Publications by authors named "Takahiro Maruno"

36 Publications

A Multi-Method Approach to Assess the Self-Interaction Behavior of Infliximab.

J Pharm Sci 2021 Feb 5. Epub 2021 Feb 5.

Department of Pharmacy, Ludwig-Maximilians-Universität München, Pharmaceutical Technology and Biopharmaceutics, 81377 Munich, Germany. Electronic address:

Attractive self-interaction processes in antibody formulations increase the risk of aggregation and extraordinarily elevated viscosity at high protein concentrations. These challenges affect manufacturing and application. This study aimed to understand the self-interaction process of Infliximab as a model system with pronounced attractive self-interaction. The association mechanism was studied by a multi-method approach comprising analytical ultracentrifugation, dynamic light scattering, small angle X-ray scattering, self-interaction bio-layer interferometry and hydrogen-deuterium exchange mass spectrometry. Based on our results, both Fab and Fc regions of Infliximab are involved in self-interaction. We hypothesize a mechanism based on electrostatic interactions of polar and charged residues within the identified areas of the heavy and the light chain of the mAb. The combination of fast and reliable screening methods and low throughput but high resolution methods can contribute to detailed characterization and deeper understanding of specific self-interaction processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2021.02.002DOI Listing
February 2021

An influenza HA stalk reactive polymeric IgA antibody exhibits anti-viral function regulated by binary interaction between HA and the antibody.

PLoS One 2021 7;16(1):e0245244. Epub 2021 Jan 7.

Department of Pathology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0245244PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790537PMC
January 2021

Physicochemical Characterization of Sabin Inactivated Poliovirus Vaccine for Process Development.

J Pharm Sci 2020 Dec 17. Epub 2020 Dec 17.

Vaccine Operations, Global Vaccine Business Unit, Takeda Pharmaceutical Company Limited, 4720 Takeda, Mitsui, Hikari, Yamaguchi 743-0011, Japan.

Upscaling the production capacity of inactivated poliovirus vaccines (IPV) is urgently needed to eradicate polio worldwide. For the development of a robust manufacturing process for IPV, the impact of stresses on the properties of the poliovirus during manufacturing needs to be carefully evaluated. In this study, the physicochemical properties of Sabin poliovirus after low pH exposure were analyzed by asymmetrical flow field-flow fractionation coupled to multi-angle laser light scattering (AF4-MALS), sedimentation velocity analytical ultracentrifugation (SV-AUC), transmission electron microscopy (TEM), dynamic light scattering (DLS) and surface plasmon resonance (SPR). Low pH stress caused structural changes and aggregation of inactivated poliovirus virions, whereas degraded virion particles would not revert to native virions even after neutralization. Importantly, a complete loss of the D-antigenicity of IPV by low pH stress, followed by neutralization, was observed in SPR. These results suggest that the exposure of poliovirus particle to low pH stress would induce irreversible denaturation and aggregation of virus particles and lead to the loss of D-antigenicity; thus, low pH stress during the manufacturing of poliovirus vaccine should be minimized. The analytical methods above can be efficiently utilized in the development of high-integrity manufacturing processes and high-quality vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2020.12.012DOI Listing
December 2020

Spatiotemporal regulation of PEDF signaling by type I collagen remodeling.

Proc Natl Acad Sci U S A 2020 05 8;117(21):11450-11458. Epub 2020 May 8.

Graduate School of Engineering, Osaka University, Suita, 565-0871 Osaka, Japan;

Dynamic remodeling of the extracellular matrix affects many cellular processes, either directly or indirectly, through the regulation of soluble ligands; however, the mechanistic details of this process remain largely unknown. Here we propose that type I collagen remodeling regulates the receptor-binding activity of pigment epithelium-derived factor (PEDF), a widely expressed secreted glycoprotein that has multiple important biological functions in tissue and organ homeostasis. We determined the crystal structure of PEDF in complex with a disulfide cross-linked heterotrimeric collagen peptide, in which the α(I) chain segments-each containing the respective PEDF-binding region (residues 930 to 938)-are assembled with an α2α1α1 staggered configuration. The complex structure revealed that PEDF specifically interacts with a unique amphiphilic sequence, KGHRGFSGL, of the type I collagen α1 chain, with its proposed receptor-binding sites buried extensively. Molecular docking demonstrated that the PEDF-binding surface of type I collagen contains the cross-link-susceptible Lys930 residue of the α1 chain and provides a good foothold for stable docking with the α1(I) N-telopeptide of an adjacent triple helix in the fibril. Therefore, the binding surface is completely inaccessible if intermolecular crosslinking between two crosslink-susceptible lysyl residues, Lys9 in the N-telopeptide and Lys930, is present. These structural analyses demonstrate that PEDF molecules, once sequestered around newly synthesized pericellular collagen fibrils, are gradually liberated as collagen crosslinking increases, making them accessible for interaction with their target cell surface receptors in a spatiotemporally regulated manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2004034117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260941PMC
May 2020

