Tomohiro Fukuda

Amyloid-β detection with saccharide immobilized gold nanoparticle on carbon electrode

The electrochemical sensing of saccharide-protein interactions using a couple of sialic acid derivatives and Alzheimers amyloid-beta (Abeta) is described. The densely-packed saccharide area for recognition of protein was fabricated onto a carbon electrode by three steps, which were electrochemical deposition of Au nanoparticles on a screen printed strip, self-assembled monolayer (SAM) formation of the acetylenyl group on Au nanoparticles, and the cycloaddition reaction of an azide-terminated sialic acid to the acetylenyl group. The attachment of Abeta peptides to the sialic acid layer was confirmed by electrochemistry and atomic force microscopy imaging. The intrinsic oxidation signal of the captured Abeta(1-40) and (1-42) peptides, containing a single tyrosine (Tyr) residues, was monitored at a peak potential of 0.6 V (vs Ag/AgCl within this sensor) in connection with differential pulse voltammetry. The peak current intensities were concentration dependent. The proposed process provides new routes for analysis of saccharide-protein interactions and electrochemical biosensor development.

Aggregation of Alzheimer Amyloid β Peptide (1−42) on the Multivalent Sulfonated Sugar Interface

The mechanism of amyloidosis of amyloid beta (1-42) (Abeta (1-42)) was investigated by the well-defined glycocluster interface. We prepared monovalent, divalent, and trivalent 6-sulfo-N-acetyl-d-glucosamine (6S-GlcNAc) immobilized substrates. The morphology and secondary structure of Abeta (1-42) aggregates on the substrates were investigated by dynamic-mode AFM and FTIR-RAS. Abeta (1-42) interactions with multivalent sugars were evaluated by surface plasmon resonance, and the cytotoxicity of Abeta (1-42) to HeLa cells was evaluated by MTT assay. Morphological images showed, interestingly, that Abeta (1-42) aggregates had a tendency to form globules rather than fibrils as the valency of 6S-GlcNAc on the substrate was increased. The SPR measurements indicated that this morphological change of Abeta (1-42) was related to the change of binding mode, and the binding mode was dependent on the multivalency of the sugar. Globular Abeta (1-42) was more toxic than fibrillar Abeta (1-42) to HeLa cells. These results suggested that the multivalency of sugars for the amyloidosis of Abeta (1-42) was significant in its morphology and aggregation effects at the surface of the cell membrane mimic.

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1–42)

Abstract Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs) on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide–protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

Syntheses of Sulfo-Glycodendrimers Using Click Chemistry and Their Biological Evaluation

A series of novel glycol-clusters containing sulfonated N-acetyl-D-glucosamine (GlcNAc) have been synthesized using click chemistry. Three dendrimers with aromatic dendrons were synthesized using chlorination, azidation and click chemistries. The resulting dendrimers were modified with azide-terminated sulfonated GlcNAc using click chemistry. The sulfonated dendrimers showed affinity for proteins, including the lectin wheat germ agglutinin and amyloid beta peptide (1-42). The dendrimers of G1 and G2 in particular showed the largest affinity for the proteins. The addition of the sulfonated GlcNAc dendrimers of G1 and G2 exhibited an inhibition effect on the aggregation of the amyloid beta peptide, reduced the β-sheet conformation, and led to a reduction in the level of nanofiber formation.

Bioinert surface to protein adsorption with higher generation of dendrimer SAMs

Interactions between proteins and biomaterial surfaces correlate with many important phenomena in biological systems. Such interactions have been used to develop various artificial biomaterials and applications, in which regulation of non-specific protein adsorption has been achieved with bioinert properties. In this research, we investigated the protein adsorption behavior of polymer brushes of dendrimer self-assembled monolayers (SAMs) with other generations. The surface adsorption properties of proteins with different pI values were examined on gold substrates modified with poly(amidoamine) dendrimer SAMs. The amount of fibrinogen adsorption was greater than that of lysozyme, potentially because of the surface electric charge. However, as the generations increased, protein adsorption decreased regardless of the surface charge, suggesting that protein adsorption was also affected by density of terminal group.

