PEDS Advance Access published March 27, 2012 Protein Engineering, Design & Selection pp. 1 –9, 2012 doi:10.1093/protein/gzs012
Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin J.W.Back1,6, C.Frisch2, K.Van Pee3, V.Boschert3, R.van Vught1,4, W.Puijk1, T.D.Mueller3, A.Knappik2 and P.Timmerman1,5
6
To whom correspondence should be addressed. E-mail:
[email protected] Received November 4, 2011; revised February 20, 2012; accepted February 27, 2012 Edited by Paul Carter
Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystineknot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein. Keywords: cystine knot/epitope specific antibodies/oxidative folding/sclerostin
Introduction The bone remodeling protein sclerostin (human uniprot ID Q9BQB4) is thought to interfere with canonical Wnt/ b-catenin signaling by binding to the Wnt co-receptors
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1 Pepscan Therapeutics, Zuidersluisweg 2, 8203RC Lelystad, The Netherlands, 2AbD Serotec, Division of MorphoSys, Zeppelinstr. 4, 82178 Puchheim, Germany, 3Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute of the University Wuerzburg, Juliusvon-Sachs-Platz 2, D-97082 Wuerzburg, Germany, 4Present address: Department of Biochemistry of Membranes, Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht, The Netherlands and 5Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam, SciencePark 904, 1098 XH, The Netherlands
LRP5 or LRP6 (Bourhis et al., 2011), and inhibiting downstream signaling (Li et al., 2005; Agholme and Aspenberg, 2011). Canonical Wnt signaling has been shown to have an important role in bone homeostasis and maintenance in the adult organism and the downregulation of bone growth often seen in women after menopause might be due to a decreased Wnt signaling. The fact that Sclerostin is a negative regulator of Wnt activity together with sclerostin’s unique restricted expression pattern in bone acting only on osteocytes and osteoblasts (Poole et al., 2005) make it a highly attractive target for treating osteoporosis (Tian et al., 2010; Paszty et al., 2010; Papapoulos, 2011). Recently, the solution structures of human (Veverka et al., 2009) and murine (Weidauer et al., 2009) sclerostin have been determined ( pdb accession numbers 2K8P and 2KD3 respectively). These studies confirmed that the protein harbors a cystine knot motif as has been predicted from bioinformatics for all members of the DAN modulator superfamily to which sclerostin belongs (Avsian-Kretchmer and Hsueh, 2004). Six cysteine residues bridge in a characteristic pattern whereby a ring formed by two disulfides is penetrated by a third cystine bridge (Vitt et al., 2001). The cystine knot divides the intervening sequence into three loops. Two loops, the first and third, protruding to one end of the knot, each consist of two pairs of twisted beta sheets, and in the case of DAN family member sclerostin are connected by an extra inter-loop disulfide, so the mature protein contains four disulfide bridges. The second loop, consisting of 24 residues and facing the opposite side of the cystine knot, was found to be largely unstructured in solution, as are the N and C termini (that were included in the NMR structure analysis of the human protein). Murine sclerostin had been produced in a truncated form, lacking most of the N-terminus as well as the entire C-terminal segment (Weidauer et al., 2009), yet was found to be active in a Wnt reporter assay. Antibodies mainly binding to the flexible second loop have been found to block sclerostin-mediated inhibition of Wnt activity (Veverka et al., 2009), suggesting that this loop might be required for sclerostin’s bioactivity. Studies of these antibodies showed an effective increase in bone mineral density and strength, both in animal models (Li et al., 2009; Tian et al., 2010) and in clinical trials in human subjects (Padhi et al., 2011; Papapoulos, 2011). To further dissect the functional domains of this protein, and to provide antibodies directed at defined sites for diagnostic purposes as well as for analysis of sclerostin’s Wnt inhibition mechanism, we decided to produce by synthetic means truncated sclerostin, containing the intact cystine knot structure, and 10 variants of the truncated protein differing in the second flexible loop. The influence of the second loop on the folding of the protein into a native cystine knot was investigated. As it has been reported that antibodies raised against peptides often do not bind native proteins with
J.W.Back et al.
sufficient affinity (Jemmerson, 1987; Spangler, 1991; Brown et al., 2011), we used our construct that lacks both termini and a truncated second loop (tSOSTDb2) as bait in phage display panning using the HuCAL antibody library (Knappik et al., 2000), providing antibodies selective for the native protein. Materials and methods
Materials All Fmoc-amino acids and SPPS resins were purchased from Bachem, solvents were obtained from Biosolve, and other chemicals were from Sigma-Aldrich, and used without further purification.
Peptide synthesis
HPLC/MS Analyses were performed on an AquityTM HPLC/MS system using a BEH C18 column (Waters) with a linear gradient 5 –55% B in A in 2 min, where solvent A is 0.05% TFA in water and solvent B was 0.05% TFA in acetonitrile. All UV chromatograms were recorded at 215 nm.
