A Prodomain Fragment from the Proteolytic Activation of Growth

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A prodomain fragment from the proteolytic activation of growth differentiation factor 11 (GDF11) remains associated with the mature growth factor and keeps it soluble R. Blake Pepinsky, Bang Jian Gong, Yan Gao, Andreas Lehmann, Janine Ferrant, Joseph Amatucci, Yaping Sun, Martin Bush, Thomas Walz, Nels Pederson, Thomas Cameron, and Dingyi Wen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00302 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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A prodomain fragment from the proteolytic activation of growth differentiation factor 11 (GDF11) remains associated with the mature growth factor and keeps it soluble

Blake Pepinsky,‡* Bang-Jian Gong,‡ Yan Gao,‡ Andreas Lehmann,‡ Janine Ferrant,‡ Joseph Amatucci,‡ Yaping Sun,‡ Martin Bush,§ Thomas Walz,§ Nels Pederson,‡ Thomas Cameron,‡ and Dingyi Wen‡

‡

Department of Biotherapeutics and Medicinal Sciences, Biogen, 115 Broadway, Cambridge, MA 02142; § Laboratory of Molecular Electron Microscopy, Rockefeller University, 1230 York Avenue, New York, NY 10065

*Corresponding Author: Blake Pepinsky Department of Biotherapeutics and Medicinal Sciences Biogen 115 Broadway Cambridge, MA 02142 Tel: 617 679 3310 Fax: 617 679 3148 Email: [email protected]

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2 ABBREVIATIONS GDF, growth differentiation factor; TGF-β, transforming growth factor β; BMP, bone morphogenetic protein; AMH, anti-Müllerian hormone; ALK, activin receptor-like kinase; KO, knock out; CHO, Chinese hamster ovary; CMV, cytomegalovirus; DHFR, dihydrofolate reductase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC, size-exclusion chromatography; AspN, endoproteinase AspN; EDTA, ethylenediaminetetraacetic acid; MS, mass spectrometry; UPLC, ultraperformance liquid chromatography; CID, collision-induced dissociation; KIRA, kinaseinduced receptor activation; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; DTT, dithiothreitol; AT, Aldrithiol™; TRE, transcriptional response element; LTBP, latent TGF-β binding protein; GASP, GDF-associated serum protein, PDP60-114, prodomain peptide containing residues 60-114; NGF, nerve growth factor.


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3 ABSTRACT Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exits as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), while the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-β family that form latent pro/mature domain complexes.


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4 INTRODUCTION Growth differentiation factor 11 (GDF11), also known as bone morphogenetic protein 11 (BMP11), is a member of the transforming growth factor β (TGF-β) family of proteins, members of which play diverse roles in regulation of embryonic patterning and morphogenesis, cell proliferation and differentiation, adhesion, immune responses, cell growth arrest and tissue or organ regeneration and maintenance.1-4 This family contains over 30 members, including TGF-βs, BMPs, GDFs, activins, inhibins, nodal and antiMüllerian hormone (AMH).5,6 All these proteins are expressed as large precursor proteins that undergo proteolytic processing to form non-covalently associated N-terminal pro and C-terminal mature domains. Processing of the precursors at mono and/or dibasic cleavage sites at the junction of the pro and mature domains can result in the formation of an active complex or a latent complex that requires further processing/activation steps. Most members of the family form active complexes. However, like TGF-β1-3, BMP10, and myostatin, processing of GDF11 forms a latent complex. Activation of this latent pro-GDF11 requires subsequent cleavage at a BMP1 site within the prodomain.7 Figure 1 shows a schematic drawing of human GDF11 precursor protein summarizing its structural domains and key processing sites. The 407 amino acid transcript contains a 24 amino acid signal sequence and a pro region corresponding to amino acids 25-298. The mature domain of GDF11, which carries no glycosylation sites, consists of amino acids 299-407, and contains 9 cysteines that form 4 intramolecular disulfide bridges and one interchain disulfide bridge that stabilizes the homodimer structure. The mature domains of all members of the TGF-β family contain the characteristic growth factor cystine-knot structural motif that confers extraordinary stability to the domain. Most exist as disulfide-linked homodimers that are recruited by type I and II signaling receptors. Key structural attributes of mature GDF11 were confirmed in the recently solved crystal structures.8,9 The mature domain of GDF11 shares 90% sequence identity with myostatin (also referred to as GDF8, BMP8). Both GDF11 and myostatin predominantly use the type II receptors activin receptor kinase IIA and II-B, and the type I receptors activin receptor-like kinase (ALK) 4 and 5 to elicit signal transduction through phosphorylation of SMADs 2 and 3.10 Myostatin is a negative regulator of muscle mass and has been extensively studied because of its


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5 essential role regulating muscle growth.11,12 Elucidation of the inhibitory role of myostatin in skeletal muscle growth was aided by the availability of knock-out (KO) mice.11 Several companies are investigating myostatin inhibitors in clinical trials as treatments for muscular dystrophy and other atrophy disorders.12 The biology of GDF11 is less well developed although it is believed to contribute to early mesoderm development and anterior-posterior patterning of the axonal skeleton. Other studies have identified roles for GDF11 in inhibiting neurogenesis in the olfactory epithelium, preventing nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells, and in promoting vascular remodeling and neurogenesis.7,13 GDF11 KO mice are perinatal lethal, preventing a more extensive assessment of GDF11 function. While GDF11 is essential for development and has been suggested to regulate aging of multiple tissues, its roles in these aging processes have recently been the focus of intense debate.13-20 Given the ligand–receptor promiscuity of many TGF-β family members21 and the contextdependency of TGF-β family signaling and biology, an important challenge in developing pathway modulators is to identify product candidates or treatment regimens that can preferentially target more restricted aspects of the signaling pathway.22 Poor solubility at neutral pH is a common issue of the mature domains of most TGF-β family members that is routinely addressed by using acidic formulations. These formulations do not fix the intrinsic properties of the proteins and frequently lead to precipitation/deposition at sites of injection in vivo due to the rapid increase in pH upon delivery. In an attempt to produce GDF11 to support biochemical evaluation and animal studies, we expressed pro-GDF11 in CHO cells and characterized its processing. Our studies provide further insights into proteolytic events that lead to activation of proGDF11 and identify the complex of mature GDF11 with prodomain peptide residues 60114, PDP60-114, as a novel form of GDF11 that renders it soluble under physiological pH conditions.


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MATERIALS AND METHODS Expression of GDF11. For expression in Chinese hamster ovary (CHO) cells, plasmid pACE378 was engineered, which contains the full-length human GDF11 gene under the cytomegalovirus (CMV) promoter and an IRES-mDHFR element for selection. Suspension-adapted CHO cells were transfected with the plasmid and selected for integration in serum-free medium deficient of nucleosides. Once established, this stable pool was cryopreserved. For production runs, cultures were expanded in serum-free medium up to a final volume of 20 L, grown for 4 days at 36.5°C to high density (5 x 106 cells/mL) with appropriate feeds, and then shifted to 28°C. After 11 days at this reduced temperature, the cells were pelleted by centrifugation and the supernatant was clarified through a 0.2-micron 4-inch PolysepII Millipore cartridge filter. The full-length sequence of GDF11 is shown below. The signal peptide is shown in grey in lower case italics, the mature domain is highlighted in bold, and the solubility-enhancing peptide PDP60-114 identified in this study is underlined. mvlaaplllgflllalelrprgeaAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEP DGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHD FQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLK AQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQ SIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKR SRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYP HTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS Purification of pro-GDF11. 17 L of clarified conditioned medium from the culture of CHO cells expressing full-length human GDF11 (ACE378) was concentrated to 4.5 L using a Millipore prepscale tangential flow filtration unit equipped with a 10K cellulose membrane. NaCl and Na2HPO4 pH 7.0 were added to final concentrations of 0.5 M and 20 mM, respectively. Based on our prior success in purifying pro-BMP7 by metal chelating chromatography,23,24 the GDF11 preparation was loaded onto a 220-mL column of Ni Sepharose excel (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) at room temperature. The column was washed with 20 mM Na2HPO4, pH 7.0, 0.5 M NaCl,


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7 and then with 5 mM NaH2PO4, pH 6.0, 100 mM NaCl. Bound protein was eluted stepwise using 30 mM, 300 mM and 500 mM imidazole in 5 mM NaH2PO4, pH 6.0, 100 mM NaCl. Column fractions were analyzed for purity by SDS-PAGE. Fractions 26-42 from the 300 mM imidazole elution step (680 mL at 2.7 mg/mL, ~1840 mg total protein) were pooled. NaCl and (NH4)2SO4 were added to the elution pool to final concentrations of 0.25 M and 1.2 M, respectively. The preparation was subjected to centrifugation at 12,000 rpm for 20 min and the clarified supernatant was loaded onto a 120-mL column of Butyl Sepharose (GE Healthcare) at room temperature. The column was washed with 5 mM NaH2PO4, pH 6.0, 0.25 M NaCl, 1.2 M (NH4)2SO4, and bound protein was eluted step-wise with 0.45 M and 0 M (NH4)2SO4 in 5 mM NaH2PO4, pH 6.0, 0.25 M NaCl. Column fractions were analyzed for purity by SDS-PAGE. Fractions 4-7 from the 0.45 M (NH4)2SO4 elution step (120 mL, 4.2 mg/mL, 500 mg) were pooled. The concentration of GDF11 was determined using an extinction coefficient at 280 nm of 1.27 per cm for a 1-mg/mL solution. The protein was dialyzed at 4°C against 5 mM NaH2PO4, pH 6.0, 150 mM NaCl with 4 x 3.5 L changes of dialysis buffer, then filtered, aliquoted, and stored at -70°C. Endoproteinase AspN/Furin digestion of pro-GDF11. Pro-GDF11 (ACE378, 10 mg, 3.6 mg/mL) in 5 mM NaH2PO4, 50 mM Tris HCl, pH 7.5, 150 mM NaCl was treated with 10 µg of endoproteinase AspN (Roche Diagnostics, Indianapolis, IN) for 2.5 hr at 37°C. NaCl was added to a final concentration of 0.5 M and the sample was loaded onto a 2-mL Ni Sepharose excel column. The column was washed with 20 mM Na2HPO4, pH 7.0, 0.5 M NaCl, then with NaH2PO4, pH 6.0, 100 mM NaCl, and bound protein was eluted with 300 mM imidazole in 5 mM NaH2PO4, pH 6.0, 100 mM NaCl. Elution fractions were pooled and concentrated to ~3 mL at 1.7 mg/mL. The protein was dialyzed at 4°C against 5 mM NaH2PO4, pH 6.0, 150 mM NaCl with 2 x 1.8 L changes of dialysis buffer, aliquoted, and stored at -70°C. 4 mg of AspN-digested pro-GDF11 was adjusted to 50 mM Tris HCl, pH 7.5, containing 1 mM CaCl2. 72 µL of furin (Sigma F2677-50UN, >2U/µL, St. Louis, MO) was added and the sample was incubated at 37°C. Aliquots were removed after 4 hr, 7 hr, and 24 hr for SDS-PAGE analysis and activity measurements. After 24 hr, the remainder of the digest was aliquoted and stored at 70°C. Ethylenediaminetetraacetic acid (EDTA) was added to 5 mM at each time point to


