| 1. |
Saeko Yanaka, Four-dimensional Structures and Molecular Designs of Glycans, Trends in Glycoscience and Glycotechnology, 10.4052/tigg.2042.1e, 34, 201, E85-E90, 2022.09. |
| 2. |
Saeko Yanaka, Maho Yagi-Utsumi, Koichi Kato, Kunihiro Kuwajima, The B Domain of Protein A Retains Residual Structures in 6 M Guanidinium Chloride as Revealed by Hydrogen/Deuterium-Exchange NMR Spectroscopy., Protein science : a publication of the Protein Society, 10.1002/pro.4569, e4569, 2023.01, The characterization of residual structures persistent in unfolded proteins is an important issue in studies of protein folding, because the residual structures present, if any, may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the residual structures of the isolated B domain (BDPA) of staphylococcal protein A in 6 M guanidinium chloride. BDPA is a small three-helix-bundle protein, and until recently its folding/unfolding reaction has been treated as a simple two-state process between the native and the fully unfolded states. We employed a dimethylsulfoxide (DMSO)-quenched hydrogen/deuterium (H/D)-exchange 2D NMR techniques with the use of spin desalting columns, which allowed us to investigate the H/D-exchange behavior of individually identified peptide amide (NH) protons. We obtained H/D-exchange protection factors of the 21 NH protons that form an α-helical hydrogen bond in the native structure, and the majority of these NH protons were significantly protected with a protection factor of 2.0-5.2 in 6 M guanidinium chloride, strongly suggesting that these weakly protected NH protons form much stronger hydrogen bonds under native folding conditions. The results can be used to deduce the structure of an early folding intermediate, when such an intermediate is shown by other methods. Among three native helical regions, the third helix in the C-terminal side was highly protected and stabilized by side-chain salt bridges, probably acting as the folding initiation site of BDPA. The present results are discussed in relation to previous experimental and computational findings on the folding mechanisms of BDPA. This article is protected by copyright. All rights reserved.. |
| 3. |
Maho Yagi-Utsumi, Saeko Yanaka, Chihong Song, Tadashi Satoh, Chiaki Yamazaki, Haruo Kasahara, Toru Shimazu, Kazuyoshi Murata, Koichi Kato, Characterization of amyloid β fibril formation under microgravity conditions., NPJ microgravity, 10.1038/s41526-020-0107-y, 6, 17-17, 2020.06, Amyloid fibrils are self-assembled and ordered proteinaceous supramolecules structurally characterized by the cross-β spine. Amyloid formation is known to be related to various diseases typified by neurogenerative disorders and involved in a variety of functional roles. Whereas common mechanisms for amyloid formation have been postulated across diverse systems, the mesoscopic morphology of the fibrils is significantly affected by the type of solution condition in which it grows. Amyloid formation is also thought to share a phenomenological similarity with protein crystallization. Although many studies have demonstrated the effect of gravity on protein crystallization, its effect on amyloid formation has not been reported. In this study, we conducted an experiment at the International Space Station (ISS) to characterize fibril formation of 40-residue amyloid β (Aβ(1-40)) under microgravity conditions. Our comparative analyses revealed that the Aβ(1-40) fibrilization progresses much more slowly on the ISS than on the ground, similarly to protein crystallization. Furthermore, microgravity promoted the formation of distinct morphologies of Aβ(1-40) fibrils. Our findings demonstrate that the ISS provides an ideal experimental environment for detailed investigations of amyloid formation mechanisms by eliminating the conventionally uncontrollable factors derived from gravity.. |
| 4. |
Ryu Hashimoto, Masafumi Minoshima, Souhei Sakata, Fumihito Ono, Hirokazu Ishii, Yuki Watakabe, Tomomi Nemoto, Saeko Yanaka, Koichi Kato, Kazuya Kikuchi, Efficient Visible/NIR Light-driven Uncaging of Hydroxylated Thiazole Orange-based Caged Compounds in Aqueous Media, Chemical Science, 10.1039/d2sc02364d, 13, 25, 7462-7467, 2022.02, In the photoactivation strategies with bioactive molecules, one-photon visible or two-photon near-infrared light-sensitive caged compounds are desirable tools for biological applications because they offer reduced phototoxicity and deep tissue penetration.... . |
| 5. |
Regina Ragasa, Eiji Nakamura, Lisa Marrone, Saeko Yanaka, Shusaku Hayashi, Koji Takeuchi, Susan J. Hagen, Isothiocyanate inhibits restitution and wound repair after injury in the stomach: Ex vivo and in vitro studies, JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, 10.1124/jpet.107.121640, 323, 1, 1-9, 2007.10, The role of isothiocyanate (ITC) in blocking epithelial restitution after injury and in the recovery of round wounds was examined in the ex vivo guinea pig stomach and in rat gastric mucosal-1 (RGM1) cells, respectively. For this, recovery of transepithelial electrical resistance and morphology after injury or the closure of round wounds was evaluated in the presence of 4,4'-diisothiocyanatostilbene2,2'- disulfonic acid ( DIDS) or 4,4-diisothiocyanatodihydrostilbene2,2 '- disulfonic acid ( H 2 DIDS) ( two ITC groups), 4-acetamido-4-isothiocyanatostilbene-2,2'-disulfonicacid ( SITS) ( one ITC group), or 