Published papers

Viruses are versatile colloidal materials in their biofunctions, monodispersed and periodic structures, and high surface designability. For expanding the applicability of virus-based materials, spatiotemporally controlled immobilization and dispersion of viruses with retained activity should be useful, though control of the dynamic nature of viruses hybridized with commonly used polymers has been difficult due to their strong interactions. Here, we report a self-assembling peptide (A2Az) enabling photo control of adhesion and dispersion of M13 bacteriophage virus (M13 phage) and successfully demonstrate patterning of localization and infection of the virus. A2Az is a cationic peptide with amphiphilicity that consists of eight amino acid residues containing a photo-responsive azobenzene group at the second position and self-assembles into a helical supramolecular fiber to form a hydrogel. The helical fibrillar morphology of A2Az exhibits strong interaction with M13 phage, allowing for immobilization not only on a two-dimensional surface but also in a three-dimensional hydrogel with suppression of infectivity. The A2Az fiber undergoes a light-triggered fiber-to-particle transition and releases the immobilized M13 phage with retained infectivity for the photo-controlled patterning of localization and infection. This approach has potential applicability to various virus-based biomaterials, such as structural materials and materials for photo-selective gene transfection to cells.

Glycoconjugates on animal cell surfaces are involved in numerous biological functions and diseases, especially the adhesion/metastasis of cancer cells, infection, and the onset of glycan-related diseases. In addition to glycoantigen detection, the regulation of glycan (carbohydrate)-protein interactions is needed to develop therapeutic strategies for glycan-related diseases. Preparation of a diverse range of glycan derivatives requires a massive effort, but the preparation and identification of alternative glycan-mimetic peptide mimotopes may provide a solution to this issue. Peptide mimotopes are recognized by glycan-binding proteins, such as lectins, enzymes, and antibodies, alternative to glycan ligands. Phage-display technology is the first choice in the selection of “glycan (carbohydrate)-mimetic peptide mimotopes” from a large repertoire of library sequences. This tutorial review describes the advantages of peptide mimotopes in comparison to glycan ligands, as well as their structural and functional mimicry. The detailed library design is followed by a description of the strategy used to improve affinity, and finally, an outline of the vaccine application of glycan-mimetic peptides is provided.

Alzheimer's disease (AD), a progressive neurodegenerative condition, is one of the most common causes of dementia. Senile plaques, a hallmark of AD, are formed by the accumulation of amyloid β protein (Aβ), which starts to aggregate before the onset of the disease. Gangliosides, sialic acid-containing glycosphingolipids, play a key role in the formation of toxic Aβ aggregates. In membrane rafts, ganglioside-bound complexes (GAβ) act as nuclei for Aβ assembly, suggesting that GAβ is a promising target for AD therapy. The formation of GAβ-induced Aβ assemblies has been evaluated using reconstituted planar lipid membranes composed of synaptosomal plasma membrane (SPM) lipids extracted from human and mouse brains. Although the effects of gangliosides on Aβ accumulation in the precuneus have been established, effects on Aβ fibrils have not been determined. In this study, Aβ42 fibrils on reconstituted membranes composed of SPM lipids prepared from the precuneus cortex of human autopsied brains were evaluated by atomic force microscopy. In particular, Aβ42 accumulation, as well as the fibril number and size were higher for membranes with precuneus lipids than for membranes with calcarine cortex lipids. In addition, artificial peptide inhibitors targeting Aβ-sensitive ganglioside nanoclusters cleared Aβ assemblies on synaptic membranes in the brain, providing a novel therapeutic strategy for AD.

The hemagglutination inhibition (HAI) assay is one of the detection methods for influenza virus (IFV) under global influenza surveillance, which uses freshly prepared animal red blood cells (RBCs). Here, we demonstrate that a mixed glycan-modified polystyrene microparticle, which can be chemically prepared in advance, can replace animal RBCs in the HAI assay. A mixture of azide-conjugated glycans containing sialyl- and sulfated-lactose moieties was produced from Madin-Darby canine kidney (MDCK) cells, which are used for IFV isolation, and then immobilized on the surface of a polystyrene microparticle using click chemistry. Human HA and IFV were detected with high sensitivity when using the mixed glycan-immobilized particle.

