methods [30]. Soluble VLDLR was purified from serum-free conditioned medium from transfected cells by affinity chromatography at 4 °C on a 5 mL RAP-coupled Sepharose column. HBS supplemented with 2 mM CaCl 2 and 1 mM MgCl 2 was used for equilibration and washing of the column while elution was carried out with 0.1 M CH 3 COOH, pH 3.5, 0.5 M NaCl, 10 mM EDTA. Fractions were neutralised immediately with 0.1 volume 1 M Tris, pH 9, 25 mM CaCl 2 and those containing soluble VLDLR selected based on 125 I-RAP ligand blots [31]. Purified soluble VLDLR was concentrated with a Centriplus YM-10 spin column, dialysed into PBS, and stored at -80 °C.

FIGURE 2

 2 FIGURE 2 Competition of paionin-1 phage binding in ELISAs Binding of paionin-1 phage clone B to 10 nM PAI-1 from HT-1080 cells was detected in ELISA format 1 (see Experimental) in the presence of additions as indicated for each experiment. (A) Paionin-1: NH 2 -DVPCFGWCQDA-COOH. Paionin-1, linear: NH 2 -DVPSFGWSQDA-COOH. Bis-ANS and XR-5118: see [13]. (B) Effects from monoclonal anti-PAI-1 antibodies Mab-1, Mab-2 and Mab-6 and vitronectin on paionin-1 phage binding.

FIGURE 3

 3 FIGURE 3 The structure of paionin-1 determined by NMR spectroscopy (A) Overlay of the 15 low-energy structures in conformational group number 1, aligned according to the backbone of the CFGWC ring, shown in red. (B) The CFGWC ring of one representative structure from each of the 9 conformational groups, each with a distinct colour, aligned according to the backbone.

FIGURE 4 FIGURE 5 1 FIGURE 5 Biochemical

 4515 FIGURE 4 Docking of paionin-1 into the structure of latent PAI-1 and residues important for paionin-1 binding in the flexible joint region Latent PAI-1 shown as a ribbon diagram with the inserted RCL in red, the large serpin fragment in orange, the small serpin fragment including hF in blue and hD and hE from the flexible joint region in green. (A) The docked structure of paionin-1 from conformational group number 1 is shown as a stick model. (B) Residues which based on binding assays with mutated PAI-1 variants are implicated in paionin-1 binding are indicated. . FIGURE 5 Conductometric determination of the CMC for biotin-paionin-1

Biochemical Journal Immediate Publication. Published on 3 Jul 2006 as manuscript BJ20060533 Copyright 2006 Biochemical Society Kem

  -En-Tech Diagnostics) in 50 mM citric acid (pH 5) supplemented with 0.03% H 2 O 2 and quenched with one volume 1 M H 2 SO 4 before measuring the absorbance at 492 nm in a microplate reader.

Biochemical Journal Immediate Publication. Published on 3 Jul 2006 as manuscript BJ20060533

TABLE 1 Selected PAI-1 binding peptides

 1 Sequence alignment and EC 50 for the PAI-1 binding peptides isolated by phage display.Consensus motifs are underlined. The frequency indicates the occurrence of each clone among the sequenced binders. The EC 50 values were determined as the amount of PAI-1 required for half-maximal binding in ELISA format 1 (see Experimental).

	Clone	Sequence	Frequency EC 50 (nM)
	A	CDPPCFGWCQDAC	2	10
	B	CDVPCFGWCVAWC	12	8.2
	C	CQGGCRVQCFGFC	1	30
	F	CTGACPVVCFGFC	1	17
	G	ILCFGMC	1	58
	H	CEGVCRVSCFGWC	3	6.0
	I	CDVPCFGWCVSWC	1	13
	J	CVCFGYCRYEC	1	9.9
	N	CHILCFGYCGDLC	1	4.2
	O	CEGKCVVSCFGFC	1	9.8

TABLE 2 NMR structure characteristics

 2 Among a total of 100 calculated low-energy structures of paionin-1 (NH 2 -DVPCFGWCQDA-COOH), 88 could be grouped based on the orientation of the Phe 5 and Trp 7 sidechains. The average RMSDs for the CFGWC ring is given for backbone and for all atoms for each group and for all 100 structures. Ribbon diagrams of active PAI-1 14-1B (a stable, active PAI-1 variant carrying four mutations [42], PDB-ID 1DVM

	Group Number of structures RMSD, backbone (Å) RMSD, all atoms (Å)
	1	15	0.47 ± 0.27	1.08 ± 0.58
	2	7	0.31 ± 0.16	0.78 ± 0.27
	3	16	0.50 ± 0.24	1.23 ± 0.40
	4	19	0.56 ± 0.22	1.19 ± 0.37
	5	9	0.58 ± 0.26	1.39 ± 0.53
	6	10	0.64 ± 0.36	1.47 ± 0.59
	7	4	0.28 ± 0.08	0.80 ± 0.29
	8	4	0.33 ± 0.15	0.70 ± 0.25
	9	4	0.15 ± 0.08	0.53 ± 0.22
	-	100	1.08 ± 0.51	2.34 ± 0.65

