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News Article | September 14, 2016
Site: www.nature.com

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. The human ET receptor gene encoding a hexa-histidine tag in the amino-terminal tail (6hNET R)11, 48 was used as the starting template, and we systematically surveyed stabilizing mutations according to the previously described method49. The established thermostabilized ET receptor construct contained five mutations (ET R-Y5: R124Y1.55, D154A2.57, K270A5.35, S342A6.54, I381A7.48). The thermostability of the ET R-Y5 construct was confirmed by a fluorescence detection size-exclusion chromatography-based thermostability assay (FSEC-TS)50 as follows. The ET R-Y5 protein fused with green fluorescent protein (GFP) at the C terminus was expressed in SF+ insect cells (expresSF+ cell, Protein Sciences). The cells were solubilized in a buffer containing 10 mM HEPES, pH 7.5, 200 mM NaCl and 1% n-dodecyl-β-d-maltopyranoside (DDM). After removing insoluble materials by centrifugation at 100,000g for 20 min, 100-μl aliquots of the supernatant were placed into polymerase chain reaction tubes and incubated at the respective temperatures for 10 min The heat-treated samples were centrifuged at 100,000g for 20 min and loaded onto a Superdex200 10/150 column pre-equilibrated with 10 mM HEPES, pH 7.5, 200 mM NaCl and 0.05% DDM. Each fluorescent signal intensity at the monomeric peak was normalized to that of the unheated sample as 100% (Extended Data Fig. 1c). Each measurement was performed three times. Melting temperatures (T ) were determined by fitting the curves to a sigmoidal dose–response equation, using the SigmaPlot 12 software (Systat Software). The haemagglutinin signal peptide, followed by the Flag epitope tag (DYKDDDD) and a nine-amino-acid linker, was added to the N terminus of the receptor, and a tobacco etch virus (TEV) protease recognition sequence was introduced between Gly57 and Leu66, to remove the disordered N terminus during the purification process. The C terminus was truncated after Ser407, and three cysteine residues were mutated to alanine (C396A, C400A and C405A) to avoid heterogeneous palmitoylation51. To improve crystallogenesis, T4 lysozyme containing the C54T and C97A mutations52 was introduced into intracellular loop 3, between Lys3035.68 and Leu3116.23 (ET R-Y5-T4L) (Extended Data Figs 1 and 2b). The recombinant baculovirus was prepared using the Bac-to-Bac baculovirus expression system (Invitrogen). SF+ insect cells were infected with the virus at a cell density of 1.5 × 106 cells per millilitre in Sf900 II medium (Invitrogen), supplemented with 50 units ml−1 penicillin, 50 μg ml−1 streptomycin and 0.125 μg ml−1 amphotericin B, and grown for 48 h at 27 °C. The harvested cells were disrupted using a high-pressure homogenizer, EmulsiFlex-C5 (Avestin), in buffer containing 10 mM HEPES-NaOH, pH 7.5, 10 mM EDTA, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg ml−1 aprotinin, 10 μg ml−1 leupeptin and 10 μg ml−1 soybean trypsin inhibitor. The cell debris was removed by centrifugation at 4,00g for 30 min, and the crude membrane fraction was collected by ultracentrifugation at 100,000g for 1 h. The membrane fraction was solubilized in buffer, containing 50 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, 2% DDM (Anatrace), 0.4% cholesterol hemisuccinate (CHS, Sigma) and 1 μM ET-1 (Peptide Institute Inc.), for 3 h at 4 °C. Afterwards, 2 mg ml−1 iodoacetamide was added to block reactive cysteines. The supernatant containing the solubilized receptor was separated from the insoluble material by ultracentrifugation at 100,000g for 1 h, and incubated with anti-Flag M2 affinity resin (Sigma) overnight at 4 °C. After binding, the resin was washed with ten column volumes of wash I buffer, containing 20 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 0.1% lauryl maltose neopentyl glycol (LMNG, Anatrace) and 0.01% CHS, followed by ten column volumes of wash II buffer, containing 10 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, 0.01% LMNG and 0.001% CHS. The receptor was eluted from the resin with 200 μg ml−1 Flag peptide (Sigma) in the presence of 100 nM ET-1, and then treated with TEV protease (prepared in our laboratory) overnight at 4 °C, to cleave the N-terminal flexible region of the receptor. The