Native chemical ligation Native chemical ligation (NCL) from two segments was performed in a one-step procedure where the C-terminal fragment was mixed with the N-terminal fragment in a 1:1.2 molar ratio in NCL buffer (6 M guanidine-HCl, 20 mM Tris(2-carboxyethyl)phosphine (TCEP), 200 mM 4mercapto-phenylacetic acid (MPAA), 0.2 M disodium hydrogenphosphate) that was manually adjusted with 10 M sodium hydroxide to pH 6.5 and the reaction was monitored by HPLC-MS, typically complete after 24 h. For NCL with three segments first the C-terminal segment was mixed with the thiaproline-protected middle segment in an equimolar ratio in NCL buffer (Bang and Kent, 2004). Once these segments were joined, methoxylamine was added to a final concentration of 20 mM (we lowered the concentration by a factor of 10 with respect to the original publication (Bang and Kent, 2004) to improve the yield in the second ligation step at the expense of a longer duration for the deprotection of the thiaproline), and the pH was lowered with TFA to pH 4.0. Deprotection of the thiaproline was monitored by 2
Preparative isolation Peptides were purified by reversed phase HPLC on a Deltapak C18 column. Material from NCL was first depleted of MPAA before HPLC by strong cation exchange on a 2-ml HiTrap SP FF column (GE Healthcare) in batch mode. Samples in NCL buffer were diluted with 60 volumes water and dithiothreitol was added to a final concentration of 5 mM before application to the column. The column was then washed with 10 mM sodium phosphate buffer pH 7.4, and subsequently bound protein was eluted with 5 ml of a solution 1 mM sodium hydroxide with 1 M NaCl, that was immediately acidified with TFA upon collection, to a pH , 4. Afterwards, the sample was desalted by HPLC.
Oxidative folding Standard folding was performed by dissolving 2 mg/ml of reduced protein in folding buffer (55 mM Tris – HCl, pH 8.0; 150 mM NaCl), followed by rapid dilution into 11 volumes of the same buffer, supplemented with reduced glutathione (GSH) to reach a 2 mM and oxidized glutathione (GSSG) to reach a 0.5 mM final concentration. The mixture was kept in narrow micronic tubes at 208C, and at time points along the folding interval, 67 ml of this mixture was mixed with 3 ml of 10% TFA to quench further oxidation. Ten microliters of this mixture was injected onto HPLC-MS. The peak area of absorbance at 215 nm was used to quantify the different folded populations.
Phage display library panning and Fab generation The HuCAL PLATINUM library (Prassler et al., 2011) was used for the generation of recombinant antibodies. Biotinylated tSOSTDb2 (Table I), both in a linear and in a conformationally constraint form, were either coupled to streptavidin-coated beads (Dynal) and incubated with the phage antibody library or incubated with the phage in solution and captured with streptavidin-coated beads. Binding Fabs were enriched in three consecutive panning rounds, the pool of Fab genes was isolated and inserted into Escherichia coli expression vectors that lead to functional periplasmic expression of monovalent Fab equipped with two peptide tags, the so-called myc tag (EQKLISEEDL) and a His6 tag, which was used for antibody purification. After transformation of E. coli TG1F2 (TG1 without the F-plasmid) with the expression vectors, individual colonies were picked and grown in microtiter plates. After induction of antibody expression with 1 mM IPTG overnight at 228C, the cultures were chemically lysed and the crude extracts were tested in enzyme-linked immunosorbent assay (ELISA) with immobilized antigens (recombinantly produced sclerostin and biotinylated peptides used in the panning) for the presence of antibody fragments that bind to both the peptide and the recombinantly produced sclerostin protein. The sequence of the antibody VH CDR regions was determined for clones that gave a strong (at least 5-fold over background, FOB) signal on the antigens in the ELISA, and colonies containing antibodies with unique CDR3 sequence were chosen for subsequent purification. Production of recombinant Fabs was as described by Jarutat et al. (2006).
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Peptides were synthesized by standard Fmoc solid-phase peptide synthesis using Rink resin or in protected form on Sasrin resin (Bachem) on a Symphony or Prelude-synthesizer (Protein Technologies), respectively. N-terminal biotin coupling was done as the last step in synthesis. The crude peptides were purified by reversed phase high-performance liquid chromatography (HPLC). The correct molecular masses of the peptides were confirmed by electro-spray ionization mass spectrometry on an AquityTM SQD mass spectrometer (Waters). Peptide 4-acetamidothiophenol thio-esters were prepared from the protected peptides by addition of two equivalents of 4-acetamidothiophenol and activation with PyBop (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) in dichloromethane with 1% N,N-diisopropylethylamine. After conversion into the desired thio-ester, the peptides were deprotected in trifluoroacetic acid (TFA):water:triisopropylsilane in a ratio 95:2.5:2.5.
HPLC-MS. After deprotection was complete, the pH was raised to pH 6.5 and 1.2 molar equivalents of the N-terminal segment were added for the second ligation step.
