210

Medical Proteomics Laboratory疾患プロテオミクスラボラトリー
Professor
Project Professor
Associate Professor
Associate Professor

MutsuhiroTakekawa, M.D., Ph.D.
Kouhei Tsumoto, Ph.D.
Masaaki Oyama, Ph.D.
Satoru Nagatoishi, Ph.D.

Senior Assistant Professor Makoto Nakakido, Ph.D.
Assistant Professor

Ryo Matsunaga, Ph.D.

Project Assistant Professor Hiroshi Sagara, Ph.D.

教 授
博士（医学）
特任教授 博士（工学）准教授
博士（医学）
准教授
博士（生命科学）（大学院工学系研究科）
講 師
博士（生命科学）（大学院工学系研究科）
助 教
博士（生命科学）（大学院工学系研究科）
特任助教 博士（医学）

武 川
津 本
尾 山
長門石

睦
浩
大

寛
平
明
曉

中木戸

誠

松

長

遼

相

良

洋

The mission of our laboratory is to develop advanced technologies for integrative
proteomic analyses from a physicochemical, structural and systems biology point ofview. Currently, we mainly focus on functional protein-protein interaction networks
related to a variety of diseases including cancer and infection. We are also
engaged in collaborative researches regarding mass spectrometry and electron
microscopy, which have made a substantial contribution to many scientificachievements.

<Group I>
1.  Integrative analysis of cancer cell signaling
networks by high-resolution proteomics andsystems biology
Post-translational modifications (PTMs), such as
phosphorylation, ubiquitination and acetylation, are
known to be widely involved in the regulation of various biological processes through extensive diversification of each protein function at the cellular network
level. Previous functional analyses of cancer cell signaling under a variety of experimental conditions revealed many of the key molecules and their associated protein modifications in relation to each type ofcancer. In order to systematically discover criticalmodulators from diversified signaling molecules, wehave developed a high-resolution mass spectrometry-based proteomics platform for integrative identification and quantification of multiple post-translational modifications from various types of cancer
cells.

1-1. 
High-resolution proteomic analysis of
EGF-regulated ubiquitination dynamics in human cancer cells
Hiroko Kozuka-Hata, Tomoko Hiroki, Aya Kitamura, Aiko Aizawa, Naoaki Miyamura, Kouhei Tsumoto, Jun-ichiro Inoue and Masaaki Oyama.
Protein ubiquitination is one of the most prevalentpost-translational modifications (PTMs) and plays
critical roles in regulating protein degradation, signaltransduction and DNA repair in cooperation withother PTMs such as phosphorylation and acetylation.
Recent mass spectrometry-based proteomics coupledwith efficient enrichment technologies for each typeof the modified peptides has enabled us to identify
precise modification sites and measure their quantitative changes on a global scale. Our previous lysine-modification proteomic analyses of thirteen representative human cancer cell lines led us to identify
thousands of ubiquitination (Ub) and acetylation (Ac)sites in total and revealed that their system-wide
modification status was mutually different at the cel-

211
lular network level. In this study, we further applied
SILAC (Stable Isotope Labeling by Amino acids in
Cell culture) for quantitative description of EGF-dependent lysine-modification site dynamics in HeLa
cells in a time-resolved manner. Through integrationof large-scale SILAC-encoded data on six time points
upon EGF stimulation, we successfully quantified approximately 1,000 kinds of Ub-sites as well as 700
kinds of Ac-sites and found that one-third of these
Ub-modified molecules, including several EGF signaling effectors, were subjected to downregulation byproteasomal inhibition.
1-2. Proteome-wide analysis of lysine acetylation
and ubiquitination reveals critical signaling
regulation in cancer cells
Hiroko Kozuka-Hata, Aya Kitamura, Tomoko Hiroki, Aiko Aizawa, Kouhei Tsumoto, Jun-ichiro Inoue and Masaaki Oyama.
Post-translational modifications (PTMs), such as
phosphorylation, ubiquitination and acetylation, are
known to be widely involved in the regulation of various biological processes through extensive diversification of each protein function at the cellular network
level. Previous functional analyses of cancer cell signaling under a variety of experimental conditions revealed many of the key molecules and their associated protein modifications in relation to each type ofcancer. In order to systematically discover criticalmodulators from diversified signaling molecules, wehave developed a high-resolution mass spectrometry-based proteomics platform for integrative identification and quantification of multiple post-translational modifications from various types of cancer
cells. Our large-scale proteomic analysis enabled us to
identify more than 5,000 kinds of ubiquitinated sites
and 1,600 kinds of acetylated sites from representative human cancer cell lines, leading to identificationof approximately 900 novel lysine modification sites
in total. Very interestingly, 236 lysine residues derived from 141 proteins were found to be modified
with both ubiquitination and acetylation. As a consequence of the subsequent motif extraction analyses,
glutamic acid (E) was found to be highly enriched at
the position (-1) for the lysine acetylation sites, whereas the same amino acid was relatively dispersed along
the neighboring residues of the lysine ubiquitinationsites.
1-3. 
System-wide perturbation of the proteome
and phosphoproteome dynamics in glioblastoma stem cells through mTOR signaling inhibition
Hiroko Kozuka-Hata, Tomoko Hiroki, Ryo KoyamaNasu, Kouhei Tsumoto, Jun-ichiro Inoue, Tetsu Akiyama and Masaaki Oyama.

As glioblastoma is the most common and aggressive brain tumor with poor prognosis, systematic elucidation of signaling networks causally linked to thetumorigenesis is very crucial for developing more effective treatments for this intractable cancer. In ourprevious study, we applied a high-resolution mass
spectrometry-based proteomics technology in combination with SILAC quantitative methods to understand EGF-dependent phosphoproteome dynamics
in patient-derived glioblastoma stem cells. We
demonstrated that the phosphorylation levels of therepresentative mTOR signaling molecules such as
RPS6 and PRAS40 were dramatically up-regulated
upon EGF stimulation. As EGFR signaling has beenreported to play a pivotal role in regulating the maintenance of cancer stem cells, we next carried out
mTOR inhibitor-dependent signaling perturbations
to unravel stemness-related pathways at the network
level.
In the present study, we identified a total of 3,726
proteins including 49 up-regulated and 436 down-regulated factors by Torin 1 treatment. Interestingly, we
found that one of the well-known cancer stem cellmarkers was significantly down-regulated throughmTOR signaling inhibition. Our in-depth phosphoproteome analysis also led to identification of 6,250
unique phosphopeptides derived from 2,221 proteins
and unveiled a variety of dynamic changes regarding
phosphorylation levels of cancer and neural stem cellmarkers in a comprehensive manner. The integrative
view of the mTOR inhibitor-dependent proteome andphosphoproteome dynamics in glioblastoma stem
cells presents us with further prospects towards understanding previously unrecognized regulations at
the system level.
1-4. 
System-level analysis of CagA-dependent
signaling network dynamics by Helicobacter
pylori infection
Hiroko Kozuka-Hata, Masato Suzuki, Kotaro Kiga,
Shinya Tasaki, Jun-ichiro Inoue, Tadashi Yamamoto, Chihiro Sasakawa and Masaaki Oyama.
The signal transduction system within a cell regulates complex biological events in response to bacterial infection. The previous analyses of cell signaling in
Helicobacter pylori-infected gastric epithelial cells
have revealed that CagA, a major virulence factor of
Helicobacter pylori, is delivered into cells via the typeIV secretion system and perturbs signaling networks
through the interaction with the key signaling molecules such as SHP-2, Grb2, Crk/Crk-L, Csk, Met, andZO-1. Although the biological activity of tyrosine-phosphorylated CagA has intensively been studied, system-wide effects of the virulence factor on cellular signaling have yet to be analyzed. Here we
performed time-resolved analyses of phosphoproteome and CagA-interactome in human gastric AGS

212
cells by CagA-positive/negative Helicobacter pylori
infection. Our highly sensitive nanoLC-MS/MS analyses in combination with the Stable Isotope Labelingby Amino acids in Cell culture (SILAC) technology
defined CagA-dependent perturbation of signaling
dynamics along with a subset of CagA-associatedpossible modulators on a network-wide scale. Our result indicated that the activation level of the phosphotyrosine-related signaling molecules in AGS cells was
suppressed overall in the presence of CagA during
Helicobacter pylori infection. As Helicobacter pylori
infection plays pivotal roles in the progression of gastric diseases including carcinogenesis, a comprehensive and fine description of the signaling dynamics
would serve as a fundamental platform to theoretically explore for the potential drug targets through analyzing the regulatory mechanisms at the system-level.

These findings reveal a novel post-transcriptional system that can augment the human proteome via thealternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by thehuman transcriptome (Oyama et al., Mol Cell Proteomics, 6: 1000-1006, 2007).

2.  Mass spectrometry-based annotation of thehuman short ORFeome

The honeybee (Apis mellifera L.) uses various
chemical signals produced by the worker exocrine
glands to maintain the functioning of its colony. The
roles of worker postcerebral glands (PcGs), thoracic
glands (TGs), and mandibular glands (MGs) and thefunctional changes they undergo according to the division of labor from nursing to foraging are not aswell studied. To comprehensively characterize themolecular roles of these glands in workers and theirchanges according to the division of labor of workers,
we analyzed the proteomes of PcGs, TGs, and MGs
from nurse bees and foragers using shotgun proteomics technology. We identified approximately 2000
proteins from each of the nurse bee or forager glands
and highlighted the features of these glands at themolecular level by semiquantitative enrichment analyses of frequently detected, gland-selective, and labor-selective proteins. First, we found the high potential to produce lipids in PcGs and MGs, suggesting
their relation to pheromone production. Second, we
also found the proton pumps abundant in TGs andpropose some transporters possibly related to the saliva production. Finally, our data unveiled candidate
enzymes involved in labor-dependent acid production in MGs.

Masaaki Oyama, Hiroko Kozuka-Hata, Sumio Sugano, Tadashi Yamamoto and Jun-ichiro Inoue.
In parallel with the human genome projects, human full-length cDNA data has also been intensively
accumulated. Large-scale analysis of their 5’-UTRs revealed that about half of these had a short ORF upstream of the coding region. Experimental verification as to whether such upstream ORFs are translated
is essential to reconsider the generality of the classicalscanning mechanism for initiation of translation anddefine the real outline of the human proteome. Our
previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct
evidence of translation of upstream ORFs in humanfull-length cDNAs (Oyama et al., Genome Res, 14:2048-2052, 2004). In order to grasp an expanded landscape of the human short ORFeome, we have performed an in-depth proteomics analysis of human
K562 and HEK293 cells using a two-dimensional nanoLC-MS/MS system. The results led to the identification of eight protein-coding regions besides 197 small
proteins with a theoretical mass less than 20 kDa thatwere already annotated coding sequences in the curated mRNA database. In addition to the upstream
ORFs in the presumed 5’-untranslated regions of mRNAs, bioinformatics analysis based on accumulated
5’-end cDNA sequence data provided evidence ofnovel short coding regions that were likely to betranslated from the upstream non-AUG start site orfrom the new short transcript variants generated byutilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream startsite occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on
the major mRNA, resulting in translation of thedownstream ORF from the second initiation codon.

