Teruhiko Matsubara

Age-dependent high-density clustering of GM1 ganglioside at presynaptic neuritic terminals promotes amyloid β-protein fibrillogenesis

The deposition of amyloid beta-protein (Abeta) is an invariable feature of Alzheimers disease (AD); however, the biological mechanism underlying Abeta assembly into fibrils in the brain remains unclear. Here, we show that a high-density cluster of GM1 ganglioside (GM1), which was detected by the specific binding of a novel peptide (p3), appeared selectively on synaptosomes prepared from aged mouse brains. Notably, the synaptosomes bearing the high-density GM1 cluster showed extraordinary potency to induce Abeta assembly, which was suppressed by an antibody specific to GM1-bound Abeta, an endogenous seed for AD amyloid. Together with evidence that Abeta deposition starts at presynaptic terminals in the AD brain and that GM1 levels significantly increase in amyloid-positive synaptosomes prepared from the AD brain, our results suggest that the age-dependent high-density GM1 clustering at presynaptic neuritic terminals is a critical step for Abeta deposition in AD.

Sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenza therapy

Influenza is an infectious disease caused by the influenza virus, and each year many people suffer from this disease. Hemagglutinin (HA) in the membrane of type A influenza viruses recognizes sialylglycoconjugate receptors on the host cell surface at an initial step in the infection process; consequently, HA inhibitors are considered potential candidates for antiviral drugs. We identified peptides that bind to receptor-binding sites through a multiple serial selection from phage-displayed random peptide libraries. Using the HA of the H1 and H3 strains as target proteins, we obtained peptides that bind to both HAs. The binding affinities of peptides for these HAs were improved by secondary and tertiary selections from the corresponding sublibraries. A docking simulation suggested that, similar to sialic acid, the peptides are recognized by the receptor-binding site in HA, which indicates that these peptides mimic the sialic acid structure. N-stearoyl peptides inhibited infections by the A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains of influenza virus. Such HA-inhibitors are promising candidates for novel antiviral drugs.

Brain insulin resistance accelerates Aβ fibrillogenesis by inducing GM1 ganglioside clustering in the presynaptic membranes

J. Neurochem. (2012) 121, 619–628.

Density of GM1 in Nanoclusters Is a Critical Factor in the Formation of a Spherical Assembly of Amyloid β-Protein on Synaptic Plasma Membranes

The deposition of amyloid β-protein (Aβ) is a pathological hallmark of Alzheimers disease (AD). We previously found that the ganglioside-enriched microdomains (ganglioside clusters) in presynaptic neuronal membranes play a key role in the initiation of the Aβ assembly process. However, not all ganglioside clusters accelerate Aβ assembly. In the present study, we directly observed a spherical Aβ in an atomic force microscopic study on the morphology of a reconstituted lipid bilayer composed of lipids that were extracted from a detergent-resistant membrane microdomain (DRM) fraction of synaptosomes prepared from aged mouse brain. The Aβ assembly was generated on a distinctive GM1 domain, which was characterized as the Aβ-sensitive ganglioside nanocluster (ASIGN). By using an artificial GM1 cluster-binding peptide, ASIGN was found to have a high density of GM1; therefore, there would be a critical density of GM1 in nanoclusters to induce Aβ binding and assembly. These results suggest that ganglioside-bound Aβ (GAβ), which acts as an endogenous seed for Aβ fibril formation in AD brains, is generated on ASIGN on synaptosomal membranes.

Selection of novel structural zinc sites from a random peptide library

Zinc ion (Zn2+) can be coordinated with four or three amino acid residues to stabilize a proteins structure or to form a catalytic active center. We used phage display selection of a dodecamer random peptide library with Zn2+ to identify structural zinc sites. The binding specificity for Zn2+ of selected sequences was confirmed using enzyme‐linked immunosorbent and competitive inhibition assays. Circular dichroism spectra indicated that the interaction with Zn2+ induced a change in conformation, which means the peptide acts as a structural zinc site. Furthermore, a search of protein databases revealed that two selected sequences corresponded to parts of natural zinc sites of copper/zinc superoxide dismutase and zinc‐containing ferredoxin. We demonstrated that Zn2+‐binding sequences selected from the random combinatorial library would be candidates for artificial structural zinc sites.

Synthesis and Influenza Virus Inhibitory Activities of Carbosilane Dendrimers Peripherally Functionalized with Hemagglutinin-Binding Peptide

A series of carbosilane dendrimers uniformly functionalized with hemagglutinin (HA) binding peptide (sialic acid-mimic peptide, Ala-Arg-Leu-Pro-Arg) was systematically synthesized, and their anti-influenza virus activity was evaluated. The carbosilane-based peptide dendrimers, unlike sialylated dendrimers, cannot be digested by virus neuraminidases. The peptide dendrimers exhibited intriguing biological activities depending on the form of their core frame, with a dumbbell-type peptide dendrimer showing particularly strong inhibitory activities against two human influenza viruses, A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2). The IC50 values of the dumbbell-type peptide dendrimer for both strains were 0.60 μM, the highest activity among the HA-binding peptide derivatives. The results suggest that a dumbbell-shaped carbosilane dendrimer is the most suitable core scaffold for HA-binding peptide dendrimers.

