[Show abstract][Hide abstract] ABSTRACT: Microbial secretory phospholipases A(2) (sPLA(2)s) are among the last discovered and least known members of this functionally diverse family of enzymes. We analyzed here two sPLA(2)s, named sPlaA and sPlaB, of the filamentous ascomycete Aspergillus oryzae. sPlaA and sPlaB consist of 222 and 160 amino acids, respectively, and share the conserved Cys and catalytic His-Asp residues typical of microbial sPLA(2)s. Two sPLA(2)s differ in pH optimum, Ca(2+) requirement and expression profile. The splaA mRNA was strongly upregulated in response to carbon starvation, oxidative stress and during conidiation, while splaB was constitutively expressed at low levels and was weakly upregulated by heat shock. Experiments with sPLA(2) overexpressing strains demonstrated that two enzymes produce subtly different phospholipid composition variations and also differ in their subcellular localization: sPlaA is most abundant in hyphal tips and secreted to the medium, whereas sPlaB predominantly localizes to the ER-like intracellular compartment. Both sPLA(2) overexpressing strains were defective in conidiation, which was more pronounced for sPlaB overexpressors. Although no major morphological abnormality was detected in either DeltasplaA or DeltasplaB mutants, hyphal growth of DeltasplaB, but not that of DeltasplaA, displayed increased sensitivity to H(2)O(2) treatment. These data indicate that two A. oryzae sPLA(2) enzymes display distinct, presumably non-redundant, physiological functions.
[Show abstract][Hide abstract] ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
[Show abstract][Hide abstract] ABSTRACT: In mammalian cells, cytosolic phospholipase A(2) (cPLA(2)) displays a variety of activities through the release of arachidonic acid in response to cellular stimuli. In this study, we characterize the putative cPLA(2)-like protein, AoPlaA, in the filamentous fungus Aspergillus oryzae. When AoPlaA-EGFP was expressed in A. oryzae, it localized to the tubular structures that was costained by the marker dye for the mitochondria. A biochemical fractionation experiment showed that AoPlaA was present in the mitochondria-enriched fraction. The presence of an N-terminal cleavable mitochondrial targeting signal in AoPlaA was demonstrated by N-terminal amino acid sequence analysis, and by showing that chimeric proteins consisting of N-terminal 65 or 50 amino acids of AoPlaA fused to enhanced green fluorescent protein (EGFP) localized to the mitochondria. Submitochondrial fractionation of AoPlaA expressed in Saccharomyces cerevisiae demonstrated that AoPlaA localizes to the intramembrane space of the mitochondria. Taken together, the results presented here demonstrate a novel, submitochondrial localization of the cPLA(2)-like protein in the filamentous fungi.
[Show abstract][Hide abstract] ABSTRACT: Recent live cell imaging analyzing the components required for endocytosis has elucidated that endocytosis actively occurs at the hyphal tip region in filamentous fungi. To examine further the physiological roles of endocytosis we investigated a conditional mutant of endocytosis in Aspergillus oryzae. Endocytosis-deficient hyphae displayed retarded apical growth, abnormal hyphal morphology, mislocalization of a vesicle- SNARE, which is thought to undergo endocytic recycling to the tip region, and aberrant accumulation of cell wall components at large invaginated structures. These results suggest that endocytosis is crucial for apical growth and for recycling components, which should be re-transported to the tip region. In this report, we discuss the endocytic recycling pathway and present its possible mechanism in filamentous fungi.
[Show abstract][Hide abstract] ABSTRACT: RsSymEG, an endoglucanase of glycosyl hydrolase family (GHF) 7 encoded by a transcript isolated from the symbiotic protist of the termite Reticulitermes speratus, is expressed in Aspergillus oryzae. Interestingly, purified RsSymEG1 has a relatively higher specific activity (603 micromol min(-1) mg(-1) protein) and V(max) value (769.6 unit/mg protein) than previously reported data for GHF7 endoglucanase of Trichoderma ressei. It also has the same K(m) value (1.97 mg/ml) with Clostridium cellulolyticum enzymes that contain cellulose binding module, a property indicative of high affinity to substrate, though no cellulose binding module is found within it. Thin-layer chromatography analysis revealed that RsSymEG1 preferentially hydrolyzes the beta-1,4-cellulosic linkage of cellodextrins into cellobiose and glucose.