3.  Shotgun proteomics deciphered age/divisionof labor-related functional specification of
three honeybee (Apis mellifera L.) exocrine
glandsToshiyuki Fujita, Hiroko Kozuka-Hata, Yutaro
Hori, Jun Takeuchi, Takeo Kubo and Masaaki
Oyama.

4.  In-depth proteomic analysis of drug-responsive signaling pathway elements in humancancer cells
Wei QI, Aya Kitamura, Naoaki Miyamura, Tomoko
Hiroki, Aiko Aizawa, Kazuki Mori, Hiroko Kozuka-Hata and Masaaki Oyama.
Abnormal expression of histone deacetylases(HDACs) in human cancer cells was reported to be
associated with angiogenesis, migration, chemotherapy resistance as well as cell differentiation and apoptosis in a wide range of previous studies. Therefore,clinical use of HDAC inhibitors has been discussed asa new therapeutic approach against cancer for a long213
period. In 2006, suberoylanilide hydroxamic acid
(SAHA), a pan-inhibitor targeting HDACs and also
known as Vorinostat, was approved by the US Foodand Drug Administration (FDA) for the treatment of
cutaneous T-cell lymphoma. In addition to the anticancer activity against hematologic cancers, SAHA
also shows a significant antitumor effect on solid tumors through inducing apoptosis, arresting cell cycle
or elevating radiation sensitization. In order to unveil
the underlying complex mechanism, we used human
HeLa cells as the model platform for analyzing SAHA-responsive elements on a proteomic scale. According to the experimental pre-evaluation throughwestern blotting for acetylated histone H3 and microscopic observation of cell growth under a variety ofdrug-perturbed conditions, we determined to treat
cultured cells with SAHA for 24 h to perform an indepth quantitative proteomic analysis of SAHA-responsive elements in human HeLa cells. After SAHA
treatment, the cells were lysed, trypsin-digested andanalyzed by high-resolution nanoflow liquid chromatography- tandem mass spectrometry. As a result of
ultra-deep proteomic analysis by Orbitrap Eclipse
Tribrid system coupled with Ultimate3000 RSLCnano
liquid chromatography technology, a total of 5,135
proteins was identified using Proteome Discoverer
software. Approximately 8 % of the identified proteins were found to be differentially regulated withmore than two-fold changes in response to SAHA
treatment by Label Free Quantification (LFQ). The
subsequent pathway analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) indicatedthat cell cycle and anti-apoptotic pathway elements
including p27 and HO-1 were prominently correlatedwith SAHA-dependent regulation in human HeLa
cells.
5.  Real-Time Search-Assisted Multiplexed Quantitative Proteomics Reveals System-Wide
Translational Regulation of Non-Canonical
Short Open Reading Frames
Hiroko Kozuka-Hata, Tomoko Hiroki, Naoaki Miyamura, Aya Kitamura, Kouhei Tsumoto, Jun-ichiro Inoue and Masaaki Oyama.
Abnormal expression of histone deacetylases(HDACs) is reported to be associated with angiogenesis, metastasis and chemotherapy resistance regarding cancer in a wide range of previous studies. Suberoylanilide hydroxamic acid (SAHA) is well known tofunction as a pan-inhibitor for HDACs and recognized as one of the therapeutic drug candidates toepigenetically coordinate cancer cell fate regulation
on a genomic scale. Here, we established a Real-Time
Search-assisted mass spectrometric platform for system-wide quantification of translated products encoded by non-canonical short open reading frames(ORFs) as well as already annotated protein coding

sequences (CDSs) on the human transcriptome andapplied this methodology to quantitative proteomic
analyses of SAHA-treated human HeLa cells to evaluate proteome-wide regulation in response to drug
perturbation. Very intriguingly, our RTS-based indepth proteomic analysis enabled us to identify approximately 5000 novel peptides from the ribosome
profiling-based short ORFs encoded in the diversified
regions on presumed ‘non-coding’ nucleotide sequences of mRNAs as well as lncRNAs and nonsense
mediated decay (NMD) transcripts. Furthermore,
TMT-based multiplex large-scale quantification of thewhole proteome changes upon differential SAHA
treatment unveiled dose-dependent selective translational regulation of a limited fraction of the non-canonical short ORFs in addition to key cell cycle/proliferation-related molecules such as UBE2C, CENPF
and PRC1. Our study provided the first system-wide
landscape of drug-perturbed translational modulation on both canonical and non-canonical proteome
dynamics in human cancer cells.
<Group II>
Biomolecular recognition is based on collective
and specific non-covalent interactions between discrete biological molecules. Our laboratory studies
various protein systems, for instance, antibody-antigen and protein-ligand complexes, to understandquantitatively how these coordinated non-covalentinteractions contribute to their specific recognition in
biological and artificial systems. We seek to elucidate
the molecular mechanisms by which biological molecules obtain high-specificity and affinity from multiple angles using advanced instrumentation. To produce functional molecules with higher performanceand better properties, we aim to build a solid foundation from which to develop drugs that modulate specific interactions between biomolecules and ultimately to understand the principles of molecularinteractions in our lives.
1.  Analytical Method for Experimental Validationof Computer-Designed Antibody
Tanabe A and Tsumoto K.
In the computational design of antibodies, the interaction analysis between target antigen and antibody is an essential process to obtain feedback forvalidation and optimization of the design. Kinetic
and thermodynamic parameters as well as binding
affinity (KD) allow for a more detailed evaluation andunderstanding of the molecular recognition. In thischapter, we summarize the conventional experimental methods which can calculate KD value (ELISA,FP), analyze a binding activity to actual cells (FCM),
and evaluate the kinetic and thermodynamic parameters (ITC, SPR, BLI), including high-throughput analysis and a recently developed experimental tech-

214nique.
2.  Structural Classification of CDR-H3 in Single-Domain VHH Antibodies
Kuroda D and Tsumoto K.
The immune systems protect vertebrates from foreign molecules or antigens, and antibodies are important mediators of this system. The sequences andstructural features of antibodies vary depending on
species. Many of antibodies from vertebrates, including camelids, have both heavy and light chain variable domains, but camelids also have antibodies thatlack the light chains. In antibodies that lack lightchains, the C-terminal variable region is called the
VHH domain. Antibodies recognize antigens throughsix complementarity-determining regions (CDRs).
The third CDR of the heavy chain (CDR-H3) is at thecenter of the antigen-binding site and is diverse interms of sequence and structure. Due to the importance of antibodies in basic science as well as in medical applications, there have been many studies of
CDR-H3s of antibodies that possess both light andheavy chains. However, nature of CDR-H3s of single-domain VHH antibodies is less well studied. In
this chapter, we describe current knowledge of sequence-structure-function correlations of single-domain VHH antibodies with emphasis on CDR-H3.
Based on the 370 crystal structures in the Protein Data
Bank, we also attempt structural classification of
CDR-H3 in single-domain VHH antibodies and discuss lessons learned from the ever-increasing numberof the structures.
3. Biophysical Characterization of the Contribution of the Fab Region to the IgG-FcγRIIIa Interaction
Kosuge H, Nagatoishi S, Kiyoshi M, Ishii-Watabe A,
Terao Y, Ide T and Tsumoto K
The cell-surface receptor FcγRIIIa is crucial to theefficacy of therapeutic antibodies as well as the immune response. The interaction of the Fc region of
IgG molecules with FcγRIIIa has been characterized,
but until recently, it was thought that the Fab regions
were not involved in the interaction. To evaluate theinfluence of the Fab regions in a biophysical context,
we carried out surface plasmon resonance analyses
using recombinant FcγRIIIa ligands. A van’t Hoffanalysis revealed that compared to the interaction of
the papain-digested Fc fragment with FcγRIIIa, the interaction of commercially available, full-length rituximab with FcγRIIIa had a more favorable binding enthalpy, a less favorable binding entropy, and a slower
off rate. Similar results were obtained from analysesof IgG1 molecules and an IgG1-Fc fragment produced
by Expi293 cells. For further validation, we also pre-pared a maltose-binding protein-linked IgG1-Fc fragment (MBP-Fc). The binding enthalpy of MBP-Fc wasnearly equal to that of the IgG1-Fc fragment for theinteraction with FcγRIIIa, indicating that such alternatives to the Fab domains as MBP do not positively
contribute to the IgG-FcγRIIIa interactions. Our investigation strongly suggests that the Fab region directlyinteracts with FcγRIIIa, resulting in an increase in thebinding enthalpy and a decrease in the dissociation
rate, at the expense of favorable binding entropy.
4. Nucleic acid-triggered tumoral immunity propagates pH-selective therapeutic antibodies
through tumor-driven epitope spreading
Furuya G, Katoh H, Atsumi S, Hashimoto I, Komura D, Hatanaka R, Senga S, Hayashi S, Akita S, Matsumura H, Miura A, Mita H, Nakakido M, Nagatoishi S, Sugiyama A, Suzuki R, Konishi H, Yamamoto
A, Abe H, Hiraoka N, Aoki K, Kato Y, Seto Y, Yoshimura C, Miyadera K, Tsumoto K, Ushiku T and
Ishikawa S.
Important roles of humoral tumor immunity areoften pointed out; however, precise profiles of dominant antigens and developmental mechanisms remain elusive. We systematically investigated thehumoral antigens of dominant intratumor immunoglobulin clones found in human cancers. We foundthat approximately half of the corresponding antigens were restricted to strongly and densely negatively charged polymers, resulting in simultaneous
reactivities of the antibodies to both densely sulfatedglycosaminoglycans (dsGAGs) and nucleic acids(NAs). These anti-dsGAG/NA antibodies matured
and expanded via intratumoral immunological driving force of innate immunity via NAs. These humancancer-derived antibodies exhibited acidic pH-selective affinity across both antigens and showed specificreactivity to diverse spectrums of human tumor cells.
The antibody-drug conjugate exerted therapeutic effects against multiple cancers in vivo by targeting cellsurface dsGAG antigens. This study reveals that intratumoral immunological reactions propagate tumor-oriented immunoglobulin clones and demonstrates a new therapeutic modality for the universal
treatment of human malignancies.
5.  Structural insights into 3Fe-4S ferredoxins diversity in M. tuberculosis highlighted by a first
redox complex with P450.
Gilep A, Varaksa T, Bukhdruker S, Kavaleuski A,
Ryzhykau Y, Smolskaya S, Sushko T, Tsumoto K,
Grabovec I, Kapranov I, Okhrimenko I, Marin E,
Shevtsov M, Mishin A, Kovalev K, Kuklin A, Gordeliy V, Kaluzhskiy L, Gnedenko O, Yablokov E,
Ivanov A, Borshchevskiy V and Strushkevich N.