4,4-diinitrostilbene-2,2'- disulfonic acid ( DNDS) ( no ITC groups). Wounded RGM1 cells were also incubated with bicarbonate-free buffer, ATP, barium, or phloretin to determine the mechanism of ITC inhibition. At 300 mu M, DIDS or H 2 DIDS blocked restitution and wound repair by 100%, SITS blocked wound repair by 50%, and DNDS blocked wound repair by 2%. These results demonstrate the dependence of restitution and wound repair on ITC. ITC-binding purino ( ATP) receptors and K ATP channels were investigated as potential sites of inhibition, but they were found not to be the target of ITC in wound repair. Phloretin, blocking the monocarboxylate transporter (MCT), dose-dependently inhibited wound repair, and this result was exacerbated when the sodium bicarbonate cotransporter (NBC) was also blocked by bicarbonate-free conditions, resulting in 100% inhibition of wound repair with no reduction in viability when both transporters were blocked simultaneously. ITC potently inhibits both MCT and NBC, which may account for the inhibitory action of DIDS during restitution and wound repair. Reverse transcription-polymerase chain reaction data verified that MCT-1 is expressed in RGM1 cells. In conclusion, our results suggest that bicarbonate and monocarboxylate transport may work cooperatively to facilitate restitution of the gastric mucosa after injury.. |
| 6. |
Chihiro Motozono, Saeko Yanaka, Kouhei Tsumoto, Masafumi Takiguchi, Takamasa Ueno, Impact of Intrinsic Cooperative Thermodynamics of Peptide-MHC Complexes on Antiviral Activity of HIV-Specific CTL, JOURNAL OF IMMUNOLOGY, 10.4049/jimmunol.0803471, 182, 9, 5528-5536, 2009.05, The antiviral activity of HIV-specific CTL is not equally potent but rather is dependent on their specificity. But what characteristic of targeted peptides influences CTL antiviral activity remains elusive. We addressed this issue based on HLA-B35-restrieted CTLs specific for two overlapping immunodominant Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). VY8-specific CTLs were more potently cytotoxic toward HIV-infected primary CD4(+) cells than RY11-specific CTLs. Reconstruction of their TCR revealed no substantial difference in their functional avidity toward cognate Ags. Instead, the decay analysis of the peptide-MHC complex (pMHC) revealed that the VY8/HLA-B35 complex could maintain its capacity to sensitize T cells much longer than its RY11 counterpart. Corroboratively, the introduction of a mutation in the epitopes; that substantially delayed pMHC decay rendered Nef-expressing target cells more susceptible to CTL, killing. Moreover, by using differential scanning calorimetry and circular dichroism analyses, we found that the susceptible pMHC ligands for CTL, killing showed interdependent and cooperative, rather than separate or sequential, transitions within their heterotrimer components under the thermally induced unfolding process. Collectively, our results highlight the significant effects of intrinsic peptide factors that support cooperative thermodynamics within pMHC on the efficient CTL. killing of HIV-infected cells, thus providing us better insight into vaccine design. The Journal of Immunology, 2009, 182: 5528-5536.. |
| 7. |
Saeko Yanaka, Motonori Kudou, Yoshikazu Tanaka, Takumi Sasaki, Sumiyo Takemoto, Atsuko Sakata, Yukio Hattori, Tomoyuki Koshi, Shiro Futaki, Kouhei Tsumoto, Toshihiro Nakashima, Contribution of the flexible loop region to the function of staphylococcal enterotoxin B, PROTEIN ENGINEERING DESIGN & SELECTION, 10.1093/protein/gzq006, 23, 5, 415-421, 2010.05, Staphylococcal enterotoxin B (SEB), a toxin produced by Staphylococcus aureus, causes food poisoning and other fatal diseases by inducing high levels of pro-inflammatory cytokines. These cytokines are released from CD4(+) T cells and major histocompatibility complex (MHC) class II antigen-presenting cells, which are activated through binding of wild-type (WT) SEB to both the MHC class II molecule and specific T-cell receptor V beta chains. Here, we focused on a trypsin/cathepsin cleavage site of WT SEB, which is known to be cleaved in vivo between Lys97 and Lys98, located within the loop region. To know the function of the cleavage, an SEB mutant, in which both of these Lys residues have been changed to Ser, was examined. This mutant showed prolonged tolerance to protease cleavage at a different site between Thr107 and Asp108, and structural analyses revealed no major conformational differences between WT SEB and the mutant protein. However, differential scanning calorimetric analysis showed an increase in enthalpy upon thermal denaturation of the mutant protein, which correlated with the speed of cleavage between Thr107 and Asp108. The mutant protein also had slightly increased affinity for MHC. In the in vivo experiment, the SEB mutant showed lower proliferative response in peripheral blood mononuclear cells and had lower cytokine-induction activity, compared with WT SEB. These results highlight the importance of the flexible loop region for the functional, physical and chemical properties of WT SEB, thus providing insight into the nature of WT SEB that was unrevealed previously.. |
| 8. |
