Synthetic Human NOTCH1 EGF Modules Unraveled Molecular

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Synthetic Human NOTCH1 EGF Modules Unraveled Molecular Mechanisms for the Structural and Functional Roles of Calcium Ions and O‑Glycans in the Ligand-Binding Region Shun Hayakawa,† Ryosuke Koide,† Hiroshi Hinou,†,‡ and Shin-Ichiro Nishimura*,†,‡ †

Graduate School of Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo 001-0021, Japan Medicinal Chemistry Pharmaceuticals, Company Ltd., N21, W12, Kita-ku, Sapporo 001-0021, Japan

‡

S Supporting Information *

ABSTRACT: The Notch signaling pathway is an evolutionarily highly conserved mechanism that operates across multicellular organisms and is critical for cell-fate decisions during development and homeostasis in most tissues. Notch signaling is modified by posttranslational glycosylations of the Notch extracellular EGF-like domain. To evaluate the structural and functional roles of various glycoforms at multiple EGF domains in the human Notch transmembrane receptor, we established a universal method for the construction of NOTCH1 EGF modules displaying the desired O-glycans at the designated glycosylation sites. The versatility of this strategy was demonstrated by the rapid and highly efficient synthesis of NOTCH1 EGF12 concurrently having a β-D-glucopyranose-initiated glycan (Xylα1→3Xylα1→3Glcβ1→) at Ser458 and α-L-fucopyranose-initiated glycan (Neu5Acα2→3Galβ1→4GlcNAcβ1→3Fucα1→) at Thr466. The efficiency of the proper folding of the glycosylated EGF12 was markedly enhanced in the presence of 5 mM CaCl2. A nuclear magnetic resonance study revealed the existence of strong nuclear Overhauser effects between key sugar moieties and neighboring amino acid residues, indicating that both O-glycans contribute independently to the intramolecular stabilization of the antiparallel β-sheet structure in the ligand-binding region of EGF12. A preliminary test using synthetic human NOTCH1 EGF modules showed significant inhibitory effects on the proliferation and adhesiveness of human breast cancer cell line MCF-7 and lung adenocarcinoma epithelial cell line A549, demonstrating for the first time evidence that exogenously applied synthetic EGF modules have the ability to interact with intrinsic Notch ligands on the surface of cancer cells.

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he Notch signaling pathway, which is similar to the Wnt and Hedgehog pathways, is a universally conserved metazoan cell-fate-determination pathway that strongly influences multiple aspects of cancer biology, including cancer stem cells, angiogenesis, cancer immunity, and embryonic differentiation.1 The human Notch signaling pathway is an essential cell−cell communication system, in which five membraneassociated Notch ligands [Jagged1, Jagged2, Delta-like ligand1 (DLL-1), DLL-3, and DLL-4] on the surface of a cell (the signal-sending cell) bind with the Notch (NOTCH1−4) extracellular domain (NECD) on another cell (the signalreceiving cell). This ligand−receptor interaction induces proteolytic cleavage by ADAM10/17 metalloproteases and γsecretase to liberate the Notch intracellular domain (NICD), which travels to the nucleus and regulates the transcription of the Notch downstream target gene.2 A growing body of evidence linking perturbations in the Notch signaling pathway to various human diseases justifies the pursuit of this pathway as a potential therapeutic target.3 A previous study reported that blockade of the DLL4-mediated Notch pathway by antiDLL4 antibodies inhibited tumor growth through the control of angiogenesis.4 Antibodies directed to the negative regulatory region (NRR) of Notch receptors are also potent paraloguespecific inhibitors of signaling from individual Notch receptors.5 © XXXX American Chemical Society

In contrast, as shown in cases of T cell acute lymphoblastic leukemia6 and myeloid leukemia,7 it seems likely that Notch signaling has potential as a tumor suppressor in many cancers.8 NOTCH1 and NOTCH2 have been shown to exert opposite effects on embryonic brain tumor growth.9 Therefore, the intricacies of the Notch signaling mechanism due to the presence of several paralogues and multiple regulatory components have made understanding of the biology and pathology of the Notch signaling pathway challenging.10 Modification with O-glycans at Thr/Ser residues in multiple tandem epidermal growth factor (EGF)-like repeats in the NECD may change their binding specificities and affinities for Notch ligands and contribute to regulating the Notch signaling pathway.11 Extensive efforts over the past decade have revealed that the O-fucosylation (O-Fuc)12 and O-glucosylation (OGlc)13 of EGF repeats in NECD are essential for the functions and activities of the Notch receptors. Recent studies revealed that O-glycosylation at the Ser/Thr residues of mammalian Notch1 EGF repeats were initiated by 2-acetamido-2-deoxy-βReceived: November 30, 2015 Revised: January 9, 2016

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Figure 1. Universal synthetic strategy toward various EGF modules in the NECD modified by O-Glc- and O-Fuc-type glycans at Ser/Thr residues in each consensus sequence. Human NOTCH1 EGF12 module 1 was designed as a key intermediate and prepared on the basis of the microwaveassisted solid-phase glycopeptide synthetic protocol using the two sugar amino acid derivatives 4 and 5 as versatile starting materials, and further enzymatic sugar elongation reactions were performed to give compounds 2 and 3.