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Ferredoxins are small iron-sulfur proteins and key
players in essential metabolic pathways. Among all
types, 3Fe-4S ferredoxins are less studied mostly due
to anaerobic requirements. Their complexes with cytochrome P450 redox partners have not been structurally characterized. In the present work, we solved thestructures of both 3Fe-4S ferredoxins from M. tuberculosis-Fdx alone and the fusion FdxE-CYP143. Our
SPR analysis demonstrated a high-affinity binding of
FdxE to CYP143. According to SAXS data, the same
complex is present in solution. The structure reveals
extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes
in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE-CYP143 andmodeled Fdx-CYP51 complexes further revealed thespecificity of ferredoxins. Our results illuminate thediversity of electron transfer complexes for the production of different secondary metabolites.
6.  Peripheral administration of nanomicelle-encapsulated anti-Aβ oligomer fragment antibody reduces various toxic Aβ species in thebrain
Amano A, Sanjo N, Araki W, Anraku Y, Nakakido
M, Matsubara E, Tomiyama T, Nagata T, Tsumoto
K, Kataoka K and Yokota T.
Although a large amount of evidence has revealed
that amyloid β (Aβ), especially Aβ oligomers, protofibrils, and pyroglutamated Aβs, participate primarily
in the pathophysiological processes of Alzheimer’s
disease, most clinical trials of anti-Aβ antibody therapy have never acquired successful efficacy in humanclinical trials, partly because peripheral administration of antibody medications was unable to deliver
sufficient amounts of the molecules to the brain. Recently, we developed polymeric nanomicelles capableof passing through the blood-brain barrier that function as chaperones to deliver larger amounts of heavy
molecules to the brain. Herein, we aimed to evaluate
the efficacy of newly developed antibody 6H4 fragments specific to Aβ oligomers encapsulated in polymeric nanomicelles on the development of Alzheimer’s disease pathology in Alzheimer’s disease model
mice at the age of emergence of early Alzheimer’s disease pathology. During the 10-week administration of
6H4 antibody fragments in polymeric nanomicelles, asignificant reduction in the amounts of various toxic
Aβ species, such as Aβ oligomers, toxic Aβ conformers, and pyroglutamated Aβs in the brain was observed. In addition, immunohistochemistry indicatedinhibition of diameters of Aβ plaques, Aβ-antibody
immunoreactive areas, and also plaque core formation. Behavioral analysis of the mice model revealed
that the 6H4 fragments-polymeric nanomicelle groupwas significantly better at maintaining long-term spa-tial reference memory in the probe and platform testsof the water maze, thereby indicating inhibition of thepathophysiological process of Alzheimer’s disease.
The results indicated that the strategy of reducing
toxic Aβ species in early dementia owing to Alzheimer’s disease by providing sufficient antibodies in thebrain may modify Alzheimer’s disease progression.
7.  Non-Affinity Purification of Antibodies
Arakawa T, Tomioka Y, Nakagawa M, Sakuma C,
Kurosawa Y, Ejima D, Tsumoto K and Akuta T.
Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture
step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which aresummarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale
antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties ofProtein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.
8.  Inactivation and spike protein denaturation ofnovel coronavirus variants by CuxO/TiO2 nano-photocatalysts
Tatsuma T, Nakakido M, Ichinohe T, Kuroiwa Y, Tomioka K, Liu C, Miyamae N, Onuki T, Tsumoto K,
Hashimoto K and Wakihara T.
In order to reduce infection risk of novel coronavirus (SARS-CoV-2), we developed nano-photocatalysts with nanoscale rutile TiO2 (4-8 nm) and CuxO
(1-2 nm or less). Their extraordinarily small size leads
to high dispersity and good optical transparency, besides large active surface area. Those photocatalysts
can be applied to white and translucent latex paints.
Although Cu2O clusters involved in the paint coating
undergo gradual aerobic oxidation in the dark, theoxidized clusters are re-reduced under > 380 nm light.
The paint coating inactivated the original and alphavariant of novel coronavirus under irradiation withfluorescent light for 3 h. The photocatalysts greatly
suppressed binding ability of the receptor binding
domain (RBD) of coronavirus (the original, alpha anddelta variants) spike protein to the receptor of humancells. The coating also exhibited antivirus effects on
influenza A virus, feline calicivirus, bacteriophage Qβ
and bacteriophage M13. The photocatalysts would beapplied to practical coatings and lower the risk ofcoronavirus infection via solid surfaces.

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9.  Ligand Installation to Polymeric Micelles for
Pediatric Brain Tumor Targeting
Watanabe T, Mizuno HL, Norimatsu J, Obara T,
Cabral H, Tsumoto K, Nakakido M, Kawauchi D
and Anraku Y
Medulloblastoma is a life-threatening disease
with poor therapeutic outcomes. In chemotherapy,
low drug accumulation has been a cause of these outcomes. Such inadequate response to treatments has
been associated with low drug accumulation, particularly with a limited cellular uptake of drugs. Recently,
the conjugation of drugs to ligand molecules withhigh affinity to tumor cells has attracted much attention for enhancing drug internalization into target
cells. Moreover, combining tumor-targeting ligandswith nano-scaled drug carriers can potentially improve drug loading capacity and the versatility of thedelivery. Herein, we focused on the possibility of targeting CD276/B7-H3, which is highly expressed on
the medulloblastoma cell membrane, as a strategy forenhancing the cellular uptake of ligand-installed nanocarriers. Thus, anti-CD276 antibodies were conjugated on the surface of model nanocarriers based onpolyion complex micelles (PIC/m) via click chemistry.
The results showed that the anti-CD276 antibody-installed PIC/m improved intracellular delivery into
CD276-expressing medulloblastoma cells in a
CD276-dependent manner. Moreover, increasing thenumber of antibodies on the surface of micelles improved the cellular uptake efficiency. These observations indicate the potential of anti-CD276 antibody-installed nanocarriers for promoting drug delivery in
medulloblastoma.
10.  
Bromodomain protein BRD8 regulates cell
cycle progression in colorectal cancer cells
through a TIP60-independent regulation of
the pre-RC complex
Yamaguchi K, Nakagawa S, Saku A, Isobe Y, Yamaguchi R, Sheridan P, Takane K, Ikenoue T, Zhu C,
Miura M, Okawara Y, Nagatoishi S, Kozuka-Hata
H, Oyama M, Aikou S, Ahiko Y, Shida D, Tsumoto
K, Miyano S, Imoto S and Furukawa Y.
Bromodomain-containing protein 8 (BRD8) is a
subunit of the NuA4/TIP60-histone acetyltransferase
complex. Although BRD8 has been considered to act
as a co-activator of the complex, its biological role remains to be elucidated. Here, we uncovered that
BRD8 accumulates in colorectal cancer cells throughthe inhibition of ubiquitin-dependent protein degradation by the interaction with MRG domain binding
protein. Transcriptome analysis coupled with genome-wide mapping of BRD8-binding sites disclosedthat BRD8 transactivates a set of genes independently
of TIP60, and that BRD8 regulates the expression ofmultiple subunits of the pre-replicative complex inconcert with the activator protein-1. Depletion of
BRD8 induced cell-cycle arrest at the G1 phase andsuppressed cell proliferation. We have also shownthat the bromodomain of BRD8 is indispensable for
not only the interaction with histone H4 or transcriptional regulation but also its own protein stability.
These findings highlight the importance of bromodomain as a therapeutic target.
11.  
Molecular mechanism underlying the increased risk of colorectal cancer metastasis
caused by single nucleotide polymorphisms
in LI-cadherin gene
Yui A, Kuroda D, Maruno T, Nakakido M, Nagatoishi S, Uchiyama S and Tsumoto K.
LI-cadherin is a member of the cadherin superfamily. LI-cadherin mediates Ca2+-dependent cellcell adhesion through homodimerization. A previous
study reported two single nucleotide polymorphisms(SNPs) in the LI-cadherin-coding gene (CDH17).
These SNPs correspond to the amino acid changes of
Lys115 to Glu and Glu739 to Ala. Patients withcolorectal cancer carrying these SNPs are reported tohave a higher risk of lymph node metastasis than patients without the SNPs. Although proteins associated with metastasis have been identified, the molecular mechanisms underlying the functions of these
proteins remain unclear, making it difficult to develop effective strategies to prevent metastasis. In thisstudy, we employed biochemical assays and molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which the amino acid changes
caused by the SNPs in the LI-cadherin-coding geneincrease the risk of metastasis. Cell aggregation assays showed that the amino acid changes weakened
the LI-cadherin-dependent cell-cell adhesion. In vitro
assays demonstrated a decrease in homodimerization
tendency and MD simulations suggested an alteration in the intramolecular hydrogen bond network by
the mutation of Lys115. Taken together, our results
indicate that the increased risk of lymph node metastasis is due to weakened cell-cell adhesion caused by
the decrease in homodimerization tendency.
12.  
An Engineered Synthetic Receptor-Aptamer
Pair for an Artificial Signal Transduction System.
Liu H, Baeumler TA, Nakamura K, Okada Y, Cho S,
Eguchi A, Kuroda D, Tsumoto K, Ueki R and Sando
S.
Cell membrane receptors regulate cellular responses through sensing extracellular environmental
signals and subsequently transducing them. Receptor
engineering provides a means of directing cells to re-