Saeko Yanaka, Emiko Sano, Norio Naruse, Kin-ichiro Miura, Mutsumi Futatsumori-Sugai, Jose M. M. Caaveiro, Kouhei Tsumoto, Non-core Region Modulates Interleukin-11 Signaling Activity GENERATION OF AGONIST AND ANTAGONIST VARIANTS, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M110.152561, 286, 10, 8085-8093, 2011.03, Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients with low platelet counts. From a structural point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting of four alpha-helices. We have investigated the region of hIL-11 that does not belong to the alpha-helical bundle motif, and that for the purpose of brevity we have termed "non-core region." The primary sequence of the interleukin was altered at various locations within the non-core region by introducing glycosylation sites. Functional consequences of these modifications were examined in cell-based as well as biophysical assays. Overall, the data indicated that the non-core region modulates the function of hIL-11 in two ways. First, the majority of muteins displayed enhanced cell-stimulatory properties (superagonist behavior) in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the full potential of hIL-11. Second, specific modification of a predicted mini alpha-helix led to cytokine inactivation, demonstrating that this putative structural element belongs to site III engaging a second copy of cell-receptor gp130. These findings have unveiled new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile medications based on this important cytokine.. |
| 9. |
Takashi Nishina, Sachiko Komazawa-Sakon, Saeko Yanaka, Xuehua Piao, Dong-Mei Zheng, Jiang-Hu Piao, Yuko Kojima, Shunhei Yamashina, Emiko Sano, Tracy Putoczki, Takahiro Doi, Takashi Ueno, Junji Ezaki, Hiroko Ushio, Matthias Ernst, Kouhei Tsumoto, Ko Okumura, Hiroyasu Nakano, Interleukin-11 Links Oxidative Stress and Compensatory Proliferation, SCIENCE SIGNALING, 10.1126/scisignal.2002056, 5, 207, ra5, 2012.01, Apoptotic cells can stimulate the compensatory proliferation of surrounding cells to maintain tissue homeostasis. Although oxidative stress is associated with apoptosis and necrosis, whether it contributes to compensatory proliferation is unknown. Here, we showed that interleukin-11 (IL-11), a member of the IL-6 family of proinflammatory cytokines, was produced by cells in an oxidative stress-dependent manner. IL-11 production depended on the activation in dying cells of extracellular signal-regulated kinase 2, which in turn caused the phosphorylation and accumulation of the transcription factor Fra-1 by preventing its proteasome-dependent degradation. Fra-1 was subsequently recruited to the Il11 promoter and activated gene transcription. Upon acute liver injury in mice, IL-11 was mainly produced by hepatocytes in response to reactive oxygen species that were presumably released from dying hepatocytes. IL-11 that was secreted by the dying cells then induced the phosphorylation of the transcription factor STAT3 in adjacent healthy hepatocytes, which resulted in their compensatory proliferation. Furthermore, an IL-11 receptor (IL-11R) agonist enhanced the proliferation of hepatocytes and ameliorated oxidative stress upon acetaminophen-induced liver injury. Conversely, the effects of acetaminophen were exacerbated in mice deficient in the IL-11R a subunit. Together, these results suggest that IL-11 provides a functional link between oxidative stress and compensatory proliferation.. |
| 10. |
Ryo Matsunaga, Saeko Yanaka, Satoru Nagatoishi, Kouhei Tsumoto, Hyperthin nanochains composed of self-polymerizing protein shackles, NATURE COMMUNICATIONS, 10.1038/ncomms3211, 4, 2211, 2013.07, Protein fibrils are expected to have applications as functional nanomaterials because of their sophisticated structures; however, nanoscale ordering of the functional units of protein fibrils remains challenging. Here we design a series of self-polymerizing protein monomers, referred to as protein shackles, derived from modified recombinant subunits of pili from Streptococcus pyogenes. The monomers polymerize into nanochains through spontaneous irreversible covalent bond formation. We design the protein shackles so that their reactions can be controlled by altering redox conditions, which affect disulphide bond formation between engineered cysteine residues. The interaction between the monomers improves their polymerization reactivity and determines morphologies of the polymers. In addition, green fluorescent protein-tagged protein shackles can polymerize, indicating proteins can be stably attached to the nanochains with its functionality preserved. Furthermore we demonstrate that a molecular-recognizable nanochain binds to its partner with an enhanced binding ability in solution. These characteristics are expected to be applied for novel protein nanomaterials.. |
| 11. |
Saeko Yanaka, Takamasa Ueno, Yi Shi, Jianxun Qi, George F. Gao, Kouhei Tsumoto, Kenji Sugase, Peptide-dependent Conformational Fluctuation Determines the Stability of the Human Leukocyte Antigen Class I Complex, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M114.566174, 289, 35, 24680-24690, 2014.08, Background: The stable presentation of HLA is necessary for effective T-cell activity in infectious diseases. Results: Conformational fluctuation analysis revealed a minor state of HLA. Conclusion: The minor state avoids the disintegration of HLA by tightly packing toward the peptide. The minor population correlates with the elongated presentation of HLA on the cell surface. Significance: We revealed the stabilization mechanism for HLA.