Stirring rate affects thermodynamics and unfolding kinetics in isothermal titration calorimetry.

J Biochem 2020 Jul;168(1):53-62

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Isothermal titration calorimetry (ITC) directly provides thermodynamic parameters depicting the energetics of intermolecular interactions in solution. During ITC experiments, a titration syringe with a paddle is continuously rotating to promote a homogeneous mixing. Here, we clarified that the shape of the paddles (flat, corkscrew and small-pitched corkscrew) and the stirring rates influence on the thermodynamic parameters of protein-ligand interaction. Stirring with the flat paddle at lower and higher rate both yielded a lower exothermic heat due to different reasons. The complete reaction with no incompetent fractions was achieved only when the stirring was performed at 500 or 750 rpm using the small-pitched corkscrew paddle. The evaluation of the protein solution after 1,500 rpm stirring indicated that proteins in the soluble fraction decreased to 94% of the initial amount, among which 6% was at an unfolded state. In addition, a significant increase of micron aggregates was confirmed. Furthermore, a new approach for the determination of the unfolding kinetics based on the time dependence of the total reaction heat was developed. This study demonstrates that a proper stirring rate and paddle shape are essential for the reliable estimation of thermodynamic parameters in ITC experiments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jb/mvaa028DOI Listing
July 2020

Structural and functional evaluation of single-chain Fv antibody HyC1 recognizing the residual native structure of hen egg lysozyme.

Biosci Biotechnol Biochem 2020 Feb 29;84(2):358-364. Epub 2019 Oct 29.

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan.

Evaluation of the molecular mechanisms by which an antibody recognizes a specific antigen could help in better understanding of the protein recognition mechanisms. We previously showed that anti-hen egg lysozyme (HEL) monoclonal antibody, HyC1, recognized the structural and hydrodynamic change in HEL. Here, we generated HyC1 single-chain Fv (scFv), and characterized it using different structural and biophysical methods. Similar to HyC1 monoclonal antibody, HyC1 scFv could recognize native HEL from carboxymethylated Cys6 and Cys127 HEL (CM-HEL). Comparison of the binding thermodynamics of HyC1 scFv between HEL and CM-HEL showed that the binding enthalpy change was different, while the binding entropy was remained unchanged. The results indicated that the fluctuation of the residual native structure in both HEL and CM-HEL was similar. The NMR experiments for N-labeled HyC1 scFv indicated that the flexibility of HyC1 scFv decreased upon the binding to HEL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09168451.2019.1683441DOI Listing
February 2020

The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III.

Sci Rep 2019 08 16;9(1):11957. Epub 2019 Aug 16.

Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, 444-8787, Japan.

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-48323-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697678PMC
August 2019

Publisher Correction: Dynamic structural states of ClpB involved in its disaggregation function.

Nat Commun 2019 Jul 12;10(1):3079. Epub 2019 Jul 12.

Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, 920-1192, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11204-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626010PMC
July 2019

An Assessment of the Ability of Submicron- and Micron-Size Silicone Oil Droplets in Dropped Prefillable Syringes to Invoke Early- and Late-Stage Immune Responses.

J Pharm Sci 2019 07 18;108(7):2278-2287. Epub 2019 Feb 18.

U-Medico Inc, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Japan, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan; Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2019.02.002DOI Listing
July 2019

SDS-induced oligomerization of Lys49-phospholipase A from snake venom.

Sci Rep 2019 02 20;9(1):2330. Epub 2019 Feb 20.

Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi, 980-8577, Japan.