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8 quench the furin reaction. The soluble and precipitated fractions were separated by centrifugation in an Eppendorf 5415C centrifuge at top speed for 4 min. SDS-PAGE. Samples were subjected to SDS-PAGE on a 4-20% gradient gel (Novex Life Technologies, Carlsbad, CA) under reducing and non-reducing conditions. The gels were stained with SimplyBlueTM SafeStain (Novex Life Technologies). Non-reduced samples were diluted with Laemmli non-reducing sample buffer, and heated at 75°C for 5 min prior to analysis. Reduced samples were treated with sample buffer containing 2% 2-mercaptoethanol and heated at 95°C for 4 min. Western blots of reduced SDS-PAGE samples were developed using a goat polyclonal antibody against a GDF8/11 C-terminal peptide sc6884 (C-20), from Santa Cruz Biotechnology (Dallas, Texas). Size-exclusion chromatography. Samples (100 µg for analytical and 500 µg for

preparative analysis) were subjected to size-exclusion chromatography (SEC) at room temperature on a GE Superdex 200 30/10 FPLC column in 10 mM sodium succinate, 75 mM NaCl, 100 mM L-arginine HCl, pH 5.5, at a flow rate of 0.5 mL/min. The column effluent was monitored for absorbance at 280 nm. Molecular weight standards were run as controls and their chromatograms overlaid on the test sample chromatograms. For preparative runs, 1-mL fractions were collected. Fluorescently labeled samples (0.2-1 µg) were analyzed on a GE Superdex 200 5/150 GL column in the same buffer containing 1 mg/mL bovine serum albumin at a flow rate of 0.3 mL/min. The column effluent was monitored on an Agilent 1200 Series with Fluorescence Detector. The GDF11 samples were labeled with Alexa Fluor-488 (Invitrogen-Molecular Probes A10235) following the manufacturer’s instructions. Mass spectrometry. Samples were deglycosylated with PNGase F overnight at 37°C. 50 pmol of AspN-treated pro-GDF11 and 50 pmol of SEC-purified AspN/furin-treated GDF11 were reduced with 40 mM dithiothreitol (DTT) for 1 hr at 37°C after deglycosylation. The reduced, deglycosylated samples and 75 pmol of the non-reduced, deglycosylated AspN-treated GDF11 were analyzed on an UPLC-LCT Premier mass spectrometer system (Waters), using a BEH 1.7 µm 2.1 x 15 mm C4 column (Waters) run at 0.07 mL/min for separation, with the following gradient: t=0 min 10% Buffer B, 0-40 min 10-50% Buffer B, 40-45 min 50-70% Buffer B, 45-50 min 70% Buffer B, 50-55 min 70%-10% Buffer B (Buffer A: water with 0.03% trifluoroacetic acid; Buffer B:


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9 acetonitrile with 0.024% trifluoroacetic acid). Molecular masses were generated by deconvolution using the MaxEnt 1 program. LC-MS/MS analysis was carried out using an UPLC (Waters)-Obitrap-Elite/ETD mass spectrometer system (Thermo Scientific). The separation was the same as described above. For the collision-induced dissociation experiment, the collision energy was set at 35% and the activation time at 30 ms. Kinase-induced receptor activation (KIRA) assay. The Neuroscreen derivative of PC12 cells were plated at 2.2 x 105 cells/mL per well in 24-well plates coated with collagen Type IV in Dulbecco’s modified Eagle medium (DMEM), 10% heat-inactivated horse serum, 5% fetal bovine serum, 4 mM L-glutamine, and cultured overnight for 20 hr at 37°C and 5% CO2. The medium was discarded and cells were washed with 1 mL/well phosphate-buffered saline (PBS). Test samples, 300 µL, were prepared containing 1:3 serial dilutions of mature GDF11 (PeproTech, Rocky Hill, NJ), which was included as a reference standard for all studies, full-length pro-GDF11 ACE378, and AspN-treated ACE378, all starting at 400 ng/mL. A 250-µL aliquot of each sample was added to the wells and incubated for 1 hr at 37°C and 5% CO2. The sample cocktails were discarded and the cells were washed with 1 mL of PBS. 300 µL of lysis buffer (10 mM Tris HCl, pH 8.0, 0.5% Nonidet-P40, 0.2% sodium deoxycholate, 50 mM NaF, 0.1 mM Na3VO4) was added, and after 15 min at room temperature, plates were frozen at -70°C. Samples were analyzed for pSMAD2/3 levels using the PathScan® Phospho-Smad2 (Ser465/467)/ Smad3 (Ser423/425) sandwich Enzyme-linked Immunosorbent Assay (ELISA) kit from Cell Signaling Technology (#12001, Danvers, MA) following the manufacturer’s protocol. After the microwell stripes reached room temperature, the number of microwells required for each experiment was broken off. The 24-well plates were thawed at room temperature during the blocking period. Lysates were pipetted up and down 5 times with a multi-channel pipet to break up cell debris. 260 µL of lysate was added to the blocked ELISA plates. Then 20 µL of sample dilution buffer from the kit was added and plates were shaken slowly for 2 hr at room temperature. Plates were washed 3 times with Tris-buffered saline containing Tween (TBST; 10 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20). 100 µL of detection antibody was added and plates were shaken slowly for 1 hr at 37°C. Plates were washed 3 times with TBST. 100 µL of TMB buffer (Thermo Product # 34028) was added and after 10 min, 100 µL 2 N


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10 H2SO4 was added to stop the reaction. Plates were read at 450 nm. Samples were analyzed in duplicate. Data points were fitted with a 4-parameter curve fit, and EC50 values were determined from the curves. Luciferase reporter assay on SMAD reporter cells. Neuroscreen SMAD2/3 reporter (Luciferase) cells were generated by transducing Neuroscreen PC12 cells with lentivirus expressing the firefly luciferase gene under the control of a CMV promoter and SMAD transcriptional response element (TRE) using the Cignal Lenti SMAD reporter (luc) kit CLS-017L from Qiagen (Germantown, MD). After transduction, the Neuroscreen cells were cultured under puromycin selection, and once established, the reporter line was cryopreserved. To assess GDF11 function, the cells were plated at 2.0 x 105 cells/mL per well in 24-well plates coated with collagen Type IV in DMEM, 10% heat-inactivated horse serum, 5% fetal bovine serum, 5 µg/mL puromycin, and 100 ng/mL NGF, and cultured overnight for 20 hr at 37°C and 5% CO2. The medium was discarded and cells were washed with 1 mL/well PBS. Serial 1:3 dilutions of test samples were prepared in 450 µL DMEM without serum with 100 ng/ml NGF. 250 µL of each sample was added to the wells and incubated for 6 hr at 37°C and 5% CO2. Sample cocktails were discarded, and the cells were washed with 1 mL of PBS. 100 µL of 1x lysis buffer from Promega was added and plates were shaken for 15 min at room temperature. Lysates were pipetted up and down 5 times with a multi-channel pipet to break up cell debris. 20 µL of lysate and 100 µL/well substrate (Promega #E4550) were added to a black ELISA plate. After 1-2 min, luciferase activity was read out on TR717 Microplate Lumimeter with WINGLOW software. Samples were analyzed in duplicate. Data were fitted with a 4-parameter curve fit, and EC50 values were determined from the curves. The time dependence of luciferase expression was assessed and maximal activity was detected after 6 hr. No GDF11-induced luciferase activity was detected after 1.5 or 3 hr, consistent with the need for transcription of the luciferase gene. Biotinylation of GDF11. Pro-GDF11 (2.9 mg/mL) in 15 mM sodium succinate, pH 5.5, 150 mM NaCl was incubated in the dark with 2 mM sodium meta-periodate (Thermo Scientific) for 30 min at room temperature and immediately desalted on a Zeba spin desalting column (Thermo Scientific) equilibrated in 5 mM NaH2PO4, pH 6.0, 150 mM NaCl. EZ-LinkHydrazide-LC-Biotin (Thermo Scientific) was added to a final