In immune-mediated control of pathogens, human leukocyte antigen (HLA) class I presents various antigenic peptides to CD8(+) T-cells. Long-lived peptide presentation is important for efficient antigen-specific T-cell activation. Presentation time depends on the peptide sequence and the stability of the peptide-HLA complex (pHLA). However, the determinant of peptide-dependent pHLA stability remains elusive. Here, to reveal the pHLA stabilization mechanism, we examined the crystal structures of an HLA class I allomorph in complex with HIV-derived peptides and evaluated site-specific conformational fluctuations using NMR. Although the crystal structures of various pHLAs were almost identical independent of the peptides, fluctuation analyses identified a peptide-dependent minor state that would be more tightly packed toward the peptide. The minor population correlated well with the thermostability and cell surface presentation of pHLA, indicating that this newly identified minor state is important for stabilizing the pHLA and facilitating T-cell recognition.. |
| 12. |
Ayako Furukawa, Tsuyoshi Konuma, Saeko Yanaka, Kenji Sugase, Quantitative analysis of protein-ligand interactions by NMR, PROGRESS IN NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY, 10.1016/j.pnmrs.2016.02.002, 96, 47-57, 2016.08, Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (K-D), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R-2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R-2 dispersion or ZZ-exchange, methods that can determine the association rate, k(on), dissociation rate, k(off) and K-D have been developed. In these approaches, R-2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R-2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used to analyze population-averaged NMR quantities. Essentially, to apply NMR successfully, both the type of experiment and equation to fit the data must be carefully and specifically chosen for the protein-ligand interaction under analysis. In this review, we first explain the exchange regimes and kinetic models of protein-ligand interactions, and then describe the NMR methods that quantitatively analyze these specific interactions. (C) 2016 Elsevier B.V. All rights reserved.. |
| 13. |
Gengwei Yan, Takumi Yamaguchi, Tatsuya Suzuki, Saeko Yanaka, Sota Sato, Makoto Fujita, Koichi Kato, Hyper-Assembly of Self-Assembled Glycoclusters Mediated by Specific Carbohydrate-Carbohydrate Interactions, CHEMISTRY-AN ASIAN JOURNAL, 10.1002/asia.201700202, 12, 9, 968-972, 2017.05, Hybridization of a self-assembled, spherical complex with oligosaccharides containing Lewis X, a functional trisaccharide displayed on various cell surfaces, yielded well-defined glycoclusters. The self-assembled glycoclusters exhibited homophilic hyper-assembly in aqueous solution in a Ca2+-dependent manner through specific carbohydrate-carbohydrate interactions, offering a structural scaffold for functional biomimetic systems.. |
| 14. |
Saeko Yanaka, Kenji Sugase, Exploration of the Conformational Dynamics of Major Histocompatibility Complex Molecules, FRONTIERS IN IMMUNOLOGY, 10.3389/fimmu.2017.00632, 8, 632, 2017.05, Major histocompatibility complex (MHC) molecules are loaded with a wide variety of self- and non-self-peptides in their binding grooves and present these to T cell receptors (TCRs) in order to activate the adaptive immune system. A large number of crystal structures of different MHC alleles with different bound peptides have been determined, and they have been found to be quite similar to one another regardless of the bound peptide sequence. The structures do not change markedly even when forming complexes with TCRs. Nonetheless, the degree of TCR activation does differ markedly depending on the peptide presented by the MHC. Recent structural studies in solution rather than as crystals have suggested that the conformational dynamics of MHC molecules may be responsible for the MHC stability differences. Furthermore, it was shown that the conformational dynamics of MHC molecules is important for peptide loading and presentation to TCR. Here, we describe the static and dynamic structures of MHC molecules and appropriate methods to analyze them. We focus particularly on nuclear magnetic resonance (NMR), one of the most powerful tools to study dynamic properties of proteins. The number of such studies in the literature is limited, but in this review, we show that NMR is valuable for elucidating the structural dynamics of MHC molecules.. |
| 15. |
Saeko Yanaka, Yoshitaka Moriwaki, Kouhei Tsumoto, Kenji Sugase, Elucidation of potential sites for antibody engineering by fluctuation editing, SCIENTIFIC REPORTS, 10.1038/s41598-017-10246-9, 7, 1, 9597, 2017.08, Target-specific monoclonal antibodies can be routinely acquired, but the sequences of naturally acquired antibodies are not always affinity-matured and methods that increase antigen affinity are desirable. Most biophysical studies have focused on the complementary determining region (CDR), which directly contacts the antigen; however, it remains difficult to increase the affinity as much as desired. While strategies to alter the CDR to increase antibody affinity are abundant, those that target non-CDR regions are scarce. Here we describe a new method, designated fluctuation editing, which identifies potential mutation sites and engineers a high-affinity antibody based on conformational fluctuations observed by NMR relaxation dispersion. Our data show that relaxation dispersion detects important fluctuating residues that are not located in the CDR and that increase antigen-antibody affinity by point mutation. The affinity-increased mutants are shown to fluctuate less in their free form and to form a more packed structure in their antigen-bound form.. |
