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International Vaccine Design Center

Division of Vaccine Engineering(New Dimensional Vaccine Design Team)
新次元ワクチンデザイン系・ワクチン工学分野
Project Professor

Kouhei Tsumoto, Ph.D.

特任教授

博士（工学）津本浩

平Biomolecular recognition is based on collective and specific non-covalentinteractions between discrete biological molecules. Our laboratory studies various
protein systems, for instance, antibody-antigen and protein-ligand complexes, tounderstand quantitatively how these coordinated non-covalent interactions
contribute to their specific recognition in biological and artificial systems. We seek
to elucidate the molecular mechanisms by which biological molecules obtain highspecificity and affinity from multiple angles using advanced instrumentation. To
produce functional molecules with higher performance and better properties, we
aim to build a solid foundation from which to develop drugs that modulate specificinteractions between biomolecules and ultimately to understand the principles ofmolecular interactions in our lives.

1.  Analytical Method for Experimental Validationof Computer-Designed Antibody
Tanabe A, 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 technique.

2. Structural Classification of CDR-H3 in Single-Domain VHH Antibodies
Kuroda D, 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 and177
heavy 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, 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, theinteraction of commercially available, full-length
rituximab with FcγRIIIa had a more favorable binding enthalpy, a less favorable binding entropy, and aslower off rate. Similar results were obtained fromanalyses of IgG1 molecules and an IgG1-Fc fragment
produced by Expi293 cells. For further validation, we
also prepared a maltose-binding protein-linked IgG1Fc fragment (MBP-Fc). The binding enthalpy of MBPFc was nearly equal to that of the IgG1-Fc fragment
for the interaction with FcγRIIIa, indicating that suchalternatives to the Fab domains as MBP do not positively contribute to the IgG-FcγRIIIa interactions. Ourinvestigation strongly suggests that the Fab region
directly interacts with FcγRIIIa, resulting in an increase in the binding 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, 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, Strushkevich N.
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.

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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, Yokota
Background: 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 human clinical trials, partly because peripheral administration of antibody medications was unable
to deliver sufficient amounts of the molecules to thebrain. Recently, we developed polymeric nanomicelles capable of 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.
Results: 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 spatial reference memory in the probe and platform testsof the water maze, thereby indicating inhibition of thepathophysiological process of Alzheimer’s disease.
Conclusions: The results indicated that the strategy of reducing toxic Aβ species in early dementia owing to Alzheimer’s disease by providing sufficient antibodies in the brain may modify Alzheimer’s diseaseprogression.
7. Non-Affinity Purification of Antibodies
Arakawa T, Tomioka Y, Nakagawa M, Sakuma C,
Kurosawa Y, Ejima D, Tsumoto K, 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 chro-

matography 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, 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.
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,
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 the179
delivery. 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, 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, Tsumoto K.
LI-cadherin is a member of the cadherin super-family. 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, 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 react 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.

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13. Agarose native gel electrophoresis analysis ofthermal aggregation controlled by HofmeisterseriesTomioka Y, Sato R, Takahashi R, Nagatoishi S, Shiba K, Tsumoto K, Arakawa T, 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, 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 to increase the binding activity in SPR amine coupling
Hirao A, Nagatoishi S, Ikeuchi E, Yamawaki T, MoriC, Nakakido M, 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, andmutating 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, Oyama M.
Abnormal expression of histone deacetylases(HDACs) is reported to be associated with angiogen-181
esis, 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 IsdBof Staphylococcus aureus with a single VHH
antibody inhibits bacterial growth
Valenciano-Bellido S, Caaveiro JMM, Nakakido M,
Kuroda D, Aikawa C, Nakagawa I, 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 na-

nomolar 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, 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.
19. Raman Spectroscopic Analysis of Highly-Concentrated Antibodies under the Acid-TreatedConditions
Sato Y, Nagatoishi S, Noguchi S, Tsumoto K.
Purpose: 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 under high concentration conditions is important.
Methods: 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 human serum IgG and recombinant
rituximab.
Results: We detected the evident changes at pH 3

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in Tyr and Trp bands, which are the sensitive markers
of intermolecular interactions. Thermal transition
analysis over the pH range demonstrated that thethermal transition temperature (Tm) was highest at
pH 3. Acid-treated and neutralized one showed higher Tm than that of pH 7, indicating that their extent ofintermolecular interactions correlated with the Tm
values. Onset temperature was clearly different between concentrated and diluted samples. Colloidal
analyses confirmed the findings of the Raman analysis.
Conclusion: 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, Zhou MM.