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11 concentration of 5 mM from a 50 mM stock prepared in dimethylsulfoxide. HEPES, pH 7.2, was added to a final concentration of 50 mM and the sample was incubated in the dark for 2 hr at room temperature and then desalted on a Zeba spin desalting column equilibrated in 5 mM NaH2PO4, pH 6.0, 150 mM NaCl. 12 mg of biotinylated proGDF11 in 5 mM NaH2PO4, 50 mM Tris HCl, pH 7.5, 150 mM NaCl, was treated for 2.5 hr at 37°C with 6 µg of endoproteinase AspN, then purified on a Ni Sepharose excel column as described above for the non-biotinylated sample. The protein was dialyzed at 4°C against 5 mM NaH2PO4, pH 6.0, 150 mM NaCl with two 1-L changes of dialysis buffer, aliquoted, and stored at -70°C. To produce mature GDF11 in complex with a biotinylated form of PDP60-114, an aliquot of the biotinylated sample was digested with furin as described above. To produce free biotinylated PDP60-114, an aliquot of the biotinylated AspN-treated pro-GDF11 was heated at 95°C for 10 min and centrifuged in an Eppendorf centrifuge at top speed for 4 min. Following this treatment, PDP60-114 remained in the supernatant whereas the rest of the protein precipitated and was in the pellet. The specificity of labeling of pro-GDF11 with biotin hydrazide was confirmed by Western blotting using Streptavidin horseradish peroxidase for detection. Only fulllength GDF11 and PDP60-114 were detected, but not the major fragments 122-407 and 299-407. Octet binding studies. Binding characteristics of the biotinylated samples were evaluated by Octet on an Octet RED System (FortéBio™, Menlo Park, CA) using Dip and Read™ Streptavidin (SA) Biosensors (FortéBio™). Samples were prepared in 5 mM NaH2PO4, pH 6.0, 150 mM NaCl, 0.005% Tween-20. Loading and dissociation measurements were performed in the same buffer. The ability of biotinylated AspN/furin-cleaved pro-GDF11 to bind type I and II receptors was also assessed by Octet using the same loading conditions, followed by treatment with 10 µg/mL of the receptors. Recombinant human activin RIB (ALK-4)-Fc chimeric protein and recombinant human activin RIIA-Fc chimeric protein were obtained from R&D systems and reconstituted at 200 µg/mL. The Streptavidin tips were presoaked in Octet buffer for 15 min. The tips were loaded into the instrument and washed for 1 min with the buffer, before biotinylated samples and controls were loaded for 5 min. The tips were then washed for 1 min and dissociation was monitored for 30 min in the presence of 20 µM free biotin. For


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12 secondary binding studies with the receptors, the tips were loaded with biotinylated GDF11 and controls, and washed as described above, before the receptor samples were loaded for 15 min. After the tips were washed for 1 min, dissociation was monitored for 5 min. Computational modeling of the PDP60-114–mature GDF11 complex. We generated a model to illustrate how the PDP60-114 peptide might interact with mature GDF11, which is largely based on the structure of the mature domain of latent pro-TGF-β1 (PDB id: 3RJR).25 Details of the modeling procedure are provided in the supplemental methods. Reduction-oxidation dimerization studies. Eleven GDF11 constructs, containing truncated prodomains linked to the mature domain of GDF11 with G4S repeats, were engineered and transiently expressed in CHO cells using growth conditions similar to those described for production of ACE378. Plausible linker lengths and orientations were evaluated using the computational model. Table 1 lists the designs for all the constructs. All were expressed in 300-mL cultures. ACE490 and ACE498 were purified on 10-mL Ni Sepharose excel columns using the same wash and elution conditions described for ACE378, then aliquoted, and stored at -70°C. For reduction of ACE490 and ACE498, samples were first concentrated to ~1.5 mg/mL in Amicon Ultra-4 centrifugal filter units (10K cellulose membranes), then treated with 3 mM DTT for 30 min at room temperature and desalted into 25 mM Na2HPO4, pH 7.0, 100 mM NaCl on Zeba spin desalting columns. Half of each of the reduced protein samples was treated with 1 mM Aldrithiol™ (AT; Sigma-Aldrich) for 30 min at room temperature and again desalted into 25 mM Na2HPO4, pH 7.0, 100 mM NaCl on Zeba spin desalting columns. To induce dimer formation, equal amounts of the DTT only and DTT/AT-treated samples were mixed and incubated for 20 hr at room temperature without guanidine, or for 70 hr at 4°C in the presence of 1 M guanidine HCl. The extent of dimer formation was assessed by SDS-PAGE. A portion of the DTT/AT-treated ACE498 preparation was further treated with endoproteinase AspN at a protein:enzyme ratio of 1:1000 (w/w) for 60 min at 37°C. Production of BMP7. The full-length gene for human BMP7 (osteogenic protein-1, OP1) was expressed in CHO cells. For production runs, cultures were expanded for 3 days to high density at 36.5°C, then shifted to 32°C and cultured for an additional 5 days,


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13 achieving a final BMP7 expression level of ~40 mg/L. Cultures were harvested by centrifugation and the supernatant was clarified by filtration. BMP7 was purified from the conditioned medium by sequential chromatography steps on Zn-chelating Sepharose and SP-Sepharose FF (GE Healthcare). Unlike with production of GDF11, where the product was largely intact, ~90% of the BMP7 was cleaved at the junction of the pro- and mature domains. N-terminal sequencing analysis of the pro- and mature domains revealed sequences of DFSLDNEV and STGSKQRS, respectively, consistent with known cleavage sites. The mature domain contained two partially clipped forms, representing 22% N-7 cleavage and 29% N-22 cleavage. The molecular mass of the prodomain was 31,420 Da and that of the mature domain was 15683 Da. For digestion with endoproteinase AspN, BMP7 was desalted into 20 mM Na2HPO4, pH 7.5, 150 mM NaCl, and then treated with AspN at a 1:1000 AspN:BMP7 ratio for 2 hr at 37°C. EDTA was added to a final concentration of 10 mM to quench the reaction, and soluble and precipitated fractions were separated by centrifugation in an Eppendorf centrifuge at top speed for 4 min. The precipitated sample was characterized on an Orbitrap Fusion mass spectrometer. Three peptides were detected, corresponding to prodomain residues 34-84 and 30-84, and mature domain residues 325-431.


Biochemistry

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14 RESULTS Proteolytic activation of pro-GDF11. A construct encoding full-length human proGDF11 was expressed in CHO cells and purified from the conditioned medium by sequential column chromatography steps on Ni Sepharose excel and Butyl Sepharose. SDS-PAGE analysis of the purified product revealed a single prominent band with molecular mass of 110 kDa under non-reducing conditions (Figure 2A, lane 1 on the left) and 55 kDa under reducing conditions (Figure 2A, lane 1 on the right). These bands are consistent with the molecular mass of the monomeric full-length pro-GDF11 precursor protein containing both the pro- and mature domains under reducing conditions and the molecular mass of the disulfide-bridged homodimer under oxidizing conditions. When analyzed by SDS-PAGE/Western blotting, the 55-kDa band seen under reducing conditions was immunoreactive with anti-GDF11 antibody specific for mature GDF11 (Figure 2B). The apparent purity of the full-length pro-GDF11 was approximately 90%. An additional 90-kDa band (non-reducing) or 40-kDa band (reducing) that represented ~5% of the total protein and several minor bands of less intensity of varying molecular weights were also observed. The reduced 40-kDa band was also detected on Western blots with the anti-GDF11 antibody (Figure 2B). Mass spectrometry (MS) revealed that the 40-kDa band contained GDF11 residues 122-407, and thus was produced by cleavage at the BMP1 site (Figure 1). By size-exclusion chromatography (SEC), the full-length GDF11 migrated as a single homogeneous peak with apparent molecular weight of 100 kDa (Figure 3A). No aggregates were detected. From a 20-L culture, ~500 mg of the purified protein was recovered. GDF11 samples were tested for function in a kinase-induced receptor activation (KIRA) assay, monitoring SMAD 2/3 phosphorylation in PC12 cells. Figure 4A shows a time course of GDF11-induced SMAD 2/3 phosphorylation as a result of exposure to mature GDF11. The maximum efficacious response occurred at 60 min. Comparison of the activity of mature GDF11 with that of recombinant full-length pro-GDF11 (Figure 4B) revealed that mature GDF11 was a potent activator of SMAD 2/3 phosphorylation with an EC50 of 20 ng/mL (~1 nM), whereas recombinant full-length pro-GDF11 was inactive, consistent with a requirement for proteolytic activation (Figure 1). In an attempt to activate the pro-GDF11, we treated the sample with furin, but were unable to promote


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Biochemistry

15 cleavage as assessed by SDS-PAGE (Figure S2, lane 3). In subsequent studies, we discovered that cleavage at the furin site required that pro-GDF11 is first cleaved at Asp122, a reaction that we were able to drive efficiently using endoproteinase AspN (see below). Figure 2A, lane 2 shows an SDS-PAGE analysis of pro-GDF11 treated with endoproteinase AspN for 2.5 hr at 37°C. A single major cleavage product with molecular weights of 65 kDa under non-reducing and 40 kDa under reducing conditions was detected, with near quantitative cleavage. A diffuse low-molecular weight band ranging in size from 6-16 kDa was also detected that showed a similar electrophoretic pattern under reducing and non-reducing conditions. No additional cleavage products were generated with 10-fold higher concentrations of the enzyme under the same conditions or following longer incubation (data not shown). Extensive MS analysis of the pro-GDF11 sample treated only with AspN (Figure 5) revealed that the major cleavage products were a fragment corresponding to residues 122-407 (predicted mass 32,383.1 Da, observed mass 32386 Da) and the glycopeptide including residues 60-114 (predicted mass of the deglycosylated peptide 6,344.5 Da, observed mass 6,344 Da). In addition, fragments corresponding to residues 128-407 (predicted mass 31,729.4 Da, observed mass 31,732 Da), residues 133-407 (predicted mass 31095.7Da, observed mass 31,098 Da) and residues 60-117 (predicted mass of the deglycosylated peptide 6,710.0 Da, observed mass 6,708 Da) were detected. In total, under the limiting conditions used for digestion with endoproteinase AspN, only 5 of 17 aspartic acids were digested and all were in or near the N-terminal glycopeptide. None of the 11 downstream sites, including 6 in the mature domain were targeted. Pro-GDF11 contains a single N-linked glycosylation site at Asn-94. From MS analysis of the sample, various glycoforms of the glycopeptide containing residues 60-114/117 were detected ranging in mass from 7951 Da to 8769 Da (residues 60-117, 6709 Da without glycan) along with a small amount of the glycopeptide containing residues 25-114/117 with glycoforms ranging in mass from 9958-12369 Da. These glycoforms account for the diffuse 6-16 kDa banding pattern seen by SDS-PAGE (Figure 2A, lane 2). The MS data also revealed an intra-chain Cys-62−Cys-65 disulfide linkage. In pro-TGF-β1, Cys-4, which corresponds to Cys-62 in pro-GDF11, is disulfide linked to latent TGF-β binding protein (LTBP).25 This association tethers pro-TGF-β1 to the extracellular matrix, where