ACKNOWLEDGEMENTS

We acknowledge Erkki Koivunen, Division of Biochemistry, University of Helsinki, Finland, Ann Gills, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium; and Jørgen Gliemann, Institute of Medical Biochemistry, University of Aarhus, Denmark, for materials. Axel Meissner, the Carlsberg Laboratory, Copenhagen, Denmark, is acknowledged for help with NMR experimental setup and for use of the Bruker 600 MHz NMR spectrometer, and Ken Howard, Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Denmark, for help with the dynamic light scattering experiments. The Danish Cancer Research Foundation, iNANO, the Danish Cancer Society, the Danish National Research Foundation, Carlsbergfondet, the Danish Natural Science Research Council, and the Danish Biotechnology Instrument Centre (DABIC) are thanked for financial support. T.W. is the recipient of a fellowship from Carlsbergfondet (ANS-1289/20).

The electric conductance was monitored while titrating biotin-paionin-1 into water. The presented data have been corrected for the conductance resulting from adding equal volumes of buffer without peptide. The two stipulated lines represent linear regressions of data points below and above 100 µM biotin-paionin-1, respectively, crossing each other at 107 µM.


Meng Lin

Stefan Rose-John

Joachim Grötzinger

Udo Conrad

Jürgen Scheller
email: jscheller@biochem.uni-kiel.de

The functional expression of a biologically active fragment of soluble gp130 as an ELP fusion protein in transgenic plants: purification via inverse-transition-cycling

Keywords: gp130, ELP, transgenic plants, IL-6, inverse transition cycling

In murine models of Crohn's disease, rheumatoid arthritis and colon cancer, interleukin (IL)-6 signalling via the soluble IL-6 receptor (sIL-6R, termed IL-6 trans-signalling) has been shown to promote the pathology associated with these conditions. These detrimental activities can however be selectively blocked by soluble forms of gp130, which acts as the universal signalling receptor subunit for all IL-6-related cytokines. Although soluble gp130 (sgp130) therefore represents a viable therapeutic modality for the treatment of these conditions, the mass manufacture of such biologics is often expensive. The advent of molecular farming has however provided an extremely cost effective strategy for the engineering of recombinant proteins. Here we describe the expression and production of a biologically active sgp130 variant which is expressed in transgenic tobacco plants as an elastin-like pentapeptide (ELP) fusion protein (mini-gp130-ELP). Mini-gp130-ELP consists of the first three domains of gp130 (Ig-like domain and cytokine binding module) fused to 100 repeats of ELP. Expression of mini-gp130-ELP did not affect the growth rate or morphology of the transgenic plants, and purification was achieved using inverse transition cycling. This approach led to an overall yield of 141 µg of purified protein per gram of fresh leaf weight. The purified mini-gp130-ELP specifically inhibited sIL-6R-mediated transsignalling as measured by binding to the IL-6/sIL-6R complex and through its ability to block sIL-6R-mediated activation of STAT3 phosphorylation and proliferation in human hepatoma cells and murine pre-B-cells.

Consequently, this study validates the potential application of molecular farming in transgenic tobacco plants as a strategy for the expression and purification of therapeutically advantageous biologics such as sgp130.

INTRODUCTION

Cytokines are a series of small soluble polypeptides, which control cellular functions like proliferation, differentiation and viability. Dysregulation of cytokine activity is often associated with the progression of chronic disease, and selective targeting of these proteins in various disease states has proven to be clinically beneficial. Such an approach is exemplified by the use of blocking anti-TNFα regimes in rheumatoid arthritis and Crohn's disease.

Cytokines of the gp130-family, including interleukin 6 (IL-6), IL-11, leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary inhibitory factor (CNTF), cardiotropin-1 (CT-1), cardiotrophin-like related cytokine/neurotrophin-1/B-cell stimulating factor 3 (CLC/NNT-1/BSF-3), neuropoietin (NPN) and IL-27 share the membrane glycoprotein gp130 as a common signal transducer subunit (reviewed in [START_REF] Heinrich | Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway[END_REF] and [START_REF] Jones | The role of soluble receptors in cytokine biology: The agonistic properties of the sIL-6R/IL-6 complex[END_REF]).

On target cells, IL-6 first binds to the IL-6 alpha receptor (IL-6R). The resulting complex of IL-6 and IL-6R then associates with the signal transducing membrane protein gp130, thereby initiating intracellular signalling [START_REF] Rose-John | Coordination of interleukin-6 biology by membrane bound and soluble receptors[END_REF]. The gp130 subunit is expressed by most, if not all cells in the body whereas IL-6R expression is mainly limited to hepatocytes, neutrophils, monocytes/macrophages and specific lymphocyte subsets. Moreover, a soluble IL-6R (sIL-6R) has been found in various body fluids [4; 5; 6; 7; 8; 9; 10; 11]. Soluble IL-6R forms an agonistic complex with IL-6 that stimulates cells which only express gp130 [12; 13]. A process which we have termed trans-signalling [12; 14]. Interestingly, a naturally occurring soluble gp130 (sgp130) possesses antagonistic properties on IL-6 acting in complex with the soluble IL-6R but not on IL-6 acting via the membrane IL-6R. At 100-fold higher concentrations soluble gp130 also inhibits LIF and OSM but not CNTF and IL-27 [15; 16; 17]. Therefore, we have concluded that sgp130 is the natural inhibitor of IL-6-trans-signalling. Moreover, in mouse models of human diseases, sgp130 has been shown to be a potent candidate for therapy of e.g. Crohn´s disease, rheumatoid arthritis and colon cancer [18; 19; 20; 21].