Complex epitopes on the cystine knot protein sclerostin
Table I. Sequences of human sclerostin and the constructs that were used in this study SOST_HUMAN
MQLPLALCLVCLLVHTAFRVVEGQGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAE
tSOST tSOSTdB2 tSOSTdB2var1 tSOSTdB2var2 tSOSTdB2var3 tSOSTdB2var4 tSOSTdB2Var5 tSOSTdB2Var6 tSOSTdB2Var7 tSOSTdB2Var8 tSOSTdB2Var9
–––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––––––– Loop 1
NGGWPGGRPPSRAPLSTDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLP – – – – – – – – – – – – – – – –$GGGCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLP – – – – – – – – – – – – – – – –$GGGCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – – – – – – – – – – – – –*DYKDDDDKCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – – – – – – – – – – – – –*DYKDDDDKCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – – – – – – – – – – – – –*DYKDDDDKCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – – – – – – – – – – – – –*DYKDDDDKCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – – – – – – – – – – – – –*DYKDDDDKCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – – – – – – – – – – – – –*DYKDDDDKCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – – – – – – – – – – – – –*DYKDDDDKCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – – – – – – – – – – – – –*DYKDDDDKCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – – – – – – – – – – – – –*DYKDDDDKCRELHFTRYVTDGPCRSAKPVTELVCSGQCG– – – – – Loop 2
Loop 3
SOST_HUMAN tSOST tSOSTdB2 tSOSTdB2var1 tSOSTdB2var2 tSOSTdB2var3 tSOSTdB2var4 tSOSTdB2Var5 tSOSTdB2Var6 tSOSTdB2Var7 tSOSTdB2Var8 tSOSTdB2Var9
NAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCKRLTRFHNQ NAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – PSGpDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – – – PADFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – – – – –PFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – – – – –SGSCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – – – GSGGSCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – – – SGSGSCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – GSGGSGGSCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – – – – – – GGCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – – – – – – –GCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – – – – – – – – – – – – – – – – – CIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKC# – – – – – – –
SOST_HUMAN tSOST tSOSTdB2 tSOSTdB2var1 tSOSTdB2var2 tSOSTdB2var3 tSOSTdB2var4 tSOSTdB2Var5 tSOSTdB2Var6 tSOSTdB2Var7 tSOSTdB2Var8 tSOSTdB2Var9
SELKDFGTEAARPQKGRKPRPRARSAKANQAELENAY –––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––– ––––––––––––––––––––––––––––––––
Alignment of human sclerostin to the synthetic constructs used in this study. Amino acids in one-letter notation, with the following symbols * ¼ N-terminal acetyl; $ ¼ N-terminal biotinyl; # ¼ C-terminal amide; p ¼ D proline. Constructs tSOSTb2, tSOSTb2var7, tSOSTb2var8 and tSOSTb2var9 were prepared in a one-step native chemical ligation, the other constructs were prepared in a two-step synthesis, as described in methods and based on Bang and Kent (2004).
ELISA MaxiSorp plates (NUNC) were coated overnight at 48C with a 5-mg/ml solution of protein in phosphate-buffered saline (PBS). For immobilization of the biotinylated peptides and biotinylated bovine serum albumin (BSA), neutravidin (Pierce) was coated. After blocking with 5% BSA in PBS containing 0.05% Tween 20 (PBST), biotinylated antigens at 2 mg/ml were incubated on neutravidin-coated plates for
30 min at room temperature. Plates were then washed. Subsequently, an aliquot of Fab at 2 mg/ml was added and incubated for 1 h at room temperature. Detection was performed using an anti-human Fab-alkaline phosphatase conjugate (AbD Serotec) using AttoPhos (Roche) as a substrate. The signals on the control proteins BSA, CD33 and glutathione-S-transferase (GST) were used for calculation of the background. 3
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SOST_HUMAN tSOST tSOSTdB2 tSOSTdB2var1 tSOSTdB2var2 tSOSTdB2var3 tSOSTdB2var4 tSOSTdB2Var5 tSOSTdB2Var6 tSOSTdB2Var7 tSOSTdB2Var8 tSOSTdB2Var9
Loop 2
J.W.Back et al.
monitored, by perfusing the chip only with HBS150T buffer for 200 s. The chip surface was regenerated with a short 60 s pulse of 10 mM glycine pH 2.0 at a flowrate of 30 ml/min. Binding affinities were determined by using the ProteOnTM Manager 2.1 software (BioRad). To remove bulk face effects interaction data to a blank (non-coated) channel was used for background subtraction. Binding data were analyzed by fitting the kinetics for association and dissociation employing a 1:1 Langmuir or a 1:1 Langmuir Mass Transfer model. To account for Fab antibodies showing only weak binding the maximal binding density was kept constant during data analysis with Rmax of 600 RU for human sclerostin and 500 RU for murine sclerostin which were determined using two standard antibodies raised against sclerostin. Equilibrium binding constants were calculated from the equation KD ¼ koff/kon and are expressed as mean values from six experiments. Wherever possible, interaction data were also analyzed using the dose dependency of equilibrium binding and KD values obtained were compared with equilibrium binding constants from the kinetic binding data.