Biochemistry

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16 it plays a critical role in local activation of TGF-β. For TGF-β1, there is no second intrachain cysteine to pair with the one that corresponds to Cys-65 in pro-GDF11. MS also provided confirmation of the processing site of the signal peptide, leading to the predicted N terminus of Ala-25, and identified two O-linked glycosylation sites at Ser-49 (>75% occupied) and Ser-54 (99% occupied). Like the untreated full-length pro-GDF11, the AspN-treated sample migrated by SEC as a single peak at a molecular weight of 100 kDa (Figure 3A), with no detectable aggregates in the preparation. No low-molecular weight components were detected from the SEC analysis despite the presence of the 60-114 glycopeptide in the SDS-PAGE and MS analyses (Figure 2A, lane 2, Figure 5), indicating that the fragment is non-covalently associated with the rest of the protein. Furin treatment of the AspN-digestion product led to further processing of GDF11. Eighty percent of the AspN-cleaved fragment containing residues 122-407 was digested by furin after 6 hr, and >95% was digested after 23 hr (Figure 2A, lanes 3 and 5). Three new major cleavage products were produced with molecular weights of 45, 25, and 23/22 kDa under non-reducing conditions, and 40, 23/22, and 14 kDa under reducing conditions. The broad low-molecular weight 6-16 kDa band seen after AspN treatment was also present in the double digest. Mature GDF11 exists as a disulfide-linked homodimer with apparent molecular weights of 25 kDa and 14 kDa under non-reducing and reducing conditions, respectively. To aid in the identification of bands corresponding to mature GDF11, a preparation of mature GDF11 was included in the analysis (lanes 6 and 7, arrows denote the position of the mature GDF11 under reducing and non-reducing conditions). Following 23 hr of digestion, mature GDF11 was the major cleavage product, present at about 60% of the theoretical yield (lane 5). The identity of the fragment was confirmed by MS (Figure 5, predicted mass for mature GDF11 residues 299-407 is 12,457.4 Da, observed mass was 12,458 Da). Cleavage at the furin site also produced the 23/22 kDa doublet of bands seen under reducing and non-reducing conditions that corresponds to the C-terminal fragment of the prodomain (residues 122298, 128-298, and 133-298). While the upper band of the doublet is partially obscured by mature GDF11 under non-reducing conditions in the analysis shown in Figure 2A, it is readily visible in the subsequent study shown in Figure S2, lane 7 (discussed below). Other bands seen in the AspN/furin digest result from incomplete cleavage at the furin


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Biochemistry

17 site, where the 45 kDa band under non-reducing conditions results from cleavage at one of the two furin sites in the 122-407 homodimer and the 40 kDa band observed under reducing conditions is the undigested 122-407 AspN fragment. The AspN/furin-cleavage product was tested for function in the SMAD 2/3 phosphorylation assay as well as in a SMAD reporter luciferase assay, in which luciferase expression in PC12 cells is under the control of the SMAD transcriptional response element (TRE) (Figure 4B and C). Both assays confirmed proteolytic activation of proGDF11. The potency of the processed product (EC50 = ~1 nM) was identical to that of the mature GDF11 standard. No activation occurred when pro-GDF11 that was treated only with AspN was analyzed (Figure 4B). Together these studies revealed that the proteolytic activation of pro- GDF11 in vitro required two steps; first a priming cleavage at Asp-122 that makes the furin-cleavage site accessible, and then cleavage at the furin site that leads to the activation of GDF11. Without cleavage at the BMP1 site by AspN, the precursor was resistant to cleavage by furin. Similar results were obtained using plasmin and trypsin in place of furin to drive cleavage at the furin site (Figure 2S). Both proteases also depended on a priming cleavage at Asp-122 to cleave at the furin site. Proteolytic activation of pro-GDF11 generates a complex that is soluble at neutral pH. Visual examination of the pro-GDF11 sample treated first with AspN and then with furin for 23 hr revealed that a precipitate had formed. SDS-PAGE analysis of the supernatant (Figure S2, lane 8) and precipitate (Figure S2, lane 9) under reducing and non-reducing conditions revealed that all of the mature GDF11 was in the soluble fraction. The precipitate contained the major prodomain fragment corresponding to residues 122-298. The solubility of the proteolytically activated GDF11 at neutral pH was unexpected, as poor solubility under non-acidic conditions is a characteristic of most members of the TGF-β family. Commercial preparations of these proteins thus routinely utilize formulations that are 50 kDa. No high-molecular weight aggregates were detected in the preparation. The >50 kDa size was significantly larger than the expected mass for mature GDF11 alone of 25 kDa. In column fractions that contain the peak (lanes 3-5), mature GDF11 (25 kDa non-reducing, 14 kDa reducing) as well as the broad band with molecular weight of 6-16 kDa under reducing and nonreducing conditions were detected. When deglycosylated and analyzed by MS, the 6-16 kDa band was identified as a fragment of the prodomain containing residues 60-114 and residues 60-112 resulting from secondary cleavage of the same peptide at amino acid 112 (Figure 5B and C). The identity of the peptides containing residues 60-112 and 60-114 were confirmed by collision-induced dissociation (CID) tandem MS/MS. The presence of the PDP60-114–mature GDF11 complex accounts in part for the larger than anticipated size of the AspN/furin cleavage product by SEC. In addition, the elongated structure of the complex creates a larger hydrodynamic radius than for a globular protein of the same size, which results in a larger apparent molecular weight estimate by SEC than would be predicted by mass alone.26,27 The glycoforms in PDP60-114 contribute to the broadness of the SEC peak since the hydrodynamic radius is also impacted by the heterogeneity in the sizes of the glycans. To explore potential biochemical features of the pro-GDF11 60-114 peptide that might account for its impact on the solubility of mature GDF11, we utilized published sequence and structure-guided alignments from other TGF-β family members, which had included the pro-GD11 sequence as part of the analysis.25 Based on these alignments, the GDF11 PDP60-114 sequence contains the entire α1 helix and latency lasso domain, and a portion of the α2 helix (see Table SI for alignments of the subset of TGF-β family members discussed in this paper). These conserved elements have been characterized in most of the TGF-β family members, and map to a structural feature seen in the latent proTGF-β1 crystal structure coined as the straight jacket that wraps around the fingers of the mature domain.25 In fact, the contacts between the α1 helix and latency lasso with the mature growth factor, which were seen in the latent TGF-β1 structure and more recently in the pro-activin-A structure, are likely to account for the stability of the PDP60114/mature

GDF11 complex. The latent TGF-β1 structure is unlikely to be representative


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Biochemistry

19 of the interactions of PDP60-114 with mature GDF11, because the GDF11/ PDP60-114 complex we produced is in an active state whereas the latent TGF-β1 complex is in an inactive state. Electron microscopy. To gain further insights into structural attributes of the GDF11 precursor protein, pro-GDF11, we subjected samples to negative-strain electron microscopy (EM) (Figure S1). As previously seen in EM studies of other precursor proteins of TGF-β family members,28 the pro-GDF11 particles were small, elongated, and V-shaped with the longest dimension being about 12 nm and the shortest dimension being about 4 nm (Figure S1A). 2D classification yielded 140 classes averages of negatively stained pro-GDF11 (Figure S1B). These averages were used to calculate a 3D map into which the crystal structures of pro-BMP9,28 pro-activin-A,29 and pro-TGF-β125 were placed. The BMP9 crystal structure was selected as a representative of a TGF-β family member, for which the precursor protein adopts an open-armed conformation with the prodomains extending away from the mature domains. The pro-TGF-β1 structure was selected as a representative, for which the precursor protein adopts a cross-armed conformation with the two prodomains in contact with each other. Finally, the proactivin-A structure was selected as a representative, for which the precursor protein adopts a structure in between those of pro-BMP9 and pro-TGF-β1. From the placing of the crystal structures into the EM density map, pro-GDF11 clearly adopts a conformation that is more similar to that of pro-BMP9 than those of pro-TGF-β1 or pro-activin-A (Figure S1C-E). AspN-treated pro-GDF11 was also imaged by negative-stain EM. Visual inspection of the particles revealed no gross changes in structure from that seen for uncleaved pro-GDF11 (data not shown). Despite structural similarities, the proBMP9 structure was not useful for investigating potential contacts between mature GDF11 and PDP60-114, since the X-ray map of pro-BMP9 showed no density for the corresponding α1 helix or latency lasso sequences. Binding studies. Binding characteristics of PDP60-114 for mature GDF11 (residues 299-407) and for the AspN fragment (residues 122-407) were assessed using an Octet Red system. For these studies, pro-GDF11 was biotinylated through its single glycan on Asn-94, which fortuitously is present in PDP60-114 (see Figure 1). Preparations of the biotinylated pro-GDF11 that had been treated with AspN alone, treated with AspN and


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20 furin, or purified biotinylated PDP60-114 following AspN treatment (Figure S2, lane 11) were captured on Streptavidin biosensors and evaluated for dissociation over time (Figure 3B). As expected, the amplitude of the response on the sensor was proportional to the size of the complex, yielding signals of 0.5 nm for PDP60-114 itself (6-11 kDa), 1.2 nm for the complex of PDP60-114 with mature GDF11 (40 kDa), and 4.7 nm for pro-GDF11 digested only with AspN (100 kDa). The dissociation kinetics of the samples were very slow, as 10% or less of the signal was lost following 60 min incubation in dissociation buffer. The dissociation rates for the sample treated only with AspN and that treated with both AspN and furin were indistinguishable indicating high-affinity binding of PDP60-114 to both forms of the protein. The binding characteristics of pro-GDF11 and AspN/furin-treated pro-GDF11 for type I and II receptors, activin RIB-Fc and RIIA-Fc, respectively, were also assessed using an Octet readout (Figure 3C). Biotinylated GDF11 samples were loaded onto Streptavidin Octet tips and then treated with the recombinant receptor-Fc fusion proteins. As expected, activin RIIA-Fc bound to the pro-GDF11 sample that was treated with both AspN and furin. No binding occurred for activin RIB-Fc. Binding of activin RIIA-Fc to intact pro-GDF11 was reduced relative to that seen for the protease-activated GDF11 sample and there was no binding of activin RIB-Fc to pro-GDF11. To confirm this finding, we labeled AspN- and furin-treated pro-GDF11 with Alexa488, mixed it with activin RIIA-Fc, and subjected the sample to SEC with on-line fluorescence detection. Alexa488-containing column fractions were collected and analyzed by SDS-PAGE, monitoring the fluorescence of the GDF11 fragments. Association of the receptor with the AspN- and furin-treated pro-GDF11 resulted in the formation of a large complex that migrated on the SEC column with a molecular weight of >100 kDa with baseline separation from free PDP60-114/mature GDF11 alone. Fractions corresponding to the activin RIIA-Fc/GDF11 complex contained both fluorescently labeled mature GDF11 and labeled PDP60-114 (data not shown). The presence of PDP60-114 in the complex indicates that binding to the receptor does not displace the N-terminal prodomain peptide. The same analysis showed no binding of the Alexa488-labeled, AspN- and furin-treated pro-GDF11 with activin RIB-Fc. Potential therapeutic uses of myostatin inhibitors for treatment of muscular


