Synthesis of Sucrose-Mimicking Disaccharide by Intramolecular Aglycone Delivery.

Rare sugars are known for their ability to suppress postprandial blood glucose levels. Therefore, oligosaccharides and disaccharides derived from rare sugars could potentially serve as functional sweeteners. A disaccharide [α-d-allopyranosyl-(1→2)-β-d-psicofuranoside] mimicking sucrose was synthesized from rare monosaccharides D-allose and D-psicose.

Synthesis of azide-modified glycerophospholipid precursor analogs for detection of enzymatic reactions.

Glycerophospholipids (GPLs) are major cell membrane components. Although various phosphorylated molecules are attached to lipid moieties as their headgroups, GPLs are biosynthesized from phosphatidic acid (PA) via its derivatives, diacylglycerol (DAG) or cytidine diphosphate diacylglycerol (CDP-DAG). A variety of molecular probes capable of introducing detection tags have been developed to investigate biological events involved in GPLs.

Squaryl group-modified UDP analogs as inhibitors of the endoplasmic reticulum-resident folding sensor enzyme UGGT.

UDP-Glc:glycoprotein glucosyltransferase (UGGT) has a central role to retain quality control of correctly folded -glycoprotein in the endoplasmic reticulum (ER). A selective and potent inhibitor against UGGT could lead to elucidation of UGGT-related events, but such a molecule has not been identified so far. Examples of small molecules with UGGT inhibitory activity are scarce.

AeiA is a novel autophagy-related protein that promotes peroxisome degradation by pexophagy in Aspergillus oryzae.

Autophagy is classified into nonselective and selective autophagy, depending on the specificity of substrate degradation. In the filamentous fungus Aspergillus oryzae, selective autophagy, which includes pexophagy and mitophagy, has been observed. However, the molecular mechanism underlying selective autophagy in filamentous fungi remains unclear.

Analysis of Selenoprotein F Binding to UDP-Glucose:Glycoprotein Glucosyltransferase (UGGT) by a Photoreactive Crosslinker.

In the endoplasmic reticulum glycoprotein quality control system, UDP-glucose : glycoprotein glucosyltransferase (UGGT) functions as a folding sensor. Although it is known to form a heterodimer with selenoprotein F (SelenoF), the details of the complex formation remain obscure. A pulldown assay using co-transfected SelenoF and truncated mutants of human UGGT1 (HUGT1) revealed that SelenoF binds to the TRXL2 domain of HUGT1.

Phenotypic analysis of α1,2-mannosidase-like protein deletion mutants in .

α1,2‑mannosidase-like proteins mediate quality control of glycoproteins in the endoplasmic reticulum. This study explored α1,2‑mannosidase-like protein functions in Single disruptants in targeted protein-coding genes were found to be viable; however, deletion of resulted in declined yeast growth at 37 °C. The normal growth rate was recovered in double-deletion strains where one of the deletions was in .

In vitro mannosidase activity of EDEM3 against asparagine-linked oligomannose-type glycans.

Oligomannose-type glycans on glycoproteins play an important role in the endoplasmic reticulum (ER)-protein quality control. Mannose trimming of the glycans triggers the ER-associated protein degradation pathway. In mammals, ER mannosyl-oligosaccharide 1,2-α-mannosidase 1 and three ER degradation -enhancing α-mannosidase-like proteins (EDEMs) are responsible for mannose trimming.

Analysis of an acyl-CoA binding protein in Aspergillus oryzae that undergoes unconventional secretion.

Acyl-CoA binding protein (ACBP) plays important roles in the metabolism of lipids in eukaryotic cells. In the industrially important filamentous fungus Aspergillus oryzae, although we have previously demonstrated that the A. oryzae ACBP (AoACBP) localizes to punctate structures and exhibits long-range motility, which is dependent on autophagy-related proteins, the physiological role of AoACBP remains elusive.

Carbon and nitrogen depletion-induced nucleophagy and selective autophagic sequestration of a whole nucleus in multinucleate cells of the filamentous fungus Aspergillus oryzae.

Autophagy is a conserved cellular degradation process in eukaryotes, in which cytoplasmic components and organelles are digested in vacuoles/lysosomes. Recently, autophagic degradation of nuclear materials, termed "nucleophagy", has been reported. In the multinucleate filamentous fungus Aspergillus oryzae, a whole nucleus is degraded by nucleophagy after prolonged culture.

Subcellular localization of acyl-CoA binding protein in Aspergillus oryzae is regulated by autophagy machinery.

In eukaryotic cells, acyl-CoA binding protein (ACBP) is important for cellular activities, such as in lipid metabolism. In the industrially important fungus Aspergillus oryzae, the ACBP, known as AoACBP, has been biochemically characterized, but its physiological function is not known. In the present study, although we could not find any phenotype of AoACBP disruptants in the normal growth conditions, we examined the subcellular localization of AoACBP to understand its physiological function.

AoAtg26, a putative sterol glucosyltransferase, is required for autophagic degradation of peroxisomes, mitochondria, and nuclei in the filamentous fungus Aspergillus oryzae.

Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus Aspergillus oryzae AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast Pichia pastoris.

AoRim15 is involved in conidial stress tolerance, conidiation and sclerotia formation in the filamentous fungus Aspergillus oryzae.

The serine-threonine kinase Rim15p is a master regulator of stress signaling and is required for stress tolerance and sexual sporulation in the yeast Saccharomyces cerevisiae. However, in filamentous fungi that reproduce asexually via conidiation, the physiological function of Rim15p homologs has not been extensively analyzed. Here, we functionally characterized the protein homolog of Rim15p in the filamentous fungus Aspergillus oryzae, by deleting and overexpressing the corresponding Aorim15 gene and examining the role of this protein in stress tolerance and development.