[Show abstract][Hide abstract] ABSTRACT: Cultured cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing stimulation and provide an in vitro model system with which to study the effects of neurotrophic substances. Our previous results showed that secretory phospholipases A(2) (sPLA(2)s) protect CGNs from apoptotic cell death under the nondepolarizing condition. In this study, we further analyzed the mechanism whereby sPLA(2) exhibits this effect. Among the primary metabolites of sPLA(2) tested, lysophosphatidylcholine (LPC), but not other lysophospholipids, remarkably rescued CGNs from apoptosis. In contrast, neither arachidonic nor oleic acids displayed neurotrophic effect. Release of LPC into the culture media occurred in response to sPLA(2) treatment, and degradation or sequestration of LPC attenuated the survival-promoting effects of sPLA(2) and LPC. The neurotrophic effect of LPC required the presence of extracellular Ca(2+) and L-type Ca(2+) channel activity, suggesting that Ca(2+) influx across the plasma membrane is evoked by LPC. sPLA(2)- or LPC-induced promotion of CGN survival was suppressed by inhibitors of protein kinase A and phospholipase C, suggesting that they play a role in mediating survival-promoting signal of sPLA(2). The results presented here demonstrate a novel, unexpected neurotrophin-like effect of LPC in the central nervous system.
Journal of Neuroscience Research 01/2009; 87(1):190-9. DOI:10.1002/jnr.21821 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Establishing the occurrence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis. Recently, however, it was shown that the fluorescent dye N-(3-triethylammoniumpropyl)-4-(p-diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC (Aspergillus oryzae UapC) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis. Although the occurrence of endocytosis was clearly demonstrated, its physiological importance in filamentous fungi still remains largely unaddressed. We generated a strain in which A. oryzae end4 (Aoend4), the A. oryzae homolog of Saccharomyces cerevisiae END4/SLA2, was expressed from the Aoend4 locus under the control of a regulatable thiA promoter. The growth of this strain was severely impaired, and its hyphal morphology was altered in the Aoend4-repressed condition. Moreover, in the Aoend4-repressed condition, neither FM4-64 nor AoUapC-EGFP was internalized, indicating defective endocytosis. Furthermore, the localization of a secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) was abnormal in the Aoend4-repressed condition. Aberrant accumulation of cell wall components was also observed by calcofluor white staining and transmission electron microscopy analysis, and several genes that encode cell wall-building enzymes were upregulated, indicating that the regulation of cell wall synthesis is abnormal in the Aoend4-repressed condition, whereas Aopil1 disruptants do not display the phenotype exhibited in the Aoend4-repressed condition. Our results strongly suggest that endocytosis is crucial for the hyphal tip growth in filamentous fungi.
[Show abstract][Hide abstract] ABSTRACT: To achieve high expression of glycoside hydrolase family 45 endoglucanase (RsSym45EG1) from a symbiotic protist of the termite Reticulitermes speratus, synthetic sequence RsSym45eg1-co, in which the codon usage was adjusted to that of the highly-expressed tef1 gene encoding translation elongation factor 1alpha, was prepared and introduced into A. oryzae. The transcript level of RsSym45eg1-co was 1.8-fold higher than that of RsSym45eg1. In cells harboring RsSym45eg1, but not RsSym45eg1-co, truncated transcripts in which the coding region was prematurely terminated and followed by a poly A chain were found. The production of endoglucanase in the culture supernatant was improved by codon optimization. Truncated transcripts were also found when cellobiohydrolase and beta-glucosidase from R. speratus symbionts were expressed, and the transcript level of the former was increased by codon-optimization. Our findings suggest that premature polyadenylation frequently occurs in heterologous protein expression in A. oryzae, which might result in the poor yield of expressed proteins.
The Journal of General and Applied Microbiology 02/2008; 54(6):343-51. DOI:10.2323/jgam.54.343 · 0.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Studies on protein production using filamentous fungi have mostly focused on improvement of the protein yields by genetic modifications such as overexpression. Recent genome sequencing in several filamentous fungal species now enables more systematic approaches based on reverse genetics and molecular biology of the secretion pathway. In this review, we summarize recent molecular-based advances in our understanding of vesicular trafficking in filamentous fungi, and discuss insights into their high secretion ability and application for protein production.