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Biochemistry

21 dystrophy and other atrophy disorders has led to the engineering of many pathway antagonists that directly bind myostatin or inactivate its receptor, ActRIIB.12 Recently, new inhibitors have been discovered that are peptides derived from within residues 19-47 of the of murine myostatin prodomain and have IC50 values for myostatin of ~30 nM.30 GDF11 PDP60-114 is twice as long as the 24-residue minimum inhibitory peptide located entirely within the putative α1 helix of myostatin (Table SI).30 In contrast, we found that the association of PDP60-114 with mature GDF11 did not impact its activity. As seen in Figure 4C, the activities of the mature GDF11/PDP60-114 complex and of mature GDF11 alone are indistinguishable. As a positive control for the study, Fc fusion proteins of the prodomain were produced, designed essentially as described previously,7 and tested for their ability to inhibit GDF11-induced signaling in the luciferase reporter assay (Figure 4D). Both prodomain fusion proteins were potent inhibitors of GDF11 activity. Pretreatment of mature GDF11 with the prodomains led to an ~100-fold decrease in activity. Designing prodomain peptide/mature GDF11 fusion proteins. No GDF11 was detected when the mature domain was expressed in CHO cells alone in the absence of the prodomain (Table 1). To test if a genetic fusion of PDP60-114 to the mature domain of GDF11 could improve the expression of GDF11, we designed a series of 11 different constructs and expressed them in CHO cells (Table 1). G4S spacers of varying lengths were incorporated into the designs between PDP60-114 and the mature domain to allow proper assembly of the domains. Some of the constructs contained deletions of up to 11 amino acids from the N terminus of the peptide. Other constructs contained additional amino acids at the C terminus that completed the putative α2 helix. An N-terminal 8-His tag was added to all constructs to facilitate their purification. Figure 6A and B show Coomassie blue-stained and Western blotted SDS-PAGE gels from conditioned medium of CHO cells expressing the 11 constructs. Expression levels of the prodomain peptide fusions varied widely (Table 1). Surprisingly, greatest expression only occurred when the full α2 helix (containing D122) was incorporated (ACE490 and ACE498). In fact, 20-fold lower levels occurred when truncated versions ending in L114 were used. Greater expression was observed when the propeptide was attached to the N terminus of the mature GDF11 domain, although expression was significant with the one C-terminal


Biochemistry

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22 version that contains a complete α2 helix design. The expression levels achieved with the ACE490 and ACE498 designs, each exceeding 50 mg/L, were greater than that observed when the full-length pro-GDF11 protein was expressed. The ACE490 and ACE498 proteins were purified from the conditioned medium by chromatography on a Ni Sepharose excel column. Non-reducing SDS-PAGE analysis of the purified preparations (Figure 7B and D) revealed that neither sample had formed the interchain disulfide that covalently links the mature GDF11 dimer when it is assembled properly. Further assessment by SEC showed that both products eluted from the sizing column as monomers (data not shown). When these samples were tested for function in the reporter assay, they were both inactive (Figures 7C and E). We next asked if we could promote dimer formation by performing redox reactions. For these studies, the proteins were reduced with DTT, then half of the preparation was treated with Aldrithiol to activate the cysteine. This half was then added back to the other half of the protein to drive dimerization. Figure 7A shows a schematic summarizing the redox steps. Indeed, this treatment of ACE490 led to significant dimer formation. Figure 7B shows an SDS-PAGE analysis of the redox product. No dimer formation occurred without the Aldrithiol activation step. When this preparation was tested in the SMAD 2/3 reporter assay, the sample recovered about 10% of the activity observed for mature GDF11 (Figure 7C). Aldrithiol/redox treatment of ACE 498 also led to dimer formation, but the preparation was inactive in the SMAD 2/3 reporter assay (Figure 7D and E). Interestingly, when this preparation was treated with AspN, the specific activity in the reporter assay was increased to about 50% of the GDF11 standard, indicating that dimerization followed by cleavage at the interface between the prodomain and mature domain was required for activation. SDS-PAGE analysis of the AspN-treated ACE498 sample (Figure 7D, lane 3) revealed a diffuse band with molecular weight of 6-16 kDa, consistent with release of the prodomain peptide and a prominent band of 16 kDa corresponding to the mature domain fragment that was only observed under reducing conditions. The slightly larger size of this band (16 kDa versus 14 kDa for mature GDF11 that was released from full-length pro-GDF11) is consistent with the presence of the 2G4S linker, which would be retained on the mature domain fragment following AspN treatment. From the characteristics of the ACE490 and ACE498 constructs and


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Biochemistry

23 activation following a simple redox reaction without the need for a refolding step, we can infer that the cystine-knot motif properly formed during expression, but the absence of the Asp-122−Arg-298 region of the prodomain or improper alignment of the Asp60−Asp-120 sequences in its complex with the mature domain prevented dimerization. The ability to restore activity through biochemical manipulation of the products suggests that one should be able to optimize the process. Processing of pro-BMP7 with endoproteinase AspN. Pro-BMP7 processing was studied to test if other members of the TGF-β family can form a soluble complex analogous to the one we observed for mature GDF11 with PDP60-114. Despite sharing only 20% sequence identity with pro-GDF11, an alignment of the pro-BMP7/pro-GDF11 sequences using annotated secondary structural motifs28 revealed AspN cleavage sites in pro-BMP7 at exactly the same positions as Asp-60 and 115 in pro-GDF11 (Table SI). Treatment with the protease would thus be expected to release a similar peptide (residues 34-85) containing the entire α1 helix, lasso, and a portion of the α2 helix. Also like GDF11 PDP60-114, the corresponding BMP7 prodomain peptide is very basic with >25% of its sequence consisting of charged amino acids. When full-length pro-BMP7 was expressed in CHO cells and purified, ~90% was processed at the monobasic cleavage site at the prodomain/mature domain interface analogous to the dibasic furin site in proGDF11. AspN treatment of pro-BMP7 indeed generated specific cleavage at the predicted sites and the 5-kDa propeptide fragment remained associated with mature BMP7, but in this case, the complex formed by mature BMP7 with the prodomain peptide precipitated and the C-terminal portion of the prodomain remained in solution. The identity of the prodomain peptide in complex with mature BMP7 was confirmed by MS (residues 34-84, predicted mass 6136.20 Da, observed mass 6136.21 Da). One significant difference between pro-BMP7 and pro-GDF11 is that GDF11 PDP60-114 contains an N-linked glycan with complex sugars attached whereas the corresponding BMP7 prodomain peptide is not glycosylated. This difference could contribute to the solubility differences between prodomain peptide complexes formed with mature GDF11 and BMP7. Our data for BMP7 indicate that the ability of the prodomain peptide to enhance the solubility of GDF11 is not a general phenomenon and will need to be evaluated for other TGF-β family members on a case-by-case basis.


Biochemistry

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24 DISCUSSION We have expressed full-length pro-GDF11 in CHO cells and characterized its proteolytic processing and activation in vitro. To our surprise the protein was resistant to proteolysis with furin and two other proteases, plasmin and trypsin, that should be able to cleave at the K294RSRR298 furin site (Figure 1). However, when pro-GDF11 was first cleaved at Asp-122, the protein was sensitive to treatments with furin, plasmin, and trypsin, efficiently releasing the mature domain. Proteolytic activation following treatment with AspN and furin was confirmed by measuring treatment-induced phosphorylation of SMAD 2/3 as well as the engagement of downstream signaling. Whereas commercial sources of mature GDF11 require formulation in acidic buffers to maintain their solubility, PDP60-114 remained non-covalently associated with the mature domain, rendering the complex soluble at neutral pH. To investigate how PDP60-114 might improve the solubility of mature GDF11, we used PIPER31 to computationally dock the crystal structure of the GDF11 mature domain homodimer8 against another copy of itself in the absence of PDP60-114. These models overwhelmingly cluster into a conformation, in which the two homodimers contact each other, largely via aromatic residues in their respective concave type I receptor-binding sites. This finding suggests that coverage of this binding site with other protein elements will aid solubility of the complex and provides a model for how PDP60-114 can prevent aggregation/precipitation of GDF11. For most TGF-β family members, proteolytic processing at the prodomain/mature domain junction produces non-covalent complexes that are active and provide simple formulations that render the growth factors soluble.29,32,33 However, TGF-β1-3, BMP10, myostatin, and GDF11 form latent complexes when they are processed at this site. Evidence for latency of GDF11 was first demonstrated in an experiment by Ge and coworkers,7 in which mature GDF11 was mixed with GDF11 prodomain, and the resulting complex was tested for activity by assessing SMAD 2/3 phosphorylation in PC12 cells. The complex was inactive, a finding we confirmed essentially as described using an Fc fusion protein containing GDF11 prodomain residues 25-298 as a source of the prodomain. Processing data presented by Ge and coworkers support a model that cleavage occurs first at the furin site.7 In their study, ~60% of the protein was cleaved at