Syntheses and Functions of Glycosaminoglycan Mimicking Polymers

Glycosaminoglycans (GAGs) are important polysaccharides in the living system. Though total syntheses of GAGs oligosaccharides have been reported, it is still difficult to obtain GAGs. In this investigation, GAGs mimetics were prepared by polymerization of vinyl sugars instead of total synthesis. Glycopolymers are polymers with pendant saccharides, and exhibit a strong molecular recognition due to the multivalency. GAGs mimicking glycopolymers were prepared by polymerizing acrylamide derivatives carrying sulfated N-acetyl glucosamine (GlcNAc). The polymer interactions with proteins were investigated. The glycopolymer libraries were prepared with varying molecular weight, sugar structure, and sugar ratios. The GAG glycopolymers with biodegradable backbone, dendrimer, and arrays were also prepared. The inhibitory activity of Alzheimer amyloid beta peptides by the glycopolymers was investigated in detail. The glycopolymer with sulfated GlcNAc inhibited the aggregation of amyloid beta (Aβ) and the multivalency of sulfated GlcNAc was the key of the interaction. The activity depends on the chemical structure of glycopolymers. Also, the sulfated saccharide function was correlated to the functions of native GAGs.

Molecular Recognition of Glycopolymer Interface

Saccharides on the cell surfaces play important roles in the living systems. For example, it mediate the cell-cell adhesion, fertilization, protein transportation, infection of pathogens and cancer metastasis etc [1, 2]. The saccharide-protein interactions also involve the various biological events (Table 1). Actualy, the saccharides are the model compounds of some of the medicines like oseltamivir [3]. The interaction between galactose and asialoglycoprotein receptor is a possible mechanism for the hepatocyte-specific drug delivery systems [4]. Therefore, it has been pointed out that the saccharide-protein interaction can be utilized for the novel bio-functional mateials such as cell cultivation, medicine target, and drug deliver‐ ly systems.

Interaction and Aggregation of Amyloid β Peptide with Multivalent Sulfonated Sugar

Proteins have native functional states but can also form amyloid fibrils (Fowler et al .,2007, Maji et al., 2009, Dobson et al., 2003). In the amyloid state, proteins are denaturated and aggregated with -sheet-rich structures. Accumulation of amyloids can result in protein amyloidosis, which has attracted much attention. Amyloidosis is associated with many serious neurodegenerative diseases, such as Alzheimer’s disease (Sipe et al., 1992), dialysisrelated amyloidosis (Rochet et al., 2000), prion disease (Prusiner, 1998), and type II diabetes (Ahmad et al., 2004). Consequently, it is important to clarify the mechanism of amyloidosis, so that it can be inhibited or controlled. Amyloid structures have been analyzed by various methods including nuclear magnetic resonance (NMR) spectroscopy, electron microscopy, X-ray spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Theoretical calculations of amyloidosis have also been conducted (Miller et al., 2010). Protein amyloidosis is affected by environmental conditions such as the temperature (Kusumoto et al., 1998), pH (Petkova et al., 2004), and ionic strength (Zidar et al., 2011), and by nucleation (Kanji et al., 2008). Because amyloidosis occurs at the cell surface, the interaction between the protein and cell interface is also important (Xu et al., 2005). Therefore, the amyloidosis of proteins can be controlled by manipulating the environmental conditions. However, it has been reported that artificial additives, such as metal ions (Chanki et al., 2007), peptides (Suzuki et al., 2010), sugars (Anubhav et al., 2004) and nanoparticles (Saraiva et al., 2010), can alter the environmental conditions and affect amyloidosis. Because amyloidosis occurs in vivo, the molecules on the cell surfaces are of interest as additives to control amyloidosis. It has been reported that protein amyloidosis with Amyloid beta (A) (Alzheimer’s disease) (McLaurin et al., 1996), 2-microglobulin (dialysisrelated amyloidosis) (Bourgault et al., 2011), and prion protein (Pan et al., 2002) formation is affected by interactions with glycosaminoglycans (GAGs) on the cell surfaces. This affects all these proteins, even though they have different amino acid sequences and native protein structures. GAGs are long unbranched polysaccharides of repeating disaccharide subunits of hexosamines (glucosamine and galactosamine) and uronic acid (glucuronic acid and iduronic acid) (Rudd et al., 2010). Well-known GAGs are heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate and hyaluronic acid. Most GAGs are highly sulfonated. The interaction of proteins with GAGs is important in amyloidosis, but it is difficult to analyze the detail of this interaction because GAGs have complex structures and high molecular weights.