Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1007/s12104-020-10004-5

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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.

DOI： 10.1007/s12551-020-00698-1

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Language：English  

DOI： 10.1038/s41598-019-48323-w

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Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

DOI： 10.3390/ijms21010147

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DOI： 10.3390/antib8030039

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DOI： 10.1007/s10858-018-0169-2

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DOI： 10.3390/molecules22101619

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DOI： 10.1016/j.jbc.2025.111012

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DOI： 10.1002/pro.70404

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/ijms26157266

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The amyloid β (Aβ) Tottori variant (D7N) exhibits unique aggregation behaviors and altered fibril formation, posing challenges for structural characterization. To overcome this, the microgravity environment on the International Space Station was employed to study Tottori-type Aβ40 fibril formation and structure. Under Earth gravity, Tottori-type Aβ40 primarily formed nonfibrillar aggregates, hindering detailed structural analysis. In contrast, microgravity significantly enhanced fibril formation and minimized amorphous aggregates. Cryo-electron microscopy revealed two structurally distinct fibril types, each comprising different protomer conformations. In both types, the N-terminal segment was disordered and nor resolved in the density maps. The D7N mutation disrupts the protection of the core by the N-terminal segment often observed in wild-type Aβ40 fibrils, enhancing the hydrophobicity-mediated aggregation propensity. However, microgravity suppressed kinetic traps and facilitated high-quality fibril formation suitable for structural studies that can explore the free energy landscape of Aβ fibril formation. These findings demonstrate the utility of microgravity for studying familial Aβ variants and potentially accelerate our understanding of Aβ aggregation mechanisms in Alzheimer's disease.

DOI： 10.1021/acschemneuro.5c00217

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DOI： 10.1016/j.sbi.2025.103049

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UNLABELLED: Ammonia-oxidizing archaea of the phylum Nitrososphaerota, formerly known as Thaumarchaeota, are globally distributed and play critical roles in the nitrogen and carbon cycles, particularly in environments with low ammonia concentrations. Like most archaea, Nitrososphaerota cells are enveloped by S-layer proteins, implicated in concentrating ammonium ions. These proteins are typically modified post-translationally by N-glycans, which often play significant roles in various biological processes, including protein function regulation, protection from phages, and environmental adaptation. Nevertheless, the glycobiological characteristics of Nitrososphaerota remain largely unexplored. Here, we investigated the glycoproteome of ammonia-oxidizing Nitrososphaerota, specifically focusing on the terrestrial Nitrososphaera viennensis and the marine Nitrosopumilus piranensis. Both species exhibited similar protein arrays throughout their growth phases, including those associated with N-glycosylation. Ns. viennensis consistently exhibited N-glycosylation predominantly on an S-layer protein and multicopper oxidase domain-containing proteins throughout all growth phases, with a marked increase during and after the late exponential phase. The glycan, characterized as a novel hexasaccharide with a chitobiose core, is hypothesized to play a role in nitrogen storage due to its probable nitrogen-rich composition, modifying asparagine residues within the conserved triplet sequence (Asn-X-Ser or -Thr). In contrast, Np. piranensis also showed a high abundance of S-layer protein but displayed no apparent N-glycosylation on any protein, suggesting variability in cell surface physical properties between these archaea. Despite similarities in their proteomes and energy metabolism, these two archaea exhibited significant differences in post-translational modification of proteins, revealing previously unrecognized diversity that may have implications for understanding their adaptive transitions to diverse environments. IMPORTANCE: Autotrophic ammonia-oxidizing archaea of the phylum Nitrososphaerota, formerly known as Thaumarchaeota, are notoriously difficult to culture yet play important roles in the global nitrogen and carbon cycles. Inhabiting environments with extremely low ammonia concentrations, these archaea are expected to conserve ammonia strictly for energy production. However, using advanced liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance techniques, we discovered that one of these archaea decorates its cell surface proteins with the most nitrogen-rich glycan identified to date, suggesting a previously unrecognized function of protein glycosylation in nitrogen storage. This newly identified N-glycan, with a chitobiose core similar to those in Thermoproteota and eukaryotes, not only deepens our understanding of archaeal evolution but also underscores the molecular adaptations enabling these archaea to thrive in diverse environments.