24 μmol) was swollen with dichloromethane (DCM) at ambient temperature for 1 h. The Fmoc group was removed with 20% piperidine in DMF (1 mL) for 3 min under microwave irradiation. Nα-Fmoc-amino acid (4.0 equiv) was coupled with 1-[bis(dimethylamino)methyliumyl]-1H-benzotriazole-3-oxide hexafluorophosphate (HBTU) (4.0 equiv), 1hydroxybenzotriazole monohydrate (HOBt) (4.0 equiv), and N,N-diisopropylethylamine (DIEA) (8.0 equiv) in DMF (240 μL) for 10 min under microwave irradiation. In the case of glycoamino acids, Fmoc-Ser[Ac3Xyl(α1→3)Ac2Xyl(α1→3)Ac3Glcβ1→]-OH (4) at Ser458 (1.2 equiv each) and FmocThr[Ac3GlcNAc(β1→3)Fucα1→]-OH (5) at Thr466 (1.2 equiv each) were coupled with 1H-benzotriazol-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) (1.2 equiv), 1-hydroxybenzotriazole (HOAt) (1.2 equiv), and DIEA (3.0 equiv) in a DMF/DCM mixture (70 μL each) for 10 min under microwave irradiation (as for the synthesis of compound 4, see Figure 2 and the Supporting Information). After agitation for 10 min under microwave irradiation, the extra PyBOP and HOAt (1.2 equiv each) were treated for an additional 10 min in the same microwave-assisted fashion.19,20 At the end of solidphase synthesis, an N-terminal amino group of peptidyl-resin was capped with acetic anhydride with DIEA in DMF for 1 min at ambient temperature. For cleavage of the peptide from the resin and removal of acid-labile protective groups, 1.5 mL of Reagent H (81:5:5:2.5:3:2:1.5 TFA:phenol:thioanisole:1,2ethanedithiol:H2O:dimethyl sulfide:ammonium iodide)21 was subjected to treatment with resin at ambient temperature for 2 h. The resin was filtered off, and the peptide was precipitated

(O-GlcNAc)14 and 2-acetamido-2-deoxy-α-Dgalactopyranose (O-GalNAc),15 respectively. The NECD of mammalian Notch1 consists of 36 tandem EGF repeats, and although the full portrait of the glycosylation status at multiple O-glycosylation sites remains elusive, O-fucosylation at EGF12, -26, and -27 within mouse Notch1 has been shown to play important roles in Notch functions.16 To elucidate the molecular mechanisms responsible for the structural and functional roles of multiple O-glycosylation sites at individual EGF repeats, it is important to note that an efficient and versatile method for the construction of EGF modules having the desired O-glycans at the designated Thr/Ser residues is definitely needed. In our previous study,17 we were the first to chemically synthesize and structurally characterize mouse Notch1 EGF12 bearing an α-L-fucopyranose-initiated O-glycan. The emerging importance of multiple O-glycosylations at putative glycosylation sites involved in Notch1 EGF12 that modulates the interaction with DLL416,18 motivated us to challenge the synthesis of human NOTCH1 EGF12 having two crucial O-glycans, Xylα1→3Xylα1→3Glcβ1→ at Ser458 and Neu5Acα2→3Galβ1→4GlcNAcβ1→3Fucα1→ at Thr466. D-glucopyranose

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EXPERIMENTAL PROCEDURES Solid-Phase Synthesis of Linear Human NOTCH1 EGF12 Intermediates. Solid-phase syntheses of human NOTCH1 EGF12 modules, naked and glycosylated EGF12 (1), were performed by a standardized protocol indicated in Figure 1.17,19,20 Rink amide ChemMatrix resin (0.48 mmol/g, B

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Figure 2. Reagents and conditions: (a) NIS (1.3 equiv), TfOH (0.1 equiv), THF/DCM (2:1), −20 °C, 92%; (b) 80% AcOH aq, 40 °C, 41%; (c) NaIO4 (2.0 equiv), MeOH, 0 °C; (d) NaBH4 (4.0 equiv), MeOH, 0 °C; (e) 60% TFA aq, room temperature (rt); (f) Ac2O, pyridine, rt, 94%; (g) H2NNH2·H2O (1.3 equiv), AcOH (1.3 equiv), 0 °C; (h) CCl3CN (5.0 equiv), DBU (0.1 equiv), rt, 74%; (i) HSC12H25 (3.0 equiv), TMSOTf (0.1 equiv), DCM, −20 °C, 64%; (j) NaOMe (0.1 equiv), MeOH, rt; (k) BnBr (5.0 equiv), NaH (5.0 equiv), rt, 96%; (l) 7 (1.2 equiv), NIS (1.5 equiv), TfOH (0.1 equiv), THF/DCM (2:1), −20 °C, 81%; (m) 80% AcOH aq, 40 °C, 40%; (n) 60% TFA aq, r.t.; (o) Ac2O, pyridine, r.t., 63%; (p) 20% Pd(OH)2/C (50 wt %), H2, THF/MeOH (1:3), r.t.; (q) Ac2O, pyridine, DMAP (0.1 equiv), 98%; (r) H2NNH2H2O (1.3 equiv), AcOH (1.3 equiv), 0 °C; (s) CCl3CN (5.0 equiv), DBU (0.1 equiv), r.t., 84%; (t) Fmoc-Ser-OtBu (1.2 equiv), TMSOTf (0.1 equiv), DCM, −20 °C, 80%; (u) 90% TFA aq, r.t., 97%.

using a mixture of tert-butylmethyl ether (20 mL) and hexane (20 mL) in an ice bath without removal of the TFA solution. After centrifugation (3000 rpm at 4 °C for 10 min), the supernatant was carefully removed by decantation and the process repeated three times. The precipitate was dissolved in 50% aqueous acetonitrile (5 mL) and then lyophilized. The dried precipitate was dissolved in methanol containing 6.5 mM dithiothreitol (DTT), and the solution was adjusted and kept to pH 12.5 with a 1 N NaOH aqueous solution. After being stirred at ambient temperature for 24 h under a N2 gas atmosphere, the solution was neutralized with 30% acetic acid in methanol. The precipitates formed by neutralization were directly subjected to oxidative folding. Folding of Human NOTCH1 EGF12 Modules in the Presence of Calcium Ions. The crude precipitates containing

linear glycopeptide intermediates were dissolved in redox buffer (0.1 mg/mL) containing 50 mM Tris-HCl (pH 8.0), 5 mM CaCl2, 1 mM reduced glutathione, and 0.2 mM oxidized glutathione.10b,22 The reaction mixtures were routinely stirred for 24 h. Reaction progress was monitored by analytical reversephase high-performance liquid chromatography (RP-HPLC). After the reaction had proceeded, the reaction mixture was acidified by addition of TFA (1% volume of the reaction mixture), then lyophilized, and subjected to RP-HPLC for purification. RP-HPLC conditions were as follows: ambient temperature and flow rate of 1 mL/min, UV detection at 220 nm, eluent A consisting of 0.1% TFA in water, eluent B consisting of 0.1% TFA in acetonitrile, and a liner gradient from 25 to 35% B over 30 min. The purity of the products was tested by using analytical RP-HPLC and ESI-HRMS. Glycosylated C