Production of recombinant sclerostin
The truncated sclerostin protein fragment tSOST—the sequence as listed in Table I—was generated in a one-pot, two-step NCL procedure as described in Materials and Methods. After reversed phase purification of the intact reduced fragment, we attempted to fold the protein by rapid dilution into an oxidative buffer. When proteins spontaneously fold into their native structure, not only do they lose two Dalton in mass per disulfide bond formed, but their retention time in an RP-HPLC is also reduced, likely the effect of burial of the more hydrophobic groups in the protein interior and exposure of the more hydrophilic side chains (Lu et al., 1992; Chatrenet and Chang, 1993; Arolas et al., 2006). Spontaneous oxidative folding can give rise to two discernable populations that emerge over time, as is depicted in Fig. 1. One population is detected in RP-HPLC as a sharp peak that is significantly shifted to the left, and the product that it contains, readily dissolving in aqueous buffers, is known to correspond to the native disulfide knotted structure (Lu et al., 1992; Cˇemazˇar et al., 2003). The other peak is usually a less resolved area in between the retention time of the peaks containing the native disulfide bridged and the reduced forms of the polypeptide. This less resolved peak contains misfolded products and aggregates, which after preparative RP-HPLC will not dissolve in aqueous buffers or in organic solvents, unless reducing agents are added. To optimize folding, we tested an array of chaotropes and additives (guanidine, L-arginine, L-aspartate up to 1 M, different GSH/ GSSG ratios) and found that the optimal yield (30% relative to the amount of reduced input material) of correctly folded protein was obtained when 0.4 M Larginine was used during refolding (data not shown). A neutralizing antibody against sclerostin has been described and its binding epitope has been mapped to the unstructured second loop (Veverka et al., 2009; Li et al., 2009), suggesting that this site is responsible for sclerostin’s inhibition on Wnt-activity. To challenge this hypothesis and to test whether additional sites or epitopes are possibly required for sclerostin-mediated Wnt-inhibition, we designed emasculated versions of sclerostin with altered or truncated loop
Recombinant human and mouse sclerostin were produced as full-length proteins containing an N-terminal His6-Tag followed by a thrombin protease cleavage site. Expression in E. coli strain Rosetta (DE3) and purification was performed as published (Weidauer et al., 2009). Proteins were purified via His6-Tag from solubilized inclusion bodies and refolded by rapid dilution for 4 days in 2 M lithium chloride, 50 mM Tris – HCl, pH 8.0, 30 mM Chaps, 500 mM arginine, 2 mM GSH and 1 mM GSSG. Active sclerostin protein was then purified to homogeneity by a two-step purification procedure. First, cation exchange chromatography was performed using CM Sepharose (GE Healthcare) and a linear gradient of 0 –1 M NaCl in 20 mM Tris – HCl pH 7.5. Finally, remaining non-natively folded protein was removed by reversed-phase HPLC using a C8 column (Machery and Nagel) employing a linear gradient of 0.1% TFA in H2O to 100% acetonitrile.
Interaction analysis using surface plasmon resonance All analyses were performed using the ProteOnTM XPR36 surface plasmon resonance (SPR) system (BioRad) at 258C. HBS150T buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.005% Tween20) was used as running buffer with a standard flow rate of 100 ml/min. The surface of a ProteOnTM GLC sensor chip (BioRad) was activated using a mixture of 100 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 25 mM N-hydroxysulfosuccinimide (Sulfo-NHS) for 60 s and a flowrate of 30 ml/min. Subsequently, recombinant human and mouse sclerostin proteins were immobilized using a 100-nM solution in 10 mM sodium acetate pH 4.5 to achieve a surface density of about 600 resonance units (RU) on two different channels in the vertical direction. For subsequent surface deactivation, 1 M ethanolamine-HCl pH 8.5 solution was injected for 200 s. Interaction data with different Fab antibody proteins were measured by injecting six different concentrations of Fab solutions (100, 75, 50, 25, 12.5 and 6.25 nM in HBS150T) on different channels in the horizontal direction (interaction time 200 s, flowrate 100 ml/min). The dissociation was 4
Results and discussion
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For the ELISA shown in Fig. 4, Maxisorp plates (NUNC) were coated overnight at 48C with a 1-mg/ml solution of protein in PBS. For coating either refolded, purified recombinant sclerostin or the raw mixture from the folding buffer was used. Plates were then blocked with 5% skim milk for 2 h at room temperature. Subsequently, an aliquot of Fab protein was added and the mixture was incubated and the ELISA was developed as described above. For competitive ELISA plates were coated with a 1-mg/ml solution of avidin (Pierce) and blocked with 5% horse serum in PBS. The biotinylated sclerostin fragment was then loaded in PBS Tween and allowed to bind to avidin for 1 h at room temperature. Subsequently, a mixture of the Fab with recombinant sclerostin in PBST plus 5% horse serum was loaded and allowed to equilibrate. Binding of Fab protein was detected by incubation with anti-Fab HRP conjugate (AbD Serotec) in a 1:1000 or 1:2500 dilution (as determined in pilot experiments) in PBST, and plate development with 2-20 -azino-di(3-ethylbenzthiazoline sulfonic acid). Absorbance at 405 nm was monitored using a Spectramax M5 plate reader (Molecular Devices, USA).