| 16. |
Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa.. |
| 17. |
Tatsuya Suzuki, Megumi Kajino, Saeko Yanaka, Tong Zhu, Hirokazu Yagi, Tadashi Satoh, Takumi Yamaguchi, Koichi Kato, Conformational Analysis of a High-Mannose-Type Oligosaccharide Displaying Glucosyl Determinant Recognised by Molecular Chaperones Using NMR-Validated Molecular Dynamics Simulation., Chembiochem : a European journal of chemical biology, 10.1002/cbic.201600595, 18, 4, 396-401, 2017.02, Exploration of the conformational spaces of flexible oligosaccharides is essential to gain deeper insights into their functional mechanisms. Here we characterised dynamic conformation of a high-mannose-type dodecasaccharide with a terminal glucose residue, a critical determinant recognised by molecular chaperones. The dodecasaccharide was prepared by our developed chemoenzymatic technique, which uses 13 C labelling and lanthanide tagging to detect conformation-dependent paramagnetic effects by NMR spectroscopy. The NMR-validated molecular dynamics simulation produced the dynamic conformational ensemble of the dodecasaccharide. This determined its spatial distribution as well as the glycosidic linkage conformation of the terminal glucose determinant. Moreover, comparison of our results with previously reported crystallographic data indicates that the chaperone binding to its target oligosaccharides involves an induced-fit mechanism.. |
| 18. |
Characterization of conformational deformation-coupled interaction between immunoglobulin G1 Fc glycoprotein and a low-affinity Fcγ receptor by deuteration-assisted small-angle neutron scattering.. |
| 19. |
Theoretical and Experimental Studies on Inclusion Complexes of Pinostrobin and β-Cyclodextrins.. |
| 20. |
Yogo R, Yanaka S, Kato K, Backbone 1H, 13C, and 15N assignments of the extracellular region of human Fcγ receptor IIIb., Biomolecular NMR assignments, 10.1007/s12104-018-9809-4, 12, 1, 201-204, 2018.02. |
| 21. |
Yogo R, Yamaguchi Y, Watanabe H, Yagi H, Satoh T, Nakanishi M, Onitsuka M, Omasa T, Shimada M, Maruno T, Torisu T, Watanabe S, Higo D, Uchihashi T, Yanaka S, Uchiyama S, Kato K, The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III, Scientific Reports, 10.1038/s41598-019-48323-w, 9, 1, 11957, 2019.12. |
| 22. |
Tatsuya Suzuki, Saeko Yanaka, Tokio Watanabe, Gengwei Yan, Tadashi Satoh, Hirokazu Yagi, Takumi Yamaguchi, Koichi Kato, Remodeling of the Oligosaccharide Conformational Space in the Prebound State To Improve Lectin-Binding Affinity., Biochemistry, 10.1021/acs.biochem.9b00594, 59, 34, 3180-3185, 2019.10, We developed an approach to improve the lectin-binding affinity of an oligosaccharide by remodeling its conformational space in the precomplexed state. To develop this approach, we used a Lewis X-containing oligosaccharide interacting with RSL as a model system. Using an experimentally validated molecular dynamics simulation, we designed a Lewis X analogue with an increased population of conformational species that were originally very minor but exclusively accessible to the target lectin without steric hindrance by modifying the nonreducing terminal galactose, which does not directly contact the lectin in the complex. This Lewis X mimetic showed 17 times higher affinity for the lectin than the native counterpart. Our approach, complementing the lectin-bound-state optimizations, offers an alternative strategy to create high-affinity oligosaccharides by increasing populations of on-pathway metastable conformers.. |
| 23. |
Mutational and Combinatorial Control of Self-Assembling and Disassembling of Human Proteasome α Subunits. |
| 24. |
Hirokazu Yagi, Saeko Yanaka, Rina Yogo, Akari Ikeda, Masayoshi Onitsuka, Toshio Yamazaki, Tatsuya Kato, Enoch Y Park, Jun Yokoyama, Koichi Kato, Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling., Biomolecules, 10.3390/biom10111482, 10, 11, 2020.10, Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.. |
| 25. |
Maho Yagi-Utsumi, Mahesh S Chandak, Saeko Yanaka, Methanee Hiranyakorn, Takashi Nakamura, Koichi Kato, Kunihiro Kuwajima, Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR., Biophysical journal, 10.1016/j.bpj.2020.10.003, 119, 10, 2029-2038, 2020.10, The characterization of residual structures persistent in unfolded proteins in concentrated denaturant solution is currently an important issue in studies of protein folding because the residual structure present, if any, in the unfolded state may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the hydrogen/deuterium (H/D)-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. We employed a dimethylsulfoxide (DMSO)-quenched H/D-exchange NMR technique with the use of spin desalting columns, which allowed us to perform a quick medium exchange from 6 M guanidinium chloride to a quenching DMSO solution. Based on the backbone resonance assignment of ubiquitin in the DMSO solution, we successfully investigated the H/D-exchange kinetics of 60 identified peptide amide groups in the ubiquitin sequence. Although a majority of these amide groups were not protected, certain amide groups involved in a middle helix (residues 23-34) and an N-terminal β-hairpin (residues 2-16) were significantly protected with a protection factor of 2.1-4.2, indicating that there were residual structures in unfolded ubiquitin and that these amide groups were more than 52% hydrogen bonded in the residual structures. We show that the hydrogen-bonded residual structures in the α-helix and the β-hairpin are formed even in 6 M guanidinium chloride, suggesting that these residual structures may function as a folding initiation site to guide the subsequent folding reactions of ubiquitin.. |
