Kazuhide Totani

Glucosamine induces autophagy via an mTOR-independent pathway

Autophagy is a cellular process that nonspecifically degrades cytosolic components and is involved in many cellular responses. We found that amino sugars with a free amino group such as glucosamine, galactosamine and mannosamine induced autophagy via an mTOR-independent pathway. Glucosamine-induced autophagy at concentrations of at least 500 microM to over 40 mM. In the presence of 40 mM glucosamine, autophagy induction was initiated at 6h and reached a plateau at 36 h. Glucosamine-induced autophagy could remove accumulated ubiquitin-conjugated proteins as well as 79-glutamine repeats. Therefore, orally administered glucosamine could contribute to the prevention of neurodegenerative diseases and promotion of antiaging effects.

Non-catalytic synthesis of Chromogen I and III from N-acetyl-D-glucosamine in high-temperature water

Non-catalytic synthesis of 2-acetamido-2,3-dideoxy-D-erythro-hex-2-enofuranose (Chromogen I) and 3-acetamido-5-(1′,2′-dihydroxyethyl)furan (Chromogen III) from N-acetyl-D-glucosamine (GlcNAc) was achieved, with the highest yields of 23.0% and 23.1%, respectively, in high-temperature water at 120–220 °C and 25 MPa with a reaction time of 7–39 s.

A small lytic polysaccharide monooxygenase from Streptomyces griseus targeting α- and β-chitin.

The lytic polysaccharide monooxygenases (LPMOs) have received considerable attention subsequent to their discovery because of their ability to boost the enzymatic conversion of recalcitrant polysaccharides. In the present study, we describe the enzymatic properties of SgLPMO10F, a small (15 kDa) auxilliary activity (AA) family 10 LPMO from Streptomyces griseus belonging to a clade of the phylogenetic tree without any characterized representative. The protein was expressed using a Brevibacillus‐based expression system that had not been used previously for LPMO expression and that also ensures correct processing of the N‐terminus crucial for LPMO activity. The enzyme was active towards both α‐ and β‐chitin and showed stronger binding and a greater release of soluble oxidized products for the latter allomorph. In chitinase synergy assays, however, SgLPMO10F worked slightly better for α‐chitin, increasing chitin solubilization yields by up to 30‐fold and 20‐fold for α‐ and β‐chitin, respectively. Synergy experiments with various chitinases showed that the addition of SgLPMO10F leads to a substantial increase in the (GlcNAc)2:GlcNAc product ratio, in reactions with α‐chitin only. This underpins the structural differences between the substrates and also shows that, on α‐chitin, SgLPMO10F affects the binding mode and/or degree of processivity of the chitinases tested. Variation in the only exposed aromatic residue in the substrate‐binding surface of LPMO10s has previously been linked to preferential binding for α‐chitin (exposed Trp) or β‐chitin (exposed Tyr). Mutation of this residue, Tyr56, in SgLPMO10F to Trp had no detectable effect on substrate‐binding preferences but, in synergy experiments, the mutant appeared to be more efficient on α‐chitin.

Design and facile synthesis of neoglycolipids as lactosylceramide mimetics and their transformation into glycoliposomes.

Neoglycolipids composed of disaccharide glycoside and phospholipid were designed and prepared as mimetics of lactosylceramide. The lactosyl- and N-acetyllactosaminyl-phospholipids (Lac-DPPA and LacNAc-DPPA) were enzymatically synthesized from lactose and LacNAc respectively by cellulase-mediated condensation with 1,6-hexanediol, followed by conjugation of the resulting glycosides and dipalmitoylphosphatidyl choline (DPPC) mediated by Streptomyces phospholipase D. Alternatively, allyl β-lactoside was ozonolyzed to give an aldehyde, which was condensed with dipalmytoyl phosphatidyl ethanolamine to afford a second type of glycolipid (Lac-DPPE). NMR spectroscopy indicated that the neoglycolipids behave differently in different solvent systems. X-ray diffraction clearly showed that multilamellar vesicles (MLVs) of Lac-DPPE and Lac-DPPA-MLV are in the bilayer gel phase at 20 °C, whereas those of Lac-DPPE-MLV were in the lamellar liquid-crystalline phase at 50 °C. Differential scanning calorimetry showed that Lac-DPPE-MLV had complex thermotropic behavior depending on the incubation conditions. After a long incubation at 10 °C, endothermic transitions are observed at 39.6, 42.3 °C, and 42.9 °C. These neoglycolipids have the ability to trap calcein, a chelating derivative of fluorescein, in MLVs and showed specific binding to lectin in plate assays using fluorescently labeled compounds.

Conversion of α-chitin substrates with varying particle size and crystallinity reveals substrate preferences of the chitinases and lytic polysaccharide monooxygenase of Serratia marcescens.

Industrial depolymerization of chitinous biomass generally requires numerous steps and the use of deleterious substances. Enzymatic methods provide an alternative, but fundamental knowledge that could direct potential development of industrial enzyme cocktails is scarce. We have studied the contribution of monocomponent chitinases (ChiA, -B, and -C) and the lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens on depolymerization of α-chitin substrates with varying particle size and crystallinity that were generated using a converge mill. For all chitinases activity was positively correlated to a decline in particle size and crystallinity. Especially ChiC, the only nonprocessive endochitinase from the S. marcescens chitinolytic machinery, benefited from mechanical pretreatment. Combining the chitinases revealed clear synergies for all substrates tested. CBP21, the chitin-active LPMO from S. marcescens, increased solubilization of substrates with high degrees of crystallinity when combined with each of the three chitinases, but this synergy was reduced upon decline in crystallinity.