Complex epitopes on the cystine knot protein sclerostin
variations. We thus produced several variants which all still contain the cystine knot but have differently shortened versions of loop 2 and we additionally also produced a control (tSOST) that contains the complete second loop. These constructs were used either in direct biochemical studies (as will be reported elsewhere) or to elicit position-specific antibodies. Hence, we designed tSOSTDb2 (see Table I for loop insert) by replacing residues 112 through 127 in the native sequence by the dipeptide sequence D-proline—glycine, a b-turn nucleating motif (Balaram, 1999), although the formation of a b-turn will depend on sequence context and cannot be proven in this polypeptide. The miniprotein was readily produced in a one-step NCL, and the resulting protein was subjected to the same folding screen as the wildtype-like tSOST peptide had been, which has full-length loops but lacks the N- and C-termini shown not to be required for bioactivity of sclerostin (Weidauer et al., 2009). It was found that in this case, the addition of chaotropes did not improve or even slightly worsen the folding of tSOSTDb2, and that the maximum yield (38%) was attained in a Tris – HCl buffer (55 mM, pH 8.0) that also contained 10 mM NaCl, and a GSH/GSSG redox couple (1.67 and 0.33 mM final concentrations, respectively). To test the suitability of these truncated forms of sclerostin as bait in an antibody phage display library selection strategy, the proteins were provided with an N-terminal biotin, spaced from the cystine knot region with a triglycine motif. As a control, an aliquot of the correctly folded protein was reduced with TCEP followed by S-alkylation of the cysteines with iodoacetamide, in order to prevent cystine formation
leading to a linearized form of the sclerostin variant. The HuCAL PLATINUM library was screened with both forms of truncated variants of sclerostin. Three consecutive rounds of panning with increasing selection stringency were used. Ultimately, 30 unique Fabs recognizing the oxidatively folded tSOSTDb2 and 12 unique Fabs raised against the S-alkylated version of tSOSTDb2 were produced. These Fabs were tested for binding to human and murine forms of recombinant sclerostin by ELISA, both the folded and S-alkylated linear version of the antigen, and a panel of control proteins (BSA, CD33 and GST). In Table II, the binding (FOB) that was observed with the Fabs is displayed. Whereas both tSOSTDb2 in the folded and the S-alkylated linearized version could be used to generate antibodies capable of selectively recognizing the cognate bait and recombinant protein, it is remarkable that the Fabs against oxidatively folded protein do not recognize the S-alkylated version, yet those Fabs raised against the linearized version can bind all forms of the target protein. Collectively, these results suggest that the Fabs directed against the alkylated version recognize a linear epitope, which is present and accessible in all forms of the protein, and that the Fabs elicited with the folded version recognize a conformational and/or discontinuous epitope, present only in the folded version and the native protein. Furthermore, the binding affinities of all Fabs toward the human and murine form of recombinant sclerostin were determined by in vitro binding studies using surface plasmon resonance. Table II shows the apparent KD values of the antibodies directed against oxidatively folded tSOSTDb2 or linearized tSOSTDb2. A 1:1 Langmuir 5
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Fig. 1. Oxidative folding followed by HPLC-MS. (A) LC-UV trace at 215 nm of reduced tSOSTDb2 (upper trace) and the mixture after 24 h oxidation (lower trace). (B) ESI-mass spectrum at t ¼ 1.09 min of the reduced tSOSTDb2 after native chemical ligation, showing the mass to be 8364 Da (upper spectrum) and ESI-mass spectrum at t ¼ 0.8 min shows an oxidized product mass of 8356 Da (lower spectrum).
J.W.Back et al.
Table II. Characterization data of the Fabs against the oxidatively folded tSOSTDb2 or the S-alkylated linearized version of tSOSTDb2 Antigen
Antibody
ELISA Controls
Oxidatively folded tSOST Db2
SPR
Sclerostin species
Sclerostin species
BSA
N1-CD33-His6
GST
BSA-bio
tSOST Db2
tSOST Db2-alk
h
m
h
m
h
m
1.3 1.0 0.9 0.9 0.9 0.9 0.9 1.0 0.9 0.9 0.9 0.9 0.7 1.2 1.0 1.0 1.0 1.0 0.9 1.0 0.9 1.0 1.1 1.0 0.9 1.0 0.9 0.9 0.9 0.9 2.5 3.6 1.8 2.0 2.2 2.0 1.4 1.4 1.2 1.3 1.3 1.2
1.7 0.9 1.0 1.1 1.3 1.1 0.9 1.0 1.0 1.1 0.9 1.2 0.9 2.1 0.9 0.9 0.9 0.9 0.9 0.9 0.8 0.9 1.0 1.0 0.9 1.0 0.9 0.9 0.9 0.9 3.7 10.4 3.0 2.1 4.1 5.7 3.2 1.7 1.6 1.7 1.6 1.8
1.6 0.9 1.0 0.8 1.1 0.9 0.9 0.9 0.9 1.0 0.9 1.0 0.8 2.0 0.9 1.0 0.9 1.0 0.9 1.0 0.9 1.0 1.0 1.0 0.9 1.0 0.9 0.9 0.9 1.0 1.5 1.8 1.3 1.7 2.3 3.0 2.3 1.4 1.6 1.2 1.2 1.2
1.4 0.9 1.4 0.8 1.2 0.9 0.9 0.9 0.9 0.8 0.9 0.9 0.8 1.1 1.2 0.9 0.9 1.0 0.9 1.0 0.9 1.1 0.9 1.9 1.0 1.0 0.9 0.9 0.9 1.0 1.3 1.7 1.7 1.7 2.0 1.8 2.2 1.5 1.3 1.2 1.2 1.5
66.4 46.3 45.7 46.7 5.1 67.4 67.1 78.7 57.0 67.0 72.3 86.2 82.8 10.2 14.5 26.1 30.1 21.9 25.1 21.7 25.6 21.3 25.1 14.8 17.0 11.9 15.4 16.0 22.5 15.9 31.1 48 16.3 10.6 19.6 25 21.2 13 7.0 11.9 10.6 18.1
2.1 2.0 1.0 1.4 1.4 1.7 1.1 1.6 1.8 1.6 1.2 1.2 0.9 1.5 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 21.1 31.8 11.1 9.0 16 19.4 15.5 9.3 4.6 6.3 7.3 15.7
76.2 72.5 52.2 50.4 7.0 63.3 79.6 82.3 58.1 34.2 90.6 90.4 86.7 25.5 18.7 17.2 29.6 18.0 24.0 21.6 22.8 30.3 23.2 14.9 13.0 13.4 12.1 13.4 18.9 15.0 29.1 74.5 23.2 10.9 26.1 34 26.1 13.6 5.8 9.8 9.6 19
86.9 70.2 61.0 43.8 3.7 46.2 37.9 73.1 29.0 19.4 88.3 94.6 1.2 6.8 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 30.9 56.5 28.1 12.6 31.4 34.2 29.6 13.7 6.8 11.1 8.5 20.5
ND 20 ND ND ND ND ND ND ND ND ND ND 25 ND 24 ND ND ND ND 10 ND 10 ND ND ND 28 3.9 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND 131 ND ND ND ND ND ND ND ND ND ND ND ND 24 ND ND ND ND 20 ND ND ND ND ND 155 2.9 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
11 14 774 NB 147 117 41 166 1000 1000 643 134 128 1000 6 317 360 89 38 7 141 286 107 242 840 25 12 29 666 49 NB NB NB NB NB NB NB NB NB NB NB NB
3 732 1000 NB 1000 NB NB NB NB NB NB 167 NB NB 10 NB NB 55 90 24 NB NB 500 1000 NB 1000 28 12 NB 1000 NB NB NB NB NB NB NB NB NB NB NB NB
ELISA for determining specific binding Fab-antigen interaction using a panel of control proteins and sclerostin variants. Numbers are signal/noise ratios of ELISA (fold over background) as described in methods for Fabs generated. IC50 values determined in competitive ELISA, and equilibrium binding constants KD (nM) obtained by surface plasmon resonance for the binding of Fab antibody proteins to immobilized recombinant human and murine sclerostin. m, mouse; h, human; ND, not determined; NB, no binding, under the conditions measured KD 5 mM.