DOI： 10.1128/mbio.03859-24

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DOI： 10.3390/ijms26031179

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DOI： 10.1007/978-981-97-6830-1_7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.isci.2024.111457

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Members of the kingdom Nanobdellati, previously known as DPANN archaea, are characterized by ultrasmall cell sizes and reduced genomes. They primarily thrive through ectosymbiotic interactions with specific hosts in diverse environments. Recent successful cultivations have emphasized the importance of adhesion to host cells for understanding the ecophysiology of Nanobdellati. Cell adhesion is often mediated by cell surface carbohydrates, and in archaea, this may be facilitated by the glycosylated S-layer protein that typically coats their cell surface. In this study, we conducted glycoproteomic analyses on two co-cultures of Nanobdellati with their host archaea, as well as on pure cultures of both host and non-host archaea. Nanobdellati exhibited various glycoproteins, including archaellins and hypothetical proteins, with glycans that were structurally distinct from those of their hosts. This indicated that Nanobdellati autonomously synthesize their glycans for protein modifications probably using host-derived substrates, despite the high energy cost. Glycan modifications on Nanobdellati proteins consistently occurred on asparagine residues within the N-X-S/T sequon, consistent with patterns observed across archaea, bacteria, and eukaryotes. In both host and non-host archaea, S-layer proteins were commonly modified with hexose, N-acetylhexosamine, and sulfonated deoxyhexose. However, the N-glycan structures of host archaea, characterized by distinct sugars such as deoxyhexose, nonulosonate sugar, and pentose at the nonreducing ends, were implicated in enabling Nanobdellati to differentiate between host and non-host cells. Interestingly, the specific sugar, xylose, was eliminated from the N-glycan in a host archaeon when co-cultured with Nanobdella. These findings enhance our understanding of the role of protein glycosylation in archaeal interactions.IMPORTANCENanobdellati archaea, formerly known as DPANN, are phylogenetically diverse, widely distributed, and obligately ectosymbiotic. The molecular mechanisms by which Nanobdellati recognize and adhere to their specific hosts remain largely unexplored. Protein glycosylation, a fundamental biological mechanism observed across all domains of life, is often crucial for various cell-cell interactions. This study provides the first insights into the glycoproteome of Nanobdellati and their host and non-host archaea. We discovered that Nanobdellati autonomously synthesize glycans for protein modifications, probably utilizing substrates derived from their hosts. Additionally, we identified distinctive glycosylation patterns that suggest mechanisms through which Nanobdellati differentiate between host and non-host cells. This research significantly advances our understanding of the molecular basis of microbial interactions in extreme environments.

DOI： 10.1128/jb.00205-24

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DOI： 10.1101/2024.05.11.593658

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

IgG molecules that bind antigen on the membrane of target cells spontaneously form hexameric rings, thus recruiting C1 to initiate the complement pathway. However, our previous report indicated that a mouse IgG mutant lacking the Cγ1 domain activates the pathway independently of antigen presence through its monomeric interaction with C1q via the CL domain, as well as Fc. In this study, we investigated the potential interaction between C1q and human CL isoforms. Quantitative single molecule observations using high-speed atomic force microscopy revealed that human Cκ exhibited comparable C1q binding capabilities with its mouse counterpart, surpassing the Cλ types, which have a higher isoelectric point than the Cκ domains. Nuclear magnetic resonance and mutation experiments indicated that the human and mouse Cκ domains share a common primary binding site for C1q, centered on Glu194, a residue conserved in the Cκ domains but absent in the Cλ domains. Additionally, the Cγ1 domain, with its high isoelectric point, can cause electrostatic repulsion to the C1q head and impede the C1q-interaction adjustability of the Cκ domain in Fab. The removal of the Cγ1 domain is considered to eliminate these factors and thus promote Cκ interaction with C1q with the potential risk of uncontrolled activation of the complement pathway in vivo in the absence of antigen. However, this research underscores the presence of potential subsites in Fab for C1q binding, offering promising targets for antibody engineering to refine therapeutic antibody design.

DOI： 10.1093/intimm/dxae017

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This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.