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Figure 3. Oxidative folding of the human NOTCH1 EGF12 module having O-glycans at the Ser458 and Thr466 residues, and subsequent enzymatic modification of the O-glycan at the Thr466 residue. (a) C18 RP-HPLC profiles of the reaction mixtures after a 24 h incubation to demonstrate the effects of the coordination of Ca2+ in the efficiency of proper folding of the glycosylated linear EGF12 compared to that conducted in the absence of calcium ions. (b) NOESY spectrum of glycosylated EGF12 (1) obtained after the folding reaction in the presence of calcium ions. NOEs between two β-protons of cross-linked cysteine residues were observed in Cys461−Cys476 and Cys478−Cys487. (c) MALDI-TOFMS and RP-HPLC profiles of EGF12 modules 2 and 3 derived by enzymatic modifications to glycopeptide 1 using recombinant human β1,4GalT and recombinant P. phosphoreum α2,3SiaT in the presence of UDP-Gal and CMP-Neu5Ac.

1421.8713, found m/z 1421.8721. MALDI-TOFMS: [M + H]+ m/z 4200.677 (Figure 3 and Figure S1). Enzymatic Sugar Elongation of Synthetic Human NOTCH1 EGF12 (1).17 Compound 1 (1.0 mg, 0.2 μmol) was dissolved in a total volume of 100 μL of 25 mM HEPES buffer (pH 7.0), 10 mM MnCl2, and 0.1% Triton X-100. To the

EGF12 (1) was obtained in 4.1% overall yield from the solidphase synthesis (4.9 mg, 0.98 μmol, tR = 14.88 min). ESIHRMS for 1: C202H306N48Na3O84S7 [M + 3Na]3+ calcd m/z 1680.2962, found m/z 1680.2916. ESI-HRMS for naked EGF12: C172H257N47Na3O62S7 [M + 3Na]3+ calcd m/z D

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and very weak (1.6−6.0 Å). In the first stage of structure determination, the structures of glycopeptides were calculated using only interproton distance information. After the validation of fulfilling distance restraints for the obtained structures, the restraints of dihedral angle ϕ were adopted for further structural calculation. When the coupling constant 3JHNα was more than 8.0 Hz, dihedral angle ϕ was constricted to −120 ± 30°. The conformation of the sugar ring was fixed to the chair conformation. All analyses of root-mean-square deviation values and the solution structures of glycopeptides were performed with PROCHECK32 and MOLMOL.33 Cell Culture. Human breast cancer cells MCF-7 were obtained from American Type Culture Collection (Manassas, VA). A549 human lung adenocarcinoma epithelial cells were obtained from Health Science Research Resource Bank (Osaka, Japan). These cells were maintained in D-MEM High-glucose (Gibco, Carlsbad, CA) supplemented in 10% fetal bovine serum (FBS, Gibco). The cells were cultured in a humidified incubator at 37 °C and 5% CO2. Cell Viability Assay.34 For the measurement of cell viability, A549 and MCF-7 cells were basically plated at a density of 5 × 103 cells per 100 μL per well in a 96-well plate, and they were preincubated for 24 h. Then cells were treated with synthetic naked EGF12, glycosylated EGF12 (1), or cisdiammineplatinum(II) dichloride (cisplatin, Sigma) dissolved in sterile water. The final concentration of each molecule in culture medium was adjusted to 100 μM. A549 and MCF-7 cells were incubated for 48 and 96 h, respectively. After the incubation period, cells were washed with medium, and then 10 μL of a Cell Counting Kit-8 solution (Dojindo, Kumamoto, Japan) was added and incubated for 2 h at 37 °C and 5% CO2. The absorbance at 450 nm was measured with a SUNRIZE Thermo microplate reader (Tecan Japan Co., Ltd., Kawasaki, Japan). All values are expressed as means ± the standard error of the mean (SEM), and the assays were repeated three times. The bar graphs in each picture were generated with Prism 4.0 (GraphPad Software, Inc., San Diego, CA). Fluorescence Microscopy Showing Cell Morphology.35 The fluorescence microscope used for image acquisition and image analysis was an all-in-one fluorescence microscope BIOREV BZ-9000 series generation II (Keyence, Osaka, Japan) equipped with a 4× lens. Images were taken using a standard filter set for DAPI. Merge images were generated and analyzed using BZ-II software. After incubation with compounds, the MCF-7 and A549 cells were washed three times with OptiMEM (Gibco). The nuclei were stained with a 100 nM Hoechst 33342 (Invitrogen) solution and incubated at 37 °C and 5% CO2 for 15 min. The cells were washed three times with Opti-MEM and observed under a fluorescent microscope.