Following the initial report on expression of complete antibodies in transgenic plants [START_REF] Hiatt | Production of antibodies in transgenic plants[END_REF], several alternative expression strategies have been described for the high yield production of recombinant proteins in transgenic plants [23; 24]. However for many proteins, increased ER retention leads to a trapping of the recombinant protein within the plant [START_REF] Artsaenko | Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco[END_REF]. Moreover, we have demonstrated a twofold increase of spider silk protein accumulation in tobacco leaves and a 40-fold increase of single chain antibody accumulation in tobacco seeds after fusion to 100 repeats of an elastin-like pentapeptide (ELP), with one peptide unit made up of Val-Pro-Gly-Xaa-Gly (Xaa is any amino acid except proline; [26; 27, 28]). This amino acid stretch is derived from the characteristic repeat motif VPGVG found in the native sequence for mammalian elastin. ELPs and ELP-fusion proteins are soluble in water below their transition temperature and become reversibly insoluble when the temperature is raised above the transition temperature (inverse transition cycling) [27]. Since we have shown that inverse transition cycling can be used to purify heat stable spider silk proteins from transgenic tobacco leaves [26], we have now asked whether this principle could be transferred to nonheat stable proteins with therapeutic potential. We therefore generated transgenic tobacco plants, which express a sgp130-ELP (mini-gp130-ELP) fusion protein under the transcriptional control of the plant virus promoter CaMV35S. Such an approach led to the accumulation of large amounts of mini-gp130-ELP in the endoplasmic reticulum, which could be purified as a functionally active variant using inverse transition cycling.

MAERIAL AND METHODS

Cells and Reagents

HepG2 cells (human hepatoma cells) were obtained from the American Type Culture Collection (Rockville, MD/Manassas, VA, USA). BA/F3 cells (murine pre-B-cells) stably transfected with human gp130 (Ba/F3-gp130 cells) or gp130 and IL-6R cDNAs (Ba/F3-gp130-IL-6R cells) have been previously described [29; 30]. All cells were grown in DMEM High Glucose Culture Medium (PAA Laboratories, Pasching, Austria) supplemented with 10% foetal calf serum, penicillin (60 mg/l) and streptomycin (100 mg/l) at 37°C with 5% CO 2 in a humidified atmosphere. For the cultivation of Ba/F3-gp130-IL-6R cells the standard DMEM medium was supplemented with 10% FCS, penicillin (60 mg/l), streptomycin (100 mg/l) and 10ng/ml IL-6 or 10ng/ml Hyper-IL-6. Hyper-IL-6 is a fusion protein of IL-6 and sIL-6R connected by a flexible polypeptide linker. Soluble gp130Fc is a fusion protein of the extracellular portion of gp130 and the Fc-portion of IgG1 [16; 29]. Both recombinant proteins were expressed and purified as previously described [16; 29]. Hyper-IL-6-Fc is a fusion protein of the Hyper-IL-6 and the Fc-portion of an IgG antibody, which was expressed in transfected EBNA-HEK-293 cells and was directly used as conditioned supernatant.

Mouse anti-STAT3 mAb was purchased from Cell signalling technology (Beverly, MA, USA), rabbit anti-phospho-STAT3 mAb (Y705) was from Cell signalling technology (Beverly, MA, USA). Mouse anti human IL-6R mAb M91 was from Immunotech (Marseille, France). Mouse anti c-myc mAb 9E10 was from Abcam (Cambridge, UK). Sheep anti-mouse IgG horseradish peroxidase and goat anti-rabbit IgG horseradish peroxidase were from Amersham Biosciences (Buckinghamshire, UK). All restriction enzymes were obtained from MBI Fermentas (St. Leon-Rot, Germany). [ 3 H]thymidine was purchased from Amersham Biosciences (Freiburg, Germany).

Construction of the binary vector for mini-gp130-ELP under the control of the CaMV-

35S-promoter for the transformation of tobacco plants

The cytokine binding portion (D1-D3) of the cDNA coding for sgp130Fc-opt (see below) was amplified by PCR using the sense strand oligonucleotide primer gp130-Δsignal (5´ GACGTCACAT GTTTAGCAGA GCTGCTGGAT CCTTGC 3´) and the gp130-NaeI (5´ CAGTGCCGGC TCTGTCCTCG TAGGTGATGC 3´) anti-sense oligonucleotide primer.

The plasmid pCR-Script-sgp130Fc-opt served as template (GeneArt, Munich, Germany).