receptor was concentrated and loaded onto a Superdex200 10/300 size-exclusion column, equilibrated in buffer containing 10 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, 0.003% MNG, 0.0003% CHS and 100 nM ET-1. Peak fractions were pooled, concentrated to 40 mg ml−1 using a Vivaspin sample concentrator with a 50 kDa molecular mass cut-off (Sartorius), and frozen until crystallization. During the concentration, ET-1 was added to a final concentration of 100 μM. The crystallization construct was further modified to obtain the ligand-free structure of ET (Extended Data Fig. 2a). T4L was modified according to the previous report53, as follows. Amino-acid residues 13–60 were removed, and the linker sequence (–GGSGG–) was inserted at the corresponding site (ET R-Y5-mT4L). The EGFP–His tag and the TEV protease cleavage site were introduced at the C terminus. The recombinant baculovirus was prepared as described above. Sf9 insect cells were infected with the virus at a cell density of 4.0 × 106 cells per millilitre in Sf900 II medium, and grown for 72 h at 27 °C. The harvested cells were disrupted by sonication, in buffer containing 20 mM Tris-HCl, pH 7.5, and 20% glycerol. The crude membrane fraction was collected by ultracentrifugation at 180,000g for 1 h. The membrane fraction was solubilized in buffer, containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% DDM, 0.2% cholesterol hemisuccinate and 2 mg ml−1 iodoacetamide, for 2 h at 4 °C. The supernatant was separated from the insoluble material by ultracentrifugation at 180,000g for 30 min, and incubated with TALON resin (Clontech) for 30 min. The resin was washed with ten column volumes of buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% LMNG, 0.01% CHS and 20 mM imidazole. The receptor was eluted in buffer, containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.1% LMNG, 0.01% CHS and 200 mM imidazole. The eluate was treated with TEV protease and dialysed against buffer (50 mM Tris-HCl, pH 7.5, and 500 mM NaCl). The cleaved GFP–His tag and the TEV protease were removed with Ni+-NTA resin. The receptor was concentrated and loaded onto a Superdex200 10/300 Increase size-exclusion column, equilibrated in buffer containing 10 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, 0.01% LMNG and 0.001% CHS. Peak fractions were pooled, concentrated to 40 mg ml−1 using a centrifugal filter device (Millipore 50 kDa MW cutoff) and frozen until crystallization. The purified receptors were reconstituted into the lipidic cubic phase (LCP) of monoolein (Nucheck), supplemented with cholesterol at a ratio of 4:5:1 (w/w) for protein:monoolein:cholesterol54. The protein-laden mesophase was dispensed into 96-well glass plates in 30–50 nl drops and overlaid with 800–1,000 nl precipitant solution, using a mosquito LCP (TTP LabTech). Crystals of ET R-Y5-T4L in complex with ET-1 were grown at 20 °C in the precipitant conditions containing 30% PEG400, 100 mM MES-NaOH (pH 6.0), 100 mM (NH ) SO and 5% 1,4-butanediol. The crystals of ET R-Y5-mT4L in the ligand-free form were grown in the precipitant conditions containing 18–25% PEG350MME, 100 mM MES-NaOH (pH 6.3), 80 mM (NH ) SO , and 8% 1,4-butanediol. The crystals were harvested directly from the LCP using micromounts (MiTeGen) or LithoLoops (Protein Wave) and frozen in liquid nitrogen, without adding any extra cryoprotectant. X-ray diffraction data were collected at the SPring-8 beamline BL32XU, using a 10 μm × 15 μm (width × height) micro-focused beam. Diffraction data were processed using XDS55. The ET-1 bound structure was determined by molecular replacement with PHASER56, using the T4L from chemokine receptor CXCR4 (PDB accession number 3OE0) and the poly-alanine model derived from the β -adrenergic receptor (PDB accession number 3NYA). Subsequently, the model was rebuilt and refined using COOT57 and PHENIX58, respectively. The ligand-free structure was determined by molecular replacement, using the ET-1 bound structure, and subsequently rebuilt and refined as described above. The final model of ET-1-bound ET R-Y5-T4L contained residues 88–129, 135–206, 217–303 and 311–401 of ET , all residues of T4L, all residues of ET-1, and 4 water molecules, and the model of ET R-Y5-mT4L contained residues 85–304 and 311–402 of ET , all residues of mT4L, 3 monoolein molecules, 4 sulfate