217
act to a designated external cue and exert programmed functions. However, rational design andprecise modulation of receptor signaling activity remain challenging. Here, we report an aptamer-based
signal transduction system and its applications incontrolling and customizing the functions of engineered receptors. A previously reported membrane
receptor-aptamer pair was used to design a synthetic
receptor system that transduces cell signaling depending on exogenous aptamer input. To eliminate
the cross-reactivity of the receptor with its native ligand, the extracellular domain of the receptor was
engineered to ensure that the receptor was solely activated by the DNA aptamer. The present system features tunability in the signaling output level using
aptamer ligands with different receptor dimerization
propensities. In addition, the functional programmability of DNA aptamers enables the modular sensingof extracellular molecules without the need for genetic engineering of the receptor.
13.  Agarose native gel electrophoresis analysisof thermal aggregation controlled by Hofmeister series
Tomioka Y, Sato R, Takahashi R, Nagatoishi S, Shiba K, Tsumoto K, Arakawa T and Akuta T.
The effects of salting-in and salting-out salts defined by Hofmeister series on the solution state of bovine serum albumin (BSA) in 50 mM Tris-HCl buffer
at pH 7.4 before and after thermal unfolding at 80 °C
for 5 min were examined using agarose native gelelectrophoresis and mass photometry. Gel electrophoresis showed that salting-in MgCl2, CaCl2 andNaSCN resulted in formation of intermediate structures of BSA upon heating on native gel, while heating in buffer alone resulted in aggregated bands.
Mass photometry showed large loss of monomer andoligomers when heated in this buffer, but retaining
these structures in the presence of 1 M MgCl2 andNaSCN. To our surprise, salting-out MgSO4 also
showed a similar effect on gel electrophoresis andmass photometry. Salting-out NaCl and (NH4)2SO4
resulted in smearing and aggregated bands, whichwere supported by mass photometry. Aggregation-suppressive ArgHCl also showed oligomer aggregates upon gel electrophoresis and mass photometry.
14.  
Elucidating Conformational Dynamics andThermostability of Designed Aromatic Clusters by Using Protein Cages
Hishikawa Y, Noya H, Nagatoishi S, Yoshidome T,
Maity B, Tsumoto K, Abe S and Ueno T.
Multiple aromatic residues assemble to form higher ordered structures known as “aromatic clusters” inproteins and play essential roles in biological systems.
However, the stabilization mechanism and dynamic
behavior of aromatic clusters remain unclear. This
study describes designed aromatic interactions confined within a protein cage to reveal how aromaticclusters affect protein stability. The crystal structuresand calorimetric measurements indicate that the formation of inter-subunit phenylalanine clusters enhance the interhelix interactions and increase themelting temperature. Theoretical calculations suggestthat this is caused by the transformation of the
T-shaped geometry into π-π stacking at high temperatures, and the hydration entropic gain. Thus, the isolated nanoenvironment in a protein cage allows reconstruction and detailed analysis of multiple
clustering residues for elucidating the mechanisms ofvarious biomolecular interactions in nature which canbe applied to design of bionanomaterials.
15.  Design of single-domain VHH antibodies toincrease the binding activity in SPR amine
coupling
Hirao A, Nagatoishi S, Ikeuchi E, Yamawaki T, MoriC, Nakakido M and Tsumoto K.
Single-domain antibodies, or VHH, nanobodies,
are attractive tools in biotechnology and pharmaceuticals due to their favorable biophysical properties.
Single-domain antibodies have potential for use insensing materials to detect antigens, and in this paper, we propose a generic design strategy of single-domain antibodies for the highly efficient use ofimmobilized antibodies on a sensing substrate. Amine
coupling was used to immobilize the single-domain
antibodies on the substrate through a robust covalentbond. First, for two model single-domain antibodies
with lysines at four highly conserved positions (K48,
K72, K84, and K95), we mutated the lysines to alanine
and measured the binding activity of the mutants (thepercentage of immobilized antibodies that can bind
antigen) using surface plasmon resonance. The twomodel single-domain antibodies tended to have higher binding activities when K72, which is close to theantigen binding site, was mutated. Adding a Lys-tag
to the C-terminus of single-domain antibodies also
increased the binding activity. We also mutated thelysine for another model single-domain antibodies
with the lysine in a different position than the four
residues mentioned above and measured the binding
activity. Thus, single-domain antibodies immobilized
in an orientation accessible to the antigen tended tohave a high binding activity, provided that the physical properties of the single-domain antibodies themselves (affinity and structural stability) were not significantly reduced. Specifically, the design strategy ofsingle-domain antibodies with high binding activity
included mutating the lysine at or near the antigen
binding site, adding a Lys-tag to the C-terminus, and218
mutating a residue away from the antigen bindingsite to lysine. It is noteworthy that mutating K72 close
to the antigen binding site was more effective in increasing the binding activity than Lys-tag addition,and immobilization at the N-terminus close to the antigen binding site did not have such a negative effecton the binding activity compared to immobilization
at the K72.
16.  Real-Time Search-Assisted Multiplexed Quantitative Proteomics Reveals System-Wide
Translational Regulation of Non-Canonical
Short Open Reading Frames
Kozuka-Hata H, Hiroki T, Miyamura N, Kitamura
A, Tsumoto K, Inoue JI and Oyama M.
Abnormal expression of histone deacetylases(HDACs) is reported to be associated with angiogenesis, metastasis and chemotherapy resistance regarding cancer in a wide range of previous studies. Suberoylanilide hydroxamic acid (SAHA) is well known tofunction as a pan-inhibitor for HDACs and recognized as one of the therapeutic drug candidates toepigenetically coordinate cancer cell fate regulation
on a genomic scale. Here, we established a Real-Time
Search (RTS)-assisted mass spectrometric platform
for system-wide quantification of translated products
encoded by non-canonical short open reading frames(ORFs) as well as already annotated protein coding
sequences (CDSs) on the human transciptome andapplied this methodology to quantitative proteomic
analyses of suberoylanilide hydroxamic acid (SAHA)-treated human HeLa cells to evaluate proteome-wide regulation in response to drug perturbation. Very intriguingly, our RTS-based in-depth
proteomic analysis enabled us to identify approximately 5000 novel peptides from the ribosome profiling-based short ORFs encoded in the diversified regions on presumed ‘non-coding’ nucleotide sequencesof mRNAs as well as lncRNAs and nonsense mediated decay (NMD) transcripts. Furthermore, TMTbased multiplex large-scale quantification of thewhole proteome changes upon differential SAHA
treatment unveiled dose-dependent selective translational regulation of a limited fraction of the non-canonical short ORFs in addition to key cell cycle/proliferation-related molecules such as UBE2C, CENPF
and PRC1. Our study provided the first system-wide
landscape of drug-perturbed translational modulation on both canonical and non-canonical proteome
dynamics in human cancer cells.
17.  
Targeting hemoglobin receptors IsdH and
IsdB of Staphylococcus aureus with a single
VHH antibody inhibits bacterial growth
Valenciano-Bellido S, Caaveiro JMM, Nakakido M,

Kuroda D, Aikawa C, Nakagawa I and Tsumoto K.
Methicillin-resistant Staphylococcus aureus, or
MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistantstrains are necessary and not only against S. aureus.
Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize thehost, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd(iron surface determinant) system. In particular, thehemoglobin receptors IsdH and IsdB located on thesurface of the bacterium are necessary to acquire theheme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition.
We determined that the antibody recognized theheme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third
complementary-determining regions. The mechanism explaining the inhibition of acquisition of hemein vitro could be described as a competitive process inwhich the complementary-determining region 3 fromthe antibody blocked the acquisition of heme by thebacterial receptor. Moreover, this antibody markedly
reduced the growth of three different pathogenic
strains of MRSA. Collectively, our results highlight amechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.
18.  
A Proximity-Induced Fluorogenic Reaction
Triggered by Antibody-Antigen Interactions
with Adjacent Epitopes
Nishiyama K, Akiba H, Nagata S, Tsumoto K, Kamada H and Ohno H.
Proximity-induced chemical reactions are site-specific and rapid by taking advantage of their high affinity and highly selective interactions with the template.
However, reactions induced solely by antibody-antigen interactions have not been developed. Herein, we
propose a biepitopic antigen-templated chemical reaction (BATER) as a novel template reaction. In BATER, reactive functional groups are conjugated to two
antibodies that interact with two epitopes of the same
antigen to accelerate the reaction. We developed amethod for visualizing the progress of BATER using
fluorogenic click chemistry for optimal antibody selection and linker design. The reaction is accelerated
in the presence of a specific antigen in a linker
length-dependent manner. The choice of the antibody
epitope is important for a rapid reaction. This design
will lead to various applications of BATER in living
systems.

219
19.  
Raman Spectroscopic Analysis of Highly-Concentrated Antibodies under the Acid-Treated Conditions
Sato Y, Nagatoishi S, Noguchi S and Tsumoto K.
Antibody drugs are usually formulated as highly-concentrated solutions, which would easily generate aggregates, resulting in loss of efficacy. Although
low pH increases the colloidal dispersion of antibodies, acid denaturation can be an issue. Therefore,knowing the physical properties at low pH underhigh concentration conditions is important. Raman
spectroscopy was used to investigate pH-induced
conformational changes of antibodies at 50 mg/ml.
Experiments in pH 3 to 7 were performed for humanserum IgG and recombinant rituximab. We detected
the evident changes at pH 3 in Tyr and Trp bands,
which are the sensitive markers of intermolecular interactions. Thermal transition analysis over the pH
range demonstrated that the thermal transition temperature (Tm) was highest at pH 3. Acid-treated andneutralized one showed higher Tm than that of pH 7,
indicating that their extent of intermolecular interactions correlated with the Tm values. Onset temperature was clearly different between concentrated anddiluted samples. Colloidal analyses confirmed thefindings of the Raman analysis. Our studies demonstrated the positive correlation between Raman analysis and colloidal information, validating as a method
for evaluating antibody conformation associated withaggregation propensities.
20.  Histone H3 lysine 27 crotonylation mediates
gene transcriptional repression in chromatin
Liu N, Konuma T, Sharma R, Wang D, Zhao N, Cao
L, Ju Y, Liu D, Wang S, Bosch A, Sun Y, Zhang S, Ji
D, Nagatoishi S, Suzuki N, Kikuchi M, Wakamori
M, Zhao C, Ren C, Zhou TJ, Xu Y, Meslamani J, Fu
S, Umehara T, Tsumoto K, Akashi S, Zeng L, Roeder RG, Walsh MJ, Zhang Q and Zhou MM.
Histone lysine acylation, including acetylationand crotonylation, plays a pivotal role in gene transcription in health and diseases. However, our understanding of histone lysine acylation has been limited
to gene transcriptional activation. Here, we reportthat histone H3 lysine 27 crotonylation (H3K27cr) directs gene transcriptional repression rather than activation. Specifically, H3K27cr in chromatin is selectively recognized by the YEATS domain of GAS41 incomplex with SIN3A-HDAC1 co-repressors. Proto-oncogenic transcription factor MYC recruits
GAS41/SIN3A-HDAC1 complex to repress genes inchromatin, including cell-cycle inhibitor p21. GAS41
knockout or H3K27cr-binding depletion results in
p21 de-repression, cell-cycle arrest, and tumor growth
inhibition in mice, explaining a causal relationshipbetween GAS41 and MYC gene amplification and p21
downregulation in colorectal cancer. Our study suggests that H3K27 crotonylation signifies a previously
unrecognized, distinct chromatin state for gene transcriptional repression in contrast to H3K27 trimethylation for transcriptional silencing and H3K27 acetylation for transcriptional activation.
21.  Dispersion Function of a Protein, DP-1, Identified in Collimonas sp. D-25, for the Synthesis of Gold Nanoparticles
Tang D, Kato Y, Zhang D, Negishi L, Kurumizaka
H, Hirata T, Nakakido M, Tsumoto K, Shuji F, Tsuguyuki S, Okumura T, Nagata K and Suzuki M.
Collimonas sp. (D-25), found in the soil of Akita
Prefecture, is a gram-negative bacterium with theability to synthesize gold nanoparticles (AuNPs).
During the synthesis of AuNPs, one specific protein(DP-1) was found to have disappeared in the sonicated solution of the bacterium. Recombinant DP-1
(rDP-1) from Escherichia coli BL21 (DE3) was used to
study the effect of DP-1 on the synthesis of AuNPs.
AuNPs synthesized with rDP-1 result in small, stabilized nanoparticles. AuNPs synthesized by DP-1 retained the stability of both the dispersion and nano-size particles under high salt concentrations.
Isothermal titration calorimetry was employed to investigate the bonding ratio of rDP-1 to AuNPs. Several thousand rDP-1 proteins are attached to the surface
of an AuNP to form a protein corona containing multiple layers. These results suggest that DP-1 obtained
from D-25 has a size and stability control function
during AuNP synthesis.
22.  Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling
activity through epitope selection
Akiba H, Fujita J, Ise T, Nishiyama K, Miyata T,
Kato T, Namba K, Ohno H, Kamada H, Nagata S
and Tsumoto K
Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different
epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against
tumor necrosis factor receptor 2 (TNFR2), which is an
attractive immune checkpoint target. Using different
pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully
regulate the size of BpAb-TNFR2 immunocomplexes
to result in controlled agonistic activities. Our seriesof results indicate that the relative positions of thetwo epitopes recognized by the BpAb are critical forcontrolling its signaling activity. One particular an-