Here, the codon usage of sgp130Fc was adapted to the codon bias for Cricetulus griseus (Chinese hamster) genes (for sequence information of the cDNA coding for mini-gp130-ELP and the protein sequence of mini-gp130-ELP see supplementary Figure 1A andB). The purified PCR-product was digested with AflIII and NaeI and cloned into the plasmid pRTRA7/3-35S-anti-KRES-c-myc-100xELP [27], which was linearised with AflIII and NaeI.

The generation of the 100xELP fusion protein was described previously [26]. The resulting plasmid pRTRA7/3-35S-mini-gp130-ELP was digested with HindIII and the fragment containing the expression cassette (35S promoter/LeB4 signal peptide/mini-gp130/c-myctag/ELP/KDEL/CaMV 35S terminator) cloned into the binary plasmid pCB301-Kan. The vector pCB301-Kan is based on the vector pCB301 [START_REF] Xiang | A mini binary vector series for plant transformation[END_REF] and was produced by the transfer of a BglII-BamHI-T-DNA-fragment containing the kanamycin resistance gene of the pBIN19 vector [START_REF] Bevan | Binary Agrobacterium vectors for plant transformation[END_REF].

Transformation of tobacco

The mini-gp130-ELP encoding construct was transferred into Agrobacterium tumefaciens C58C1 (pGV2260) by electroporation. Tobacco (Nicotiana tabacum cv. SNN) leaf discs were transformed as described elsewhere [START_REF] Zambriski | Ti-plasmid vector for introduction of DNA into plant cells without altering their normal regeneration capacity[END_REF]. Regenerated transgenic plants were grown in vitro on Murashige-Skoog medium containing 100 mg/l kanamycin. Regenerated plants were grown to maturity in the greenhouse and were screened for high expression by Western blot analysis using the anti c-myc mAB 9E10.

Purification of recombinant mini-gp130-ELP

Green leaves from transgenic tobacco plants were ground in a mortar under liquid nitrogen. 50 ml PBS was added to 10 g ground leaves and the suspension was stirred for 5 min at RT. The extract was cleared by centrifugation at 8,000 g for 30 min. The supernatant was filtered (Syringe Filter 0.22 µM pore diameter (Roth GmbH, Karlsruhe, Germany)) and sodium chloride added to a final concentration of 2 M. The solution was incubated in a water bath at 40°C for 30 min to allow for aggregation of the ELP-fusion proteins. The aggregates were precipitated by centrifugation at 8,000 g for 30 min at 40°C. The precipitate was dissolved on a vertical shaker at 200 rpm in 50 ml PBS for 15 min at 20°C. Insoluble material was removed by centrifugation at 8,000 g for 15 min at 20°C. The supernatant was supplemented with sodium chloride to a final concentration of 2 M. The solution was again incubated in a water bath at 40°C for 30 min. The precipitate was removed by centrifugation at 8,000 g for 30 min at 40°C. The precipitate was dissolved on a vertical shaker at 200 rpm in 2 ml PBS for 15 min at 20°C. Insoluble material was removed by centrifugation at 8,000 g for 15 min at 20°C.

Size exclusion chromatography

The mini-gp130-ELP protein was further purified on a calibrated HiPrep 26/60 Sephacryl S-300 High Resolution column (Amersham Biosciences, Freiburg, Germany) using PBS as the mobile phase with a constant flow-rate of 1.0 ml/minute. For calibration, the high molecular mass standards (Amersham Biosciences, Freiburg, Germany) were used (see supplementary Figure 4A andB). Fractions of 2.5 ml were collected, analyzed by SDS-PAGE, pooled and concentrated.

Protein concentration

The method of Waxman [START_REF] Waxman | Determination of the tryptophan:tyrosine ratio in proteins[END_REF] was used to determine the protein concentration. The extinction coefficient of mini-gp130-ELP at 280 nm was calculated to be 64890 l/mol x cm. The absorption spectra were recorded in the range of 240-320 nm (supplementary Figure 5A).

Edman sequencing

10µg of the purified mini-sgp130-ELP dissolved in 10µl PBS was sent for N-terminal sequence determination by automated Edman degradation to Sequence laboratories Göttingen GmbH (Seqlab, Göttingen, Germany).

Immunoprecipitation

Purified mini-gp130-ELP protein was incubated with Hyper-IL-6-Fc in DMEM-10% FCS overnight at 4°C followed by addition of 50 µl of protein A-Sepharose (50% slurry, CL-4B from Amersham Biosciences, Freiburg, Germany) for at least 1 h at 4°C. Immunoprecipitates were washed twice with washing buffer (10mM Tris pH 7.6, 150mM NaCl, 0.2 % Tween, 2mM EDTA) and once with PBS prior to addition of Laemmli sample buffer (2xLaemmli:100 mM Tris pH 6.8; 4% SDS; 200 mM DTT; 20 % glycerol; 0.1% Bromophenol Blue) and boiling at 95°C for 5 min.