model of binding was applied to analyze the SPR sensorgrams. Wherever necessary, the Langmuir Mass Transfer model was used, to cope with a limited transfer of the analyte onto the ligand immobilized on the biosensor surface, which was the case for Fabs displaying very fast binding to the immobilized sclerostin protein. If binding of the Fab proteins to sclerostin reached steady state, the data were additionally fitted using an equilibrium model (data not shown). Differences between the apparent KD values obtained from both, kinetic and equilibrium, data fitting routines were usually less than 2-fold. Binding affinities up to the low nanomolar range were obtained for various Fabs. Interestingly, some antibodies exhibit high species specificity, whereas others reveal almost identical affinities as well as binding kinetics to human and murine 6
recombinant sclerostin (Supplementary Figs S1 and S2, Table II). For the antibodies derived from the panning using the S-alkylated version of tSOSTDb2 binding data could not be analyzed due to poor binding of these antibodies to recombinant sclerostin, usually displaying biphasic binding kinetics. As these Fabs show binding in the ELISA (albeit less than those obtained against the oxidatively folded peptide), we assume that the binding affinities are too low (KD 1 – 5 mM) to be measured by our SPR setup. Another rather unlikely possibility would be that the linear epitope of sclerostin, which is recognized by the antibodies, is not accessible due to the immobilization on the chip via amine coupling. As none of the 12 antibodies against the S-alkylated version of the miniprotein have dissociation constants below the micromolar range, it is likely
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Linear alkylated tSOST Db2
AbD10723 AbD10724 AbD10725 AbD10726 AbD10727 AbD10728 AbD10729 AbD10730 AbD10731 AbD10732 AbD10733 AbD10734 AbD10735 AbD10736 AbD11369 AbD11370 AbD11371 AbD11372 AbD11373 AbD11374 AbD11375 AbD11376 AbD11377 AbD11378 AbD11379 AbD11380 AbD11381 AbD11382 AbD11383 AbD11384 AbD10737 AbD10738 AbD10739 AbD10740 AbD10741 AbD10742 AbD10743 AbD10744 AbD10745 AbD10746 AbD10747 AbD10748
Sclerostin species
IC50
Complex epitopes on the cystine knot protein sclerostin
that the superior binding of the antibodies against folded tSOSTDb2 is in part due to the complex and discontinuous epitopes that allow burial of a larger surface area into the paratope. It is also interesting to note that the Fabs against the S-alkylated form of tSOSTDb2 show no discrimination between the human and murine recombinant sclerostin in the ELISA, whereas the antibodies obtained against the folded peptide often display species specificity in the ELISA and also the SPR study. To benchmark the quality of the antibodies that we derived from pannings against our mimics versus those elicited against a recombinant form of the protein we performed a competitive ELISA in which we immobilized the bait on an avidin-coated plate, and competed binding of the Fabs with recombinant human and murine sclerostin protein (Fig. 2 and Table II). For comparison, Fab AbD09101 (Boschert, V and Mueller T.D., manuscript in preparation) that has been selected against recombinant human sclerostin was also included. From non-linear regression analysis using GraphPad Prism 5, we obtained IC50 values of 2.5 nM for AbD09101 (anti-recombinant human sclerostin), 10 nM for AbD11374, 28 nM for AbD11380, and 3.9 nM for AbD 11381 (all anti-tSOSTDb2), respectively, indicating that antibodies of similar potency can be discovered by selection on structured peptide mimics as by selection on recombinant proteins. SPR measurements of these antibodies on recombinant human sclerostin show that compared with AbD09101 the peptide-derived antibodies do exhibit similar binding kinetics and binding affinities (Fig. 2C, Table II, KD of AbD09101 is 45 nM). Again the antibodies that were raised against the linear version of the antigen showed very poor binding in direct ELISA to be considered for competition
experiments to derive an IC50, suggesting low binding affinity (data not shown). As tSOSTDb2 showed a superior folding efficiency to a fragment of the protein that contained the native sequence, we decided to investigate the influence of sequence substitutions on folding of the cystine knot, in an effort to delineate the boundaries for productive folding. Nine additional variants were designed, whose sequences are given in Table I. The loops include monoglycine, diglycine and triglycine that ought to be too short to span the distance between the third and fourth cystinyl residue in a native cystine knot structure, and furthermore comprise six artificial sequences ranging in length from four to nine residues. N-terminally, these tSOSTDb2 variant proteins were given a FLAG-tag, to be able to detect them in an orthogonal manner in assay conditions beyond the scope of this study. However, as the additional tag is outside the cystine knot region, it is not expected to impose hugely on their folding, and moreover these sequences can be compared with one another due to their identical N-terminus. As a benchmark, we included the construct tSOST, containing the native loop 2 sequence, which is N-terminally biotinylated. The proteins were harvested in reduced form, and subjected to folding as described in methods. At intervals a sample was taken, and the fraction folded (defined as peak area at 215 nm of the prominent sharp and early eluting peak relative to the peak area of an equivalent amount of peptide in fully reduced form at t ¼ 0) was determined and plotted against time in Fig. 3A. Evidently, those peptides with an insert of four or more residues all fold to significant relative yields under the conditions that were chosen. Peptides containing monoglycine, diglycine or triglycine inserts do 7