| 26. |
Ryo Ohtani, Kenichi Kawano, Masanao Kinoshita, Saeko Yanaka, Hikaru Watanabe, Kenji Hirai, Shiroh Futaki, Nobuaki Matsumori, Hiroshi Uji-I, Masaaki Ohba, Koichi Kato, Shinya Hayami, Pseudo-membrane jackets: Two-dimensional coordination polymers achieving visible phase separation in cell membrane., Angewandte Chemie (International ed. in English), 10.1002/anie.202006600, 59, 41, 17931-17937, 2020.06, Cell membranes contain lateral systems that consist of various lipid compositions and actin cytoskeleton, providing two-dimensional (2D) platforms for chemical reactions. However, such complex 2D environments have not yet been used as a synthetic platform for artificial 2D nanomaterials. Herein, we demonstrate the direct synthesis of 2D coordination polymers (CPs) at the liquid-cell interface of the plasma membrane of living cells. The coordination-driven self-assembly of networking metal complex lipids produces cyanide-bridged CP layers with metal ions, enabling 'pseudo-membrane jackets' that produce long-lived micro-domains with a size of 1-5 μm. The resultant artificial and visible phase separation systems remain stable even in the absence of actin skeletons in cells. Moreover, the jackets achieved the enhancement of cellular calcium response to ATP. This confirms that the artificial nanomaterials formed via moderate coordinative intermolecular interactions create a 2D scaffold on complex cell membrane environments, thereby providing a unique tool for the chemical control of cell functions.. |
| 27. |
Tamiki Komatsuzaki, Haruki Nakamura, Jeremy Tame, Saeko Yanaka, Takeharu Nagai, Kuniaki Nagayama, Editorial for the Special Issue of Biophysical Reviews focused on the Biophysical Society of Japan with select scientific content from the 57th BSJ annual meeting, Miyazaki, Japan., Biophysical reviews, 10.1007/s12551-020-00691-8, 12, 2, 183-185, 2020.03. |
| 28. |
Saeko Yanaka, Susumu Uchiyama, Current status and issues of protein solution biophysics-Session 1SDP., Biophysical reviews, 10.1007/s12551-020-00671-y, 12, 2, 263-264, 2020.04. |
| 29. |
Supa Hannongbua, Siriluk Ratanabunyong, Niran Aeksiri, Saeko Yanaka, Maho Yagi-Utsumi, Koichi Kato, Kiattawee Choowongkomol, Characterization of new DNA Aptamers for anti-HIV-1 Reverse Transcriptase., Chembiochem : a European journal of chemical biology, 10.1002/cbic.202000633, 22, 5, 915-923, 2020.10, Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a necessary enzyme for retroviral replication which is the main target for antiviral therapy against AIDs. The effective anti-HIV-1 RT drugs are classified into two groups; nucleoside inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI) which inhibit the DNA polymerase function. In this study, new DNA aptamers were isolated as anti-HIV-1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild type (WT) HIV-1 RT and gave a K D value of 75.10 ± 0.29 nM and an IC 50 value of 84.81 ± 8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103N/Y181C double mutant (KY) HIV-1 RT by around 80%. Furthermore, the ITC results showed that this aptamer has slightly small binding enthalpies with both WT and KY HIV-1 RTs through which the complex may form a hydrophobic interaction or non-covalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV-1 RT at the connection domain.. |
| 30. |
Saeko Yanaka, Rina Yogo, Koichi Kato, Biophysical characterization of dynamic structures of immunoglobulin G., Biophysical reviews, 10.1007/s12551-020-00698-1, 2020.05, Immunoglobulin G (IgG) is a major antibody and functions as a hub linking specific antigen binding and recruitment of effector molecules typified by Fcγ receptors (FcγRs). These activities are associated primarily with interactions involving its Fab and Fc sites, respectively. An IgG molecule is characterized by a multiple domain modular structure with conserved N-glycosylation in Fc. The molecule displays significant freedom in internal motion on various spatiotemporal scales. The consequent conformational flexibility and plasticity of IgG glycoproteins are functionally significant and potentially important factors for design and engineering of antibodies with enhanced functionality. In this article, experimental and computational approaches are outlined for characterizing the conformational dynamics of IgG molecules in solution. In particular, the importance of integration of these approaches is highlighted, as illustrated by dynamic intramolecular interactions between the pair of N-glycans and their proximal amino acid residues in Fc. These interactions can critically affect effector functions mediated by human IgG1 and FcγRIII. Further improvements in individual biophysical techniques and their integration will advance understanding of dynamic behaviors of antibodies in physiological and pathological conditions. Such understanding will provide opportunities for engineering antibodies through controlling allosteric networks in IgG molecules.. |