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Biochemistry

25 the furin site. In contrast, only about 5% of the protein we produced (Figure 2) was cleaved, and MS analysis revealed that this fragment contained GDF11 residues 122-407. Thus, we found that cleavage occurred at the BMP1 site during cell culture without initial cleavage at the furin site. One significant difference was that Ge and coworkers engineered pro-GDF11 with an N-terminal tag and used this for purification, whereas we expressed the endogenous protein as is without a tag. Pro-GDF11 contains an extended connecting sequence between the end of the signal sequence, Ala-24, and prior to the start of the α1 helix at Asp-60. We found that this region is protease sensitive, with near quantitative cleavage at Asp-60 following AspN treatment and 60% cleavage at Arg-48 following plasmin cleavage. Cleavage within this connecting sequence would release the N-terminal tag used for purification in the study by Ge and coworkers and consequently could impact the product they generated. It is unclear if this contributed to the differences in the products that were isolated. Figure 8 shows a schematic illustration depicting key intermediates in the proteolytic activation of the latent GDF11 precursor protein by AspN and furin, leading to the release of active and soluble mature GDF11/PDP60-114 complex and precipitation of the GDF11 prodomain peptide 122-298. Pro-GDF11 is shown in an open-armed conformation to reflect the results from the EM studies (Figure S1). The conditions used for proteolytic activation led to quantitative precipitation of the propeptide 122-298. To date crystal structures have been solved for only three TGF-β family members in complex with their prodomains (pro-TGF-β1, pro-activin A, and pro-BMP9). Structures for mature/active TGF-β1 (PDB id: 3KFD)34 and for latent pro-TGF-β1 (PDB id: 3RJR) 25 revealed that the prodomain α1 helix in the latent TGF-β1 structure displaces the active growth factor wrist helix from its position, distorting the wrist epitope that is characteristic of the active state.25 As a result, the α1 helix distorts the type I receptor binding site that is formed by the concave interface and the wrist helix in the active conformation. In the structure of proteolytically cleaved pro-activin A complex (PDB id: 5HLZ), the α1 helix also distorts the wrist helix of the mature activin A domain, and the orientation of the α1 helix is similar to that observed in the pro-TGF-β1 structure. However, in contrast to pro-TGF-β1, the pro-activin-A complex is biologically active.29 Crystal structures of pro-activin A with and without proteolytic processing were similar


Biochemistry

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26 and support the notion that proteolytic activation does not induce a conformational change in the structures of the complex.29 By contrast, in the pro-BMP9 structure, the α1 helix was not detected, and instead, the elongated prodomain α5 helix partially occupies the undistorted type I receptor binding site. The sequences corresponding to the BMP9 α5 helix are truncated in pro-TGF-β1 and pro-activin A and do not displace the α1 helix from its contacts with the mature domain.25 From sequence alignments (Ref. 28, Table SI), the corresponding α5 helix sequences in pro-GDF11 and myostatin are even shorter. In contrast to the differences seen in the orientation of the α1 helix, the α2 helix in the crystal structures of latent pro-TGF-β1, pro-activin A and pro-BMP9 occupies a similar orientation at the convex interface on the surface of finger 2 that overlaps with the type II receptor interface for the latter two proteins.5,35 When examined relative to its position in the mature domain, the position of the α2 helix is comparatively unchanged between the open conformation of BMP9 (PDB ids: 4YCG, 4YCI)28 and the closed conformation of the TGF-β1 prodomain (PDB id: 3RJR)25 and in the cross-armed proactivin A structures with and without proteolytic activation.29 Sequence alignments of the convex interface of the finger 2 region and prodomain α2 helix of many members of the TGF-β family suggest that the α2 helix of GDF11 is likely to occupy a similar site in the finger 2 region of GDF11. Because in our studies, activin RIIA-Fc can bind the mature GDF11/PDP60-114 complex without displacing the prodomain peptide, we infer that truncation at residue 114 allows the complex to be presented in an active state. The large effect of the fusion protein designs of the propeptide fragment to the growth factor domain of GDF11 on their expression in CHO cells was unexpected (Table 1). The two lead molecules, ACE 490 and ACE498, were well-behaved proteins, but monomeric, failing to form the inter-chain Cys-372−Cys-372 disulfide bond that covalently links the mature domain dimer. Although formation of the inter-chain disulfide bond is not a prerequisite for dimer formation, dimer formation is required to engage signaling (see Li et al., 2002 for references).36 It was encouraging that we could chemically induce functional dimerization of the ACE490 and ACE498 monomeric units as it indicates that the cystine-knot motif was properly formed without the need for denaturation/renaturation strategies to promote formation of the inter-chain disulfide bond. The reduced activity of the fusion proteins is consistent with the α2 helix contacts


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27 overlapping with the putative type II receptor-binding site, which would explain why truncation of the α2 helix following AspN treatment led to activation of the ACE498 construct. Alternatively, the α1 helix may have retained some of its intercalation propensity in the genetically fused GDF11 constructs. In this scenario, binding of the α1 helix at the mature domain monomer could sterically interfere with monomer-monomer contacts, which could explain why the fusion proteins had failed to dimerize during expression. This intercalative property may also explain the inhibitory activity of the putative α1 helix peptide of mouse myostatin.30 In this and other studies, myostatin prodomain fragments were first characterized for their inhibitory activity towards mature human myostatin and if they were not inhibitory they were not characterized for their capacity to bind/stabilize the mature domain. 30,37 Studies with the inhibin α-subunit provide further insights into the complex interactions between prodomain sequences with the growth factor domain.38,39 The inhibin α-subunit contains a second furin site and cleavage at this site releases a 43-amino acid prodomain peptide analogous to GDF11 PDP60-114 (Table SI).38 A synthetic peptide of this prodomain fragment blocked inhibin function with an IC50 of 86 nM; however, characterization of inhibin A and B from biological fluids have detected complexes of which some retain their biological activity.38,39 While prodomain sequences display diverse roles in regulating activity, biosynthesis, and biodistribution of TGF-β family growth factors, there is clear specialization by the individual family members. More detailed studies are needed to understand prodomain peptide−mature domain interactions at a molecular level across the TGF-β family and their contribution to active and latent states. The development of formulations suitable for in vivo administration is a critical part of drug development, requiring individualized optimization for each product.40,41 Acidic formulations routinely used for most TGF-β family members carry liabilities in that they can promote reversible or irreversible denaturation, chemical degradation, increase surface charge, and frequently lead to precipitation/ deposition at sites of injection due to the rapid increase in pH upon delivery. In rat studies comparing mature BMP7 and soluble BMP7 (full-length pro-BMP7 that is proteolytically activated by cleavage at the prodomain/mature domain interface), we observed bone formation at the


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28 injection sites following local intramuscular administration of mature BMP7 and poor serum bioavailability, whereas injections of soluble BMP7 led to elevated serum levels and did not promote bone formation at the injection sites (unpublished data). In fact, it was these observations that prompted us to search for soluble formulations of GDF11. Here, we show that PDP60-114 enhances the solubility of mature GDF11 at neutral pH without impacting its activity and may thus provide a novel formulation opportunity for GDF11 that warrants further evaluation. Acknowledgements We thank Werner Meier, Alan Buckler, and Richelle Sopko for helpful discussions and critical reading of the manuscript, Judy George for preparing conditioned media, and Monika Vecchi for mass spectrometry analysis. Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Negative-stain EM analysis of pro-GDF11 (Figure S1), processing data of proGDF11 with plasmin and trypsin, prodomain α1 helix (Figure S2), latency lasso and α2 helix sequence alignments (Table S1), and computational modeling of the PDP60-114–mature GDF11 complex.


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29 REFERENCES (1) Massagué, J. (2000) How cells read TGF-β signals. Nat. Rev. Mol. Cell Biol. 1, 169– 178. (2) Chang, H., Brown, C.W., and Matzuk, M.M. (2002) Genetic analysis of the mammalian transforming growth factor-beta superfamily. Endocr. Rev. 23, 787–823. (3) Heldi, C.H., Landstrom, M., and Moustakas, A. (2009) Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition. Curr. Opin. Cell Biol. 21, 166–176. (4) Wakefield, L.M., and Hill, C.S. (2013) Beyond TGFbeta: roles of other TGFbeta superfamily members in cancer. Nat. Rev. Cancer 13, 328–341. (5) Hinck, A.P. (2012) Structural studies of the TGF-betas and their receptors - insights into evolution of the TGF-beta superfamily. FEBS Lett. 586, 1860–1870. (6) Weiss, A., and Attisano, L. (2013) The TGFbeta superfamily signaling pathway. Wiley Interdiscip. Rev. Dev. Biol. 2, 47–63. (7) Ge, G., Hopkins, D.R., Ho, W.B., and Greenspan, D.S. (2005) GDF11 forms a bone morphogenetic protein 1-activated latent complex that can modulate nerve growth factorinduced differentiation of PC12 cells. Mol. Cell Biol. 25, 5846–5858. (8) Padyana, A.K., Vaidialingam, B., Hayes, D.B., Gupta, P., Franti, M., and Farrow, N.A. (2016) Crystal structure of human GDF11. Acta Crystallogr. F Struct. Biol. Commun. 72, 160–164. (9) Walker, R.G., Czepnik, M., Goebel, E.J., McCoy, J.C., Vujic, A., Cho, M., Oh, J., Aykul, S., Walton, K.L., Schang, G., Bernard, D.J., Hinck, A.P., Harrison, C.A., Martinez-Hackert, E., Wagers, A.J., Lee, R.T., and Thompson, T.B. (2017) Structural basis for potency differences between GDF8 and GDF11. BMC Biol. 15, 19. (10) Mueller, T.D., and Nickel, J. (2005) Promiscuity and specificity in BMP receptor activation. FEBS Lett. 586, 1846–1859. (11) McPherron, A.C., Lawler, A.M., and Lee, S.J. (1997) Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387, 83–90. (12) Smith, R.C., and Lin, B.K. (2013) Myostatin inhibitors as therapies for muscle wasting associated with cancer and other disorders. Curr. Opin. Support Palliat. Care 7, 352-360. (13) Katsimpardi, L., Litterman, N.K., Schein, P.A., Miller, C.M., Loffredo, F.S., Wojtkiewicz, G.R., Chen, J.W., Lee, R.T., Wagers, A.J., and Rubin, L.L. (2014) Vascular