Phospholipase A (PLA) is one of the representative toxic components of snake venom. PLAs are categorized into several subgroups according to the amino acid at position 49, which comprises either Asp49, Lys49, Arg49 or Ser49. Previous studies suggested that the Lys49-PLA assembles into an extremely stable dimer. Although the behavior on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions suggested the presence of intermolecular disulfide bonds, these bonds were not observed in the crystal structure of Lys49-PLA. The reason for this discrepancy between the crystal structure and SDS-PAGE of Lys49-PLA remains unknown. In this study, we analyzed a Lys49-PLA homologue from Protobothrops flavoviridis (PflLys49-PLA BPII), by biophysical analyses including X-ray crystallography, SDS-PAGE, native-mass spectrometry, and analytical ultracentrifugation. The results demonstrated that PflLys49-PLA BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-38861-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382788PMC
February 2019

Stability of cytochromes c' from psychrophilic and piezophilic Shewanella species: implications for complex multiple adaptation to low temperature and high hydrostatic pressure.

Extremophiles 2019 Mar 28;23(2):239-248. Epub 2019 Jan 28.

Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, 1-4-4 Kagamiyama, Hiroshima, 739-8528, Japan.

The stability of dimeric cytochrome c' from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the heme and at the subunit-subunit interface, both of which are molecular interior regions. Here, we showed that interactions in the equivalent interior regions of homologous cytochromes c' from two psychrophiles, Shewanella benthica and Shewanella violacea (SBCP and SVCP, respectively) were similarly weakened as compared with those of the counterparts of psychrophilic Shewanella livingstonensis and mesophilic Shewanella amazonensis (SLCP and SACP, respectively), and consistently the stability of SVCP, SLCP, and SACP increased in that order. Therefore, the stability of cytochromes c' from the psychrophile, mesophile, and thermophile is systematically regulated in their molecular interior regions. Unexpectedly, however, the stability of SBCP was significantly higher than that of SVCP, and the former had additional molecular surface interactions. Collectively, SBCP had weakened interior interactions like SVCP did, but the former was stabilized at the molecular surface as compared with the latter, implying complex multiple adaptation of the proteins because the psychrophilic sources of SBCP and SVCP are also piezophilic, thriving in deep-sea extreme environments of low temperature and high hydrostatic pressure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00792-019-01077-9DOI Listing
March 2019

N-terminal HCV core protein fragment decreases 20S proteasome activity in the presence of PA28γ.

Biochem Biophys Res Commun 2019 02 31;509(2):590-595. Epub 2018 Dec 31.

Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan. Electronic address:

The Hepatitis C virus (HCV) core protein plays a crucial role in the development of chronic liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Its involvement in these diseases is reportedly abolished by a knockout of the proteasome activator PA28γ gene in transgenic mice, suggesting an interaction between the core protein and the PA28γ-proteasome system. This study found a direct interaction between the N-terminal 1-71 fragment of HCV core protein (Core71) and PA28γ in vitro, and that this interaction was found to enhance PA28γ-20S proteasome complex formation. While 20S proteasome activity was increased by PA28γ, it was significantly reduced by Core71 attachment in a dose-dependent manner. These results suggest that the Core-PA28γ interaction has an important role in regulating 20S proteasome activity and furthers our understanding of the pathogenesis of HCV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2018.12.167DOI Listing
February 2019

Collaborative Study for Analysis of Subvisible Particles Using Flow Imaging and Light Obscuration: Experiences in Japanese Biopharmaceutical Consortium.

J Pharm Sci 2019 02 16;108(2):832-841. Epub 2018 Aug 16.

Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan.

The evaluation of subvisible particles, including protein aggregates, in therapeutic protein products has been of great interest for both pharmaceutical manufacturers and regulatory agencies. To date, the flow imaging (FI) method has emerged as a powerful tool instead of light obscuration (LO) due to the fact that (1) protein aggregates contain highly transparent particles and thereby escape detection by LO and (2) FI provides detailed morphological characteristics of subvisible particles. However, the FI method has not yet been standardized nor listed in any compendium. In an attempt to assess the applicability of the standardization of the FI method, we conducted a collaborative study using FI and LO instruments in a Japanese biopharmaceutical consortium. Three types of subvisible particle preparations were shared across 12 laboratories and analyzed for their sizes and counts. The results were compared between the methods (FI and LO), inter-laboratories, and inter-instruments (Micro Flow Imaging and FlowCam). We clarified the marked difference between the detectability of FI and LO when counting highly transparent protein aggregates in the preparations. Although FlowCam provided a relatively higher number of particles compared with MFI, consistent results were obtained using the instrument from the same manufacturer in all 3 samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2018.08.006DOI Listing
February 2019

Interplay of a secreted protein with type IVb pilus for efficient enterotoxigenic colonization.