ions and 24 water molecules. The model quality was assessed by MolProbity59. Figures were prepared using cuemol (http://www.cuemol.org/ja/). For competitive ligand binding assays, the genes encoding wild-type (6hNET R)11 and mutant receptors were cloned in the pFastBac1 vector and the pcDNA3.1 vector, which were used for expression in insect cells and mammalian cells, respectively. Membranes from SF+ or HEK293 cells expressing 6hNET R or its mutants were prepared, and the expressed ET receptors were quantitated as described previously60, 61. Peptide binding competition was initiated by the addition of the membranes from SF+ cells (0.1–1.2 μg) or HEK293 cells (1–5 μg) to the assay mixture, composed of 0.1% bovine serum albumin (BSA), 0.03–0.05 nM 125I-labelled ET-1 (2,200 Ci mmol−1, PerkinElmer Life Sciences), and eight concentrations of unlabelled ET-1 or ET-3 (ranging from 1 pM to 1 μM, Peptide Institute) in 50 mM HEPES-NaOH, pH 7.5, and 10 mM MgCl (Mg-HEPES)62. Binding reactions were incubated at 37 °C for 1 h, terminated by dilution with ice-cold Mg-HEPES, and filtered onto glass fibre filters in 96-well plates (multiscreen HTS FB, Merck Millipore) to separate the unbound 125I-labelled ET-1. After three washes with ice-cold Mg-HEPES, the radioactivity captured by the filters was counted using a γ counter. Filters were pretreated with 0.1% BSA in Mg-HEPES. The results were analysed by nonlinear regression, using the GraphPad Prism 6 software. In the saturation binding assays, membranes containing approximately 2 fmol receptor were incubated with six different concentrations of 125I-labelled ET-1, ranging from 1.5 pM to 253 pM in 100 μl of Mg-HEPES buffer containing 0.1% BSA, at 37 °C for 2 h (ref. 62). The membranes were isolated from the unbound 125I-labelled ET-1 and washed, and the amount of receptor-bound 125I-labelled ET-1 was measured as described above. The non-specific binding of the 125I-labelled ET-1 in each reaction was assessed by including 100 nM ET-1 in the same reaction. The apparent dissociation constants (K ) of ET-1 for wild-type ET receptor, ET R-Y5, and ET R-Y5-T4L were determined by fitting to a one-site binding equation, using the GraphPad Prism 6 software. Each experiment was performed 3 or 4 times. For the G protein activation assay, the ET receptor construct containing the haemagglutinin signal peptide, followed by the Flag sequence and hexa-histidine tag at the N terminus, was used as the wild type. Reconstitution of the purified receptor and G into phospholipid vesicles was performed as described previously11. GDP/[35S]GTP-γS exchange assays were also performed as described previously, with 1.8 nM receptor, 50 nM Gα (purified from E. coli), ~140 nM Gβ γ (purified from Sf9 cells), 1 μM GDP and 55 nM [35S]GTP-γS, with or without 1 μM ET-1, at 30 °C for the indicated times, in 20 mM HEPES-NaOH, pH 8.0, 1 mM EDTA, 100 mM NaCl, 10 mM MgCl and 1 mM dithiothreitol. [35S]GTP-γS (1,250 Ci mmol−1, PerkinElmer Life Sciences) was used after dilution with unlabelled GTP-γS to 113.6 Ci mmol−1. The reactions were terminated by dilution with ice-cold stopping buffer containing 100 μM GTP, in 20 mM Tris-HCl, pH 8.0, 25 mM MgCl and 100 mM NaCl, and filtered onto cellulose-mixed ester filters in 96-well plates (multiscreen HTS haemagglutinin, Merck Millipore), to isolate the G proteins from the unbound [35S]GTP-γS. After three washes with ice-cold stopping buffer without GTP, the radioactivity of the bound [35S]GTP-γS was measured, using a liquid scintillation counter. The measured values were normalized to that of wild-type receptor-activated [35S]GTP-γS binding for 10 min in the presence of ET-1. The assays were repeated twice. In the ligand concentration-dependent GDP/[35S]GTP-γS exchange assays, the receptors, reconstituted at 1 nM in the phospholipid vesicles with G proteins as described above, were pre-incubated in the 20 μl mixture with 11 different concentrations of ET-1 (ranging from 1 pM to 1 μM) for 5 min at 30 °C. The reactions were then started by the addition of [35S]GTP-γS. After an incubation for 2 min at 30 °C, the reactions were stopped by the addition of ice-cold stopping buffer, filtered and measured as described above. The assays were repeated four or five times. The data were analysed using the GraphPad Prism 6 software (Extended Data Fig. 1f, g).