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30 and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science 344, 630–634. (14) Loffredo, F.S., Steinhauser, M.L., Jay, S.M., Gannon, J., Pancoast, J.R., Yalamanchi, P., Sinha, M., Dall'Osso, C., Khong, D., Shadrach, J.L., Miller, C.M., Singer, B.S., Stewart, A., Psychogios, N., Gerszten, R.E., Hartigan, A.J., Kim, M.J., Serwold, T., Wagers, A.J., and Lee, R.T. (2013) Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell 153, 828–839. (15) Sinha, M., Jang, Y.C., Oh, J., Khong, D., Wu, E.Y., Manohar, R., Miller, C., Regalado, S.G., Loffredo, F.S., Pancoast, J.R., Hirshman, M.F., Lebowitz, J., Shadrach, J.L., Cerletti, M., Kim, M.J., Serwold, T., Goodyear, L.J., Rosner, B., Lee, R.T., and Wagers, A.J. (2014) Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle. Science 344, 649–652. (16) Smith, S.C., Zhang, X., Zhang, X., Gross, P., Starosta, T., Mohsin, S., Franti, M., Gupta, P., Hayes, D., Myzithras, M., Kahn, J., Tanner, J., Weldon, S.M., Khalil, A., Guo, X., Sabri, A., Chen, X., MacDonnell, S., and Houser, S.R. (2015) GDF11 does not rescue aging-related pathological hypertrophy. Circ. Res. 117, 926–932. (17) Brun, C.E., and Rudnicki, M.A. (2015) GDF11 and the mythical fountain of youth. Cell Metab. 22, 54–56. (18) Egerman, M.A., Cadena, S.M., Gilbert, J.A., Meyer, A., Nelson, H.N., Swalley, S.E., Mallozzi, C., Jacobi, C., Jennings, L.L., Clay, I., Laurent, G., Ma, S., Brachat, S., Lach-Trifilieff, E., Shavlakadze, T., Trendelenburg, A.U., Brack, A.S., and Glass, D.J. (2015) GDF11 increases with age and inhibits skeletal muscle regeneration. Cell Metab. 22, 164–174. (19) Poggioli, T., Vujic, A., Yang, P., Macias-Trevino, C., Uygur, A., Loffredo, F.S., Pancoast, J.R., Cho, M., Goldstein, J., Tandias, R.M., Gonzalez, E., Walker, R.G., Thompson, T.B., Wagers, A.J., Fong, Y.W., and Lee, R.T. (2016) Circulating growth differentiation factor 11/8 levels decline with age. Circ. Res. 118, 29–37. (20) Walker, R.G., Poggioli, T., Katsimpardi, L., Buchanan, S.M., Oh, J., Wattrus, S., Heidecker, B., Fong, Y.W., Rubin, L.L., Ganz, P., Thompson, T.B., Wagers, A.J., and Lee, R.T. (2016) Biochemistry and biology of GDF11 and myostatin: similarities, differences, and questions for future investigation. Circ. Res. 118, 1125–1142. (21) Yadin, D., Knaus, P., and Mueller, T.D. (2016) Structural insights into BMP receptors: specificity, activation and inhibition. Cytokine Growth Factor Rev. 27, 13–34. (22) Akhurst, R.J., and Padgett, R.W. (2016) Matters of context guide future research in TGFβ superfamily signaling. Sci. Signal. 8, 1–10.


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32 (34) Radaev, S., Zou, Z., Huang, T., Lafer, E.M., Hinck, A.P., and Sun, P.D. (2010) Ternary complex of transforming growth factor-beta1 reveals isoform-specific ligand recognition and receptor recruitment in the superfamily. J. Biol. Chem. 285, 1480614814. (35) Allendorph, G.P., Vale, W.W., and Choe, S. (2006) Structure of the ternary signaling complex of a TGF-beta superfamily member. Proc. Natl. Acad. Sci. U.S.A. 103, 7643–7648. (36) Li, T., Yamane, H., Arakawa, T., Narhi, L.O., and Philo, J. (2002) Effect of the intermolecular disulfide bond on the conformation and stability of glial cell line-derived neurotrophic factor. Protein Eng. 15, 59–64. (37) Jiang, M-S., Liang, L-F., Wang, S., Ratovitski, T., Holmstrom, J., Barker, C., and Stotish, R. (2004) Characterization and identification of the inhibitory domain of GDF-8 propeptide. Biochem. Biophys. Res. Commun. 315, 525-531. (38) Walton, K.L., Kelly, E.K., Chan, K.L., Harrison, C.A., and Robertson, D.M. (2015) Inhibin biosynthesis and activity are limited by a prodomain-derived peptide. Endocrinology 156, 3047-3057. (39) Robertson, D.M., Cahir, N., Findlay, J.K., Burger, H.G., and Groome, N. (1997) The biological and immunological characterization of inhibin A and B forms in human follicular fluid and plasma. J. Clin. Endocrinol. Metab. 82, 889-896. (40) Wang, W., Singh, S., Zeng, D.L., King, K., and Nema, S. (2007) Antibody structure, instability, and formulation. J. Pharm. Sci. 96, 1–26. (41) Uchiyama, S. (2014) Liquid formulation for antibody drugs. Biochim. Biophys. Acta. 1844, 2041–2052.


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33 Table 1. Design and expression of GDF11 prodomain fragment-mature domain fusion proteins.a

Construct

Protein sequence attributes

Expression

pACE487

8xHis-TEV-GDF (G61-D122)-G-4xG4S-GDF (N299-S407)

++

pACE490

8xHis-TEV-GDF (S71-A123)-3xG4S-GDF (N299-S407)

pACE491

8xHis-TEV-GDF (G61-L114)-3xG4S-GDF (N299-S407)

+

pACE492

8xHis-TEV-GDF (D60-L114)-3xG4S-GDF (N299-S407)

+/-

pACE493

8xHis-TEV-GDF (D60-L114)-4xG4S-GDF (N299-S407)

+

pACE494

8xHis-TEV-GDF (N299-S407)-2xG4S-GDF (S71-L114)

-

pACE495

8xHis-TEV-GDF (N299-S407)-3xG4S-GDF (S71-L114)

-

pACE496

8xHis-TEV-GDF (N299-S407)-3xG4S-GDF (D60-L114)

-

pACE497

8xHis-TEV-GDF (N299-S407)-3xG4S-SP-GDF (R72-L114)

-

pACE498

8xHis-TEV-GDF (D60-D122)-2xG4S-GDF (N299-S407)

++++

pACE499

8xHis-TEV-GDF (N299-S407)-3xG4S-GDF (S71-D122)

+

pACE382

GDF (N299-S407)

-

++++

.

a

Eleven constructs with the GDF11 signal peptide linked to an 8-histidine affinity tag and a TEV protease cleavage site incorporated at the junction of the tag and GDF11 sequence were engineered for expression in CHO cells. Prodomain sequences are indicated in red and mature domain sequences in blue. One series of constructs was designed in the “natural” orientation, with PDP60-114 attached to the N terminus of the mature domain. Because of the close proximities between either of the C termini from both chains of mature GDF11 in the dimer, and the putative position of the N terminus of PDP60-114, another series of constructs was designed with the prodomain peptide attached at the C terminus of the mature domain. As control, a mature domain only construct, ACE382, was expressed using the GDF11 signal sequence with no His tag. Relative expression levels for each construct were assessed directly from the conditioned medium by Western blotting, using a polyclonal antibody against a C-terminal peptide of GDF11 for detection.


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34 Figure Legends Figure 1. Schematic drawing denoting key features of the human GDF11 precursor protein. Signal peptide (residues 1-24, yellow), BMP1-type protease and furin-like convertase cleavage sites (red arrows), N-terminal prodomain cleavage fragment (residues 25-122), C-terminal prodomain cleavage fragment (residues 123-298), furin recognition sequence (residues 295-298), mature domain (residues 299-407, blue), solubility enhancing N-terminal prodomain subfragment (residues 60-114, burgundy hatched pattern) within the N-terminal prodomain cleavage fragment, the single N-linked glycosylation site (N94), and the single inter-chain disulfide bridge-forming cysteine (C372). Molecular weights are deduced from the pro-GDF11 sequence and do not take into account the impact of glycosylation. Figure 2. Characterization of GDF11 samples by SDS-PAGE. Samples (5 µg/lane) were subjected to SDS-PAGE on 4-20% gradient gels under non-reducing and reducing conditions. All gels were stained with SimplyBlue Coomassie dye, except the right panel in B, which was visualized by Western blotting with a polyclonal antibody raised against the C terminus of mature GDF11. The apparent molecular masses of pre-stained molecular weight markers are indicated at the left. Red arrows denote the stained bands representing mature GDF11. A. Lane 1, full-length pro-GDF11; lane 2, full-length proGDF11 treated with endoproteinase AspN for 2.5 hr at 37°C; lane 3, AspN-treated GDF11 further digested with furin at 37°C for 6 hr; lane 4, AspN-treated GDF11 digested with furin at 37°C for 7 hr followed by digestion overnight at 4°C; lane 5, AspN-treated GDF11 digested with furin at 37°C for 23 hr; lane 6, mature GDF11standard (0.25 µg) formulated in bovine serum albumin; lane 7, mature GDF11standard (1 µg) formulated in bovine serum albumin; lane S, molecular weight standards. B. Commassie blue-stained gel (left) and Western blot analysis (right) of full-length pro-GDF11 under reducing conditions. Figure 3. Characterization of GDF11 samples by SEC. A. Full-length pro-GDF11 (100 µg), AspN-treated pro-GDF11 (100 µg), and AspN-treated GDF11 digested with furin at 37°C for 24 hr (500 µg) were subjected to SEC on a Superdex 200 column. Molecular masses of gel-filtration standards deduced from their elution profile are also shown. Insert: Column fractions from the AspN/furin digest were collected and analyzed by