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,
Tsumoto K

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.

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 antagonist, 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.

21. Dispersion Function of a Protein, DP-1, Identified in Collimonas sp. D-25, for the Synthesis
of Gold Nanoparticles

23. Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal
5’-phosphate (PLP) via Schiff base formation

Tang D, Kato Y, Zhang D, Negishi L, Kurumizaka
H, Hirata T, Nakakido M, Tsumoto K, Shuji F, Tsuguyuki S, Okumura T, Nagata K, Suzuki M.

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, Nagat-

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oishi S, Tsumoto K, Kojima H, Okabe T, Shimizu T,
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 by asmall molecule that inhibits key protein-protein interactions involved in cell adhesion
Senoo A, Nagatoishi S, Kuroda D, Ito S, Ueno G,
Caaveiro JMM, 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, 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, Nakagawa I.
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184
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
Tomioka Y, Nagatoishi S, Nakagawa M, Tsumoto K,
Arakawa T, Akuta T.
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 native-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, 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
Davaapil H, Hopkins J, Bonnin N, Papadaki V, Leung A, Kosuge H, Tashima T, Nakakido M, Sekido
R, Tsumoto K, Sagoo MS, Ohnuma SI.
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
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

185
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, 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, 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, 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
hydrogen-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, Tsumoto K.

186

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, 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 protein 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, 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.
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, Saito A.
Background: Kidney angiotensin (Ang) II is produced mainly from liver-derived, glomerular-filteredangiotensinogen (AGT). Podocyte injury has been reported to increase the kidney Ang II content and induce Na + retention depending on the function of
megalin, a proximal tubular endocytosis receptor.
However, how megalin regulates the renal content
and action of Ang II remains elusive.
Methods: We used a mass spectrometry-based,
parallel reaction-monitoring assay to quantitate Ang

187
II in plasma, urine, and kidney homogenate of kidney-specific conditional megalin knockout (MegKO)and control (Ctl) mice. We also evaluated the pathophysiological changes in both mouse genotypes under the basal condition and under the condition ofincreased glomerular filtration of AGT induced byadministration of recombinant mouse AGT (recmAGT).
Results: Under the basal condition, plasma andkidney Ang II levels were comparable in the twomouse groups. Ang II was detected abundantly infresh spot urine in conditional MegKO mice. Megalin
was also found to mediate the uptake of intravenously administered fluorescent Ang II by PTECs. Administration of 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.
Conclusion: Increased glomerular filtration of
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,
Mori 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. Dendritic 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 in-duce 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 the introduction of simultaneous disulfide bonds in Pichia
pastorisOyama K, Nakakido M, Ohkuri T, Nakamura H,
Tsumoto K, 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.
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, 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,188
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.

Publications
1.		Tanabe A, Tsumoto K. Analytical Method for Experimental Validation of Computer-Designed Antibody. Methods Mol Biol. 2552. 409-433.2023
2.		Kuroda D, Tsumoto K. Structural Classification of
CDR-H3 in Single-Domain VHH Antibodies.
Methods Mol Biol. 2552. 61-79.2023
3.		Kosuge H, Nagatoishi S, Kiyoshi M, Ishii-Watabe
A, Terao Y, Ide T, Tsumoto K. Biophysical Characterization of the Contribution of the Fab Region to
the IgG-FcγRIIIa Interaction. Biochemistry. 62.
262-269.2023
4.		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, Ishikawa S.Nucleic acid-triggered tumoral immunity propagates pH-selective therapeutic antibodies through tumor-driven epitope
spreading. Cancer Sci. 114(1). 321-338.2023
5.		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, Strushkevich N.
Structural insights into 3Fe-4S ferredoxins diversity in M. tuberculosis highlighted by a first redox
complex with P450. Front Mol Biosci. 9.2023
6.		Amano A, Sanjo N, Araki W, Anraku Y, Nakakido
M, Matsubara E, Tomiyama T, Nagata T, Tsumoto
K, Kataoka K, Yokota T. Peripheral administrationof nanomicelle-encapsulated anti-Aβ oligomer
fragment antibody reduces various toxic Aβ species in the brain. J Nanobiotechnology. 21(1).
36.2023
7.		Arakawa T, Tomioka Y, Nakagawa M, Sakuma C,
Kurosawa Y, Ejima D, Tsumoto K, Akuta T.
Non-Affinity Purification of Antibodies. Antibodies (Basel). 12(1). 15.2023
8.		Tatsuma T, Nakakido M, Ichinohe T, Kuroiwa Y,
Tomioka K, Liu C, Miyamae N, Onuki T, Tsumoto
K, Hashimoto K, Wakihara T. Inactivation andspike protein denaturation of novel coronavirus
variants by CuxO/TiO2 nano-photocatalysts. Sci
Rep. 13(1). 4033.2023