Enzyme-Linked Immunosorbent Assay (ELISA)

Microtitre plates (Greiner Microlon, Solingen, Germany) were coated with purified mini-gp130-ELP (5µg/ml) in PBS. The plate was incubated over night at room temperature. After blocking with 3% (w:v) bovine serum albumin in PBS for 2 h, 100 µl aliquots of Hyper IL-6 (0.1µg/ml, 0.5µg/ml and 1µg/ml) (with or without antagonistic sgp130Fc (5µg/ml)) were added. The plate was incubated for 1 h at room temperature. Hyper IL-6 bound to the plate was detected by the non-neutralizing mAb M91 (working concentration 0.1µg/ml) followed by anti-mouse IgG horseradish peroxidase 1/1000 diluted in PBS/3% BSA. The enzymatic reaction was performed with soluble peroxidase substrate (BM blue POD from Roche, Mannheim, Germany) at 37°C for 30 min and the absorbance was measured at 450 nm on a SLT Rainbow plate reader from Tecan (Maennedorf, Switzerland).

Immunoblotting and enhanced chemiluminescence (ECL) detection

For Western Blotting, proteins separated by SDS-PAGE were transferred to polyvinylidendifluoride membranes (Hybond-P from Amersham Biosciences, Freiburg, Germany) by a semi-dry electro-blotting procedure. Membranes were blocked in a solution of TBS (10 mM Tris, pH 8, 150 mM NaCl) supplemented with 0.02% Tween-20 and 5% skimmed milk powder, and probed overnight with the indicated antibodies at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were detected using a chemiluminescence kit (ECL plus Western Blotting Detection System, Amersham Biosciences, Freiburg, Germany) following the manufacturer's instructions. Band intensities were quantified using ImageJ software (National Institutes of Health).

Proliferation assays

Ba/F3-gp130-IL-6R cells were washed 3 times with sterile PBS and re-suspended in DMEMcontaining 10% FCS at 5 x 10 3 cells per well of a 96-well plate. The cells were cultured in a final volume of 100 µl with additional cytokines as indicated in the legend of Figure 4C for 72 h and subsequently pulse labelled with 0.25 µCi [ 3 H]-thymidine for 4 h. The specific activity of [ 3 H]thymidine was 25Ci/mmol. Cells were harvested on a glass fibre filter (Filtermat A from Wallac, Turku, Finland) and the filter was microwave-baked for 2 min. Subsequently, the filter was soaked in 4.5 ml liquid scintillation cocktail (Betaplate Scint from Wallac, Turku, Finland).

RESULTS

The mini-gp130-ELP fusion protein is expressed in high amounts in leaves of transgenic tobacco plants and can be purified by inverse transition cycling

A cDNA coding for the cytokine binding domains D1-D3 of gp130 was fused to a cDNA encoding 100 repeats of the elastin-like peptide (ELP) and placed under the control of the plant virus promoter CaMV35S (Figure 1A). For sequence information see supplementary Figure 1A and1B.

We used ELP-units that were made up of the pentapeptide Val-Pro-Gly-Xaa-Gly, where Xaa is valine, glycine or alanine [26]. The LeB4 signal peptide and the KDEL signal at the Cterminus caused ER retention. KDEL mediated ER retention was chosen because maintenance of single chain Fv-fragments and spider silk proteins as ELP-fusion proteins in the ER resulted in high level accumulation in transgenic tobacco seeds and leaves, respectively [26; 27]. The expression of mini-gp130-ELP without an ER retention signal in transgenic tobacco plants was not examined. A c-myc tag was used for detection of mini-gp130-ELP in Western blotting. Transgenic tobacco plants were generated by leaf disc transformation [START_REF] Zambrinski | Ti-plasmid vector for introduction of DNA into plant cells without altering their normal regeneration capacity[END_REF] and leaves were harvested and tested by Western blotting (data not shown). High-producers were chosen (12 out of 25 for mini-gp130-ELP, supplementary Figure 3A) and proteins were extracted from leaves with PBS. Protein expression of recombinant mini-gp130-ELP could already be detected after SDS-Page-Gel separation and Coomassie-staining of raw leaf extracts (Figure 2A, lane 1 and 2). Recombinant mini-gp130-ELP was purified by two rounds of inverse transition cycling as described in the material and methods section (Figure 1B and Figure 2A, lane 3-8).

Finally, 2 ml of pure mini-gp130-ELP-protein from 10 g of green leaves was further purified by size exclusion chromatography on a calibrated HiPrep 26/60 Sephacryl S-300 High Resolution column (for calibration curve see supplementary Figure 4). We determined the molecular mass of monomeric mini-gp130-ELP by SDS-Page analysis to be 100 kDa. The difference to the calculated molecular mass of mini-gp130-ELP (77.4 kDa) is likely due to glycosylation within the gp130 portion of the mini-gp130-ELP fusion protein. Since the majority of mini-gp130-ELP behaved on the column like a 240 kDa protein as estimated by comparison with a calibration curve of molecular mass standards, it is likely that the majority of the mini-gp130-ELP formed at least dimers (large peak in Figure 2B). A smaller fraction formed even higher ordered oligomers (650 kDa), whereas monomeric mini-gp130-ELP could not be detected. On the other hand, it could not be excluded that the presence of the An enzyme-linked immunosorbent assay (ELISA) was developed to detect the binding of mini-gp130-ELP to the IL-6/sIL-6R-complex. Mini-gp130-ELP protein was surface adsorbed and increasing amounts of Hyper IL-6 were added followed by detection via an anti-IL-6R antibody M91 (Figure 3B). The monoclonal antibody M91 directed against the human IL-6R was chosen for the immunochemical detection of Hyper IL-6. M91 recognizes the Ig-like domain of IL-6R and therefore does not affect binding of Hyper-IL-6 to mini-gp130-ELP.