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35 SDS-PAGE under non-reducing conditions. Lane 1, load; lanes 2-8 and 9-11, column fractions 12-18 and 20-22, respectively. Fractions 13-15 that correspond to lanes 3-5 were pooled and subjected to analysis by mass spectrometry and activity measurements. B. Octet assessment of the dissociation rate of the mature GDF11/PDP60-114 complex. The indicated samples, GDF11 fragments that were biotinylated through the single Nlinked glycan in the prodomain peptide and the control sample (the GDF11 peptide derived from AspN/furin treatment without biotinylation), were captured on a Streptavidin sensor for 5 min, washed for 1 min, and dissociation over time was monitored for 30 min. Insert: Binding of the biotinylated GDF11 AspN/furin-treated GDF11 at 12.5 µg/mL (yellow), 25 µg/mL (green), 50 µg/mL (cyan), 100 µg/mL (red), and 200 µg/mL (blue). C. Octet analysis of the binding characteristics of the mature GDF11/PDP60-114 complex and pro-GDF11 for activin RIB-Fc and activin RIIA-Fc. Biotinylated GDF11 samples (100 µg/mL) were captured on Streptavidin sensor tips for 5 min, washed for 1 min, then incubated for 15 min with the receptors (20 µg/mL). Figure 4. Bioactivity of GDF11 samples on PC12 cells. GDF11 samples were tested for function in a KIRA assay monitoring SMAD 2/3 phosphorylation (A and B) and in a SMAD reporter luciferase assay with luciferase expression under the control of the SMAD transcriptional response element (C and D). A. Time course of GDF11-induced phosphorylation of SMAD 2/3. PC12 cells were incubated with serial dilutions of mature GDF11 (ng/mL) for 15, 30, 60, or 120 min. Resulting phospho-SMAD 2/3 levels were quantified using a KIRA ELISA kit with horseradish peroxidase-conjugated secondary antibody and colorimetric TMB substrate monitoring absorbance at 450 nm for detection. B. SMAD 2/3 phosphorylation activity of GDF11 test samples shown in Figure 2A following the 60 min treatment and the detection method described in A. C. Signaling activity of the denoted GDF11 samples (also shown in Figure 2A) in a luciferase reporter assay. PC12-derived reporter cells were incubated with serial dilutions of test samples for 6 hr, and luciferase expression was quantified using a luminescent luciferase substrate measuring relative light units (RLU). D. Signaling activity in the luciferase reporter assay as described in C of serial dilutions of mature GDF11 with or without treatment with a fixed concentration (10 µg/mL) of GDF11 prodomain-Fc (wild-type GDF11 residues 25-298 or a modified version of the same construct carrying a D122A point


Biochemistry

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36 mutation) and serial dilutions of mature TGF-β1 with and without treatment with 10 µg/mL TGF-β1 LAP-Fc at a fixed concentration. Figure 5. Characterization of GDF11 samples by mass spectrometry. A. Reverse phase chromatography tracing of the reduced and deglycosylated SEC-purified AspN/furindigested GDF11 sample shown in Figure 3A. Deconvoluted mass spectra of the 25.0min peak (B), 21.3-min peak (C), and 23.0-min peak (D), from panel A. E. Deconvoluted mass spectrum of the reduced, deglycosylated, and AspN protease-treated GDF11 sample shown in Figure 2A, lane 2. Figure 6. Characterization of mature GDF11 domain fused to different prodomain fragments by SDS-PAGE. A. Coomassie blue-stained SDS-PAGE gel analysis of conditioned media, for the fusion proteins indicated (see Table 1 for construct designs). Arrow denotes the position of the prodomain fragment−mature GDF11 fusion proteins. Apparent molecular masses of molecular weight markers are indicated at the right. Samples were analyzed under non-reducing conditions. B. Western blot of the fusion proteins visualized using a polyclonal antibody against a C-terminal peptide for detection. The apparent molecular masses of the molecular weight markers are indicated at the right. Samples were analyzed under reducing conditions. Figure 7. Biochemical and functional analysis of the ACE490 and ACE498 fusion proteins. A. Schematic drawing summarizing the redox steps used to induce dimerization of prodomain fragment−mature GDF11 domain fusion proteins. B. Coomassie bluestained SDS-PAGE gel analysis of purified ACE490 protein before and after the redox reaction under reducing and non-reducing conditions. The apparent molecular masses of the molecular weight markers are indicated at the right. Lane 1, ACE490 alone; lane 2, ACE490 plus DTT and Aldrithiol (AT); lane 3, ACE490 plus DTT and AT in 1 M guanidine HCl; lane S, molecular weight markers. C. Signaling activity of the denoted ACE490 samples in a luciferase reporter assay following 6 hr treatment. ACE490 samples were analyzed without treatment, and following reduction with DTT and oxidization using an AT redox reaction in the absence and presence of 1 M guanidine HCl. D. Coomassie blue-stained SDS-PAGE gel analysis of purified ACE498 protein before and after the redox reaction and after treatment with AspN under reducing and non-reducing conditions. The apparent molecular masses of the molecular weight


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Biochemistry

37 markers are indicated at the right. Lane 1, ACE498 alone; lane 2, ACE498 plus DTT and AT for 20 hr at room temperature; lane 3, ACE498 plus DTT/AT and then treated with AspN protease; lane S, molecular weight markers. E. Signaling activity of the denoted ACE498 samples in a luciferase reporter assay following 6 hr treatment. ACE498 samples were analyzed without further treatment, and following reduction with DTT and oxidization using an AT redox reaction directly or after treatment with AspN protease. Figure 8. Schematic for proteolytic activation of pro-GDF11. Schematic illustration depicting how cleavage of the latent GDF11 precursor protein by AspN and furin results in the release of active and soluble mature GDF11/PDP60-114 complex and precipitation of the GDF11 prodomain peptide 122-298. The illustration of the latent GDF11 procomplex was generated by producing a computational model for the mature GDF11/PDP60-114 complex and then superimposing the mature domains of the GDF11/PDP60-114 model onto the structure of pro-BMP9. Details for the modeling procedures are provided in the supplemental information.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 46

38 Graphic for Table of Contents For table of content use only.

A prodomain fragment from the proteolytic activation of growth differentiation factor 11 (GDF11) remains associated with the mature growth factor and keeps it soluble Blake Pepinsky, Bang-Jian Gong, Yan Gao, Andreas Lehmann, Janine Ferrant, Joseph Amatucci, Yaping Sun, Martin Bush, Thomas Walz, Nels Pederson, Thomas Cameron, and Dingyi Wen

precipitate + AspN + Furin

pro-GDF11

soluble complex


Page 39 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

39

Human GDF11 precursor protein (42.5 kDa) BMP1-type protease site A25

Signal peptide

N-glycan D122 N94

D60

Furin-like convertase site Inter-chain N299 disulfide S407 C372 K294

Mature domain

L114

Prodomain cleavage fragments

Prodomain (~29 kDa)

KRSRR298

Mature (12.5 kDa) Active

Figure 1


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 46

40 kDa

A

kDa 250 148

250 150 98 64

kDa

98 64

50 36

50 36

22 16

22 16 6

6 1 2 3 4 5 6 S 1 2 3 4 5 6 7 Non-reducing

Reducing

Figure 2


60 50 40 30 20

B

Page 41 of 46

A

1.4

17

44

150

70

670

(kDa)

41

(kDa) 250 148 98 64 50 36

60 GDF11 Full-length GDF11 AspN GDF11 AspN/furin

40 30 20 10

22 16 6

injection

mAU

50

1 2

0 1

5

10

B

C 5.0

4.5

4.5

4.0

Biotin pro-GDF11

No addition +RIIA-Fc +RIB-Fc

4.0

3.5

3.5

nm

3.0 2.5 2.0

3.0 2.5

+RIIA-Fc

2.0

1.5 1.0 0.5 0.0

3 4 5 6 7 8 9 10 11

25

20

15 Fraction

Biotin GDF11 AspN

5.0

nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Biotin GDF11 AspN/furin

1.5

Biotin GDF11 PDP 60-114

1.0

Unlabeled GDF11 AspN/furin 0

400

800

1200

1600

2000

+RIB-Fc No addition

Biotin GDF11 AspN/furin

0.5 0.0

0

200

400

600

800

1000 1200

Time (sec)

Time (sec)

Figure 3


Biochemistry

42

A

8000

Mature GDF11 AspN+furin 6h AspN+furin 7h+4C AspN+furin 23h

Concentration (ng/ml)

20000

C

15000

RLU

6000

B

Mature GDF11 Full length AspN AspN + furin 6h AspN + furin 23h

OD at 450 nm

OD at 450 nm

15 min 30 min 60 min 120 min


RLU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 46

4000 2000

GDF11+ProD122A GDF11+ProWT GDF11 TGF-β1+LAP TGF-β1

D

10000 5000

0 0.0001

0.01

1

100


10000

0 0.0001

0.01

1

100


Figure 4


10000

Page 43 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

43

AU

A

Oxidized Res. 299-407

Res. 299-407

B

Res. 299-407 Res. 60-112

Res. 60-114

Oxidized Res. 299-407

Res. 60-112

C

Res. 60-114

Res. 122-407 Res. 128-407 Res. 133-407 +O

+O

+O +TFA

Figure 5


+TFA

D

E

Biochemistry

487 490 491 493 494 495 497 498 499

44

kDa

A

250250$kD$ 150148$kD$ 98 98$kD$ 64 64$kD$ 50 50$kD$ 36 36$kD$ 22 22$kD$ 16 16$kD$ 6

1$$$$$2$$$$3$$$$4$$$$$5$$$$6$$$$7$$$$8$$$$9$$$10$$$ 490 491 492 493 494 495 496 497 498 499

487

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 46

kDa 64

B

50 36 22

Figure 6


Page 45 of 46

45

A

-Cys

+"

+"

1 2 3 S 1 2 3 S

kDa

+"

B

250 148 98 64

5000

RLU

4000

50 36 22 16 6 NR

Mature GDF11 ACE490 ACE490 DTT/AT ACE490DTT/AT+Gd

2000

0 0.0001

0.01

1

100

10000


R

1 2 3 S 1 2 3 S

kDa 250 148 98 64 50 36 22 16 6

NR

3000

C

1000

R

D

10000 8000

RLU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

6000

E

Mature GDF11 ACE498 ACE498 DTT/AT ACE498 DTT/AT+AspN

4000 2000 0 0.0001

0.01

1

100


Figure 7


10000

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 46

46

Precipitate arm domain

arm domains α2helix

lasso α1helix

+AspN +Furin

mature domain lasso α1helix

Intact pro-GDF11

Soluble complex

Figure 8


A Prodomain Fragment from the Proteolytic Activation of Growth

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