Efficient synthesis of glyceroyl β-lactoside and its derivatives through a condensation reaction by cellulase

Condensation reaction between lactose and glycerol was effectively catalyzed by utilizing a commercially available cellulase preparation from Trichoderma reesei. The enzyme induced the formation of 1-O-beta-lactosyl-(R,S)-glycerol (1) and 2-O-beta-lactosyl glycerol (2) in a molar ratio of 7:3 and in a 20% yield based on lactose added. The enzyme also induced the condensation of lactose with 1,3-propanediol to produce O-beta-lactosyl propanediol (3) in a yield of 15%. When various alkanols (N: 2-8) and allyl alcohol were used in the condensation reaction, the corresponding alkyl and allyl beta-lactoside were obtained in the yields of 0.9-3.8% of the desired compounds.

Enzymatic synthesis of an α-chitin-like substance via lysozyme-mediated transglycosylation.

The enzymatic synthesis of an α-chitin-like substance via a non-biosynthetic pathway has been achieved by transglycosylation in an aqueous system of the corresponding substrate, tri-N-acetylchitotriose [(GlcNAc)(3)] for lysozyme. A significant amount of water-insoluble product precipitated out from the reaction system. MALDI-TOFMS analysis showed that the resulting precipitate had a degree of polymerization (DP) of up to 15 from (GlcNAc)(3). Solid-state (13)C NMR analysis revealed that the resulting water-insoluble product is a chitin-like substance consisting of N-acetylglucosamine (GlcNAc) residues joined exclusively in a β-(1→4)-linked chain with stringent regio-/stereoselection. X-ray diffraction (XRD) measurement as well as (13)C NMR analysis showed that the crystal structure of synthetic product corresponds to α-chitin with a high degree of crystallinity. We propose that the multiple oligomers form an α-chitin-like substance as a result of self-assembly via oligomer-oligomer interaction when they precipitate.

Purification of endo-(1→3)-β-D-glucanases lysing yeast cell walls from Rhizoctonia solani

Abstract Three forms of endo -(1→3)-β- g -glucanases lysing yeast cell walls from Rhizoctonia solani were separated by precipitation with ammonium sulfate and by successive chromatographies on CM Bio-Gel A and Bio-Gel P-60 or P-30, and were finally purified by substrate affinity chromatography on short-chain pachyman-AH-Sepharose CL 6B column. Each preparation was found to be homogeneous on gel filtration and by electrophoresis on acrylamide gel with sodium dodecyl sulfate. They exhibit high activity against insoluble pachyman, but only restricted activity against soluble short-chain pachyman. In the affinity chromatography, three enzymes were found to be strongly absorbed on the column, so that they could be easily eluted with substrate solution using biospecific counter-ligand. It was thus revealed that covalent binding of such a soluble glucan to aminohexyl-Sepharose provides a useful carrier for separation of endo -(1→3)-β- D -glucanases lysing yeast cell walls.

Enzymatic synthesis of cellulose II-like substance via cellulolytic enzyme-mediated transglycosylation in an aqueous medium

The enzymatic synthesis of cellulose-like substance via a non-biosynthetic pathway has been achieved by transglycosylation in an aqueous system of the corresponding substrate, cellotriose for cellulolytic enzyme endo-acting endoglucanase I (EG I) from Hypocrea jecorina. A significant amount of water-insoluble product precipitated out from the reaction system. MALDI-TOF mass analysis showed that the resulting precipitate had a degree of polymerization (DP) of up to 16 from cellotriose. Solid-state (13)C NMR spectrum of the resulting water-insoluble product revealed that all carbon resonance lines were assigned to two kinds of anhydroglucose residues in the corresponding structure of cellulose II. X-ray diffraction (XRD) measurement as well as (13)C NMR analysis showed that the crystal structure corresponds to cellulose II with a high degree of crystallinity. We propose the multiple oligomers form highly crystalline cellulose II as a result of self-assembly via oligomer-oligomer interaction when they precipitate.

Membrane microdomains from early gastrula embryos of medaka, Oryzias latipes, are a platform of E-cadherin- and carbohydrate-mediated cell–cell interactions during epiboly

Formation of membrane microdomain is critical for cell migration (epiboly) during gastrulation of medaka fish [Adachi et al. (Biochem. Biophys. Res. Commun. 358:848–853, 2007)]. In this study, we characterized membrane microdomain from gastrula embryos to understand its roles in epiboly. A cell adhesion molecule (E-cadherin), its associated protein (β-catenin), transducer proteins (PLCγ, cSrc), and a cytoskeleton protein (β-actin) were enriched in the membrane microdomain. LeX-containing glycolipids and glycoproteins (LeX-gp) were exclusively enriched in the membrane microdomain. Interestingly, the isolated membrane microdomain had the ability to bind to each other in the presence of Ca2+. This membrane microdomain binding was achieved through the E-cadherin homophilic and the LeX-glycan-mediated interactions. E-cadherin and LeX-gp were co-localized on the same membrane microdomain, suggesting that these two interactions are operative at the same time. Thus, the membrane microdomain functions as a platform of the E-cadherin- and LeX-glycan-mediated cell adhesion and signal transduction.