220tagonist, Bp109-92, binds TNFR2 in a 1:1 mannerwithout unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation ofregulatory T cells expressing TNFR2. Therefore, the
BpAb format would be useful in designing specificand distinct antibody functions.
23.  Discovery of a cystathionine γ-lyase (CSE)
selective inhibitor targeting active-site pyridoxal 5’-phosphate (PLP) via Schiff base formation
Echizen H, Hanaoka K, Shimamoto K, Hibi R, Toma-Fukai S, Ohno H, Sasaki E, Komatsu T, Ueno T,
Tsuchiya Y, Watanabe Y, Otsuka T, Saito H, Nagatoishi S, Tsumoto K, Kojima H, Okabe T, Shimizu T
and Urano Y.
D,L-Propargylglycine (PAG) has been widelyused as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), whichcatalyzes the formation of reactive sulfur species(RSS). However, PAG also inhibits other PLP (pyridoxal-5’-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase(ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified
oxamic hydrazide 1 as a potent inhibitor of CSE (IC50= 13 ± 1 μM (mean ± S.E.)) with high selectivity overother PLP-dependent enzymes and RSS-generating
enzymes. Inhibitor 1 inhibited the enzymatic activityof human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure
analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active
site may be a promising target for development of
selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine β-synthase (CBS) and cysteinyl-tRNA synthetase
2 (CARS2), which have unique substrate binding
pocket structures.
24.  Modulation of a conformational ensemble bya small molecule that inhibits key protein-protein interactions involved in cell adhesion
Senoo A, Nagatoishi S, Kuroda D, Ito S, Ueno G,
Caaveiro JMM and Tsumoto K.
Small molecules that regulate protein-protein interactions can be valuable drugs; however, the development of such small molecules is challenging as themolecule must interfere with an interaction that often
involves a large surface area. Herein, we propose thatmodulating the conformational ensemble of the proteins participating in a given interaction, rather thanblocking the interaction by directly binding to the interface, is a relevant strategy for interfering with aprotein-protein interaction. In this study, we applied
this concept to P-cadherin, a cell surface protein forming homodimers that are essential for cell-cell adhesion in various biological contexts. We first determined the crystal structure of P-cadherin with a small
molecule inhibitor whose inhibitory mechanism was
unknown. Molecular dynamics simulations suggestthat the inhibition of cell adhesion by this small molecule results from modulation of the conformational
ensemble of P-cadherin. Our study demonstrates thepotential of small molecules altering the conformation ensemble of a protein as inhibitors of biologicalrelevant protein-protein interactions.
25.  
Arginine cluster introduction on framework
region in anti-lysozyme antibody improved
association rate constant by changing conformational diversity of CDR loops
Maeta S, Nakakido M, Matsuura H, Sakai N, Hirata
K, Kuroda D, Fukunaga A and Tsumoto K.
Antibodies are used for many therapeutic and biotechnological purposes. Because the affinity of an
antibody to the antigen is critical for clinical efficacyof pharmaceuticals, many affinity maturation strategies have been developed. Although we previously
reported an affinity maturation strategy in which theassociation rate of the antibody toward its antigen is
improved by introducing a cluster of arginine residues into the framework region of the antibody, thedetailed molecular mechanism responsible for thisimprovement has been unknown. In this study, we
introduced five arginine residues into an anti-hen egg
white lysozyme antibody (HyHEL10) Fab fragment
to create the R5-mutant and comprehensively characterized the interaction between antibody and antigen
using thermodynamic analysis, X-ray crystallography, and molecular dynamics (MD) simulations. Our
results indicate that introduction of charged residues
strongly enhanced the association rate, as previously
reported, and the antibody-antigen complex structure
was almost the same for the R5-mutant and wild-type
Fabs. The MD simulations indicate that the mutation
increased conformational diversity in complementarity-determining region loops and thereby enhanced
the association rate. These observations provide themolecular basis of affinity maturation by R5 mutation.
26.  Group A Streptococcus cation diffusion facilitator proteins contribute to immune evasion
by regulating intracellular metal concentrations
Aikawa C, Shimizu A, Nakakido M, Murase K, Nozawa T, Tsumoto K and Nakagawa I.

221

Cation diffusion facilitators (CDFs) are a large
family of divalent metal transporters with broad specificities that contribute to intracellular metal homeostasis and toxicity in bacterial pathogens. Streptococcus pyogenes (Group A Streptococcus [GAS])
expresses two homologous CDF efflux transporters,
MntE and CzcD, which selectively transport Mn and
Zn, respectively. We discovered that the MntE- andCzcD-deficient strains exhibited a marked decrease in
the viability of macrophage-differentiated THP-1
cells and neutrophils. In addition, the viability of mice
infected with both deficient strains markedly increased. Consistent with a previous study, our results
suggest that MntE regulates the PerR-dependent oxidative stress response by maintaining intracellular
Mn levels and contributing to the growth of GAS. The
maturation and proteolytic activity of streptococcal
cysteine protease (SpeB), an important virulence factor in GAS, has been reported to be abrogated by zincand copper. Zn inhibited the maturation and proteolytic activity of SpeB in the culture supernatant of the
CzcD-deficient strain. Furthermore, Mn inhibited
SpeB maturation and proteolytic activity in a
MntE-deficient strain. Since the host pathogenicity of
the SpeB-deficient strain was significantly reduced,
maintenance of intracellular manganese and zinc levels in the GAS via MntE and CzcD may not only confer metal resistance to the bacterium, but may also
play an essential role in its virulence. These findings
provide new insights into the molecular mechanismsof pathogenicity, which allow pathogens to survive
under stressful conditions associated with elevatedmetal ion concentrations during host infection.
27.  Ferguson plot analysis of multiple intermediate species of thermally unfolded bovine serum albumin

tive-like state consistent with no SYPRO Orange
staining. The intermediate species observed at higher
temperatures with the mobility less than that of thenative BSA showed a steeper Ferguson plot and were
stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native
BSA and were unfolded.
28.  Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes
Yamazaki T, Nagatoishi S, Yamawaki T, Nozawa T,
Matsunaga R, Nakakido M, Caaveiro JMM, Nakagawa I and Tsumoto K.
Listeriosis, caused by infection with Listeria
monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host
receptor E-cadherin, is necessary to invade host cells.
Here, we isolated two single-domain antibodies(VHHs) that bind to InlA with picomolar affinities
from an alpaca immune library using the phage display method. These InlA-specific VHHs inhibited thebinding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction
analysis. Furthermore, we determined that the VHHs
inhibited the invasion of L. monocytogenes into hostcells in culture. High-resolution X-ray structure analyses of the complexes of VHHs with InlA revealed
that the VHHs bind to the same binding site as E-cadherin against InlA. We conclude that these VHHs
have the potential for use as drugs to treat listeriosis.
29.  PRELP secreted from mural cells protects thefunction of blood brain barrier through regulation of endothelial cell-cell integrity

Tomioka Y, Nagatoishi S, Nakagawa M, Tsumoto K,
Arakawa T and Akuta T.

Davaapil H, Hopkins J, Bonnin N, Papadaki V, Leung A, Kosuge H, Tashima T, Nakakido M, Sekido
R, Tsumoto K, Sagoo MS and Ohnuma SI.

Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin(BSA) upon thermal unfolding. Differential scanningcalorimetry showed an irreversible melting of BSA in
Tris-HCl and phosphate buffers with a mid-transition
temperature, Tm, of ∼68 °C. Thermally unfolded BSA
was analyzed by agarose native gel electrophoresis
stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration.
SYPRO Orange was used to stain unfolded proteins.
BSA heated at 70 and 80 °C, i.e., above the Tm, formed
multiple intermediate species, which depended on
the pH between 7.0 and 8.0, protein concentration andwhich buffer was used. These intermediate species
were analyzed by Ferguson plot, which showed that
BSA heated at 60 °C had a similar size to the native
BSA, indicating that they are either native or na-

Introduction: Proline/arginine-rich end leucine-rich repeat protein (PRELP), is a small secreted
proteoglycan expressed by pericytes and vascularsmooth muscle cells surrounding the brain vasculature of adult mouse. Methods: We utilised a Prelp
knockout (Prelp -/-) mouse model to interrogate vasculature integrity in the brain alongside performingin vitro assays to characterise PRELP application to
endothelial cells lines. Our findings were supplemented with RNA expression profiling to elucidate
the mechanism of how PRELP maintains neurovasculature function. Results: Prelp -/- mice presented withneuroinflammation and reducedneurovasculature integrity, resulting in IgG and dextran leakage in the
cerebellum and cortex. Histological analysis of Prelp-/- mice revealed reducedcell-cell integrity of theblood brain barrier, capillary attachment of pericytes

222
andastrocyte end-feet. RNA-sequencing analysis
found that cell-cell adhesion andinflammation are affected in Prelp -/- mice and gene ontology analysis as
well as gene set enrichment analysis demonstrated
that inflammation related processes and adhesion related processes such as epithelial-mesenchymal transition and apical junctions were significantly affected,
suggesting PRELP is a regulator of cell-cell adhesion.
Immunofluorescence analysis showed that adhesion
junction protein expression levels of cadherin, claudin-5, and ZO-1, was suppressed in Prelp -/- miceneurovasculature. Additionally, in vitro studies revealed that PRELP application to endothelial cells enhances cell-cell integrity, induces mesenchymal-endothelial transition and inhibits TGF-β mediateddamage to cell-cell adhesion. Discussion: Our study
indicates that PRELP is a novel endogenous secreted
regulator of neurovasculature integrity and that
PRELP application may be a potential treatment for
diseases associated with neurovascular damage.
30.  Specific peptide conjugation to a therapeutic
antibody leads to enhanced therapeutic potency and thermal stability by reduced Fc dynamics
Kiyoshi M, Nakakido M, Rafique A, Tada M,
Aoyama M, Terao Y, Nagatoishi S, Shibata H, Ide T,
Tsumoto K, Ito Y and Ishii-Watabe A.
Antibody-drug conjugates are powerful tools forcombatting a wide array of cancers. Drug conjugation
to a therapeutic antibody often alters molecular characteristics, such as hydrophobicity and effector function, resulting in quality deterioration. To develop adrug conjugation methodology that maintains themolecular characteristics of the antibody, we engineered a specific peptide for conjugation to the Fc region. We used trastuzumab and the chelator (DOTA)
as model antibody and payload, respectively. Interestingly, peptide/DOTA-conjugated trastuzumab exhibited enhanced antibody-dependent cellular cytotoxicity (ADCC) and increased thermal stability.
Detailed structural and thermodynamic analysis clarified that the conjugated peptide blocks the Fc dynamics like a “wedge.” We revealed that (1) decreased
molecular entropy results in enhanced ADCC, and (2)blockade of Fc denaturation results in increased thermal stability. Thus, we believe that our methodology
is superior not only for drug conjugation but also as
for reinforcing therapeutic antibodies to enhance
ADCC and thermal stability.
31.  One-pot preparation of mannan-coated antigen nanoparticles using human serum albumin as a matrix for tolerance induction
Li S, Murakami D, Nagatoishi S, Liu Y, Tsumoto K,
Katayama Y and Mori T.