9.		Watanabe T, Mizuno HL, Norimatsu J, Obara T,
Cabral H, Tsumoto K, Nakakido M, Kawauchi D,
Anraku Y. Ligand Installation to Polymeric Micelles for Pediatric Brain Tumor Targeting. Polymers (Basel). 15(7). 1808.2023
10.	
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, Furukawa Y. Bromodomain protein BRD8 regulates cell cycle progression in colorectal cancer cells through a
TIP60-independent regulation of the pre-RC complex. iScience. 26(4). 106563.2023
11.	Yui A, Kuroda D, Maruno T, Nakakido M, Nagatoishi S, Uchiyama S, Tsumoto K. Molecular mechanism underlying the increased risk of colorectal
cancer metastasis caused by single nucleotide polymorphisms in LI-cadherin gene. Sci Rep. 13(1).
6493.2023
12.	Liu H, Baeumler TA, Nakamura K, Okada Y, Cho
S, Eguchi A, Kuroda D, Tsumoto K, Ueki R, Sando
S. An Engineered Synthetic Receptor-Aptamer
Pair for an Artificial Signal Transduction System.
ACS Nano. 17(10). 9039-9048.2023
13.	Tomioka Y, Sato R, Takahashi R, Nagatoishi S, Shiba K, Tsumoto K, Arakawa T, Akuta T. Agarose
native gel electrophoresis analysis of thermal aggregation controlled by Hofmeister series. Biophys Chem. 296. 106977.2023
14.	Hishikawa Y, Noya H, Nagatoishi S, Yoshidome T,
Maity B, Tsumoto K, Abe S, Ueno T. Elucidating
Conformational Dynamics and Thermostability of
Designed Aromatic Clusters by Using Protein
Cages. Chemistry. 29(34) e202300488.2023
15.	Hirao A, Nagatoishi S, Ikeuchi E, Yamawaki T,
Mori C, Nakakido M, 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
16.	Kozuka-Hata H, Hiroki T, Miyamura N, Kitamura
A, Tsumoto K, Inoue JI, Oyama M. Real-Time
Search-Assisted Multiplexed Quantitative Proteomics Reveals System-Wide Translational Regulation of Non-Canonical Short Open Reading
Frames. Biomolecules. 13(6). 979.2023
17.	
Valenciano-Bellido S, Caaveiro JMM, Nakakido