Alzheimer’s disease is a progressive neurodegenerative disease and is the most common cause of dementia. It has been reported that the assembly of amyloid β-protein (Aβ) on the cell membrane is induced by the interaction of the Aβ monomer with gangliosides such as GM1. The ganglioside-bound Aβ (GAβ) complex acts as a seed to promote the toxic assembly of the Aβ fibrils. In a previous study, we found that a GM1 cluster-binding peptide (GCBP) specifically recognizes Aβ-sensitive ganglioside nanoclusters and inhibits the assembly of Aβ on a GM1-containing lipid membrane. In this study, cysteine-substituted double mutants of GCBP were designed and cyclized by intramolecular disulfide bond formation. Affinity assays indicated that one of the cyclic peptides had a higher affinity to a GM1-containing membrane compared to that of GCBP. Furthermore, surface topography analysis indicated that this peptide recognizes GM1 nanoclusters on the lipid membrane. An evaluation of the inhibitory kinetics indicated that the cyclic peptide could inhibit the formation of Aβ fibrils with an IC50 value of 1.2 fM, which is 10,000-fold higher than that of GCBP. The cyclic peptide was also shown to have a clearance effect on Aβ fibrils deposited on the lipid membrane and suppressed the formation of toxic Aβ assemblies. Our results indicate that the cyclic peptide that binds to the Aβ-sensitive ganglioside nanocluster is a potential novel inhibitor of ganglioside-induced Aβ assembly.

Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50values of 25.7 and 63.2 μM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50values (for infection of 293TA cells) of 19.7 and 9.7 μM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50concentrations.

The levitation methodology, which enables us to operate a contactless reaction without a container, is likely to be a revolutionary technology in the fields of chemistry and biology to reduce the plastic waste in life science laboratories. Here, the authors show that plasmid DNA can be effectively transfected into animal cells in a floating droplet of culture medium levitated using ultrasonic standing waves. The data indicate that there is no significant damage to the plasmid and cells during the levitating transfection time, and the transgene expression efficiency and cellular uptake in the droplet are significantly higher than those in the conventional tube, with and without shaking. These results suggest the consolidation of the endocytic uptake pathway into macropinocytosis, indicating that ultrasonic levitation induced a change in cell characteristics. This study suggests that transfection methodology using ultrasonic levitation has the potential to advance the current experimental procedures in the field of cell engineering, in addition to presenting a revolutionary containerless reactor for sustainable technology.

Although phage display selection using a library of M13 bacteriophage has become a powerful tool for finding peptides that bind to target materials on demand, a remaining concern of this method is the interference by the M13 main body, which is a huge filament >103 times larger than the displayed peptide, and therefore would nonspecifically adhere to the target or sterically inhibit the binding of the displayed peptide. Meanwhile, filamentous phages are known to be orientable by an external magnetic field. If M13 filaments are magnetically oriented during the library selection, their angular arrangement relative to the target surface would be changed, being expected to control the interference by the M13 main body. This study reports that the magnetic orientation of M13 filaments vertical to the target surface significantly affects the selection. When the target surface was affinitive to the M13 main body, this orientation notably suppressed the nonspecific adhesion. Furthermore, when the target surface was less affinitive to the M13 main body and intrinsically free from the nonspecific adhesion, this orientation drastically changed the population of M13 clones obtained through library selection. The method of using no chemicals but only a physical stimulus is simple, clean, and expected to expand the scope of phage display selection.

Biological membranes are functionalized by membrane-associated protein machinery. Membrane-associated transport processes, such as endocytosis, represent a fundamental and universal function mediated by membrane-deforming protein machines, by which small biomolecules and even micrometer-size substances can be transported via encapsulation into membrane vesicles. Although synthetic molecules that induce dynamic membrane deformation have been reported, a molecular approach enabling membrane transport in which membrane deformation is coupled with substance binding and transport remains critically lacking. Here, we developed an amphiphilic molecular machine containing a photoresponsive diazocine core (AzoMEx) that localizes in a phospholipid membrane. Upon photoirradiation, AzoMEx expands the liposomal membrane to bias vesicles toward outside-in fission in the membrane deformation process. Cargo components, including micrometer-size M13 bacteriophages that interact with AzoMEx, are efficiently incorporated into the vesicles through the outside-in fission. Encapsulated M13 bacteriophages are transiently protected from the external environment and therefore retain biological activity during distribution throughout the body via the blood following administration. This research developed a molecular approach using synthetic molecular machinery for membrane functionalization to transport micrometer-size substances and objects via vesicle encapsulation. The molecular design demonstrated in this study to expand the membrane for deformation and binding to a cargo component can lead to the development of drug delivery materials and chemical tools for controlling cellular activities.