solution were added recombinant β1,4-GalT (4 microunits) and UDP-Gal (283 μg, 0.5 μmol), and the mixture was incubated at ambient temperature for 24 h. Analytical RPHPLC gave purified EGF12 module 2 in quantitative yield (tR = 11.1 min). ESI-HRMS of compound 2: C208H316N48Na3O89S7 [M + 3Na]3+ calcd m/z 1734.3183, found m/z 1734.3169. The human NOTCH1 EGF12 module (2) (0.1 mg, 0.02 μmol) was dissolved in a total of 20 μL of 50 mM Tris-HCl (pH 6.5), 0.5 M NaCl, and 0.1% Triton X-100. To the solution were added recombinant Photobacterium phosphoreum α2,3SiaT (0.2 microunit)23 and CMP-Neu5Ac (127 μg, 0.2 μmol), and the mixture was incubated at ambient temperature for 24 h. After incubation for 24 h, to a reaction mixture was added extra CMP-Neu5Ac (127 μg, 0.2 μmol), and the reaction mixture was incubated for 5 h. Analytical RP-HPLC gave pure fully glycosylated EGF12 (3) in a quantitative yield (0.1 mg, 0.02 μmol, tR = 9.90 min). The RP-HPLC conditions were as follows: ambient temperature and flow rate of 1 mL/min, UV detection at 220 nm, eluent A consisting of 25 mM ammonium acetate buffer (pH 5.0), eluent B consisting of 10% eluent A in acetonitrile, and a linear gradient from 20 to 35% B over 25 min. ESI-HRMS of compound 3: C219H330N49O97S7 [M − 3H]3+ calcd m/z 1807.3481, found m/z 1807.3491. MALDITOFMS spectra of compounds 3 and 4 are shown in Figure 3c. Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR experiments with synthetic human NOTCH1 EGF12 (1) were performed on a Bruker AVANCE 800 MHz spectrometer to determine proton frequency. Compound 1 or the naked EGF12 module was dissolved in 99.9% D2O (300 μL) or in a mixture solution of 90% H2O and 10% D2O (300 μL) containing 25 mM CaCl2 at a concentration of 3.0 mM. These solutions were adjusted at pH 6.0 by 0.1 N aqueous NaOH. These solutions were packed in Shigemi thin-walled micro NMR tubes prior to NMR spectroscopy. All spectra were measured at 298 K. Data acquisition was performed with the Bruker TopSpin 3.1 software package. Two-dimensional24 homonuclear DQF-COSY,25 TOCSY26 with MLEV-17 sequence,27 and NOESY spectra28 were recorded in the indirect dimension using States-TPPI phase cycling. Two-dimensional heteronuclear 13C-edited HSQC and HSQC-TOCSY measurements were taken in echo−antiecho mode. TOCSY experiments were applied for a spin-lock time of 80 ms, and NOESY experiments were conducted with mixing times of 100, 200, and 400 ms. The suspension of the water signal was performed by presaturation during a 2 s relaxation delay and by a 3-9-19 WATERGATE pulse sequence with a field gradient.29 TOCSY and NOESY spectra were acquired with 2048 × 512 frequency data points and were zero-filled to yield 2048 × 2048 data matrices. DQF-COSY spectra with 16384 × 512 frequency data points were also recorded and zero-filled to yield a 16384 × 16384 matrix to measure the coupling constant. Sweep widths of 799.714 Hz were applied. Time domain data in both dimensions were multiplied by a sine bell window function with a 90° phase shift prior to Fourier transformation. All NMR data were analyzed using a Sparky program.30 Structure Calculation. Three-dimensional structures of synthetic human NOTCH1 EGF12 (1) were calculated using the CNS 1.1 program31 with standard protocols for distance geometry-simulated annealing and refinement. Distance restraints for calculations were estimated from the cross-peak intensities in NOESY spectra with a mixing time of 400 ms. The estimated restraints were classified into four categories: strong (1.6−2.6 Å), medium (1.6−3.5 Å), weak (1.6−5.0 Å),

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RESULTS AND DISCUSSION Synthesis of Human NOTCH1 EGF Modules. Figure 1 shows the synthetic route of the key EGF12 glycopeptide 1 and its derivatives 2 and 3, which concurrently have O-Glc- and OFuc-type O-glycans involved in consensus sequences C1-X-S-X(P/A)-C2 and C2-X-X-X-X-(S/T)-C3. EGF modules commonly have three disulfide bonds (C1−C3, C2−C4, and C5−C6), and two different O-glycans EGF12 are modified at Ser458 and Thr466 residues in the rigid and extremely small space within the two disulfide bonds. Therefore, we considered that the bottleneck in the total synthetic pathway may be the proper folding of a linear glycopeptide intermediate released from a solid support and the subsequent enzymatic sugar extensions of E