Proc Natl Acad Sci U S A 2018 07 25;115(28):7422-7427. Epub 2018 Jun 25.

Research Institute for Microbial Diseases, Osaka University, Suita, 565-0871 Osaka, Japan

Initial attachment and subsequent colonization of the intestinal epithelium comprise critical events allowing enteric pathogens to survive and express their pathogenesis. In enterotoxigenic (ETEC), these are mediated by a long proteinaceous fiber termed type IVb pilus (T4bP). We have reported that the colonization factor antigen/III (CFA/III), an operon-encoded T4bP of ETEC, possesses a minor pilin, CofB, that carries an H-type lectin domain at its tip. Although CofB is critical for pilus assembly by forming a trimeric initiator complex, its importance for bacterial attachment remains undefined. Here, we show that T4bP is not sufficient for bacterial attachment, which also requires a secreted protein CofJ, encoded within the same CFA/III operon. The crystal structure of CofB complexed with a peptide encompassing the binding region of CofJ showed that CofJ interacts with CofB by anchoring its flexible N-terminal extension to be embedded deeply into the expected carbohydrate recognition site of the CofB H-type lectin domain. By combining this structure and physicochemical data in solution, we built a plausible model of the CofJ-CFA/III pilus complex, which suggested that CofJ acts as a molecular bridge by binding both T4bP and the host cell membrane. The Fab fragments of a polyclonal antibody against CofJ significantly inhibited bacterial attachment by preventing the adherence of secreted CofJ proteins. These findings signify the interplay between T4bP and a secreted protein for attaching to and colonizing the host cell surface, potentially constituting a therapeutic target against ETEC infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1805671115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048534PMC
July 2018

Dynamic structural states of ClpB involved in its disaggregation function.

Nat Commun 2018 06 1;9(1):2147. Epub 2018 Jun 1.

Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, 920-1192, Japan.

The ATP-dependent bacterial protein disaggregation machine, ClpB belonging to the AAA+ superfamily, refolds toxic protein aggregates into the native state in cooperation with the cognate Hsp70 partner. The ring-shaped hexamers of ClpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize ClpB using high-speed atomic force microscopy (HS-AFM) to gain a mechanistic insight into its disaggregation function. The HS-AFM movies demonstrate massive conformational changes of the hexameric ring during ATP hydrolysis, from a round ring to a spiral and even to a pair of twisted half-spirals. HS-AFM observations of Walker-motif mutants unveil crucial roles of ATP binding and hydrolysis in the oligomer formation and structural dynamics. Furthermore, repressed and hyperactive mutations result in significantly different oligomeric forms. These results provide a comprehensive view for the ATP-driven oligomeric-state transitions that enable ClpB to disentangle protein aggregates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-04587-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984625PMC
June 2018

Sweeping of Adsorbed Therapeutic Protein on Prefillable Syringes Promotes Micron Aggregate Generation.

J Pharm Sci 2018 06 6;107(6):1521-1529. Epub 2018 Feb 6.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan. Electronic address:

This study evaluated how differences in the surface properties of prefillable syringe barrels and in-solution sampling methods affect micron aggregates and protein adsorption levels. Three syringe types (glass barrel with silicone oil coating [GLS/SO+], glass barrel without silicone oil coating [GLS/SO-], and cyclo-olefin polymer [COP] barrel syringes) were tested with 3 therapeutic proteins (adalimumab, etanercept, and infliximab) using 2 sampling methods (aspiration or ejection). After quiescent incubation, solutions sampled by aspiration exhibited no significant change in micron aggregate concentration in any syringes, whereas those sampled by ejection exhibited increased micron aggregates in both GLS syringe types. Micron aggregate concentration in ejected solutions generally increased with increasing density of adsorbed proteins. Notably, COP syringes contained the lowest micron aggregate concentrations, which were independent of the sampling method. Correspondingly, the adsorbed protein density on COP syringes was the lowest at 1-2 mg/m, which was much less compared with that on GLS syringes and was calculated to be equivalent to only 1-2 protein layers, as visually confirmed by high-speed atomic force microscopy. These data indicate that low-adsorption prefillable syringes should be used for therapeutic proteins because protein aggregate concentration in the ejected solution is elevated by increased protein adsorption to the syringe surface.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2018.01.021DOI Listing
June 2018

Stabilization of mesophilic Allochromatium vinosum cytochrome c' through specific mutations modeled by a thermophilic homologue.