DOI： 10.1248/bpb.b23-00751

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Archaeal cells are typically enveloped by glycosylated S-layer proteins. Archaeal protein glycosylation provides valuable insights not only into their adaptation to their niches but also into their evolutionary trajectory. Notably, thermophilic Thermoproteota modify proteins with N-glycans that include two GlcNAc units at the reducing end, resembling the "core structure" preserved across eukaryotes. Recently, Asgard archaea, now classified as members of the phylum Promethearchaeota, have offered unprecedented opportunities for understanding the role of archaea in eukaryogenesis. Despite the presence of genes indicative of protein N-glycosylation in this archaeal group, these have not been experimentally investigated. Here we performed a glycoproteome analysis of the firstly isolated Asgard archaeon Promethearchaeum syntrophicum. Over 700 different proteins were identified through high-resolution LC-MS/MS analysis, however, there was no evidence of either the presence or glycosylation of putative S-layer proteins. Instead, N-glycosylation in this archaeon was primarily observed in an extracellular solute-binding protein, possibly related to chemoreception or transmembrane transport of oligopeptides. The glycan modification occurred on an asparagine residue located within the conserved N-X-S/T sequon, consistent with the pattern found in other archaea, bacteria, and eukaryotes. Unexpectedly, three structurally different N-glycans lacking the conventional core structure were identified in this archaeon, presenting unique compositions that included atypical sugars. Notably, one of these sugars was likely HexNAc modified with a threonine residue, similar to modifications previously observed in mesophilic methanogens within the Methanobacteriati. Our findings advance our understanding of Asgard archaea physiology and evolutionary dynamics.

DOI： 10.1016/j.bbadva.2024.100118

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DOI： 10.3390/ijms24076075

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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.

DOI： 10.1002/pro.4569

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Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

DOI： 10.1080/19420862.2022.2038531

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Forum: Carbohydrates Coming of Age  

DOI： 10.4052/tigg.2042.1e

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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.

DOI： 10.3390/molecules27123748

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/ejoc.202200109

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/ejoc.202200109

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

<jats:p>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....</jats:p>

DOI： 10.1039/d2sc02364d

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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.

DOI： 10.3390/molecules27010285

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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.

DOI： 10.1038/s41598-021-00724-6

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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.

DOI： 10.1021/acs.jpcb.1c04850

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Publishing type：Part of collection (book)   Publisher：Elsevier  

DOI： 10.1016/b978-0-12-819475-1.00044-4

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Publishing type：Research paper (scientific journal)   Publisher：Rangsit University  

DOI： 10.14456/JCST.2021.8

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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.

DOI： 10.1002/anie.202102774

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Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

<p>Oligosaccharides play versatile roles in various biological systems but are difficult to characterize from a structural viewpoint due to their remarkable degrees of freedom in internal motion. Therefore, molecular dynamics...</p>

DOI： 10.1039/d0cp06448c

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Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1093/jb/mvab012

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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.

DOI： 10.3390/biom10111482

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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.

DOI： 10.1002/cbic.202000633

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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.

DOI： 10.1016/j.bpj.2020.10.003

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Ubiquitin (Ub) molecules can be enzymatically connected through a specific isopeptide linkage, thereby mediating various cellular processes by binding to Ub-interacting proteins through their hydrophobic surfaces. The Lys48-linked Ub chains, which serve as tags for proteasomal degradation, undergo conformational interconversions between open and closed states, in which the hydrophobic surfaces are exposed and shielded, respectively. Here, we provide a quantitative view of such dynamic processes of Lys48-linked triUb and tetraUb in solution. The native and cyclic forms of Ub chains are prepared with isotope labeling by in vitro enzymatic reactions. Our comparative NMR analyses using monomeric Ub and cyclic diUb as reference molecules enabled the quantification of populations of the open and closed states for each Ub unit of the native Ub chains. The data indicate that the most distal Ub unit in the Ub chains is the most apt to expose its hydrophobic surface, suggesting its preferential involvement in interactions with the Ub-recognizing proteins. We also demonstrate that a mutational modification of the distal end of the Ub chain can remotely affect the solvent exposure of the hydrophobic surfaces of the other Ub units, suggesting that Ub chains could be unique design frameworks for the creation of allosterically controllable multidomain proteins.

DOI： 10.3390/ijms21155351

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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.