The purpose of the present invention is to provide a method for highly accurately detecting a cancer cell. The method of the present invention is characterized by comprising imaging with the use of a fluorescently labeled L-glucose derivative. By using the method and imaging agent according to the present invention, a high contrast between a cancer cell and a normal cell can be obtained compared with the case that imaging is conducted with the use of a fluorescently labeled D-glucose derivative. According to this method, moreover, no fasting is needed for the determination. Thus, the imaging can be quickly carried out without imposing a burden on a patient.


The purpose of the present invention is to provide a method for highly accurately detecting a cancer cell. The method of the present invention is characterized by comprising imaging with the use of a fluorescently labeled L-glucose derivative. By using the method and imaging agent according to the present invention, a high contrast between a cancer cell and a normal cell can be obtained compared with the case that imaging is conducted with the use of a fluorescently labeled D-glucose derivative. According to this method, moreover, no fasting is needed for the determination. Thus, the imaging can be quickly carried out without imposing a burden on a patient.


Mochizuki M.,Peptide Institute Inc. | Hibino H.,Peptide Institute Inc. | Nishiuchi Y.,Peptide Institute Inc. | Nishiuchi Y.,Osaka University
Organic Letters | Year: 2014

Tritylation using trityl alcohol (Trt-OH) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) is a convenient and efficient procedure that can offer S-protection of the Cys located in fully unprotected peptides. The procedure simply requires Trt-OH and HFIP to selectively promote S-tritylation in the presence of peptide nucleophilic functionalities. © 2014 American Chemical Society.


Hibino H.,Peptide Institute Inc. | Miki Y.,Kinki University | Nishiuchi Y.,Peptide Institute Inc. | Nishiuchi Y.,Osaka University
Journal of Peptide Science | Year: 2012

The 4-methoxybenzyloxymethyl (MBom) group was introduced at the Nπ-position of the histidine (His) residue by using a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the microwave (MW)-assisted solid phase peptide synthesis (SPPS) with 9-fluorenylmethyoxycarbonyl (Fmoc) chemistry. The Nπ-MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side-chain-induced racemization during incorporation of the His residue even in the case of MW-assisted SPPS performed at a high temperature. In particular, the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the imidazole ring during detachment of the protected His-containing peptide segments from acid-sensitive linkers by treatment with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc-His(π-MBom)-OH with the aid of purification procedure by crystallization to effectively remove the undesired τ-isomer without resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large-scale production without any obstacles. © 2012 European Peptide Society and John Wiley & Sons, Ltd.


Hibino H.,Peptide Institute Inc. | Nishiuchi Y.,Peptide Institute Inc. | Nishiuchi Y.,Osaka University
Organic Letters | Year: 2012

The 4-methoxybenzyloxymethyl (MBom) group was introduced for sulfhydryl protection of Cys in combination with Fmoc chemistry. The MBom group proved to substantially suppress racemization of Cys during its incorporation mediated by phosphonium or uronium reagents. Furthermore, this group was found to significantly reduce racemization of the C-terminal Cys linked to a hydroxyl resin during repetitive base treatment, in comparison with the usually used trityl (Trt) and acetamidomethyl (Acm) groups. © 2012 American Chemical Society.


Hibino H.,Peptide Institute Inc. | Miki Y.,Kinki University | Nishiuchi Y.,Peptide Institute Inc. | Nishiuchi Y.,Osaka University
Journal of Peptide Science | Year: 2014

Phosphonium and uronium salt-based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine-assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6-trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4′-dimethoxydiphenylmethyl and 4-methoxybenzyloxymethyl groups were demonstrated to be acid-labile S-protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave-assisted SPPS performed at a high temperature. © 2013 The Authors. European Peptide Society published by John Wiley & Sons, Ltd.