189
M, Kuroda D, Aikawa C, Nakagawa I, Tsumoto K.
Targeting hemoglobin receptors IsdH and IsdB of
Staphylococcus aureus with a single VHH antibody inhibits bacterial growth. J Biol Chem. 299(9).
104927.2023
18.	
Nishiyama K, Akiba H, Nagata S, Tsumoto K,
Kamada H, Ohno H. A Proximity-Induced Fluorogenic Reaction Triggered by Antibody-Antigen
Interactions with Adjacent Epitopes. Angew
Chem Int Ed Engl. 62(30). e202306431.2023
19.	Sato Y, Nagatoishi S, Noguchi S, Tsumoto K. Raman Spectroscopic Analysis of Highly-Concentrated Antibodies under the Acid-Treated Conditions. Pharm Res. 40(7). 1853-1864.2023
20.	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, Zhou MM. Histone H3 lysine 27 crotonylation mediates genetranscriptional repression in chromatin. Mol Cell.
83(13). 2206-2221.2023
21.	Tang D, Kato Y, Zhang D, Negishi L, Kurumizaka
H, Hirata T, Nakakido M, Tsumoto K, Shuji F, Tsuguyuki S, Okumura T, Nagata K, Suzuki M. Dispersion Function of a Protein, DP-1, Identified in
Collimonas sp. D-25, for the Synthesis of Gold Nanoparticles. Chembiochem. 24(14). e202300221.
2023
22.	Akiba H, Fujita J, Ise T, Nishiyama K, Miyata T,
Kato T, Namba K, Ohno H, Kamada H, Nagata S,
Tsumoto K. Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling activity through epitope selection. Commun
Biol. 6(1). 987.2023
23.	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, 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(1). 16456.2023
24.	Senoo A, Nagatoishi S, Kuroda D, Ito S, Ueno G,
Caaveiro JMM, Tsumoto K. Modulation of a conformational ensemble by a small molecule that inhibits key. Protein Sci. 32(9). e4744.2023
25.	Maeta S, Nakakido M, Matsuura H, Sakai N, Hirata K, Kuroda D, Fukunaga A, Tsumoto K. Arginine
cluster introduction on framework region in anti-lysozyme antibody improved association rate
constant by changing conformational diversity of
CDR loops. Protein Sci. 32(9). e4745.2023
26.	Aikawa C, Shimizu A, Nakakido M, Murase K,
Nozawa T, Tsumoto K, 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
27.	Tomioka Y, Nagatoishi S, Nakagawa M, Tsumoto
K, Arakawa T, Akuta T. Ferguson plot analysis ofmultiple intermediate species of thermally unfolded bovine serum albumin. Biophys Chem. 301.
107095.2023
28.	Yamazaki T, Nagatoishi S, Yamawaki T, Nozawa
T, Matsunaga R, Nakakido M, Caaveiro JMM, Nakagawa I, Tsumoto K. Anti-InlA single-domain
antibodies that inhibit the cell invasion of Listeria
monocytogenes. J Biol Chem. 299(10). 105254.2023
29.	Davaapil H, Hopkins J, Bonnin N, Papadaki V, Leung A, Kosuge H, Tashima T, Nakakido M, Sekido
R, Tsumoto K, Sagoo MS, 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
30.	Kiyoshi M, Nakakido M, Rafique A, Tada M,
Aoyama M, Terao Y, Nagatoishi S, Shibata H, Ide
T, Tsumoto K, Ito Y, Ishii-Watabe A. Specific peptide conjugation to a therapeutic antibody leads toenhanced therapeutic potency and thermal stability by reduced Fc dynamics. Sci Rep. 13(1).
16561.2023
31.	Li S, Murakami D, Nagatoishi S, Liu Y, Tsumoto K,
Katayama Y, Mori T. One-pot preparation of mannan-coated antigen nanoparticles using humanserum albumin as a matrix for tolerance induction. J Colloid Interface Sci. 649. 955-965.2023
32.	Mori C, Nagatoishi S, Matsunaga R, Kuroda D,
Nakakido M, 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
33.	Matsunaga R, Ujiie K, Inagaki M, Fernández Pérez
J, Yasuda Y, Mimasu S, Soga S, Tsumoto K.
High-throughput analysis system of interaction
kinetics for data-driven antibody design. Sci Rep.
13(1). 19417.2023
34.	Hoya M, Matsunaga R, Nagatoishi S, 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
35.	Nagatoishi S, Toyoshima T, Furukawa K, Tsumoto
K. Quantitative analysis of antibody aggregates bycombination of pinched-flow fractionation andcoulter method. Anal Biochem. 681. 115331.2023
36.	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, Saito A. Megalin is involved in angiotensinogen-induced, angiotensin II-mediated ERK1/2
signaling to activate Na + -H + exchanger 3 inproximal tubules. J Hypertens. 41(11). 18311843.2023

190
37.	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,
Mori T. Safe and efficient oral allergy immunotherapy using one-pot-prepared mannan-coatedallergennanoparticles
Biomaterials.
303.
122381.2023
38.	Oyama K, Nakakido M, Ohkuri T, Nakamura H,
Tsumoto K, Ueda T. Enhancing thermal stability

in the CH2 domain to suppress aggregation
through the introduction of simultaneous disulfide bonds in Pichia pastoris. Protein Sci. 32(12).
e4831.2023
39.	Yamamoto K, Nagatoishi S, Matsunaga R, Nakakido M, Kuroda D, Tsumoto K. Conformational features and interaction mechanisms of VH H antibodies with β-hairpin CDR3: A case of Nb8-HigB2
interaction. Protein Sci. 32(12). e4827.2023