Nanoparticles (NPs) for allergen immunotherapy
have garnered attention for their high efficiency andsafety compared with naked antigen proteins. In thiswork, we present mannan-coated protein NPs, incorporating antigen proteins for antigen-specific tolerance induction. The heat-induced formation of protein NPs is a one-pot preparation method and can beapplied to various proteins. Here, the NPs wereformed spontaneously via heat denaturation of threecomponent proteins: an antigen protein, human serum albumin (HSA) as a matrix protein, and mannoprotein (MAN) as a targeting ligand for dendritic
cells (DCs). HSA is non-immunogenic, therefore suitable as a matrix protein, while MAN coats the surfaceof the NP. We applied this method to various antigen
proteins and found that the self-disperse after heat
denaturation was a requirement for incorporation
into the NPs. We also established that the NPs could
target DCs, and the incorporation of rapamycin into
the NPs enhanced the induction of a tolerogenic phenotype of DC. The MAN coating provided steric hindrance and heat denaturation destroyed recognition
structures, successfully preventing anti-antigen antibody binding, indicating the NPs may avoid anaphylaxis induction. The MAN-coated NPs proposed here,
prepared by a simple method, have the potential for
effective and safe allergies treatment for various antigens.
32.  Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex
Mori C, Nagatoishi S, Matsunaga R, Kuroda D, Nakakido M and Tsumoto K.
Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of
multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease,cancer, and fibrosis. All IL-6 family cytokines signal
through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct aswell as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the
IL-11 signaling complex, comparing it with the IL-6
complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear
difference between IL-11 and IL-6. Surface plasmon
resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy
driven, whereas that between IL-6 and IL-6 receptor α(IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results inentropy loss, but that the interaction of gp130 with
the IL-6/IL-6Rα complex results in entropy gain. Our

223hydrogen-deuterium exchange mass spectrometry
experiments suggested that the D2 domain of gp130
was not involved in IL-6-like interactions in the IL-11/
IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface locatedin its D2 domain of gp130. Our findings suggest thatunique interactions of the IL-11 signaling complex
with gp130 are responsible for the distinct biologicalactivities of IL-11 compared to IL-6.
33.  High-throughput analysis system of interaction kinetics for data-driven antibody design
Matsunaga R, Ujiie K, Inagaki M, Fernández Pérez
J, Yasuda Y, Mimasu S, Soga S and Tsumoto K.
Surface plasmon resonance (SPR) is widely used
for antigen-antibody interaction kinetics analysis.
However, it has not been used in the screening phasebecause of the low throughput of measurement andanalysis. Herein, we proposed a high-throughput
SPR analysis system named “BreviA” using the Brevibacillus expression system. Brevibacillus was transformed using a plasmid library containing various
antibody sequences, and single colonies were cultured in 96-well plates. Sequence analysis was performed using bacterial cells, and recombinant antibodies secreted in the supernatant were immobilized
on a sensor chip to analyze their interactions with
antigens using high-throughput SPR. Using this system, the process from the transformation to 384 interaction analyses can be performed within a week. Thissystem utility was tested using an interspecies specificity design of an anti-human programmed cell death
protein 1 (PD-1) antibody. A plasmid library containing alanine and tyrosine mutants of all complementarity-determining region residues was generated. A
high-throughput SPR analysis was performed against
human and mouse PD-1, showing that the mutationin the specific region enhanced the affinity for mouse
PD-1. Furthermore, deep mutational scanning of theregion revealed two mutants with > 100-fold increased affinity for mouse PD-1, demonstrating thepotential efficacy of antibody design using data-driven approach.
34.  Experimental modification in thermal stabilityof oligomers by alanine substitution and site
saturation mutagenesis of interfacial residues
Hoya M, Matsunaga R, Nagatoishi S and Tsumoto
K.
For certain industrial applications, the stability ofprotein oligomers is important. In this study, wedemonstrated an efficient method to improve thethermal stability of oligomers using the trimeric pro-tein chloramphenicol acetyltransferase (CAT) as themodel. We substituted all interfacial residues of CAT
with alanine to detect residues critical for oligomer
stability. Mutation of six of the forty-nine interfacial
residues enhanced oligomer thermal stability. Sitesaturation mutagenesis was performed on these six
residues to optimize the side chains. About 15% ofmutations enhanced thermal stability by more than
0.5 °C and most did not disrupt activity of CAT. Certain combinations of mutations further improved
thermal stability and resistance against heat treatment. The quadruple mutant, H17V/N34S/F134A/
D157C, retained the same activity as the wild-typeafter heat treatment at 9 °C higher temperature than
the wild-type CAT. Furthermore, combinations with
only alanine substitutions also improved thermal stability, suggesting the method we developed can beused for rapid modification of industrially important
proteins.
35.  Quantitative analysis of antibody aggregatesby combination of pinched-flow fractionationand coulter method
Nagatoishi S, Toyoshima T, Furukawa K and Tsumoto K.
For the pharmaceutical development of proteins,multiple methods of analysis are recommended forevaluating aggregation, and the development of morequantitative and simpler analytical techniques forsubvisible particles is expected. This study introduces
the Pinched-Flow Fractionation (PFF)-Coulter method, which combines the Pinched-flow fractionation(PFF) and Coulter methods to analyze the concentration of submicron-sized particles. The PFF method
separates the particles by size. Separated particles
were individually detected using the Coulter method.
We have utilized the PFF-Coulter method to quantitatively analyze particle concentrations using standard
particles, evaluate detection limits, variability, andcorrelation between theoretical and measured values,
and analyze mixtures of different particle sizes. The
PFF-Coulter method allows for quantitatively analyzing of particle sizes from 0.2 to 2.0 μm. The quantifiable weight concentration range was 2.5 × 10-2 - 50 μg/
mL, and the number concentration range was 1041010 particles/mL. The sample volume was small (<10μL). The PFF-Coulter method is capable of quantitative analysis that complements data from conventional measurement techniques, and when used in conjunction with existing submicron-size particle analysis
techniques, will enable more accurate particle analysis.

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36.  
Megalin is involved in angiotensinogen-induced, angiotensin II-mediated ERK1/2 signaling to activate Na + -H + exchanger 3 in proximal tubules
Goto S, Yoshida Y, Hosojima M, Kuwahara S, Kabasawa H, Aoki H, Iida T, Sawada R, Ugamura D,
Yoshizawa Y, Takemoto K, Komochi K, Kobayashi
R, Kaseda R, Yaoita E, Nagatoishi S, Narita I, Tsumoto K and Saito A.
Kidney angiotensin (Ang) II is produced mainly
from liver-derived, glomerular-filtered angiotensinogen (AGT). Podocyte injury has been reported to increase the kidney Ang II content and induce Na +retention depending on the function of megalin, aproximal tubular endocytosis receptor. However,
how megalin regulates the renal content and action of
Ang II remains elusive. We used a mass spectrometry-based, parallel reaction-monitoring assay to quantitate Ang II in plasma, urine, and kidney homogenate of kidney-specific conditional megalin knockout(MegKO) and control (Ctl) mice. We also evaluatedthe pathophysiological changes in both mouse genotypes under the basal condition and under the condition of increased glomerular filtration of AGT induced
by administration of recombinant mouse AGT (recmAGT). Under the basal condition, plasma and kidney Ang II levels were comparable in the two mousegroups. Ang II was detected abundantly in fresh spot
urine in conditional MegKO mice. Megalin was also
found to mediate the uptake of intravenously administered fluorescent Ang II by PTECs. Administrationof rec-mAGT increased kidney Ang II, exerted renal
extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, activated proximal tubular Na + -H + exchanger 3 (NHE3), and decreased urinary Na + excretion in Ctl mice, whereas these changes were
suppressed but urinary Ang II was increased in conditional MegKO mice. Increased glomerular filtrationof AGT is likely to augment Ang II production in theproximal tubular lumen. Thus, megalin-dependent
Ang II uptake should be involved in the ERK1/2 signaling that activates proximal tubular NHE3 in vivo ,thereby causing Na + retention.
37.  Safe and efficient oral allergy immunotherapy
using one-pot-prepared mannan-coated allergen nanoparticles
Li S, Toriumi H, Takahashi D, Kamasaki T, Fujioka
Y, Nagatoishi S, Li J, Liu Y, Hosokawa T, Tsumoto
K, Ohba Y, Katayama Y, Murakami D, Hase K andMori T.
Allergen immunotherapy (AIT) is the only curative treatment for allergic diseases. However, AIT has
many disadvantages related to efficiency, safety,
long-term duration, and patient compliance. Dendrit-

ic cells (DCs) have an important role in antigen-specific tolerance induction; thus, DC-targeting strategies to treat allergies such as glutaraldehyde
crosslinked antigen to mannoprotein (MAN) havebeen established. However, glutaraldehyde crosslinking may reduce the antigen presentation efficiency ofDCs. To overcome this, we developed a MAN-coatedovalbumin (OVA) nanoparticle (MDO), which uses
intermolecular disulfide bond to crosslink OVA andMAN. MDO effectively targeted DCs resulting intolerogenic DCs, and promoted higher antigen presentation efficiency by DCs compared with OVA or
glutaraldehyde crosslinked nanoparticles. In vitro
and in vivo experiments showed that DCs exposed to
MDO induced Treg cells. Moreover, MDO had low
reactivity with anti-OVA antibodies and did not induce anaphylaxis in allergic mice, demonstrating its
high safety profile. In a mouse model of allergic asthma, MDO had significant preventative and therapeutic effects when administered orally or subcutaneously. Therefore, MDO represents a promising new
approach for the efficient and safe treatment of allergies.
38.  
Enhancing thermal stability in the CH2 domain to suppress aggregation through theintroduction of simultaneous disulfide bonds
in Pichia pastoris
Oyama K, Nakakido M, Ohkuri T, Nakamura H,
Tsumoto K and Ueda T.
Protein aggregations decrease production yields
and impair the efficacy of therapeutics. The CH2 domain is a crucial part of the constant region of human
IgG. But, it is also the least stable domain in IgG,
which can result in antibody instability and aggregation problems. We created a novel mutant of the CH2
domain (T250C/L314C, mut10) by introducing a disulfide bond and expressed it using Pichia pastoris.
The mut10 variant exhibited enhanced thermal stability, resistance to enzymatic degradation, and reduced
aggregation in comparison to the original CH2 domain. However, it was less stable than mut20 (L242C/
K334C), which is the variant prepared in a previous
study (Gong et al., J. Biol. Chem., 2009). A more advanced mutant, mut25, was created by combining
mut10 and mut20. Mut25 artificially contains two disulfide bonds. The new mutant, mut25, showed enhanced thermal stability, increased resistance to enzymatic digestion, and reduced aggregation incomparison to mut20. According to our knowledge,
mut25 achieves an unprecedented level of stability
among the humanized whole CH2 domains that havebeen reported so far. Mut25 has the potential to serve
as a new platform for antibody therapeutics due to its
ability to reduce immunogenicity by decreasing aggregation.