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the novel Fmoc-Ser derivative 4 carrying an unusual peracetylated trisaccharide moiety (Xylα1→3Xylα1→ 3Glcβ1→) in addition to the Fmoc-Thr derivative 517 bearing a partially O-acetylated disaccharide moiety (GlcNAcβ1→ 3Fucα1→). The availability of such sugar amino acids, for example, Fmoc-Thr/Ser building blocks having core glycoforms involved ubiquitously in mucin glycoproteins such as GalNAcα1→ (Tn antigen), Galβ1→3GalNAcα1→ (T antigen), Galβ1→3(GlcNAcβ1→6)GalNAcα1→ (core 2), Galβ1→3(Galβ1→3GlcNAcβ1→6)GalNAcα1→, and Galβ1→ 3(GlcNAcβ1→3Galβ1→4GlcNAcβ1→6)GalNAcα1→, greatly facilitates the construction of a robust compound library of highly complex glycopeptides related to cancer-relevant neoepitopes.20 Figure 2 shows the synthetic procedure of compound 4, a new sugar amino acid derivative required for the construction of EGF modules having O-Glc-type glycans. The disaccharide intermediate 10 containing the Xylα1→3Xyl unit was derived by the regioselective manipulation of compound 9, obtained by coupling of the thiophenyl glycoside 636 and 1,2:5,6-Odiisopropylidene glucose 7 in high yield (94%, four-step overall yield). Because of the acid-labile nature of the α-glycoside linkage between the two xylose residues, the conversion of compound 10 into the penta-O-benzyl derivative 13 as a reliable donor to achieve the α-dominant glycosylation reaction was performed via the stable thioalkyl glycoside 12.37 Trisaccharide derivative 14 as an α/β anomeric mixture obtained by coupling thioalkyl donor 13 with acceptor 7 was subjected to the isolation of an α-isomer as diol 15 and converted into per-O-acetate 17. The glycosylation of trisaccharide imidate 18 with Fmoc-Ser-OtBu proceeded stereoselectively to afford β-glycoside 19, and the removal of tert-butyl protection gave compound 4 in high yield. Calcium Ions Promote the Proper Folding of Glycosylated EGF12 (1). The solid-phase chemical synthesis of the 38-mer human NOTCH1 EGF12 glycopeptide was performed by the standardized protocol depicted in Figure 1. The coupling and removal of Fmoc protection were conducted under microwave irradiation for 10 min on Rink amideChemMatrix resin using the HBTU/HOBt activation system.17,19,20 To enhance the coupling efficiency of FmocThr/Ser derivatives having a bulky oligosaccharide side chain, the incorporation of sugar amino acids 4 and 5 was performed by the double activation method using PyBOP/HOAt coupling reagents.19b,20i These procedures were repeated 38 times to allow the coupling of 38 amino acids onto the resin followed by acetylation of the N-terminus; the resulting glycopeptide was released from resin under a nitrogen atmosphere using a cleavage cocktail [Reagent H (81:5:5:2.5:3:2:1.5 TFA:phenol:thioanisole:1,2-ethanedithiol:H2O:dimethyl sulfide:ammonium iodide)]21 to prevent the formation of methionine sulfoxide. Acetyl groups on the carbohydrate moiety were removed by a treatment with 1 N NaOH in methanol (pH 12.5) containing 6.5 mM dithiothreitol to avoid undesired disulfide bond formation. Crude products were directly subjected to the oxidative folding reaction without further purification. A synthetic linear glycopeptide intermediate was dissolved (0.1 mg/mL) in redox buffer containing 50 mM TrisHCl (pH 8.0), 1.0 mM reduced glutathione, and 0.2 mM oxidized glutathione.10b,22 To assess the effects of calcium ions on the formation of proper folding of human NOTCH1 EGF12 having two different O-glycans, the reaction was performed in the presence of 5 mM CaCl2 at room temperature for 24 h. It is

Figure 4. Three-dimensional structures of synthetic human NOTCH1 EGF12 module 1. These figures were generated with MOLMOL and PyMOL. (a) Schematic representation of the most energy-minimized structures. The backbone structure of the 20 lowest-energy structures is shown at the left. These structures are superimposed on the Cys456−Asp469 and Glu473−Pro480 region, and mean structures are represented by a bold line. The structures are oriented with the Nterminus at the top. The peptide backbones are colored blue; Ser458 and Thr466 (O-glycosylation sites) are colored orange and cyan, respectively. The ribbon structures of most energy-favored structures are shown at the right. The heavy atoms of carbohydrate moieties and underlying Ser458 and Thr466 are shown as sticks. Oxygen, nitrogen, and carbon atoms are colored red, blue, and black, respectively. The βsheet strands are represented by arrows, and the sugar moieties, including sugar-linked threonine and serine, are shown as sticks. Panels b and c are schematic representations focusing on the disaccharide GlcNAcβ1→3Fucα1→ at Thr466 and the tripeptide 477Ile-Cys-Met479 region and trisaccharide Xylα1→3Xylα1→3Glcβ1→ at Ser458 and Glu473, respectively.

folded glycopeptide 1. Recombinant glycosyltransferases such as β1,4GalT, α1,3/6FucT, and α2,3/6SiaT and sugar nucleotides allowed for marked modifications to small core glycan structures to afford larger and more complex glycoforms. A previous study demonstrated that it was possible to quantitatively modify mouse Notch1 EGF12 carrying a disaccharide moiety (GlcNAcβ1→3Fucα1→) by a treatment with recombinant β1,4GalT and α2,3SiaT in the presence of UDP-Gal and CMP-Neu5Ac to afford an EGF12 domain with a mature tetrasaccharide (Neu5Acα2→3Galβ1→4GlcNAcβ1→ 3Fucα1→).17 However, in this study, we needed to synthesize F

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Figure 5. Effects of Ca2+ binding and O-glycosylation on the conformation of the human NOTCH1 EGF12 domain. (a) Comparison of NMR structures of human and mouse Notch1 EGF12 modules showing the synergistic effects of calcium ions and multiple O-glycosylation sites on the intramolecular stabilization of the antiparallel β-sheet. Mouse Notch1 EGF12 having GlcNAcβ1→3Fucα1→ at Thr466 folded, in the absence of calcium ions (yellow) (PDB entry 2RQZ),17 and human NOTCH1 EGF12, having GlcNAcβ1→3Fucα1→ at Thr466 and Xylα1→3Xylα1→ 3Glcβ1→ at Ser458 (blue), were superimposed by focusing on the antiparallel β-sheet region. These structures are represented by the backbone structure using stick lines. (b) The superimposed structure of the human NOTCH1 EGF12 domain was resolved by NMR in this study and X-ray crystallography. The blue stick is for the backbone of human EGF12 (1) having the Xylα1→3Xylα1→3Glcβ1→ and GlcNAc-Fucα1→ moieties resolved in this study, and the cyan stick is for the EGF12 region of human NOTCH1 EGF11−13 carrying GlcNAcβ1→3Fucα1→ at Thr466 and calcium ion (red) on the EGF12 domain generated by X-ray crystallography11c (PDB entry 4D0E). (c) Comparison of NMR structures of human Notch1 naked (green) and glycosylated (blue) EGF12 modules showing the effects of multiple O-glycosylation sites on the intramolecular stabilization of the antiparallel β-sheet.

