Isao Ohtsuka

Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein.

Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimers β-amyloid protein (AβP) play crucial roles in the pathogenesis of Alzheimers disease (AD). Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”), and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.

Synthetic studies on glycosphingolipids from the Protostomia phyla: Syntheses of arthro-series glycosphingolipids

Glycosphingolipids isolated from larvae of the green-bottle fly, Lucilia caesar, have quite unique structures containing GlcNAcbeta-(1 --> 3)-Man and GalNAcbeta-(1 --> 4)-GlcNAcbeta-(1 --> 3)-Man. We have synthesized two glycosphingolipids, beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer and beta-D-GalNAcp-(1 --> 4)-beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer. A key reaction in the synthetic sequence is the application of the intramolecular aglycon delivery (IAD) approach for the synthesis of the beta-mannopyranosidic linkages.

Synthetic studies on novel fucosylated glycosphingolipids from the millipede, Parafontaria laminata armigera

Abstract A novel glycosphingolipid, β- d -Man p -(1→4)-[(α- l -Fuc p -(1→3)]-β- d -Glc p -(1→1)-Cer, from the millipede, Parafontaria laminata armigera , was synthesized. A key reaction of this synthetic procedure is the formation of a spiro-orthoester and its reduction for β-selective mannosylation.

The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling

Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.

Single‐Step Multisyntheses of Glycosyl Acceptors: Benzylation of n‐1 Hydroxyl Groups of Phenylthio Glycosides of Xylose, Mannose, Glucose, Galactose, 2‐Azido‐2‐deoxy‐glucose, and 2‐Azido‐2‐deoxy‐galactose

An array of synthons is required to access an oligosaccharide library; however, multistep and thus time‐consuming synthesis is inevitable. To rapidly access such synthetic units, multiple benzylation reactions of monosaccharides under phase‐transfer conditions were examined. Multiple benzyl groups were successfully incorporated in one step, especially in the cases of reactions with triol systems.

Diversity in aconitine alkaloid profile of Aconitum plants in Hokkaido contrasts with their genetic similarity.

Aconite tuber is a representative crude drug for warming the body internally in Japanese Kampo medicine and Chinese traditional medicine. The crude drug is used in major prescriptions for the aged. Varieties of Aconitum plants are distributed throughout the Japanese Islands, especially Hokkaido. With the aim of identifying the medicinal potential of Aconitum plants from Hokkaido, 107 specimens were collected from 36 sites in the summer of 2011 and 2012. Their nuclear DNA region, internal transcribed spacer (ITS), and aconitine alkaloid contents were analyzed. Phylogenic analysis of ITS by maximum parsimony analysis showed that the majority of the specimens were grouped into one cluster (cluster I), separated from the other cluster (cluster II) consisting of alpine specimens. The aconitine alkaloid content of the tuberous roots of 76 specimens showed 2 aspects—specimens from the same collection site showed similar aconitine alkaloid profiles, and cluster I specimens from different habitats showed various alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profile of these genetically similar specimens to diversify.

The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: carbohydrate-protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar.

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate-protein interactions. We investigated whether carbohydrate-lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp-GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.

Synthesis of a new glycosphingolipid, neurosporaside, from Neurospora crassa.

The glycosphingolipid neurosporaside (α-D-Glcp-(1 → 2)-β-D-Galp-(1 → 6)-β-D-Galp-(1 → 6)-β-D-Galp-(1 →)-Cer) occurs in Neurospora crassa. We attempted to synthesize neurosporaside by block synthesis (route A) and linear synthesis (route B). Oligosaccharide derivatives were synthesized using trimethylsilyltrifluoromethanesulfonate and N-iodosuccinimide/trifluoromethane sulfonic acid as promoters. The target tetrasaccharide could not be attained via route A, but route B showed potential: glycosidic bonds (β-D-Galp-(1 → 6)-β-D-Galp-(1 → 6)-β-D-Galp) were formed stereoselectively, leading to the synthesis of glycosphingolipid 2.

Photoreactive Biomacromolecules: Installation of Photoreactive Units and Applications for Analyzing Biological Interfaces

Photoaffinity labeling enables analysis of weak biological interactions through formation of a covalent bond between ligands and receptor proteins upon photolysis of photoreactive groups. The photoreactive group, 3-trifluoromethyl-3-phenyldiazirine, has many advantages over other photoreactive groups like phenylazide and benzophenone, but its use is limited due to the difficulty to incorporate this photophore within macromolecules. The chemoselective ligation of versatile photoreactive units to macromolecules is a reliable solution for this difficulty; this approach easily attaches the useful 3-trifluoromethyl-3-phenyldiazirine photophore to biomacromolecules. Photoreactive bromide unit bearing 3-trifluoromethyl-3-phenyldiazirine is chemoselectively attached to phosphorothioate linkages of modified oligonucleotides to prepare photoreactive DNA, which is then used in a photoaffinity electrophoretic mobility shift assay for analyzing the proteins bound to a specific DNA motif. This review focuses on the preparation of photoreactive biomacromolecules such as peptides, proteins, oligosaccharides, and DNA based on our photoreactive unit technique and describes their application as novel approaches for the photoaffinity labeling of biomacromolecules.