Mini-gp130-ELP alone did not cross react with the detection antibodies. Binding of Hyper-IL-6 to mini-gp130-ELP could be competed by a molar excess of sgp130Fc. sgp130Fc consists of the complete extracellular part of gp130 (domains 1-6) fused to a dimerization domain (Fc-part of a human IgG-antibody) [16]. These experiments clearly show that mini-gp130-ELP binds to Hyper-IL-6, which can be competed with the sgp130Fc protein.

Next, we asked whether mini-gp130-ELP could inhibit IL-6 or Hyper-IL-6 induced STAT3phosphorylation. Human hepatoma cells (HepG2) were stimulated with 50 ng/ml IL-6 or 50 ng/ml Hyper-IL-6. We have shown previously that sgp130Fc exclusively inhibits the IL-6/sIL-6R complex without affecting the activity of IL-6 [16]. As shown in Figure 3C, increasing amounts of mini-gp130-ELP led to a dose-dependent inhibition of Hyper-IL-6 induced STAT3-phosphorylation. On the other hand, mini-gp130-ELP was not able to inhibit STAT-3 phosphorylation when the cells were stimulated with IL-6 alone. These results indicate that mini-gp130-ELP specifically inhibits IL-6 signals that were mediated by the soluble IL-6 receptor but not by the membrane bound IL-6 receptor.

The IL-3 dependent murine pro B-cell line Ba/F3 is the only mammalian cell line known, which completely lacks gp130 expression [START_REF] Hibi | Molecular cloning and expression of an IL-6 signal transducer, gp130[END_REF]. However, if these cells are transfected with gp130 (Ba/F3-gp130 cells) their reliance on IL-3 can be circumvented by stimulation with Hyper-IL-6 [39; 40; 41; 29]. Ba/F3-gp130 cells were stimulated with a fixed concentration of Hyper-IL-6 in the presence of increasing amounts of mini-gp130-ELP. Mini-gp130-ELP inhibited Hyper-IL-6 induced STAT3-phosphorylation of Ba/F3-gp130 cells (Figure 4A). To prove that mini-gp130-ELP selectively blocked sIL-6R-mediated responses, an identical experiment was performed in Ba/F3 cells transfected with cDNA for both gp130 and the IL-6R (Ba/F3-gp130-IL-6R cells). These cells respond to either IL-6 or Hyper-IL-6, as shown in Figure 4B mini-gp130-ELP selectively blocked Hyper-IL-6 induced activation of STAT3, but not the IL-6 mediated phosphorylation of STAT3 in these cells. The activity of sgp130Fc was used as a comparative control protein. Moreover, the proliferation of Ba/F3-gp130-IL-6R cells stimulated by Hyper-IL-6 could be blocked by mini-gp130-ELP in a dose dependent manner (Figure 4C). As expected, mini-gp130-ELP had no inhibitory effect on the proliferation of Ba/F3-gp130-IL-6R cells treated with IL-6 alone. Thus, mini-gp130-ELP specifically inhibits IL-6 trans-signalling, but not classical IL-6-signalling.

DISCUSSION

Plant expression systems have the potential to be used for the production of safe and biologically active recombinant proteins. It has been estimated that proteins can be produced 10-50 times cheaper in plants than in E. coli [START_REF] Scheller | Production of spider silk proteins in tobacco and potato[END_REF]. Therefore, the combination of safe and low cost protein production with a protein-intrinsic purification strategy would be very desirable.

So far, there have been examples of high yield production of recombinant proteins in transgenic plants, mainly in the field of molecular medicine such as antibodies, enzymes and vaccines [42; 44; 45]. Moreover, health risks that may arise from contamination of recombinant proteins with potential human pathogens or toxins are minimized by molecular farming [START_REF] Daniell | Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants[END_REF]. Recently, an improvement of the production of recombinant single chain antibodies and spider silk proteins, respectively, in seeds and leaves has been achieved by ER-retention of ELP-fusion constructs [26; 27]. Furthermore, inverse transition cycling of a spider silk ELP-fusion protein has been used for purification from leaf extracts [26]. Spider silk proteins are heat stable and therefore represent an ideal model for inverse transition cycling since the temporary heat treatment did not lead to protein denaturation. Initially, we generated transgenic tobacco plants accumulating sgp130Fc or sgp130Fc-ELP in the ER of leaves. It turned out that these proteins of around 800 and 1300 amino acids respectively were produced very poorly (< 0.01 % and 0.05 % of total soluble protein, respectively; supplementary Figure 3A). Therefore, we decided to generate transgenic plants, which accumulate a minimal mini-gp130-ELP-fusion protein in the ER of leaves. Mini-gp130-ELP only contains the cytokine-binding portion of gp130 (domains 1 -3, 303 amino acids) fused to 100 repeats of ELP and was found to be produced in high amounts in transgenic tobacco leaves by ER-retention (compare supplementary Figure 3A andB). So far, it has not been examined whether ELP-fusion proteins, which are expressed in the cytoplasm or in plastids, will also result in increased protein accumulation compared to the non-fusion proteins.