important to note that the coordination of Ca2+ may modulate the ligand binding of Notch1 by stabilizing the major β-hairpin of EGF12.10b,11c,38 However, whether Ca2+ binding to this site directly contributes to the proper folding of the EGF12 module in the presence of glycosylated Thr466 and Ser458 residues has not been elucidated. The folding processes of the glycosylated EGF12 intermediate in the presence and absence of calcium ions were monitored with a RP-HPLC analysis of aliquots of the sample solution. As shown in Figure 3a, the effects of Ca2+ binding on the conformational stabilization during the folding reaction were evident because marked differences were observed in elution profiles between the presence and absence of calcium ions. Calcium ions facilitated the proper folding of the linear unfolded glycopeptide and afforded EGF12 module 1 in high yield (4.1% overall yield based on the initial resin loading amount), the molecular ion peak of which was m/z 4976.40 as determined by MALDI-TOFMS. The folding reaction in the absence of Ca2+ gave multiple peaks, indicating the formation of a large amount of misfolded products in addition to properly folded glycopeptide 1. The NOESY spectrum of the folded glycopeptide 1 clearly showed a correlation between the βprotons of Cys461−Cys476 (C2−C4) and Cys478− Cys487Cys (C5−C6) (Figure 3b) while it was not possible to identify one of the three disulfide pairs because of overlapping peaks. Enzymatic sugar elongation of folded glycopeptide 1 proceeded smoothly in the presence of sugar nucleotides under optimized conditions17,19,23 and gave novel EGF12 module 2 having a trisaccharide moiety (Galβ1→ 4GlcNAcβ1→3Fucα1→) (m/z 5138.90) and fully glycosylated 3 having a tetrasaccharide moiety (Neu5Acα2→3Galβ1→ 4GlcNAcβ1→3Fucα1→) (m/z 5430.07) at the Thr466 residue in quantitative yield (Figure 3c). These results clearly demonstrated the introduction of a bulky trisaccharide moiety (Xylα1→3Xylα1→3Glcβ1→) at the neighboring Ser458 residue of the folded EGF12 (1) does not disturb modifications to the nonreducing GlcNAc residue of the disaccharide moiety (GlcNAcβ1→3Fucα1→) substituted at Thr466 involved in the

ligand-binding region. It is important to note that sialylation of the nonreducing Gal residue of EGF12 module 2 was also achieved by means of bacterial sialyltransferase (recombinant P. phosphoreum α2,3SiaT)23 as well as the mammalian enzyme, recombinant rat α2,3-(N)-SiaT, as reported previously.17 These results indicate the high potential of the present strategy for the construction of various Notch EGF modules having both natural and unnatural glycoforms produced by enzymatic modifications of the individual folded EGF intermediates bearing the needed core sugar residues. Molecular Mechanisms for the Intramolecular Stabilization of the EGF12 Domain. The three-dimensional structure of synthetic EGF12 glycopeptide 1 carrying Xylα1→ 3Xylα1→3Glcβ1→ and GlcNAcβ1→3Fucα1→ moieties was generated by calculations using distance and angle restrictions estimated from two-dimensional NMR experiments (Figure 4a and Tables S1−S3 of the Supporting Information). Figure 4b represents key NOEs between the C6 methyl group of the Fuc residue at the Thr466, Ile477, and Met479 residues, supporting the principle that the Fuc residue functions directly as a “bridge” in the formation of the antiparallel β-sheet to stabilize the structure through close contact with these amino acids. However, we did not observe NOEs showing potential hydrogen bonding between the GlcNAc and Asp464 residues as reported for mouse Notch1 EGF12 folded in the absence of calcium ions.17 We also found a specific NOESY correlation between Glu473 and the anomeric proton of the nonreducing Xyl residue of the trisaccharide moiety Xylα1→3Xylα1→ 3Glcβ1→ modified at the Ser458 residue, which is located far from the central β-hairpin of EGF12 (Figure 4c). It is interesting to note that these amino acid residues are all key members involved in the one of the two strands in the antiparallel β-sheet structure. These results suggest that both Oglycans modified at the Ser458 and Thr466 residues both contribute independently to the stabilization of the antiparallel β-sheet structure through an interaction with amino acids located in the ligand-binding region of human NOTCH1 EGF12. The effects of the sugar elongations at Thr466 on the G

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Figure 6. Effects of exogenously applied synthetic NOTCH1 EGF12 modules on the proliferation of human cancer cell lines. (a) Human breast cancer MCF-7 cells (5 × 103 cells/well) were cultured in semisolid medium for 96 h with 100 μM synthetic naked EGF12, glycosylated EGF12 (1), or cisplatin in the presence of 100 nM Hoechst 33342. The white scale bar indicates 200 μm. (b) Human lung adenocarcinoma A549 cells (5 × 103 cells/well) were cultured with 100 μM synthetic naked EGF12, glycosylated EGF12, or cisplatin for 48 h in the presence of 100 nM Hoechst 33342. The white scale bar indicates 200 μm. (c) Cell viability of MCF-7 cells incubated with 100 μM synthetic naked EGF12, glycosylated EGF12 (1), or cisplatin after 96 h. (d) Cell viability of A549 cells incubated with 100 μM synthetic naked EGF12, glycosylated EGF12 (1), or cisplatin after 48 h. (e) The experiment described in panel a was repeated with various concentrations of reagents. Proliferation was assessed using the MTT assay, and H

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Biochemistry Figure 6. continued

the graph shows the mean values of triplicate experiments with the standard error of the mean (SEM) in panel c−e. The control shows cell viability without any specific treatment.