Cooperative role of calnexin and TigA in Aspergillus oryzae glycoprotein folding.

Calnexin (CNX), known as a lectin chaperone located in the endoplasmic reticulum (ER), specifically recognizes G1M9GN2-proteins and facilitates their proper folding with the assistance of ERp57 in mammalian cells. However, it has been left unidentified how CNX works in Aspergillus oryzae, which is a filamentous fungus widely exploited in biotechnology. In this study, we found that a protein disulfide isomerase homolog TigA can bind with A.

Functional analysis of AoAtg11 in selective autophagy in the filamentous fungus Aspergillus oryzae.

Autophagy is a highly conserved cellular degradation process in eukaryotes and consists of both non-selective and selective types. Selective autophagic processes include pexophagy, mitophagy, and the cytoplasm-to-vacuole targeting (Cvt) pathway of yeast, in which particular vacuolar proteins, such as aminopeptidase I (Ape1), are selectively transported to vacuoles. Although selective autophagy has been mainly studied in the yeasts Saccharomyces cerevisiae and Pichia pastoris, there is evidence for selective autophagy in filamentous fungi; however, the details are poorly understood.

Vesicle trafficking, organelle functions, and unconventional secretion in fungal physiology and pathogenicity.

Specific localization of appropriate sets of proteins and lipids is central to functions and integrity of organelles, which in turn underlie cellular activities of eukaryotes. Vesicle trafficking is a conserved mechanism of intracellular transport, which ensures such a specific localization to a subset of organelles. In this review article, we summarize recent advances in our understanding of how vesicle trafficking and related organelles support physiology and pathogenicity of filamentous fungi.

Enhanced production of bovine chymosin by autophagy deficiency in the filamentous fungus Aspergillus oryzae.

Aspergillus oryzae has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, A. oryzae often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production.

Functional analysis of Abp1p-interacting proteins involved in endocytosis of the MCC component in Aspergillus oryzae.

We have investigated the functions of three endocytosis-related proteins in the filamentous fungus Aspergillus oryzae. Yeast two-hybrid screening using the endocytic marker protein AoAbp1 (A.oryzae homolog of Saccharomyces cerevisiae Abp1p) as a bait identified four interacting proteins named Aip (AoAbp1 interacting proteins).

Functional analysis of Aoatg1 and detection of the Cvt pathway in Aspergillus oryzae.

Autophagy is a degradation system in which cellular components are digested via vacuoles/lysosomes. In the budding yeast Saccharomyces cerevisiae, the induction of autophagy results from inactivation of target of rapamycin complex 1 (TORC1), promoting formation of the serine/threonine kinase Atg1, which is one of the key autophagy-related (Atg) proteins required for both nonselective and selective autophagy such as the cytoplasm-to-vacuole targeting (Cvt) pathway. Here, to understand the induction mechanism of autophagy in filamentous fungi, we first identified the ATG1 homolog Aoatg1 in Aspergillus oryzae and then analyzed the localization of an enhanced green fluorescent protein (EGFP)-AoAtg1 fusion protein.

Autophagy delivers misfolded secretory proteins accumulated in endoplasmic reticulum to vacuoles in the filamentous fungus Aspergillus oryzae.

Autophagy is a conserved intracellular degradation process of eukaryotic cells. In filamentous fungi, although autophagy has been reported to have multiple physiological roles, it is not clear whether autophagy is involved in the degradation of misfolded proteins. Here, we investigated the role of autophagy in the degradation of misfolded secretory proteins accumulated in endoplasmic reticulum (ER) in the filamentous fungus Aspergillus oryzae.

Analysis of autophagy in Aspergillus oryzae by disruption of Aoatg13, Aoatg4, and Aoatg15 genes.

Autophagy is a degradation system in which cellular components are digested via vacuoles/lysosomes, and involved in differentiation in addition to helping cells to survive starvation. The autophagic process is composed of several steps: induction of autophagy, formation of autophagosomes, transportation to vacuoles, and degradation of autophagic bodies. To further understand autophagy in the filamentous fungus Aspergillus oryzae, we first constructed A.

Macroautophagy-mediated degradation of whole nuclei in the filamentous fungus Aspergillus oryzae.

Filamentous fungi consist of continuum of multinucleate cells called hyphae, and proliferate by means of hyphal tip growth. Accordingly, research interest has been focusing on hyphal tip cells, but little is known about basal cells in colony interior that do not directly contribute to proliferation. Here, we show that autophagy mediates degradation of basal cell components in the filamentous fungus Aspergillus oryzae.

Aggregation of endosomal-vacuolar compartments in the Aovps24-deleted strain in the filamentous fungus Aspergillus oryzae.

Previously, we found that deletion of Aovps24, an ortholog of Saccharomyces cerevisiae VPS24, that encodes an ESCRT (endosomal sorting complex required for transport)-III component required for late endosomal function results in fragmented and aggregated vacuoles. Although defective late endosomal function is likely responsible for this phenotype, critical lack of our knowledge on late endosomes in filamentous fungi prevented us from further characterization. In this study, we identified late endosomes of Aspergillus oryzae, by expressing a series of fusion proteins of fluorescent proteins with orthologs of late endosomal proteins.

Autophagy during conidiation and conidial germination in filamentous fungi.

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A.

Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae.

Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus.

Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria.

We report here a development of the MultiSite Gateway(TM)-based versatile plasmid construction system applicable for the rapid and efficient preparation of Aspergillus oryzae expression plasmids. This system allows the simultaneous connection of the three DNA fragments inserted in entry clones along with a destination vector in a defined order and orientation. We prepared a variety of entry clones and destination vectors containing promoters, genes encoding carrier-proteins and fusion tags, and selectable markers, which makes it possible to generate 80 expression plasmids for each target protein.