Biosci Biotechnol Biochem 2018 Feb 12;82(2):304-311. Epub 2018 Jan 12.

a Graduate School of Biosphere Science , Hiroshima University , Higashi-Hiroshima , Japan.

AVCP cytochrome c' from mesophilic Allochromatium vinosum exhibits lower stability than a thermophilic counterpart, Hydrogenophilus thermoluteolus cytochrome c' (PHCP), in which the six specific amino acid residues that are not conserved in AVCP are responsible for its stability. Here we measured the stability of AVCP variants carrying these specific residues instead of the original AVCP ones. Among the six single AVCP variants, all of which formed a dimeric structure similar to that of the wild-type, three were successfully stabilized compared with the wild-type, while one showed lower stability than the wild-type. In addition, the most stabilized and destabilized AVCP variants could bind CO, similar to the wild-type. These results indicated that mesophilic AVCP could be stabilized through specific three mutations modeled by the thermophilic counterpart, PHCP, without changing the CO binding ability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09168451.2017.1419856DOI Listing
February 2018

Friability Testing as a New Stress-Stability Assay for Biopharmaceuticals.

J Pharm Sci 2017 10 8;106(10):2966-2978. Epub 2017 Jun 8.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan. Electronic address:

A cycle of dropping and shaking a vial containing antibody solution was reported to induce aggregation. In this study, antibody solutions in glass prefillable syringes with or without silicone oil lubrication were subjected to the combined stresses of dropping and shaking, using a friability testing apparatus. Larger numbers of subvisible particles were generated, regardless of silicone oil lubrication, upon combination stress exposure than that with shaking stress alone. Nucleation of antibody molecules upon perturbation by an impact of dropping and adsorption of antibody molecules to the syringe surface followed by film formation and antibody film desorption were considered key steps in the particle formation promoted by combination stress. A larger number of silicone oil droplets was released when silicone oil-lubricated glass syringes containing phosphate buffer saline were exposed to combination stress than that observed with shaking stress alone. Polysorbate 20, a non-ionic surfactant, effectively reduced the number of protein particles, but failed to prevent silicone oil release upon combination stress exposure. This study indicates that stress-stability assays using the friability testing apparatus are effective for assessing the stability of biopharmaceuticals under the combined stresses of dropping and shaking, which have not been tested in conventional stress-stability assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2017.05.035DOI Listing
October 2017

Tryptophan introduction can change β-glucan binding ability of the carbohydrate-binding module of endo-1,3-β-glucanase.

Biosci Biotechnol Biochem 2017 May 8;81(5):951-957. Epub 2017 Feb 8.

a Graduate School of Life and Environmental Sciences, Kyoto Prefectural University , Kyoto , Japan.

Endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1 has a carbohydrate-binding module (CBM-DK) at the C-terminal side of a catalytic domain. Out of the imperfect tandem α-, β-, and γ-repeats in CBM-DK, the α-repeat primarily contributes to β-glucan binding. This unique feature is derived from Trp273 in α-repeat, whose corresponding residues in β- and γ-repeats are Asp314 and Gly358, respectively. In this study, we generated Trp-switched mutants, W273A/D314W, D270A/W273A/D314W, W273A/G358W, and D270A/W273A/G358W, and analyzed their binding abilities toward laminarioligosaccharides and laminarin. While the binding affinities of D270A/W273A and W273A mutants were either lost or much lower than that of the wild-type, those of Trp-switched mutants recovered, indicating that a Trp introduction in β- or γ-repeat can substitute the α-repeat by primarily contributing to β-glucan binding. Thus, we have successfully engineered a CBM-DK that binds to laminarin by a mechanism different from that of the wild-type, but with similar affinity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09168451.2017.1285687DOI Listing
May 2017

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments.

MAbs 2017 May/Jun;9(4):664-679. Epub 2017 Mar 3.

a Graduate School of Engineering, Osaka University , Yamadaoka, Suita , Osaka , Japan.

A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/19420862.2017.1297909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5419078PMC
February 2018

Structural and functional insights into thermally stable cytochrome c' from a thermophile.

Protein Sci 2017 04 6;26(4):737-748. Epub 2017 Mar 6.

Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.

Thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c' (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit-subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit-subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit-subunit interactions with conservation of the ligand-binding ability.