| 31. |
Kazuhisa Miyazawa, Satoru G Itoh, Hiroki Watanabe, Takayuki Uchihashi, Saeko Yanaka, Maho Yagi-Utsumi, Koichi Kato, Kazuharu Arakawa, Hisashi Okumura, Tardigrade Secretory-Abundant Heat-Soluble Protein Has a Flexible β-Barrel Structure in Solution and Keeps This Structure in Dehydration., The journal of physical chemistry. B, 10.1021/acs.jpcb.1c04850, 125, 32, 9145-9154, 2021.08, Secretory-abundant heat-soluble (SAHS) proteins are unique heat-soluble proteins of Tardigrada and are believed to play an essential role in anhydrobiosis, a latent state of life induced by desiccation. To investigate the dynamic properties, molecular dynamics (MD) simulations of a SAHS protein, RvSAHS1, were performed in solution and under dehydrating conditions. For comparison purposes, MD simulations of a human liver-type fatty-acid binding protein (LFABP) were performed in solution. Furthermore, high-speed atomic force microscopy observations were conducted to ascertain the results of the MD simulations. Three properties of RvSAHS1 were found as follows. (1) The entrance region of RvSAHS1 is more flexible and can be more extensive in solutions compared with that of a human LFABP because there is no salt bridge between the βD and βE strands. (2) The intrinsically disordered domain in the N-terminal region significantly fluctuates and can form an amphiphilic α-helix. (3) The size of the entrance region gets smaller along with dehydration, keeping the β-barrel structure. Overall, the obtained results provide atomic-level dynamics of SAHS proteins.. |
| 32. |
Saeko Yanaka, Yoshiki Yamaguchi, Takeshi Takizawa, Yohei Miyanoiri, Rina Yogo, Ichio Shimada, Koichi Kato, NMR assignments of the N-glycans of the Fc fragment of mouse immunoglobulin G2b glycoprotein., Biomolecular NMR assignments, 10.1007/s12104-020-10004-5, 15, 1, 187-192, 2021.01, The Fc portion of immunoglobulin G (IgG) promotes defensive effector functions in the immune system by interacting with Fcγ receptors and complement component C1q. These interactions critically depend on N-glycosylation at Asn297 of each CH2 domain, where biantennary complex-type oligosaccharides contain microheterogeneities resulting primarily from the presence or absence of non-reducing terminal galactose residues. Crystal structures of Fc have shown that a pair of N-glycans is located between the two CH2 domains. Here we applied our metabolic isotope labeling technique using mammalian cells for in-solution structural characterization of mouse IgG2b-Fc glycoforms with a molecular mass of 54 kDa. Based on spectral assignments of the N-glycans as well as polypeptide backbones of Fc, we probed conformational perturbations of Fc induced by N-glycan trimming, especially enzymatic degalactosylation. The results indicated that degalactosylation structurally perturbed the Fc region through rearrangement of glycan-protein interactions. The spectral assignments of IgG2b-Fc glycoprotein will provide the basis for NMR investigation of its dynamic conformations and interactions with effector molecules in solution.. |
| 33. |
Ryo Ohtani, Yuka Anegawa, Hikaru Watanabe, Yutaro Tajima, Masanao Kinoshita, Nobuaki Matsumori, Kenichi Kawano, Saeko Yanaka, Koichi Kato, Masaaki Nakamura, Masaaki Ohba, Shinya Hayami, Metal complex lipids for fluid-fluid phase separation in co-assembled phospholipid membranes., Angewandte Chemie (International ed. in English), 10.1002/anie.202102774, 60, 24, 13603-13608, 2021.03, We demonstrate a fluid-fluid phase separation in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes using a metal complex lipid of type [Mn(L1)] (1; HL1 = 1-(2-hydroxybenzamide)-2-(2-hydroxy-3-formyl-5-hexadecyloxybenzylideneamino)ethane. Small amount of 1 produces two separated domains in DMPC, whose phase transition temperatures of lipids (Tc) are both lower than that of the pristine DMPC. Variable temperature fluorescent microscopy for giant-unilamellar vesicles of DMPC/1 hybrids demonstrates that visible phase separations remain in fluid phases up to 37 °C, which is clearly over the Tc of DMPC. This provides a new dimension for the application of metal complex lipids toward controlling lipid distributions in fluid membranes.. |
| 34. |
Maho Yagi-Utsumi, Kazuhiro Aoki, Hiroki Watanabe, Chihong Song, Seiji Nishimura, Tadashi Satoh, Saeko Yanaka, Christian Ganser, Sae Tanaka, Vincent Schnapka, Ean Wai Goh, Yuji Furutani, Kazuyoshi Murata, Takayuki Uchihashi, Kazuharu Arakawa, Koichi Kato, Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade., Scientific reports, 10.1038/s41598-021-00724-6, 11, 1, 21328-21328, 2021.11, Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.. |
| 35. |