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Fig. 2. Competitive ELISA of a panel of selected Fabs. (A) The bait protein that was used to select the Fabs was attached to avidin coated onto the microtiter plate, and antibody binding was competed with recombinant sclerostin. (B) Inhibition ELISA curves for unique antibodies AbD09091, AbD11374, AbD11380 and AbD11381. (C) Binding kinetics of these antibodies measured by surface plasmon resonance. Recombinant human sclerostin was immobilized on the surface of a ProteOnTM GLC sensor chip. Antibodies (100 nM) were injected for 200 s starting at time point zero. Dissociation (starting at time point 200) was monitored for 200 s by perfusing the biosensor surface with running buffer. All binding curves shown were deduced from the same biosensor.
J.W.Back et al.
Fig. 4. ELISA detecti on of folded sclerostin species in the folding mixture. The raw folding mixture, or the linearized form of the protein (tSOST-IAM) was coated onto an ELISA plate and probed with antibodies AbD10735 that is sensitive only for correctly folded sclerostin (mimics), and AbD10748 that detects both folded and unstructured sclerostin mimics. Plotted is the ratio of signal obtained with AbD10735 relative to the amount of signal obtained with AbD10748.
produce a peak on RP-HPLC that shifts to a retention time that corresponds to a folded form; however, we were not able to exceed a yield of 3% relative to the amount of reduced input material. We did not find a good way to verify that the disulfide bridges are correctly formed, but all evidence points in that direction. The kinetics of folding of the tSOSTDb2 variant proteins follow a sigmoidal shape, consistent with the conversion of starting material into product via (a series of ) intermediates (Mamathambika and Bardwell, 2008). It is possible to discern a log-linear phase, from which one can infer the rate of folding. It is noteworthy that those proteins folding at the 8
highest rates also give the highest relative yields upon completion of the folding reaction, and even a linear relationship between these parameters can be identified (Fig. 3B). Surprisingly, the four amino acid-looped tSOSTDb2 variant proteins fold faster than all proteins containing other intervening sequences. The longest loop 2 is the native sequence, which folds at lowest speed into lowest yield under these conditions. As the NMR structures suggest that the loop is largely unstructured in solution, apparently no positive contribution to correct folding is made by this part of the protein. Therefore, it is attractive to speculate upon a model in which the distance that is spanned by the non-structured linker negatively correlates with folding speed, as it allows larger average distances for the cysteines that are to participate in the cystine knot. Of course, in vivo the protein is folded and quality controlled in the endoplasmatic reticulum, assuring that only correctly folded and active species are secreted. However, this model must be an oversimplification, as the intervening sequence might both speed up folding—e.g. through attaining secondary structure that could properly apposition future cystines—or disallow folding by static interference (Mamathambika and Bardwell, 2008). This is also evident from our own results, as the inserts of six amino acids do not follow the same logic, the GSGSGS and GGSGGS sequences fold at nearly the same rate, but the GAPDFR insert folds slower and to a lower yield than the protein containing the nine-residue GGSGGSGGS insert. Still it is noteworthy that these unstructured fully synthetic inserts apparently allow the formation of correctly folded cystine knot proteins at rates and yields superior to the native sequence. To verify that the structured region, comprising the first and third loop, indeed exhibits the native fold, we set up an ELISA screen using an antibody that is able to detect total sclerostin protein, irrespective of structure and an antibody that can discriminate between correctly and misfolded protein. For total quantification, we chose Fab AbD10748, elicited against S-alkylated tSOSTDb2, and able to recognize
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Fig. 3. Kinetic monitoring of the folding of tSOSTDb2 variants. Different b2-loop variants were folded under standard conditions. (A) The fraction folded (area of absorbance at 215 nm of the early eluting sharp peak relative to the absorbance area of the reduced protein at t ¼ 0) plotted against time. (B) Reduction of the data of panel A into a plot of folding half time versus folding yield for the six constructs gives a linear relationship, contoured by the 95% confidence interval (dotted gray lines).