Neurotoxicity caused by peptide and protein aggregates is associated with the onset of neurodegenerative diseases. Accumulation of the amyloid β protein (Aβ) induced by neuronal ganglioside-enriched nanodomains (nanoclusters) in the presynaptic neuronal membrane, resulting in toxic oligomeric and fibrous forms, is implicated in the onset of Alzheimer's disease (AD). In the current study, we found that the ganglioside cluster-binding peptide (GCBP), a pentadecapeptide VWRLLAPPFSNRLLP that binds to ganglioside-enriched nanoclusters, inhibits the formation of Aβ assemblies with an IC50 of 12 pM and also removes Aβ fibrils deposited on the lipid membrane. Thus, in addition to inhibiting Aβ assembly formation, GCBP effectively clears toxic Aβ assemblies as well, thereby suppressing neuronal cellular damage and death induced by such assemblies. These results indicate that ganglioside cluster-binding molecules may act as novel Aβ-targeting drugs with a unique mechanism of action that may be utilized to ameliorate AD.

The assembly of amyloid ß protein (Aß) on ganglioside-enriched microdomains in presynaptic neuronal membranes is involved in the onset of Alzheimer’s disease. We previously found that a highly enriched ganglioside domain generated in a reconstituted lipid bilayer composed of synaptosomal lipids functioned as an Aß-sensitive ganglioside nanocluster (ASIGN) that may induce a spherical Aß assembly. In the present study, the major lipid components of a detergent-resistant membrane microdomain (DRM) fraction of synaptosomes prepared from an aged mouse brain were identified, and we demonstrate that the proportion of Aß-insensitive gangliosides such as GM3 directly affects the Aß assembly in synaptosomal membranes. Liquid chromatography coupled to mass spectrometry analyses indicated that ganglio a-series gangliosides (GM1, GM2, GM3, and GD1a) were abundant in DRM fractions. In addition, the GM3/GM1 ratio and the cholesterol content in the synaptosomal fraction differed from those in the non-synaptosomal fraction, in which GM1 and GM3 are sensitive and insensitive gangliosides, respectively. The highly enriched ganglioside nanocluster was identified by the surface topography of ternary mixed lipid bilayers composed of gangliosides, sphingomyelin, and cholesterol. Our results indicate that the composition of gangliosides is responsible for the characteristics of the nanocluster generated in the presynaptic neuronal membranes.

Some protein and peptide aggregates, such as those of amyloid- protein (A), are neurotoxic and have been implicated in several neurodegenerative diseases. A accumulates at nanoclusters enriched in neuronal lipids called gangliosides in the presynaptic neuronal membrane, and the resulting oligomeric and/or fibrous forms accelerate the development of Alzheimer’s disease. Although the presence of A deposits at such nanoclusters is known, the mechanism of their assembly and the relationship between A secondary structure and topography are still unclear. Here, we first confirmed by atomic force microscopy that A40 fibrils can be obtained by incubating seed-free A40 monomers with a membrane composed of sphingomyelin, cholesterol, and the ganglioside GM1. Using Fourier transform infrared (FTIR) reflection–absorption spectroscopy, we then found that these lipid-associated fibrils contained parallel -sheets, whereas self-assembled A40 molecules formed antiparallel -sheets. We also found that the fibrils obtained at GM1-rich nanoclusters were generated from turn A40. Our findings indicate that A generally self-assembles into antiparallel -structures but can also form protofibrils with parallel -sheets by interacting with ganglioside-bound A. We concluded that by promoting the formation of parallel -sheets, highly ganglioside-enriched nanoclusters help accelerate the elongation of A fibrils. These results advance our understanding of ganglioside-induced A fibril formation in neuronal membranes and may help inform the development of additional therapies for Alzheimer’s disease.

The development of a simple detection method with high sensitivity is essential for the diagnosis and surveillance of infectious diseases. Previously, we constructed a sensitive biosensor for the detection of pathological human influenza viruses using a boron-doped diamond electrode terminated with a sialyloligosaccharide receptor-mimic peptide that could bind to hemagglutinins involved in viral infection. Circulation of influenza induced by the avian virus in humans has become a major public health concern, and methods for the detection of avian viruses are urgently needed. Here, peptide density and dendrimer generation terminated on the electrode altered the efficiency of viral binding to the electrode surface, thus significantly enhancing charge-transfer resistance measured by electrochemical impedance spectroscopy. The peptide-terminated electrodes exhibited an excellent detection limit of less than one plaque-forming unit of seasonal H1N1 and H3N2 viruses. Furthermore, the improved electrode was detectable for avian viruses isolated from H5N3, H7N1, and H9N2, showing the potential for the detection of all subtypes of influenza A virus, including new subtypes. The peptide-based electrochemical architecture provided a promising approach to biosensors for ultrasensitive detection of pathogenic microorganisms.