DOI： 10.1002/anie.202006600

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Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s41526-020-0107-y

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DOI： 10.1007/s12551-020-00671-y

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DOI： 10.1007/s12551-020-00691-8

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DOI： 10.1038/s41598-019-48911-w

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DOI： 10.1038/s41598-019-50722-y

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Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1021/acs.biochem.9b00594

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Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

DOI： 10.3390/ijms20092308

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DOI： 10.1080/19420862.2018.1544454

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Language：English   Publishing type：Research paper (scientific journal)  

Fcγ receptor (FcγR) promotes various immune responses through interactions with the Fc portion of immunoglobulin G (IgG). FcγRIIIb is a glycosylphosphatidylinositol-linked protein expressed on neutrophils and triggers degranulation and opsonic phagocytosis. The extracellular region of FcγR is composed of two Ig-fold domains and can be cleaved as a soluble form (sFcγRIIIb), which is also reactive with complement receptor type 3. Although structure and Fc interaction of sFcγRIIIb have been characterized by X-ray crystallography, little has been known about its structure in solution. We herein report the backbone NMR assignments of human sFcγRIIIb to gain basic understanding of functional IgG-FcγRIII interactions of immunological and biopharmaceutical interest regarding the structural investigation.

DOI： 10.1007/s12104-018-9809-4

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DOI： 10.1007/978-981-13-2158-0_11

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DOI： 10.3390/scipharm86010005

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DOI： 10.1007/978-981-10-5966-7_15

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Language：Japanese   Publisher：The Pharmaceutical Society of Japan  

<p>Detailed structural characterization of protein biopharmaceuticals is a critical step in research and development; however, this step is often hampered by the structural complexities associated with glycosylation. Most protein biopharmaceuticals are modified with structurally heterogeneous and dynamic oligosaccharides which govern the physicochemical properties, functionality, pharmacokinetics, and potential pathogenicity of these glycoproteins. Considering this, we have developed a structural biological approach to describe the dynamic three-dimensional structures and interactions of glycoproteins as biopharmaceuticals. We developed an NMR technique assisted by metabolic stable-isotope labeling that can provide useful atomic-level probes for detecting and characterizing structural perturbations of glycoproteins caused by alterations in solution conditions and production protocols, as well as by mutagenesis. We have applied this method in conjunction with X-ray crystallography to investigate the structural impacts of varying glycoforms of the Fc region of immunoglobulin G (IgG), thereby elucidating the functional roles of the Fc glycans. In particular, we have successfully elucidated the structural mechanisms by which defucosylation of the IgG-Fc region increases its affinity for Fcγ receptor IIIa, leading to an improvement in ameliorating antibody-dependent cell-mediated cytotoxicity. In addition, we applied our stable-isotope-assisted NMR method to analyzing biomolecular interactions in serum environments, which are characterized by molecular crowding and promiscuous intermolecular interactions. An integrative structural biological approach combining NMR spectroscopy, X-ray crystallography, neutron scattering, atomic force microscopy, and molecular dynamics simulation will provide new research tools that will enable the visualization of dynamic structures and interactions of glycoproteins of pharmaceutical interest, thereby providing valuable insights for the development of biopharmaceuticals.</p>

DOI： 10.1248/yakushi.18-00020-3

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Other Link： http://search.jamas.or.jp/link/ui/2019209131

Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0187022

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DOI： 10.1038/s41598-017-13845-8

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-017-10246-9

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Other Link： http://orcid.org/0000-0002-3513-5701

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrep.2017.08.004

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/asia.201700202

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Other Link： http://orcid.org/0000-0002-3513-5701

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DOI： 10.3389/fimmu.2017.00632

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Other Link： http://orcid.org/0000-0002-3513-5701

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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.

DOI： 10.1002/cbic.201600595

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Other Link： http://orcid.org/0000-0002-3513-5701

Language：English  

DOI： 10.1016/j.pnmrs.2016.02.002

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Other Link： http://orcid.org/0000-0002-3513-5701

Language：English   Publisher：Biophysical Society of Japan  

DOI： 10.2142/biophysics.11.103

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Other Link： http://orcid.org/0000-0002-3513-5701

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M114.566174

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Other Link： http://orcid.org/0000-0002-3513-5701

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S205_4

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M114.566174

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DOI： 10.1038/ncomms3211

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Other Link： http://orcid.org/0000-0002-3513-5701

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1126/scisignal.2002056

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Other Link： http://orcid.org/0000-0002-3513-5701

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S46_4

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M110.152561

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Other Link： http://orcid.org/0000-0002-3513-5701

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/protein/gzq006

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Other Link： http://orcid.org/0000-0002-3513-5701

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S90_5

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.4049/jimmunol.0803471

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Other Link： http://orcid.org/0000-0002-3513-5701

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1124/jpet.107.121640

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Other Link： http://orcid.org/0000-0002-3513-5701

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