Nobuhiro Sato, Rina Yogo, Saeko Yanaka, Anne Martel, Lionel Porcar, Ken Morishima, Rintaro Inoue, Taiki Tominaga, Takao Arimori, Junichi Takagi, Masaaki Sugiyama, Koichi Kato, A feasibility study of inverse contrast-matching small-angle neutron scattering method combined with size exclusion chromatography using antibody interactions as model systems., Journal of biochemistry, 10.1093/jb/mvab012, 2021.02, Small-angle neutron scattering (SANS) and small- angle X-ray scattering (SAXS) are powerful techniques for the structural characterization of biomolecular complexes. In particular, SANS enables a selective observation of specific components in complexes by selective deuteration with contrast-matching techniques. In most cases, however, biomolecular interaction systems with heterogeneous oligomers often contain unfavorable aggregates and unbound species, hampering data interpretation. To overcome these problems, SAXS has been recently combined with size exclusion chromatography (SEC), which enables the isolation of the target complex in a multi-component system. By contrast, SEC-SANS is only at a preliminary stage. Hence, we herein perform a feasibility study of this method based on our newly developed inverse contrast-matching (iCM) SANS technique using antibody interactions as model systems. Immunoglobulin G (IgG) or its Fc fragment was mixed with 75% deuterated Fc-binding proteins, i.e. a mutated form of IgG-degrading enzyme of Streptococcus pyogenes and a soluble form of Fcγ receptor IIIb, and subjected to SEC-SANS as well as SEC-SAXS as reference. We successfully observe SANS from the non-deuterated IgG or Fc formed in complex with these binding partners, which were unobservable in terms of SANS in D2O, hence demonstrating the potential utility of the SEC-iCM-SANS approach.. |
| 36. |
Saeko Yanaka, Hirokazu Yagi, Rina Yogo, Masayoshi Onitsuka, Koichi Kato, Glutamine-free mammalian expression of recombinant glycoproteins with uniform isotope labeling: an application for NMR analysis of pharmaceutically relevant Fc glycoforms of human immunoglobulin G1, Journal of Biomolecular NMR, 10.1007/s10858-021-00387-5, 76, 1-2, 17-22, 2022.01, Mammalian cells are widely used for producing recombinant glycoproteins of pharmaceutical interest. However, a major drawback of using mammalian cells is the high production costs associated with uniformly isotope-labeled glycoproteins due to the large quantity of labeled L-glutamine required for their growth. To address this problem, we developed a cost-saving method for uniform isotope labeling by cultivating the mammalian cells under glutamine-free conditions, which was achieved by co-expression of glutamine synthase. We demonstrate the utility of this approach using fucosylated and non-fucosylated Fc glycoforms of human immunoglobulin G1.. |
| 37. |
Takanori Matsumaru, Kasumi Sakuratani, Saeko Yanaka, Koichi Kato, Sho Yamasaki, Yukari Fujimoto, Fungal β‐Mannosyloxymannitol Glycolipids and Their Analogues: Synthesis and Mincle‐Mediated Signaling Activity, European Journal of Organic Chemistry, 10.1002/ejoc.202200109, 2022, 20, 2022.05. |
| 38. |
Kunihiro Kuwajima, Maho Yagi-Utsumi, Saeko Yanaka, Koichi Kato, DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science., Molecules (Basel, Switzerland), 10.3390/molecules27123748, 27, 12, 2022.06, Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching techniques are indispensable, because the exchange reaction is usually too fast to follow by 2D NMR. In this article, we will describe the dimethylsulfoxide (DMSO)-quenched H/D-exchange method and its applications in protein science. In this method, the H/D-exchange buffer is replaced by an aprotic DMSO solution, which quenches the exchange reaction. We have improved the DMSO-quenched method by using spin desalting columns, which are used for medium exchange from the H/D-exchange buffer to the DMSO solution. This improvement has allowed us to monitor the H/D exchange of proteins at a high concentration of salts or denaturants. We describe methodological details of the improved DMSO-quenched method and present a case study using the improved method on the H/D-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride.. |
| 39. |
Siriluk Ratanabunyong, Supaphorn Seetaha, Supa Hannongbua, Saeko Yanaka, Maho Yagi-Utsumi, Koichi Kato, Atchara Paemanee, Kiattawee Choowongkomon, Biophysical Characterization of Novel DNA Aptamers against K103N/Y181C Double Mutant HIV-1 Reverse Transcriptase., Molecules (Basel, Switzerland), 10.3390/molecules27010285, 27, 1, 2022.01, The human immunodeficiency virus type-1 Reverse Transcriptase (HIV-1 RT) plays a pivotal role in essential viral replication and is the main target for antiviral therapy. The anti-HIV-1 RT drugs address resistance-associated mutations. This research focused on isolating the potential specific DNA aptamers against K103N/Y181C double mutant HIV-1 RT. Five DNA aptamers showed low IC50 values against both the KY-mutant HIV-1 RT and wildtype (WT) HIV-1 RT. The kinetic binding affinity forms surface plasmon resonance of both KY-mutant and WT HIV-1 RTs in the range of 0.06-2 μM and 0.15-2 μM, respectively. Among these aptamers, the KY44 aptamer was chosen to study the interaction of HIV-1 RTs-DNA aptamer complex by NMR experiments. The NMR results indicate that the aptamer could interact with both WT and KY-mutant HIV-1 RT at the NNRTI drug binding pocket by inducing a chemical shift at methionine residues. Furthermore, KY44 could inhibit pseudo-HIV particle infection in HEK293 cells with nearly 80% inhibition and showed low cytotoxicity on HEK293 cells. These together indicated that the KY44 aptamer could be a potential inhibitor of both WT and KY-mutant HIV-RT.. |