Complex epitopes on the cystine knot protein sclerostin
Funding European Union Framework Program 6 Grant TALOS. References Agholme,F. and Aspenberg,P. (2011) Acta Orthop., 82, 125–130. Arolas,J.L., Aviles,F.X., Chang,J.-Y., et al. (2006) Trends Biochem. Sci., 31, 292–301. Avsian-Kretchmer,O. and Hsueh,A.J.W. (2004) Mol. Endocrinol., 18, 1 –12. Balaram,P. (1999) J. Pept. Res., 54, 195– 199. Bang,D. and Kent,S.B. (2004) Angew. Chem. Int. Ed. Engl., 43, 2534– 2538. Bourhis,E., Wang,W., Tam,C., et al. (2011) Structure, 19, 1433–1442. Brown,M.C., Joaquim,T.R., Chambers,R., et al. (2011) PLoS ONE, 6, 12. ˇ emazˇar,M., Zahariev,S., Lopez,J.J., et al. (2003) Proc. Natl. Acad. Sci., C 100, 5754–5759. Chatrenet,B. and Chang,J.Y. (1993) J. Biol. Chem., 268, 20988–20996. Jarutat,T., Frisch,C., Nickels,C., et al. (2006) Biol. Chem., 387, 995 –1003. Jemmerson,R. (1987) Proc. Natl. Acad. Sci. USA 84, 9180–9184. Knappik,A., Ge,L., Honegger,A., et al. (2000) J. Mol. Biol., 296, 57–86. Li,X., Zhang,Y., Kang,H., et al. (2005) J. Biol. Chem., 280, 19883– 19887. Li,X., Ominsky,M.S., Warmington,K.S., et al. (2009) J. Bone. Miner. Res., 24, 578–588. Lu,H.S., Clogston,C.L., Narhi,L.O., et al. (1992) J. Biol. Chem., 267, 8770– 8777. Mamathambika,B.S. and Bardwell,J.C. (2008) Annu. Rev. Cell Dev. Biol., 24, 211–235. Padhi,D., Jang,G., Stouch,B., et al. (2011) J. Bone Miner. Res., 26, 19–26. Papapoulos,S.E. (2011) Ann. Rheum. Dis., 70(Suppl. 1), i119–i122. Paszty,C., Turner,C.H. and Robinson,M.K. (2010) J. Bone Min. Res., 25, 1897– 1904. Poole,K.E., van Bezooijen,R.L., Loveridge,N., et al. (2005) Faseb J., 19, 1842– 1844. Prassler,J., Thiel,S., Pracht,C., et al. (2011) Journal of Molecular Biology, 413, 261– 278. Spangler,B.D. (1991) J. Immunol., 146, 1591–1595. Tian,X., Jee,W.S., Li,X., et al. (2010) Bone, 48, 197– 201. Veverka,V., Henry,A.J., Slocombe,P.M., et al. (2009) J. Biol. Chem., 284, 10890–10900. Vitt,U.A., Hsu,S.Y. and Hsueh,A.J. (2001) Mol Endocrinol, 15, 681–694. Weidauer,S.E., Schmieder,P., Beerbaum,M., et al. (2009) Biochem. Biophys. Res. Commun., 380, 160– 165.
Conclusion We have shown that by using NCL, we are able to get (fragments of ) the cystine knot protein sclerostin that can adopt complex native structures. We are able to delete an intervening loop that is highly flexible and unstructured in the structures of recombinant sclerostin, but does likely contribute to sclerostin-mediated Wnt inhibition, and established the minimum length of this loop for correct folding of this protein. The resulting miniproteins were successfully applied as bait in phage library pannings, yielding antibodies with nanomolar affinities against targeted complex epitopes. This approach making use of folded fragments of a complex structured protein to raise Fabs against these fragments enables us to obtain Fabs with known predefined binding sites. Supplementary data Supplementary data are available at PEDS online. Acknowledgement The authors thank Stella Weidauer and Eva Maria Muth (University of Wuerzburg) for their help in the production of recombinant mouse and human sclerostin.
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the cognate antigen, the folded version of tSOSTDb2, as well as the folded forms of the murine and human recombinant sclerostin all with similar signal-to-noise levels, which are clearly above those obtained when a panel of irrelevant proteins is used. For the antibody selective for the correct structure, we used Fab AbD10735 that was elicited against the folded form of the variant tSOSTDb2. This antibody recognizes both the folded target antigen as well as the folded recombinant human protein, but cannot bind the alkylated version of the antigen, suggesting that it is binding to a conformational and/or non-continuous epitope. The antibody also recognizes murine sclerostin much less than the human protein, likely delineating the epitope to a region that contains differences in primary structure between human and mouse sclerostin. The ELISA was performed as described in the Methods section, and the ratio of signal obtained with AbD10735 relative to the amount of signal obtained with AbD10748 was calculated and plotted in Fig. 4, for each different construct. The signal of the ELISA with structure-insensitive Fab AbD10748 was adjusted to fall into the range of 0.2– 0.5 AU at 405 nm by diluting the secondary antibody concentration to 1:2500, in order to avoid saturation problems. The proteins that were purified after folding (tSOSTDb2 and recombinant sclerostin) get high relative ratios in this assay. All other constructs were directly coated from the folding assay, containing in addition to a population of correctly folded material clearly a fraction that is not correctly structured, and this may well explain the overall lower signals for these constructs. However, still we clearly have high amounts of correctly folded material in these sclerostin miniproteins with an artificial b2 loop. In contrast, the signals of tSOSTDb2 variants containing monoglycine, diglycine and triglycine loop inserts are lowest, indicating a low abundance of material mimicking the correct 3D structure in the loop 1 to loop 3 region.