Brief Summary: We report the X-ray crystal structure of cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus. The high thermal stability of PHCP was attributed to heme-related and subunit-subunit interactions, which were confirmed by a mutagenesis study. The ligand-binding ability of PHCP was examined by spectrophotometry. PHCP acquired the thermal stability with conservation of the ligand-binding ability. This study furthers the understanding of the stability and function of cytochromes c.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.3120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368077PMC
April 2017

Synergistic Effect of Cavitation and Agitation on Protein Aggregation.

J Pharm Sci 2017 02 23;106(2):521-529. Epub 2016 Nov 23.

Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan; Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan. Electronic address:

It was recently reported that dropping induces protein aggregation due to the occurrence of cavitation. Agitation also causes protein aggregation. In this study, vials filled with antibody solution were subjected to a cycle of dropping and shaking using the friability testing apparatus to examine the combined effect of cavitation and agitation on protein aggregation. A cycle of dropping and shaking generated a massive amount of subvisible particles. Comparison of aggregation rate at different fill volumes indicated that shaking plays an important role in protein aggregation due to combination stress. Furthermore, the impact of dropping on aggregate formation was apparent because aggregation rate under combination stress was much faster than that under shaking stress alone. Increase in aggregate concentration was observed after shaking of the antibody solution, which was freshly filled into vials that had been previously used in the dropping and shaking test. Polysorbate 80 was effective in inhibiting aggregate formation under combination stress. These results suggest the following particle formation pathway: cavitation caused by dropping promotes antibody unfolding, the unfolded antibodies adsorb on the inner surface of the vial, and subsequent shaking yields subvisible particles by desorbing the adsorbed antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2016.10.015DOI Listing
February 2017

Hesperidin interaction to collagen detected by physico-chemical techniques.

Dent Mater 2017 01 19;33(1):33-42. Epub 2016 Oct 19.

Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Objective: Dentin collagen can be modified by some plant-derived flavonoids to improve properties of dentin organic matrix. Hesperidin (HPN), a hesperetin-7-O-rutinoside flavonoid, has a potential of dentin modification for being based on evidence that a treatment with HPN may resist collagenase degradation and arrest demineralization of human dentin. In this study, biophysical and molecular-level information on the interaction of HPN and collagen was investigated.

Methods: HPN is extracted from citrus fruits. Sample collagenous solution was prepared using atelocollagen (ATCL) as a triple-helical peptide model. We have performed circular dichroism spectroscopic analysis, sedimentation velocity measurement by ultracentrifuge and saturation transfer difference measurement (STD) by NMR on HPN-collagen in solution state.

Results: The circular dichroism and sedimentation velocity measurement showed the evidence for the molecular interaction between ATCL and HPN, while HPN did not induce any conformational change of ATCL. The STD-NMR study further confirmed this interaction and suggested that HPN interacted with ATCL through its aromatic part, not through its disaccharide moiety.

Significance: These findings indicated that HPN is weakly bound to ATCL not causing structural modification of collagen. This interaction may contribute to the preservation of collagen by protecting from collagenase degradation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dental.2016.09.035DOI Listing
January 2017

Comparative study on stabilization mechanism of monomeric cytochrome c from deep-sea piezophilic Shewanella violacea.

Biosci Biotechnol Biochem 2016 Dec 20;80(12):2365-2370. Epub 2016 Sep 20.

a Graduate School of Biosphere Science , Hiroshima University , Higashi-Hiroshima , Japan.

Monomeric cytochrome c from deep-sea piezophilic Shewanella violacea (SVcytc) was stable against heat and denaturant compared with the homologous protein from shallow-sea piezo-sensitive Shewanella livingstonensis (SLcytc). Here, the SVcytc crystal structure revealed that the Lys-50 side chain on the flexible loop formed a hydrogen bond with heme whereas that of corresponding hydrophobic Leu-50 could not form such a bond in SLcytc, which appeared to be one of possible factors responsible for the difference in stability between the two proteins. This structural insight was confirmed by a reciprocal mutagenesis study on the thermal stability of these two proteins. As SVcytc was isolated from a deep-sea piezophilic bacterium, the present comparative study indicates that adaptation of monomeric SVcytc to high pressure environments results in stabilization against heat.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09168451.2016.1232155DOI Listing
December 2016

Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl.

Int J Biol Macromol 2016 Oct 21;91:151-7. Epub 2016 May 21.

