U Medico Inc.
U Medico Inc.
Zhao H.,Cellular Bioengineering |
Ghirlando R.,U.S. National Institute of Diabetes and Digestive and Kidney Diseases |
Alfonso C.,CSIC - Biological Research Center |
Arisaka F.,Nihon University |
And 124 more authors.
PLoS ONE | Year: 2015
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies. © 2015, Public Library of Science. All rights reserved. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Fujita D.,University of Tokyo |
Suzuki K.,University of Tokyo |
Sato S.,University of Tokyo |
Yagi-Utsumi M.,Japan Institute for Molecular Science |
And 14 more authors.
Nature Communications | Year: 2012
Protein encapsulation has long attracted many chemists and biologists because of its potential to control the structure and functions of proteins, but has been a daunting challenge because of their incommensurably larger size compared with common synthetic hosts. Here we report the encapsulation of a small protein, ubiquitin, within giant coordination cages. The protein was attached to one bidentate ligand and, upon addition of Pd(II) ions (M) and additional ligands (L), M 12 L 24 coordination nanocages self-assembled around the protein. Because of the well-defined host framework, the protein-encapsulated structure could be analysed by NMR spectroscopy, ultracentrifugation and X-ray crystallography. © 2012 Macmillan Publishers Limited. All rights reserved.
Kita S.,Hokkaido University |
Matsubara H.,Hokkaido University |
Matsubara H.,High Energy Accelerator Research Organization |
Kasai Y.,Hokkaido University |
And 11 more authors.
European Journal of Immunology | Year: 2015
Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended β-sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Oda M.,Kyoto Prefectural University |
Tanabe Y.,Kyoto Prefectural University |
Noda M.,U Medico Inc. |
Inaba S.,Kyoto Prefectural University |
And 4 more authors.
Carbohydrate Research | Year: 2016
One of the β-1,3-glucans, laminarin, has been widely used as a substrate for enzymes including endo-1,3-β-glucanase. To obtain quantitative information about the molecular interaction between laminarin and endo-1,3-β-glucanase, the structural properties of laminarin should be determined. The results from pioneering work using analytical ultracentrifugation for carbohydrate analysis showed that laminarin from Laminaria digitata predominantly exists as a single-chain species with approximately 5% of triple-helical species. Differential scanning calorimetry experiments did not show a peak assignable to the transition from triple-helix to single-chain, supporting the notion that a large proportion of laminarin is the single-chain species. The interaction of laminarin with an inactive variant of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, E119A, was quantitatively analyzed using isothermal titration calorimetry. The binding was enthalpically driven and the binding affinity was approximately 106 M-1. The results from binding stoichiometric analysis indicated that on average, E119A binds to laminarin in a 2:1 ratio. This seems to be reasonable, because laminarin mainly exists as a monomer, the apparent molecular mass of laminarin is 3.6 kDa, and E119A would have substrate-binding subsites corresponding to 6 glucose units. The analytical ultracentrifugation experiments could detect different complex species of laminarin and endo-1,3-β-glucanase. © 2016 Elsevier Ltd.
Ohto U.,University of Tokyo |
Shibata T.,University of Tokyo |
Shibata T.,Japan Science and Technology Agency |
Tanji H.,University of Tokyo |
And 7 more authors.
Nature | Year: 2015
Innate immunity serves as the first line of defence against invading pathogens such as bacteria and viruses. Toll-like receptors (TLRs) are examples of innate immune receptors, which sense specific molecular patterns from pathogens and activate immune responses. TLR9 recognizes bacterial and viral DNA containing the cytosine-phosphate-guanine (CpG) dideoxynucleotide motif. The molecular basis by which CpG-containing DNA (CpG-DNA) elicits immunostimulatory activity via TLR9 remains to be elucidated. Here we show the crystal structures of three forms of TLR9: unliganded, bound to agonistic CpG-DNA, and bound to inhibitory DNA (iDNA). Agonistic-CpG-DNA-bound TLR9 formed a symmetric TLR9-CpG-DNA complex with 2:2 stoichiometry, whereas iDNA-bound TLR9 was a monomer. CpG-DNA was recognized by both protomers in the dimer, in particular by the amino-terminal fragment (LRRNT-LRR10) from one protomer and the carboxy-terminal fragment (LRR20-LRR22) from the other. The iDNA, which formed a stem-loop structure suitable for binding by intramolecular base pairing, bound to the concave surface from LRR2-LRR10. This structure serves as an important basis for improving our understanding of the functional mechanisms of TLR9. © 2015 Macmillan Publishers Limited.
PubMed | Osaka University, Osaka Prefecture University, Kyoto Prefectural University and U Medico Inc.
Type: | Journal: Carbohydrate research | Year: 2016
One of the -1,3-glucans, laminarin, has been widely used as a substrate for enzymes including endo-1,3--glucanase. To obtain quantitative information about the molecular interaction between laminarin and endo-1,3--glucanase, the structural properties of laminarin should be determined. The results from pioneering work using analytical ultracentrifugation for carbohydrate analysis showed that laminarin from Laminaria digitata predominantly exists as a single-chain species with approximately 5% of triple-helical species. Differential scanning calorimetry experiments did not show a peak assignable to the transition from triple-helix to single-chain, supporting the notion that a large proportion of laminarin is the single-chain species. The interaction of laminarin with an inactive variant of endo-1,3--glucanase from Cellulosimicrobium cellulans, E119A, was quantitatively analyzed using isothermal titration calorimetry. The binding was enthalpically driven and the binding affinity was approximately 10(6)M(-1). The results from binding stoichiometric analysis indicated that on average, E119A binds to laminarin in a 2:1 ratio. This seems to be reasonable, because laminarin mainly exists as a monomer, the apparent molecular mass of laminarin is 3.6kDa, and E119A would have substrate-binding subsites corresponding to 6 glucose units. The analytical ultracentrifugation experiments could detect different complex species of laminarin and endo-1,3--glucanase.
Han W.,Osaka University |
Yamauchi M.,U medico Inc. |
Hasegawa U.,Osaka University |
Noda M.,U medico Inc. |
And 5 more authors.
Journal of Bioscience and Bioengineering | Year: 2015
Polymer-based monoliths with interconnected porous structure have attracted much attention as a high-performance stationary phase for online digestion liquid chromatography-mass spectrometry (LC-MS) system. In this study, a poly(glycidyl methacrylate-. co-methyl methacrylate) (PGM) monolith prepared via thermally induced phase separation (TIPS) was used as a solid support to covalently immobilize pepsin. The PGM monolith was modified with aminoacetal to yield an aldehyde-bearing (PGM-CHO) monolith. Pepsin was immobilized onto the PGM-CHO monolith via reductive amination. The immobilized pepsin showed better pH and thermal stability compared with free pepsin. Furthermore, the PGM-CHO monolith modified with pepsin was applied for online protein digestion followed by LC-MS and LC-MS/MS analyses. As a result, a larger number of peptides are reproducibly identified compared to those by polystyrene/divinylbenzene particle (POROS)-based online pepsin column. © 2014 The Society for Biotechnology, Japan.