activation and cis inhibition10b,c through interactions with dominant ligand molecules. To determine whether synthetic EGF12 analogues (naked EGF12 and glycosylated 1) affect endogenous human NOTCH1 receptor signaling, we assessed the effects of exogenously applied synthetic EGF12 modules on the proliferation of the MCF-7 breast cancer cell line and A549 lung adenocarcinoma epithelial cell line because Notch signaling is known to initiate and activate these cancers and negatively correlates with clinical outcomes.3a,40 Preliminary experiments revealed that synthetic EGF12 modules inhibited the formation of large aggregates of cancer cells when cells were cultured without these reagents, as shown in panels a and b of Figure 6. These results suggest that synthetic EGF12 modules influence the cellular adhesiveness of MCF-7 and A549 cells but do not induce marked changes in cellular morphology when compared with those of dead cells treated with cisplatin. In addition, the level of proliferation of these cancer cells in the presence of synthetic EGF12 modules at 96 h (MCF-7) and 48 h (A549) was significantly lower than that of the control (Figure 6c,d). The inhibitory effects of naked and glycosylated EGF12 modules on these cancer cells were similar, and the level of cell proliferation after seeding appeared to be approximately 80% of that observed in the control experiments. As indicated in Figure 6e, the inhibitory effects by EGF12 modules appeared to be saturated at 100 μM, suggesting that NOTCH1 signaling states in the single tumor cell type may be generated by the integration of expression levels between Notch receptors and ligands.10c However, the preliminary experiments performed in this study using a single tumor type did not reveal specific functional roles for O-glycans in the EGF12 domain from that of a naked EGF12 analogue. In addition, it is important to note that modification with Gal and Neu5Ac residues may influence the functional roles of EGF12 module 1. The studies using EGF12 modules 2 and 3 are under way, and the results will be communicated as soon as possible. Therefore, the molecular mechanisms responsible for the effects of glycosylation in each EGF domain on Notch receptor signaling remain unclear.11c,16a

conformation of EGF12 modules 2 and 3 will be discussed below. Significance of Ca2+-Binding and “Sugar Bridges” in the Ligand-Binding Region. Our results indicated for the first time that calcium ions significantly enhance the efficiency of the folding process from the unfolded glycosylated EGF12 polypeptide intermediate to the properly folded glycosylated EGF12 (1) as described above (Figure 3). However, on the basis of a growing body of evidence that functional fragments of human NOTCH1 EGF11−13 have the ability to interact with ligands in a Ca2+-dependent manner,10b,25b,38 the coordination of calcium ions in EGF12 appears to be crucial for the modulation of binding with Notch ligands. We hypothesized that both Ca2+ binding and O-glycosylation in EGF12 may both contribute synergistically to the intramolecular stabilization of the antiparallel β-sheet structure of this important domain. To test this hypothesis, we carefully compared the three-dimensional (3D) structures obtained in this study with those reported previously, the NMR structure of the mouse Notch1 EGF12 module17 or the structure of the EGF12 domain of the NOTCH1 EGF11−13 fragment revealed by X-ray crystallography.10b,11c The difference between the NMR structures of human and mouse EGF12 shown in Figure 5a was evident not only in the topology of the central β-hairpin region as indicated by an arrow at Ile471 but also in the adjacent O-glycosylation site at Thr466. The result suggests that the intramolecular stabilization mechanisms of O-glycosylations and Ca2+ binding are different from each other. In contrast, the superimposed structure of the human NOTCH1 EGF12 domain (1) resolved by NMR and the corresponding domain of EGF11−13 obtained by X-ray crystallography converged very well, even in the region involving two O-glycosylation sites at residues Ser458 and Thr466 (Figure 5b). This indicates that coordination of Ca2+ has a pivotal function both for the stabilization of β-hairpin structure in this region and for accelerating the ideal folding of the EGF12 domain as shown in panels a and b of Figure 3. The conformational impact of two different types of O-glycosylation on the central β-sheet was also clearly demonstrated when this structure was compared with the 3D structure of naked human NOTCH1 EGF12 folded in the presence of calcium ions (Figure 5c). Taken together, these results imply that two intramolecular “sugar bridges” of human NOTCH1 EGF12, Xyl−Glu473 and Fuc− Ile477, elicited by NOESY experiments (Figure 4) contribute to the stabilization of the antiparallel β-sheet structure in a synergistic manner with the functions of calcium ions. Val453, Glu455, Leu468, and Ile477 appeared to be critical amino acid residues for modulating the coordination of calcium ions or ligand binding.10b,11c,39 Effects of the Synthetic EGF Modules on Cancer Cell Growth. We then focused on the feasibility of the synthetic EGF modules as new tools for investigating the functional roles of posttranslational O-glycosylation in the Notch receptormediated signaling pathway. We considered that synthetic human NOTCH1 EGF modules, having a conformation in the ligand-binding region similar to that of the EGF12 domain involved in the prokaryotic NOTCH1 EGF11−13 variants, can act as soluble antagonists modulating mechanisms in trans

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CONCLUSION We herein established a standard method for the rapid and highly efficient synthesis of EGF modules having multiple Oglycans by combining chemical and enzymatic synthetic strategies. The synthetic human NOTCH1 EGF12 domain having an O-Fuc-type glycan (GlcNAcβ1→3Fucα1→) at Thr466 and O-Glc-type glycan (Xylα1→3Xylα1→3Glcβ1→) at Ser458 provided structural and functional insights into the mechanistic roles of O-glycans and calcium ions during folding and the formation of the important antiparallel β-sheet in the ligand-binding region. The versatility of the present approach is evident because soluble synthetic EGF12 analogues have the ability to modulate the Notch signaling pathway in cancer cells by interacting with intrinsic Notch ligands. Further chemical manipulations for conjugations with functional probes35,41 as well as the robust compound library of synthetic EGF modules will facilitate comprehensive studies for deciphering the structures and functions of individual EGF domains in terms I