Inverse transition cycling was applied to purify the mini-gp130-ELP to homogeneity. It has previously been shown that for sgp130Fc, a 2-5 fold molar excess was sufficient to inhibit IL-6/sIL-6R mediated BA/F3-gp130 cell proliferation by 50%. In sgp130Fc the complete extracellular part of gp130 (domains 1-6) was fused to a dimerisation domain (Fc-part of a human IgG-antibody). Thereby sgp130Fc mimics the receptor signal complex on the membrane, which also exists as a preformed dimer [47]. Interestingly, the preformed sgp130Fc-dimer has a ten-fold higher efficiency to inhibit IL-6/sIL-6R mediated responses than the sgp130 monomer [16]. For mini-gp130-ELP an 80-fold molar excess inhibited IL-6/sIL-6R mediated BA/F3-gp130 cell proliferation by 50%. Therefore, we conclude that plant produced mini-gp130-ELP antagonized the activity of Hyper-IL-6 less efficiently than the preformed sgp130Fc dimer but with comparable efficiency as a sgp130-monomer purified from mammalian cell culture supernatants [16]. In this respect, mini-gp130-ELP resembles the functional properties of the highly truncated spliced variant of sgp130 known as gp130-RAPS, which consists of D1-D3 of gp130 plus a unique COOH-terminal amino acid sequence [48; 49]. Interestingly, mini-gp130-ELP very likely formed dimers, which in contrast to the preformed sgp130Fc-dimer are non-directional, since mini-gp130-ELP exhibited antagonistic properties comparable to the monomeric sgp130 and not to dimeric sgp130Fc.

In summary, we have generated transgenic tobacco plants accumulating high amounts of biologically active mini-gp130-ELP in the endoplasmic reticulum, which could be purified to homogeneity by inverse transition cycling. The results reported in this manuscript are encouraging and will stimulate the generation of constructs with a cleavable ELP-portion, which can then be dimerised artificially and in a directed fashion, e.g. by free accessible Cterminal cysteines. This strategy might lead to plant produced mini-gp130-cytokine antagonists with even higher activity.

  [ 3 H]-thymidine incorporated into cellular DNA was determined by scintillation counting using the MicroBeta TriLux counter from PerkinElmer (Wellesley, MA, USA). Bioassays were performed with each value being determined in triplicate. Biochemical Journal Immediate Publication. Published on 23 May 2006 as manuscript BJ20060544

Figure 1

 1 Figure 1 Expression and purification of mini-gp130-ELP. (A) Schematic representation of the plant expression cassette and (B) illustration of the purification of ELP-fusion proteins from plant leaves by two rounds of ´inverse transition cycling´.

Figure 2

 2 Figure 2 Purification of mini-gp130-ELP by ´inverse transition cycling´. (A) Lane 1: raw extract of leaf material ground in a mortar under liquid nitrogen and extracted in PBS; lane 2: centrifuged and filtered (0.2 µM pore size) raw extract; lane 3: supernatant after 1 st round of inverse transition cycling; lane 4: soluble proteins after 1 st round of inverse transition cycling; lane 5: soluble proteins after 1 st round of inverse transition cycling cleared by centrifugation; ; lane 6: supernatant after 2 nd round of inverse transition cycling; lane 7: soluble proteins after 2 nd round of inverse transition cycling (concentrated by a factor of 25 when compared to raw extract); lane 8: soluble proteins after 2 nd round of inverse transition cycling cleared by centrifugation (concentrated by a factor of 25 when compared to raw extract). Proteins were separated on a 10% SDS polyacrylamide gel and stained with Coomassie. M: molecular mass marker (kDa).(B) Size exclusion chromatography of pure mini-gp130-ELP (2 ml) after 2 rounds of transition cycling on a calibrated HiPrep 26/60 Sephacryl S-300 High Resolution column using PBS as the mobile phase with a constant flow-rate of 1.0 ml/minute. Fractions of 2.5 ml were collected. Proteins were separated on a 10 % SDS polyacrylamide gel and silver stained.

Figure 3

 3 Figure 3 Plant produced mini-gp130-ELP is biologically active. (A) Immunoprecipitation of mini-gp130-ELP by Hyper-IL-6-Fc. 4 µg mini-gp130-ELP was coincubated with conditioned culture supernatant containing Hyper-IL-6-Fc or with culture supernatant without Hyper-IL-6-Fc. Precipitated proteins and supernatants were separated by SDS/PAGE and blotted onto a PVDF membrane. Proteins were detected with the c-myc specific antibody 9E10 and visualized by ECL detection. Lane 1: supernatant after precipitation of mini-gp130-ELP with Hyper-IL-6-Fc. Lane 2: Pellet after precipitation of mini-gp130-ELP with Hyper-IL-6-Fc. Lane 3: Supernatant after precipitation of mini-gp130-ELP without Hyper-IL-6-Fc. Lane 4: Pellet after precipitation of mini-gp130-ELP without Hyper-IL-6-Fc.