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39.  
Conformational features and interaction
mechanisms of VH H antibodies with β-hairpin CDR3: A case of Nb8-HigB2 interaction
Yamamoto K, Nagatoishi S, Matsunaga R, Nakakido M, Kuroda D and Tsumoto K.
The β-hairpin conformation is regarded as an important basic motif to form and regulate protein-protein interactions. Single-domain VH H antibodies arepotential therapeutic and diagnostic tools, and thethird complementarity-determining regions of theheavy chains (CDR3s) of these antibodies are criticalfor antigen recognition. Although the sequences andconformations of the CDR3s are diverse, CDR3s
sometimes adopt β-hairpin conformations. However,characteristic features and interaction mechanisms of
β-hairpin CDR3s remain to be fully elucidated. In thisstudy, we investigated the molecular recognition of
the anti-HigB2 VH H antibody Nb8, which has a
CDR3 that forms a β-hairpin conformation. The interaction was analyzed by evaluation of alanine-scanning mutants, molecular dynamics simulations, andhydrogen/deuterium exchange mass spectrometry.
These experiments demonstrated that positions 93
and 94 (Chothia numbering) in framework region 3,
which is right outside CDR3 by definition, play pivotal roles in maintaining structural stability and binding properties of Nb8. These findings will facilitate
the design and optimization of single-domain antibodies.
<Group III>
1.  Development of new methods for analyzing
neural circuits in the retina
Neural circuits in the central nervous system are
the basis of various higher-order brain functions. It isalso true in case of retina. In the retina, six main classes of neural cells connect systematically to make upcomplex neural circuits. Characteristics of the retinal
neural cell functions have been examined mainly by
the electrophysiological methods and models of cellconnectivity have been proposed. Morphologicalstudies of the actual neural connection, which constitute the physiological properties of retinal neurons,
have been desired. Until recently the only method toreveal the three-dimensional (3D) connectivity of actual neural cells morphologically was to collect ultrathin serial sections and observe them in transmission electron microscope (TEM). But the technicaldifficulties discouraged us from such a troublesome
procedure. Recent progress in scanning electron microscope (SEM) equipment allowed us to develop a
new method to observe ultrathin TEM sections in
SEM (thin section scanning electron microscopy: TSSEM). To observe thin TEM sections, we have developed new sample staining methods to enhance electron contrast. To collect huge number of serial sections

stably and efficiently, we have been developing new
equipment and techniques. By using this equipment,
it became possible to collect more than 1000 serial sections of less than 30 nm thickness much easier. We
have analyzed about 500 serial thin sections of zebrafish retinal outer plexiform layer by this method andsucceeded in tracing thin processes of bipolar cells
into the photoreceptor terminals.
Aside from getting 3D information, TSSEM method can provide us precise information of much wider
areas of thin sections more effectively and more easily
than transmission electron microscopy. Such studies
are currently in progress.
2.  Collaborative and supportive works as electron microscope core-laboratory
This group is also engaged in collaborative researches using electron microscope. We offer supports for the research projects those need electron microscopic analysis. The services available in this groupare the conventional thin section transmission electron microscopy, immuno-electron microscopy, negative staining techniques and scanning electron microscopy. By using individual technique or
combination of some of these, we can offer direct
visual evidence that cannot be acquired by othermethods. This year, 21 projects in 10 laboratories were
performed as core-laboratory works.
a.  Thin section transmission electron microscopy
Thin section transmission electron microscopy is
the most widely used technique to observe the inner
structure of cells and tissues. In this method, samples
are fixed and embedded in epoxy resin, thin sections
with about 70 nm thickness are cut and observed in
the electron microscope. In case of immuno-electron
microscopy, thin sections are obtained by similar procedure and the antigen epitopes exposed on the surface of the sections are marked by sequential reaction
with appropriate primary antibodies and colloidal
gold labeled secondary antibodies. This year, thin section electron microscopy and those combined withimmuno-electron microscopy were used in many collaborative works.
a-1. Ultrastructural analysis of entry and assembly of Herpes Simplex Virus
We have been performing several studies with research groups in Dr. Kawaguchi1’s laboratory: 1Division of Molecular Virology, Department of Microbiology and Immunology, regarding the infection/
replication processes of herpes simplex virus (HSV).
Thin section electron microscopy has been used toanalyze the function of viral proteins in trans-nuclear
membrane processes of the newly formed viruses. By

226
analyzing the virus forming processes in some mutant host cells, we could analyze viral proteins as wellas candidate host molecules those may be involved in
the trans-nuclear process of the HSV.

Life Science Dentistry, The Nippon Dental University
and so on.

a-2. Analysis of sodium iodate effects on retinaldegeneration

Negative staining techniques are simple and quick
method to observe the morphology of the macro-molecules. This year, negative staining techniques wereused to analyze exosomes in collaboration with Dr.
Hayashi6 in 6Division of Vaccine Science, Laboratoryof Adjuvant Innovation. The same techniques are also
used in the research with Dr. Isoo7, in 7Division of Infectious Diseases.

We have been performing several studies alsowith research groups in Dr. Watanabe2’s laboratory:
2
Department of Retinal Biology and Pathology, Graduate School of Medicine, The University of Tokyo.
This year, we analyzed the effects of sodium iodate(NaIO3) administration on retinal degeneration.
NaIO3 is widely used to induce retinal degenerationin model animals such as mice, rats and rabbits. With
precise morphological analysis of NaIO3 administered mouse retina, we found that NaIO3 specificallydamages retinal choriocapillaris and this leads to the
disfunction of the retinal pigment epithelial (RPE)
cells and father to the retinal degeneration. With biochemical analysis, retinal degeneration is supposed to
be induced by rapid decrease of VEGF in RPE cells.
(ref. Iwagawa et al)Some other collaborative research works using
thin section electron microscopy and/or immuno-electron microscopy were performed with Dr. Eguchi3, in
3Division of Genetics, about the roles of muscle specific kinase on formation of neuromuscular junction.
(ref. Eguchi et al) Dr. Kamioka4, in 4Division of Malaria Immunology, about the function of Paneth cells on
Malaria infection, Dr. Nakahara5 in 5Department ofb.  Negative staining techniquesc.  Conventional scanning electron microscopy
Conventional scanning electron microscopy is atechnique used to examine the surface structure of thecells, tissues or other non-biological materials. Scanning electron microscopy combined with thin section
transmission microscopy were used in collaborative
work with Dr. Ishikawa8, 8Laboratory of Reproductive Systems Biology, about the structure of young
and aged mouse oocyte zona pellucida and also about
the morphology of the sperm and spermatocyte.
Scanning electron microscopy are also used in the observation of the surface structure of electrochemically
active microorganisms with Dr. Kobayashi9, 9Frontier
Research Center for Energy and Resource (FRCER),
Graduate School of Engineering, The University ofTokyo.

Publications<Group I>
Miyashita R, Nishiyama A, Qin W, Chiba Y, Kori S,
Kato N, Konishi C, Kumamoto S, Kozuka-Hata H,
Oyama M, Kawasoe Y, Tsurimoto T, Takahashi TS,
Leonhardt H, Arita K, and Nakanishi M. The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5. Elife, 12:e79013, 2023.
Yamaguchi K, Nakagawa S, Saku A, Isobe Y, Yamaguchi R, Sheridan P, Takane K, Ikenoue T, Zhu C, Miura M, Okawara Y, Nagatoishi S, Kozuka-Hata H,
Oyama M, Aikou S, Ahiko Y, Shida D, Tsumoto K,
Miyano S, Imoto S, and Furukawa Y. Bromodomain
protein BRD8 regulates cell cycle progression incolorectal cancer cells through a TIP60-independent regulation of the pre-RC complex. iScience, 26:106563, 2023.
Fujikawa D, Nakamura T, Yoshioka D, Li Z, Moriizumi H, Taguchi M, Tokai-Nishizumi N, Kozuka-Hata H, Oyama M, and Takekawa M. Stressgranule formation inhibits stress-induced apoptosis by selectively sequestering executioner caspases. Curr Biol, 33: 1967-1981.e8, 2023.

Arakawa K, Hirose T, Inada T, Ito T, Kai T, Oyama M,
Tomari Y, Yoda T, and Nakagawa S. Nondomain
biopolymers: Flexible molecular strategies to acquire biological functions. Genes Cells, 28: 539-552,
2023.
Kozuka-Hata H, Hiroki T, Miyamura N, Kitamura A,
Tsumoto K, Inoue JI, and Oyama M. Real-Time
Search-Assisted Multiplexed Quantitative Proteomics Reveals System-Wide Translational Regulation of Non-Canonical Short Open Reading
Frames. Biomolecules, 13: 979, 2023.
Soeda S, Oyama M, Kozuka-Hata H, and Yamamoto
T. The CCR4-NOT complex suppresses untimelytranslational activation of maternal mRNAs. Development, 150: dev201773, 2023.
Homma MK, Nakato R, Niida A, Bando M, Fujiki K,
Yokota N, Yamamoto S, Shibata T, Takagi M, Yamaki J, Kozuka-Hata H, Oyama M, Shirahige K, andHomma Y. Cell cycle-dependent gene networks forcell proliferation activated by nuclear CK2α complexes. Life Sci Alliance, 7: e202302077, 2023.
Yoshimoto R, Nakayama Y, Nomura I, Yamamoto I,
Nakagawa Y, Tanaka S, Kurihara M, Suzuki Y,

227
Kobayashi T, Kozuka-Hata H, Oyama M, Mito M,
Iwasaki S, Yamazaki T, Hirose T, Araki K, and Nakagawa S. 4.5SH RNA counteracts deleterious exonization of SINE B1 in mice. Mol Cell, 83: 4479-4493.
e6, 2023.
<Group II>
Tanabe A and Tsumoto K. Analytical Method for Experimental Validation of Computer-Designed Antibody. Methods Mol Biol, 2552: 409-433, 2023.
Kuroda D and Tsumoto K. Structural Classification of
CDR-H3 in Single-Domain VHH Antibodies. Methods Mol Biol, 2552: 61-79, 2023.
Kosuge H, Nagatoishi S, Kiyoshi M, Ishii-Watabe A,
Terao Y, Ide T, and Tsumoto K. Biophysical Characterization of the Contribution of the Fab Region to
the IgG-FcγRIIIa Interaction. Biochemistry, 62: 262269, 2023.
Furuya G, Katoh H, Atsumi S, Hashimoto I, Komura
D, Hatanaka R, Senga S, Hayashi S, Akita S, Matsumura H, Miura A, Mita H, Nakakido M, Nagatoishi
S, Sugiyama A, Suzuki R, Konishi H, Yamamoto A,
Abe H, Hiraoka N, Aoki K, Kato Y, Seto Y, Yoshimura C, Miyadera K, Tsumoto K, Ushiku T, andIshikawa S. Nucleic acid-triggered tumoral immunity propagates pH-selective therapeutic antibodies through tumor-driven epitope spreading. Cancer Sci, 114: 321-338, 2023.
Gilep A, Varaksa T, Bukhdruker S, Kavaleuski A, Ryzhykau Y, Smolskaya S, Sushko T, Tsumoto K,
Grabovec I, Kapranov I, Okhrimenko I, Marin E,
Shevtsov M, Mishin A, Kovalev K, Kuklin A, Gordeliy V, Kaluzhskiy L, Gnedenko O, Yablokov E,
Ivanov A, Borshchevskiy V, and Strushkevich N.
Structural insights into 3Fe-4S ferredoxins diversity
in M. tuberculosis highlighted by a first redox complex with P450. Front Mol Biosci, 9: 1100032, 2023.
Amano A, Sanjo N, Araki W, Anraku Y, Nakakido M,
Matsubara E, Tomiyama T, Nagata T, Tsumoto K,
Kataoka K, and Yokota T. Peripheral administration of nanomicelle-encapsulated anti-Aβ oligomer
fragment antibody reduces various toxic Aβ species
in the brain. J Nanobiotechnology, 21: 36, 2023.
Arakawa T, Tomioka Y, Nakagawa M, Sakuma C,
Kurosawa Y, Ejima D, Tsumoto K, and Akuta T.
Non-Affinity Purification of Antibodies. Antibodies (Basel), 12: 15, 2023.
Tatsuma T, Nakakido M, Ichinohe T, Kuroiwa Y, Tomioka K, Liu C, Miyamae N, Onuki T, Tsumoto K,
Hashimoto K, and Wakihara T. Inactivation andspike protein denaturation of novel coronavirus
variants by CuxO/TiO2 nano-photocatalysts. Sci
Rep, 13: 4033, 2023.
Watanabe T, Mizuno HL, Norimatsu J, Obara T,
Cabral H, Tsumoto K, Nakakido M, Kawauchi D,
and Anraku Y. Ligand Installation to Polymeric Micelles for Pediatric Brain Tumor Targeting. Polymers (Basel), 15: 1808, 2023.
Yamaguchi K, Nakagawa S, Saku A, Isobe Y, Yamagu-