Sano S.,Japan National Institute of Biomedical Innovation | Tagami S.,Osaka University | Hashimoto Y.,Japan National Institute of Biomedical Innovation | Yoshizawa-Kumagaye K.,Peptide Institute Inc. | And 3 more authors.
Journal of Proteome Research | Year: 2014

Selected/multiple reaction monitoring (SRM/MRM) has been widely used for the quantification of specific proteins/peptides, although it is still challenging to quantitate low abundant proteins/peptides in complex samples such as plasma/serum. To overcome this problem, enrichment of target proteins/peptides is needed, such as immunoprecipitation; however, this is labor-intense and generation of antibodies is highly expensive. In this study, we attempted to quantify plasma low abundant APLP1-derived Aβ-like peptides (APL1β), a surrogate marker for Alzheimer's disease, by SRM/MRM using stable isotope-labeled reference peptides without immunoaffinity enrichment. A combination of Cibacron Blue dye mediated albumin removal and acetonitrile extraction followed by C18-strong cation exchange multi-StageTip purification was used to deplete plasma proteins and unnecessary peptides. Optimal and validated precursor ions to fragment ion transitions of APL1β were developed on a triple quadruple mass spectrometer, and the nanoliquid chromatography gradient for peptide separation was optimized to minimize the biological interference of plasma. Using the stable isotope-labeled (SI) peptide as an internal control, absolute concentrations of plasma APL1β peptide could be quantified as several hundred amol/mL. To our knowledge, this is the lowest detection level of endogenous plasma peptide quantified by SRM/MRM. © 2013 American Chemical Society.


Wong C.T.T.,Nanyang Technological University | Taichi M.,Peptide Institute Inc. | Nishio H.,Peptide Institute Inc. | Nishiuchi Y.,Peptide Institute Inc. | Tam J.P.,Nanyang Technological University
Biochemistry | Year: 2011

Hedyotide B1, a novel cyclotide isolated from the medicinal plant Hedyotis biflora, contains a cystine knot commonly found in toxins and plant defense peptides. The optimal oxidative folding of a cystine knot encased in the circular peptide backbone of a cyclotide poses a challenge. Here we report a systematic study of optimization of the oxidative folding of hedyotide B1, a 30-amino acid cyclic peptide with a net charge of +3. The linear precursor of hedyotide B1, synthesized as a thioester by solid phase synthesis, was cyclized quantitatively by a thia-zip cyclization to form the circular backbone and then subjected to oxidative folding in a thiol - disulfide redox system under 38 different conditions. Of the oxidative conditions examined, the nature of the organic cosolvent appeared to be critical, with the use of 70% 2-propanol affording the highest yield (48%). The disulfide connectivity of the folded hedyotide was identical to that of the native form as determined by partial acid hydrolysis. The use of such a high alcohol concentration suggests that a partial denaturation may be necessary for the oxidative folding of a cyclotide with the inverse orientation of hydrophobic side chains that are externalized to the solvent face to permit the formation of the interior cystine core in the circularized backbone. We also show that synthetic hedyotide B1 is an antimicrobial, exhibiting minimal inhibitory concentrations in the micromolar range against both Gram-positive and -negative bacteria. © 2011 American Chemical Society.


Hibino H.,Peptide Institute Inc. | Nishiuchi Y.,Peptide Institute Inc. | Nishiuchi Y.,Osaka University
Tetrahedron Letters | Year: 2011

The 4-methoxybenzyloxymethyl (MBom) group was introduced at the N π-position of histidine, and its utility was examined under the conditions for peptide synthesis by Fmoc strategy. The N π-MBom group proved to prevent the risk of racemization during incorporation of the His residue and to possess all of the chemical properties required for Fmoc chemistry. The side reaction associated with formaldehyde generated from the N π-MBom group upon acidolysis could be effectively prevented by performing the standard TFA treatment in the presence of methoxyamine· hydrochloride (MeONH 2·HCl). © 2011 Elsevier Ltd. All rights reserved.

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