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D., Dow, G. J., Shelton, A., Stawicki, S., Watts, R. J., Zhang, J., Choy, R., Howard, P., Kadyk, L., Yan, M., Zha, J., Callahan, C. A., Hymowitz, S. G., and Siebel, C. W. (2010) Therapeutic antibody targeting of individual Notch receptors. Nature 464, 1052−1057. (6) (a) Ellisen, L. W., Bird, J., West, D. C., Soreng, A. L., Reynolds, T. C., Smith, S. D., and Sklar, J. (1991) TAN-1, the human homolog of the Drosophila Notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 66, 649−661. (b) Weng, A. P., Ferrando, A. A., Lee, W., Morris, J. P., IV, Silverman, L. B., SanchezIrizarry, C., Blacklow, S. C., Look, A. T., and Aster, J. C. (2004) Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306, 269−271. (7) (a) Klinakis, A., Lobry, C., Abdel-Wahab, O., Oh, P., Haeno, H., Buonamici, S., van De Walle, I., Cathelin, S., Trimarchi, T., Araldi, E., Liu, C., Ibrahim, S., Beran, M., Zavadil, J., Efstratiadis, A., Taghon, T., Michor, F., Levine, R. L., and Aifantis, I. (2011) A novel tumoursuppressor function for the Notch pathway in myeloid leukaemia. Nature 473, 230−233. (b) Kannan, S., Sutphin, R. M., Hall, M. G., Golfman, L. S., Fang, W., Nolo, R. M., Akers, L. J., Hammitt, R. A., McMurray, J. S., Kornblau, S. M., Melnick, A. M., Figueroa, M. E., and Zweidler-McKay, P. A. (2013) Notch activation inhibits AML growth and survival: A potential therapeutic approach. J. Exp. Med. 210, 321− 337. (8) (a) Nicolas, M., Wolfer, A., Raj, K., Kummer, J. A., Mill, P., van Noort, M., Hui, C.-C., Clevers, H., Dotto, G. P., and Radtke, F. (2003) Notch1 functions as a tumor suppressor in mouse skin. Nat. Genet. 33, 416−421. (b) Zweidler-McKay, P. A., He, Y., Xu, L., Rodriguez, C. G., Kamell, F. G., Carpenter, A. C., Aster, J. C., Allman, D., and Pear, W. S. (2005) Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies. Blood 106, 3898− 3906. (c) Hanlon, L., Avila, J. L., Demarest, R. M., Troutman, S., Allen, M., Ratti, F., Rustgi, A. K., Stanger, B. Z., Radtke, F., Adsay, V., Long, F., Capobianco, A. J., and Kissil, J. L. (2010) Notch1 functions as a tumor suppressor in a model of K-ras-induced pancreatic ductal adenocarcinoma. Cancer Res. 70, 4280−4286. (d) Viatour, P., Ehmer, U., Saddic, L. A., Dorrell, C., Andersen, J. B., Lin, C., Zmoos, A.-F., Mazur, P. K., Schaffer, B. E., Ostermeier, A., Vogel, H., Sylvester, K. G., Thorgeirsson, S. S., Grompe, M., and Sage, J. (2011) Notch signaling inhibits hepatocellular carcinoma following inactivation of the RB pathway. J. Exp. Med. 208, 1963−1976. (e) Wang, N. J., Sanborn, Z., Arnett, K. L., Bayston, L. J., Liao, W., Proby, C. M., Leigh, I. M., Collisson, E. A., Gordon, P. B., Jakkula, L., Pennypacker, S., Zou, Y., Sharma, M., North, J. P., Vemula, S. S., Mauro, T. M., Neuhaus, I. M., LeBoit, P. E., Hur, J. S., Park, K., Huh, N., Kwok, P.-Y., Arron, S. T., Massion, P. P., Bale, A. E., Haussler, D., Cleaver, J. E., Gray, J. W., Spellman, P. T., South, A. P., Aster, J. C., Blacklow, S. C., and Cho, R. J. (2011) Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma. Proc. Natl. Acad. Sci. U. S. A. 108, 17761−17766. (f) Rampias, T., Vgenopoulou, P., Avgeris, M., Polyzos, A., Stravodimos, K., Valavanis, C., Scorilas, A., and Klinakis, A. (2014) A new tumor suppressor role for the Notch pathway in bladder cancer. Nat. Med. 20, 1199−1205. (9) Fan, X., Mikolaenko, I., Elhassan, I., Ni, X., Wang, Y., Ball, D., Brat, D. J., Perry, A., and Eberhart, C. G. (2004) Notch1 and Notch2 have opposite effects on embryonal brain tumor growth. Cancer Res. 64, 7787−7793. (10) (a) Guruharsha, K. G., Kankel, M. W., and Artavanis-Tsakonas, S. (2012) The Notch signalingsystem: Recent insights into the complexity of a conserved pathway. Nat. Rev. Genet. 13, 654−666. (b) Cordle, J., Johnson, S., Zi Yan Tay, J., Roversi, P., Wilkin, M. B., de Madrid, B. H., Shimizu, H., Jensen, S., Whiteman, P., Jin, B., Redfield, C., Baron, M., Lea, S. M., and Handford, P. A. (2008) A conserved face of the Jagged/Serrate DSL domain is involved in Notch transactivation and cis-inhibition. Nat. Struct. Mol. Biol. 15, 849−857. (c) Sprinzak, D., Lakhanpal, A., LeBon, L., Santat, L. A., Fontes, M. E., Anderson, G. A., Garcia-Ojalvo, J., and Elowitz, M. B. (2010) Cisinteractions between Notch and Delta generate mutually exclusive signaling states. Nature 465, 86−90. (d) Lobry, C., Oh, P., and Aifantis, I. (2011) Oncogenic and tumor suppressor functions of Notch in

of the significance of posttranslational modifications in Notch receptors and provide tools for the discovery of novel approaches to targeting the Notch signaling pathway in cancers.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01284. Materials, reagents, instrumentation, synthetic procedures, and details of sugar amino acid 4 and all intermediates, NMR spectra of all new compounds, tables of all NMR data, and MALDI-TOFMS data for the naked EGF12 module (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +81-11-706-9042. Telephone: +81-11-706-9043. Funding

This work was partly supported by JSPS KAKENHI Grant 25220206 and a grant for Innovation COE Project for Future Medicine and Medical Care from the Ministry of Education, Culture, Sports, Science, and Technology Japan (MEXT). Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.biochem.5b01284 Biochemistry XXXX, XXX, XXX−XXX