Imbalance in Fatty-Acid-Chain Length of Gangliosides Triggers Alzheimer Amyloid Deposition in the Precuneus

Amyloid deposition, a crucial event of Alzheimer’s disease (AD), emerges in distinct brain regions. A key question is what triggers the assembly of the monomeric amyloid ß-protein (Aß) into fibrils in the regions. On the basis of our previous findings that gangliosides facilitate the initiation of Aß assembly at presynaptic neuritic terminals, we investigated how lipids, including gangliosides, cholesterol and sphingomyelin, extracted from synaptic plasma membranes (SPMs) isolated from autopsy brains were involved in the Aß assembly. We focused on two regions of the cerebral cortex; precuneus and calcarine cortex, one of the most vulnerable and one of the most resistant regions to amyloid deposition, respectively. Here, we show that lipids extracted from SPMs isolated from the amyloid-bearing precuneus, but neither the amyloid-free precuneus nor the calcarine cortex, markedly accelerate the Aß assembly in vitro. Through liquid chromatography-mass spectrometry of the lipids, we identified an increase in the ratio of the level of GD1b-ganglioside containing C20:0 fatty acid to that containing C18:0 as a cause of the enhanced Aß assembly in the precuneus. Our results suggest that the local glycolipid environment play a critical role in the initiation of Alzheimer amyloid deposition.

Acrosome reaction-related steroidal saponin, Co-ARIS, from the starfish induces structural changes in microdomains.

Cofactor for acrosome reaction-inducing substance (Co-ARIS) is a steroidal saponin from the starfish Asterias amurensis. Saponins exist in many plants and few animals as self-defensive chemicals, but Co-ARIS has been identified as a cofactor for inducing the acrosome reaction (AR). In A. amurensis, the AR is induced by the cooperative action of egg coat components (ARIS, Co-ARIS, and asterosap); however, the mechanism of action of Co-ARIS is obscure. In this study we elucidated the membrane dynamics involved in the action of Co-ARIS. We found that cholesterol specifically inhibited the Co-ARIS activity for AR induction and detected the binding of labeled compounds with sperm using radioisotope-labeled Co-ARIS. Co-ARIS treatment did not reduce the content of sperm sterols, however, the condition was changed and localization of GM1 ganglioside on the periacrosomal region disappeared. We then developed a caveola-breaking assay, a novel method to detect the effect of chemicals on microdomains of culture cell, and confirmed the disturbance of somatic cell caveolae in the presence of Co-ARIS. Finally, by atomic force microscopy observations and surface plasmon resonance measurements using an artificial membrane, we revealed that Co-ARIS colocalized with GM1 clusters on the microdomains. Through this study, we revealed a capacitation-like event for AR in starfish sperm.

Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide

Significance A large number of people suffer from influenza every year. Early detection is critical for preventing severe influenza in high-risk groups. There is usually not enough time to produce vaccines against pandemic viruses before they spread to other countries. Anti-influenza therapy using neuraminidase inhibitors is effective within 30 h of the onset of symptoms. Here we show that several dozen plaque-forming units of influenza virus (IFV) are detectable by a boron-doped diamond electrode terminated with a sialic acid-mimic peptide with the ability to capture IFV. The electrode was sufficiently sensitive to detect IFV in specimens obtained in the early phase of infection. The device developed in the present work has potential applications in the highly sensitive detection of IFV to fight against influenza. The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20–500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection.

Observations of the distribution of GM3 in membrane microdomains by atomic force microscopy

Glycosphingolipids (GSLs) in membrane microdomains participate in important biological functions. In the present paper, we propose a novel model of the distribution of GSLs in membrane microdomains composed of sphingomyelin (SM) and cholesterol. We investigated the distribution of the ganglioside GM3 in a lipid membrane reconstituted with lipid extract from mouse B16 melanoma cells using an atomic force microscope (AFM). The surface topography of the reconstituted lipid bilayer showed three areas of different heights. The highest area was confirmed to be a GM3 domain by labeling with wheat germ agglutinin. To identify the lipids which are contributed to make the topography, the topographies of the artificial lipid bilayers composed of GM3, SM, 1-palmitoyl-2-oleoyl-phosphatidylcholine, and cholesterol were investigated. AFM images of the artificial lipid bilayers showed that the GM3 domain surrounded by a SM-containing phase only occurred, and its formation was found to depend on the cholesterol content.