chi R, Sheridan P, Takane K, Ikenoue T, Zhu C, Miura M, Okawara Y, Nagatoishi S, Kozuka-Hata H,
Oyama M, Aikou S, Ahiko Y, Shida D, Tsumoto K,
Miyano S, Imoto S, and Furukawa Y. Bromodomain
protein BRD8 regulates cell cycle progression incolorectal cancer cells through a TIP60-independent regulation of the pre-RC complex. iScience, 26:106563, 2023.
Yui A, Kuroda D, Maruno T, Nakakido M, Nagatoishi
S, Uchiyama S, and Tsumoto K. Molecular mechanism underlying the increased risk of colorectal
cancer metastasis caused by single nucleotide polymorphisms in LI-cadherin gene. Sci Rep, 13: 6493,
2023.
Liu H, Baeumler TA, Nakamura K, Okada Y, Cho S,
Eguchi A, Kuroda D, Tsumoto K, Ueki R, and Sando S. An Engineered Synthetic Receptor-Aptamer
Pair for an Artificial Signal Transduction System.
ACS Nano, 17: 9039-9048, 2023.
Tomioka Y, Sato R, Takahashi R, Nagatoishi S, Shiba
K, Tsumoto K, Arakawa T, and Akuta T. Agarose
native gel electrophoresis analysis of thermal aggregation controlled by Hofmeister series. Biophys
Chem, 296: 106977, 2023.
Hishikawa Y, Noya H, Nagatoishi S, Yoshidome T,
Maity B, Tsumoto K, Abe S, and Ueno T. Elucidating Conformational Dynamics and Thermostabilityof Designed Aromatic Clusters by Using Protein
Cages. Chemistry, 29: e202300488, 2023.
Hirao A, Nagatoishi S, Ikeuchi E, Yamawaki T, MoriC, Nakakido M, and Tsumoto K. Design of single-domain VHH antibodies to increase the binding activity in SPR amine coupling. Biochem Biophys Res Commun, 663: 54-60, 2023.
Kozuka-Hata H, Hiroki T, Miyamura N, Kitamura A,
Tsumoto K, Inoue JI, and Oyama M. Real-Time
Search-Assisted Multiplexed Quantitative Proteomics Reveals System-Wide Translational Regulation of Non-Canonical Short Open Reading
Frames. Biomolecules, 13: 979, 2023.
Valenciano-Bellido S, Caaveiro JMM, Nakakido M,
Kuroda D, Aikawa C, Nakagawa I, and Tsumoto K.
Targeting hemoglobin receptors IsdH and IsdB of
Staphylococcus aureus with a single VHH antibody
inhibits bacterial growth. J Biol Chem, 299: 104927,
2023.
Nishiyama K, Akiba H, Nagata S, Tsumoto K, Kamada H, and Ohno H. A Proximity-Induced Fluorogenic Reaction Triggered by Antibody-Antigen Interactions with Adjacent Epitopes. Angew ChemInt Ed Engl, 62: e202306431, 2023.
Sato Y, Nagatoishi S, Noguchi S, and Tsumoto K. Raman Spectroscopic Analysis of Highly-Concentrated Antibodies under the Acid-Treated Conditions.
Pharm Res, 40: 1853-1864, 2023.
Liu N, Konuma T, Sharma R, Wang D, Zhao N, Cao L,
Ju Y, Liu D, Wang S, Bosch A, Sun Y, Zhang S, Ji D,
Nagatoishi S, Suzuki N, Kikuchi M, Wakamori M,
Zhao C, Ren C, Zhou TJ, Xu Y, Meslamani J, Fu S,

228
Umehara T, Tsumoto K, Akashi S, Zeng L, Roeder
RG, Walsh MJ, Zhang Q, and Zhou MM. Histone
H3 lysine 27 crotonylation mediates gene transcriptional repression in chromatin. Mol Cell, 83: 22062221, 2023.
Tang D, Kato Y, Zhang D, Negishi L, Kurumizaka H,
Hirata T, Nakakido M, Tsumoto K, Shuji F, Tsuguyuki S, Okumura T, Nagata K, and Suzuki M. Dispersion Function of a Protein, DP-1, Identified in
Collimonas sp. D-25, for the Synthesis of Gold Nanoparticles. Chembiochem, 24: e202300221, 2023.
Akiba H, Fujita J, Ise T, Nishiyama K, Miyata T, Kato
T, Namba K, Ohno H, Kamada H, Nagata S, andTsumoto K. Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling
activity through epitope selection. Commun Biol,
6: 987, 2023.
Echizen H, Hanaoka K, Shimamoto K, Hibi R, Toma-Fukai S, Ohno H, Sasaki E, Komatsu T, Ueno T,
Tsuchiya Y, Watanabe Y, Otsuka T, Saito H, Nagatoishi S, Tsumoto K, Kojima H, Okabe T, Shimizu T,
and Urano Y. Discovery of a cystathionine γ-lyase(CSE) selective inhibitor targeting active-site pyridoxal 5’-phosphate (PLP) via Schiff base formation.
Sci Rep, 13: 16456, 2023.
Senoo A, Nagatoishi S, Kuroda D, Ito S, Ueno G,
Caaveiro JMM, and Tsumoto K. Modulation of aconformational ensemble by a small molecule that
inhibits key. Protein Sci, 32: e4744, 2023.
Maeta S, Nakakido M, Matsuura H, Sakai N, Hirata
K, Kuroda D, Fukunaga A, and Tsumoto K. Arginine cluster introduction on framework region inanti-lysozyme antibody improved association rate
constant by changing conformational diversity of
CDR loops. Protein Sci, 32: e4745, 2023.
Aikawa C, Shimizu A, Nakakido M, Murase K, Nozawa T, Tsumoto K, and Nakagawa I. Group A Streptococcus cation diffusion facilitator proteins
contribute to immune evasion by regulating intracellular metal concentrations. Biochem Biophys
Res Commun, 676: 141-148, 2023.
Tomioka Y, Nagatoishi S, Nakagawa M, Tsumoto K,
Arakawa T, and Akuta T. Ferguson plot analysis ofmultiple intermediate species of thermally unfolded bovine serum albumin. Biophys Chem, 301:
107095, 2023.
Yamazaki T, Nagatoishi S, Yamawaki T, Nozawa T,
Matsunaga R, Nakakido M, Caaveiro JMM, Nakagawa I, and Tsumoto K. Anti-InlA single-domain
antibodies that inhibit the cell invasion of Listeria
monocytogenes. J Biol Chem, 299: 105254, 2023.
Davaapil H, Hopkins J, Bonnin N, Papadaki V, Leung
A, Kosuge H, Tashima T, Nakakido M, Sekido R,
Tsumoto K, Sagoo MS, and Ohnuma SI. PRELP secreted from mural cells protects the function ofblood brain barrier through regulation of endothelial cell-cell integrity. Front Cell Dev Biol, 11:1147625, 2023.
Kiyoshi M, Nakakido M, Rafique A, Tada M, Aoyama

M, Terao Y, Nagatoishi S, Shibata H, Ide T, Tsumoto
K, Ito Y, and Ishii-Watabe A. Specific peptide conjugation to a therapeutic antibody leads to enhanced
therapeutic potency and thermal stability by reduced Fc dynamics. Sci Rep, 13: 16561, 2023.
Li S, Murakami D, Nagatoishi S, Liu Y, Tsumoto K,
Katayama Y, and Mori T. One-pot preparation of
mannan-coated antigen nanoparticles using human serum albumin as a matrix for tolerance induction. J Colloid Interface Sci, 649: 955-965, 2023.
Mori C, Nagatoishi S, Matsunaga R, Kuroda D, Nakakido M, and Tsumoto K. Biophysical insight into
protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex. Biochem Biophys Res Commun, 682: 174179, 2023.
Matsunaga R, Ujiie K, Inagaki M, Fernández Pérez J,
Yasuda Y, Mimasu S, Soga S, and Tsumoto K.
High-throughput analysis system of interaction kinetics for data-driven antibody design. Sci Rep, 13:
19417, 2023.
Hoya M, Matsunaga R, Nagatoishi S, and Tsumoto K.
Experimental modification in thermal stability ofoligomers by alanine substitution and site saturation mutagenesis of interfacial residues. Biochem
Biophys Res Commun, 691: 149316, 2023.
Nagatoishi S, Toyoshima T, Furukawa K, and Tsumoto K. Quantitative analysis of antibody aggregatesby combination of pinched-flow fractionation andcoulter method. Anal Biochem, 681: 115331, 2023.
Goto S, Yoshida Y, Hosojima M, Kuwahara S, Kabasawa H, Aoki H, Iida T, Sawada R, Ugamura D,
Yoshizawa Y, Takemoto K, Komochi K, Kobayashi
R, Kaseda R, Yaoita E, Nagatoishi S, Narita I, Tsumoto K, and Saito A. Megalin is involved in angiotensinogen-induced,
angiotensin
II-mediated
ERK1/2 signaling to activate Na + -H + exchanger
3 in proximal tubules. J Hypertens, 41: 1831-1843,
2023.
Li S, Toriumi H, Takahashi D, Kamasaki T, Fujioka Y,
Nagatoishi S, Li J, Liu Y, Hosokawa T, Tsumoto K,
Ohba Y, Katayama Y, Murakami D, Hase K, andMori T. Safe and efficient oral allergy immunotherapy using one-pot-prepared mannan-coated allergen nanoparticles. Biomaterials, 303: 122381, 2023.
Oyama K, Nakakido M, Ohkuri T, Nakamura H, Tsumoto K, and Ueda T. Enhancing thermal stability in
the CH2 domain to suppress aggregation throughthe introduction of simultaneous disulfide bonds in
Pichia pastoris. Protein Sci, 32: e4831, 2023.
Yamamoto K, Nagatoishi S, Matsunaga R, Nakakido
M, Kuroda D, and Tsumoto K. Conformational features and interaction mechanisms of VH H antibodies with β-hairpin CDR3: A case of Nb8-HigB2
interaction. Protein Sci, 32: e4827, 2023.
<Group III>
Yasuda T, Nakazawa T, Hirakawa K, Matsumoto I,
Nagata K, Mori S, Igarashi K, Sagara H, Oda S, and229
Mitani H. Retinal regeneration after injury induced
by gamma-ray irradiation during early embryogenesis in medaka, Oryzias latipes. Int J Radiat
Biol, 9: 1-8, 2023.
Iwagawa T, Saita K, Sagara H, and Watanabe S.
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