To ensure sustainable consumption and production patterns, alternative process design without plastics for chemical and biological reactions will benefit future generations. Reaction flasks used in chemical and biological laboratories have been made from glass, metals, and plastics so far. If containerless processing can be realized, researchers will have a next-generation reaction process, which will be reactor and plastic-free, and without risks of unforeseen issues induced by contact with reactions flasks, including contamination and alteration of the reactants. Here, polymerization, click chemistry, and enzymatic reactions can proceed effectively in a floating droplet at a node of standing wave generated by ultrasonic levitation. These results demonstrate that floating droplets levitated by acoustic waves can represent a revolutionary containerless reactor for performing various reactions in the fields of chemistry and biology.

Synthetic polymers with well-defined structures allow the development of nanomaterials with additional functions beyond biopolymers. Herein, we demonstrate de novo design of star-shaped glycoligands to interact with hemagglutinin (HA) using well-defined synthetic polymers with the aim of developing an effective inhibitor for the influenza virus. Prior to the synthesis, the length of the star polymer chains was predicted using the Gaussian model of synthetic polymers, and the degree of polymerization required to achieve multivalent binding to three carbohydrate recognition domains (CRDs) of HA was estimated. The star polymer with the predicted degree of polymerization was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, and 6′-sialyllactose was conjugated as the glycoepitope for HA. The designed glycoligand exhibited the strongest interaction with HA as a result of multivalent binding. This finding demonstrated that the biological function of the synthetic polymer could be controlled by precisely defining the polymer structures.

Abstract: Influenza virus (IFV) detection in the early phase of disease is critical for effective anti-influenza therapy using neuraminidase inhibitors. Sialyloligosaccharide receptors on the surface of respiratory cells are recognized by IFV hemagglutinin (HA) in the infection. Here, we show that agglutination of IFV is detected using poly(glycidyl methacrylate) (PGMA)-coated polystyrene nanoparticles conjugated with a sialic acid-mimic peptide. The azido peptide was immobilized onto the surface of the PGMA-coated nanoparticles by click chemistry. The distribution of particle size, determined by dynamic light scattering, indicated that the peptide-conjugated nanoparticles were agglutinated in the presence of HA and IFV. Nanoparticles conjugated with the receptor-mimic peptide may be a useful alternative to red blood cells in the global surveillance and clinical diagnosis of influenza.

The specific features of the lateral distribution of gangliosides play key roles in cell-cell communications and the onset of various diseases related to the plasma membrane. We herein demonstrated that an artificial peptide identified from a phage-displayed library is available as a molecular probe for specific ganglioside nanoclustering sites in caveolae/membrane rafts on the cell surface. Atomic force microscopy studies indicated that the peptide specifically binds to the highly enriched monosialoganglioside GM1 nanodomains of reconstituted lipid bilayers composed of GM1, sphingomyelin, cholesterol, and unsaturated phospholipids. The ganglioside-containing area recognized by the peptide on the surface of PC12 cells was part of the area recognized by the cholera toxin B subunit, which has high affinity for GM1. Furthermore, the peptide bound to the cell surface after a treatment with methyl-β-cyclodextrin (MβCD), which disrupts membrane rafts by removing cholesterol. The present results indicate that there are heterogeneous ganglioside clusters with different ganglioside densities in caveolae/membrane rafts, and the peptidyl probe selectively recognizes the high-density ganglioside nanodomain that resists the MβCD treatment. This peptidyl probe will be useful for obtaining information on the lipid organization of the cell membrane and will help clarify the mechanisms by which the lateral distribution of gangliosides affects biological functions and the onset of diseases.

Hoshinoamides A (1) and B (2), new acyclic lipopeptides, were isolated from the marine cyanobacterium Caldora penicillata. Their structures were elucidated by spectroscopic analyses and degradation reactions. Hoshinoamides A (1) and B (2) did not exhibit any cytotoxicity against HeLa cells at 10 μM, but inhibited the in vitro growth of the malarial parasite Plasmodium falciparum (IC50 = 0.52 and 1.0 μM, respectively).

An antimalarial lipopeptide, ikoamide, was isolated from an Okeania sp. marine cyanobacterium. Its gross structure was established by spectroscopic analyses, and the absolute configuration was clarified based on a combination of chiral-phase HPLC analyses, spectroscopic analyses, and derivatization reactions. Ikoamide showed strong antimalarial activity with an IC50 value of 0.14 μM without cytotoxicity against human cancer cell lines at 10 μM.

Izenamides A, B, and C (1-3), new linear depsipeptides, were isolated from a taxonomically distinct marine cyanobacterium. Izenamides A and B contain a statine moiety [(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid] and inhibited the activity of cathepsin D, an aspartic peptidase. Meanwhile, izenamides did not show growth-inhibitory activity against HeLa, HL60, or MCF-7 cells at up to 10 μM.