Biochemical Journal Immediate Publication. Published on 23 May 2006 as manuscript BJ20060544

ACKNOWLEDGEMENTS

We kindly thank Christine Helmold and Stefanie Schnell for excellent technical help and Simon Jones for discussions and the help with the preparation of the manuscript. The work in the laboratory of SRJ and JS was supported by grants from the Deutsche Forschungsgemeinschaft Bonn, Germany. UC was supported by the Pharma Planta Project supported by the European Community.

ELP-region resulted in an elongated protein that eluted in the position expected for a globular protein of higher molecular mass.

Mini-gp130-ELP from the major peak was collected and the concentration was quantified by UV-spectroscopy using the method of Waxman [START_REF] Waxman | Determination of the tryptophan:tyrosine ratio in proteins[END_REF] (see supplementary Figure 5A). Therefore, the final yield of purified mini-gp130-ELP was 141 µg purified protein per gram leaf fresh weight (supplementary Figure 5B).

The correct cleavage of the LeB4-signal peptide is important since the N-terminus of gp130 is needed for efficient binding to the IL-6/IL-6R complex [36; 37]. The four N-terminal amino acids of the mature mini-gp130-ELP-protein were determined by automated Edman sequencing (performed by Seqlab, Göttingen, Germany) to be ELLD, which corresponds to the wild type sequence of mature gp130. Purified mini-gp130-ELP protein was routinely stored at -20°C but was also stable at 4°C and room temperature without detectable loss of soluble recombinant protein integrity (supplementary Figure 2A andB).

Mini-gp130-ELP is biologically active and inhibits exclusively IL-6 trans-signalling, but not classic IL-6 signalling

4 µg purified mini-gp130-ELP was incubated with a molar excess of Hyper-IL-6-Fc and precipitated by protein A-Sepharose. Hyper-IL-6 is a fusion protein of IL-6 and the soluble IL-6R connected by a flexible linker [START_REF] Fischer | A designer cytokine with high activity on human hematopoietic progenitor cells[END_REF], which can directly bind to gp130. Here, Hyper-IL-6 was additionally fused to the Fc portion of an IgG antibody to allow for direct precipitation with protein A-Sepharose. Hyper-IL-6-Fc was produced in EBNA-HEK-293 cells and was directly used as conditioned supernatant. As depicted in Figure 3A, mini-gp130-ELP was precipitated by Hyper-IL-6-Fc (lane 2), whereas no mini-gp130-ELP remained in the cleared supernatant after immunoprecipitation with Hyper-IL-6-Fc (lane 1). Moreover, mini-gp130-ELP could not be precipitated by protein A-Sepharose alone (lane 3: supernatant; lane 4: immuno-precipitated protein). Since it was very likely that mini-gp130-ELP formed dimers at higher protein concentrations (Figure 2B), we cannot exclude that these dimers also existed at the lower mini-gp130-ELP concentrations used for the immunoprecipitation and for the following experiments. Therefore it is conceivable that the interaction of one out of two sgp130 molecules of the mini-gp130-ELP-dimer with Hyper-IL-6 is necessary for complete immunoprecipitation of mini-gp130-ELP. Since there was no detectable mini-gp130-ELP remaining in the supernatant after immunoprecipitation with Hyper-IL-6-Fc, we conclude that at least 50% of the mini-gp130-ELP molecules are biologically active in terms of binding to the complex of IL-6 and soluble IL-6R. Copyright 2006 Biochemical Society (B) Binding of Hyper-IL-6 to mini-gp130-ELP and competition with sgp130Fc as measured by enzyme-linked immunosorbent assay -Microtiter plates were coated with mini-gp130-ELP (5µg/ml) in PBS. After blocking with 3% BSA in PBS, 100µl aliquots of Hyper IL-6 (0.1 µg/ml, 0.5 µg/ml and 1 µg/ml) (with or without antagonistic sgp130Fc (5 µg/ml)) were added. Hyper IL-6 bound to the plate was detected by the non-neutralizing anti-IL-6R mAb M91 followed by anti mouse immunoglobuline-peroxidase conjugate. The enzymatic reaction was performed with soluble Peroxidase substrate at 37°C for 30 min and the absorbance was measured at 450 nm.

(C) Inhibition of Hyper-IL-6-induced STAT3-phosphorylation by mini-gp130-ELP in human hepatoma cells (HepG2). HepG2 cells were stimulated with 50 ng/ml IL-6 or Hyper-IL-6 for 5 min in the presence or absence of mini-gp130-ELP. Unstimulated cells were used as controls. STAT3 phosphorylation was detected by Western Blot using anti-phospho-STAT3 specific antibodies. Western Blotting against STAT3 served as loading control.