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan. Electronic address:

Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijbiomac.2016.05.074DOI Listing
October 2016

Taste substance binding elicits conformational change of taste receptor T1r heterodimer extracellular domains.

Sci Rep 2016 05 10;6:25745. Epub 2016 May 10.

RIKEN SPring-8 Center, 1-1-1, Kouto, Sayo, Hyogo, 679-5148, Japan.

Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-protein coupled receptors (GPCRs), and the extracellular ligand binding domains (LBDs) of T1r1/T1r3 and T1r2/T1r3 heterodimers are responsible for binding of chemical substances eliciting umami or sweet taste. However, molecular analyses of T1r have been hampered due to the difficulties in recombinant expression and protein purification, and thus little is known about mechanisms for taste perception. Here we show the first molecular view of reception of a taste substance by a taste receptor, where the binding of the taste substance elicits a different conformational state of T1r2/T1r3 LBD heterodimer. Electron microscopy has showed a characteristic dimeric structure. Förster resonance energy transfer and X-ray solution scattering have revealed the transition of the dimerization manner of the ligand binding domains, from a widely spread to compactly organized state upon taste substance binding, which may correspond to distinct receptor functional states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep25745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861910PMC
May 2016

Homo-trimeric Structure of the Type IVb Minor Pilin CofB Suggests Mechanism of CFA/III Pilus Assembly in Human Enterotoxigenic Escherichia coli.

J Mol Biol 2016 Mar 11;428(6):1209-1226. Epub 2016 Feb 11.

Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

In gram-negative bacteria, the assembly of type IV pilus (T4P) and the evolutionally related pseudopilus of type II secretion system involves specialized structural proteins called pilins and pseudopilins, respectively, and is dynamically regulated to promote bacterial pathogenesis. Previous studies have suggested that a structural "tip"-like hetero-complex formed through the interaction of at least three minor (pseudo) pilins plays an important role in this process, while some members of the pathogenic type IVb subfamily are known to have only one such minor pilin subunit whose function is still unknown. Here, we determined the crystal structure of the type IVb minor pilin CofB of colonization factor antigen/III from human enterotoxigenic Escherichia coli at 1.88-Å resolution. The crystal structure, in conjunction with physicochemical analysis in solution, reveals a symmetrical homo-trimeric arrangement distinct from the hetero-complexes of minor (pseudo) pilins observed in other T4P and type II secretion systems. Each CofB monomer adopts a unique three-domain architecture, in which the C-terminal β-sheet-rich lectin domain can effectively initiate trimer association of its pilin-like N-terminal domain through extensive hydrophobic interactions followed by domain swapping at the central hinge-like domain. Deletion of cofB produces a phenotype with no detectable pili formation on the cell surface, while molecular modeling indicates that the characteristic homo-trimeric structure of CofB is well situated at the pilus tip of colonization factor antigen/III formed by the major pilin CofA, suggesting a role for the minor pilin in the efficient initiation of T4P assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2016.02.003DOI Listing
March 2016

Ordered self-assembly of the collagenous domain of adiponectin with noncovalent interactions via glycosylated lysine residues.

FEBS Lett 2016 Jan 28;590(2):195-201. Epub 2016 Jan 28.

Graduate School of Pharmaceutical Sciences, Osaka University, Japan.

Adiponectin, an anti-atherogenic and insulin-sensitizing adipokine, forms multiple isoforms including a trimer, a hexamer and heavier oligomers (mainly octadecamer) that determine their biological activities. We designed 89-residue peptides containing modifications found in the collagenous domain of native adiponectin. Circular dichroism and analytical ultracentrifugation measurements showed that the peptide bearing glucosyl-galactosyl-hydroxylysine residues forms a stable collagen-like triple helical structure and spontaneously assembled into an octadecamer. An assembly model mediated by noncovalent interactions via glycosylated lysine residues for the octadecamer was constructed. Our findings clarified an essential role of glycosyl modifications to coordinate the ordered self-assembly of adiponectin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12034DOI Listing
January 2016

Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase.

Biochem Biophys Res Commun 2015 Dec 4-11;468(1-2):234-9. Epub 2015 Oct 27.

Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D2 (PGD2) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP(+), and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2015.10.124DOI Listing
March 2016

Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition.

J Biol Chem 2015 Dec 20;290(49):29461-77. Epub 2015 Oct 20.

From the Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ~30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.670794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705948PMC
December 2015