[Show abstract][Hide abstract] ABSTRACT: Sphingosylphosphorylcholine (SPC) is a choline-containing naturally occurring derivative of sphingolipid involved in various biological processes. Here we show that SPC displays neurotrophic effects in cerebellar granule neurons (CGNs) and in PC12 cells. When CGNs were cultured under non-depolarizing condition, they exhibited condensed and fragmented nuclei typical of apoptotic phenotype. SPC added to the culture medium rescued cells from undergoing apoptosis. The anti-apoptotic effect of SPC was dependent on the presence of extracellular Ca2+, suggesting that Ca2+ influx occurs upon SPC treatment. In PC12 cells, SPC displayed nerve growth factor-like neuritogenic effect which was sensitive to the presence of Ca2+ channel blocker and Ca2+ withdrawal from the medium. These results suggest that SPC plays novel neurotrophic effects in the nervous system.
Biochemical and Biophysical Research Communications 01/2008; 364(1):163-8. DOI:10.1016/j.bbrc.2007.09.121 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In spite of their great importance for both applied and basic biology, studies on vesicular trafficking in filamentous fungi have been so far very limited. Here, we identified 21 genes, which might be a total set, encoding putative SNARE proteins that are key factors for vesicular trafficking, taking advantage of available whole genome sequence in the filamentous fungus Aspergillus oryzae. The subsequent systematic analysis to determine the localization of putative SNAREs using EGFP-fused chimeras revealed that most putative SNAREs show similar subcellular distribution to their counterparts in the budding yeast. However, there existed some characteristic features of SNAREs in A. oryzae, such as SNARE localization at/near the septum and the presence of apparently non-redundant plasma membrane Qa-SNAREs. Overall, this analysis allowed us to provide an overview of vesicular trafficking and organelle distribution in A. oryzae.
[Show abstract][Hide abstract] ABSTRACT: 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. Using these fusion proteins as markers, we observed late endosomes in the wild type strain and the Aovps24 disruptant and demonstrated that late endosomes are aberrantly aggregated in the Aovps24 disruptant. Moreover, we revealed that the aggregated late endosomes have features of vacuoles as well. As deletion of another ESCRT-III component-encoding gene, Aovps2, resulted in similar phenotypes to that in the Aovps24 disruptant, phenotypes of the Aovps24 disruptant are probably due to defective late endosomal function.
Biochemical and Biophysical Research Communications 11/2007; 362(2):474-9. DOI:10.1016/j.bbrc.2007.08.027 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with alpha-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi.
[Show abstract][Hide abstract] ABSTRACT: 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. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)-AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.
[Show abstract][Hide abstract] ABSTRACT: Vps24 (vacuolar protein sorting) is a component of ESCRT III (endosomal sorting complex required for transport), which is required for the formation of MVB (multivesicular body). We have isolated the VPS24 homologue gene, Aovps24, from the filamentous fungus Aspergillus oryzae, and analyzed the localization of AoVps24 using EGFP. AoVps24 was localized in the cytoplasm and late endosome-like structures. Furthermore, we constructed an Aovps24 disruptant, which showed impaired growth, conidiation, and hyphal morphology. In addition, normal vacuoles were not observed in the Aovps24 disruptant. In the Saccharomyces cerevisiae vps24 disruptant, the normal vacuoles are formed and it does not show the impaired growth and abnormal cell shape as the A. oryzae Aovps24 disruptant. The results suggest that AoVps24 is required for vacuolar formation and normal vacuoles could have the function to maintain the normal hyphal elongation and conidiation in A. oryzae.
Biochemical and Biophysical Research Communications 10/2006; 347(4):970-8. DOI:10.1016/j.bbrc.2006.06.183 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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. Using this system, plasmids for expression of the EGFP fused with the mitochondrial-targeting signal of citrate synthase (AoCit1) were generated. Tubular structures of mitochondria were visualized in the transformants expressing the AoCit1-EGFP fusion protein. This plasmid construction system allows us to prepare a large number of expression plasmids without laborious DNA manipulations, which would facilitate molecular biological studies on A. oryzae.
[Show abstract][Hide abstract] ABSTRACT: 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. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures
under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed
in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores
and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating
that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy
functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.
[Show abstract][Hide abstract] ABSTRACT: Morphological analyses of vacuoles in filamentous fungi in the past decade have led to the remarkable finding that they are highly pleiomorphic organelles. Among them, tubular vacuoles have been implicated in nutrient transport between hyphal tips and the host plant surface in mycorrhizal fungi. However, a series of works suggested the presence of tubular vacuoles in other fungi that are not mycorrhizal, including Aspergillus oryzae, hinting at more general roles of the tubular vacuoles. Recently, we made two key observations by using the fusion protein of enhanced green fluorescent protein (EGFP) with a putative vacuolar t-SNARE in A. oryzae; tubular vacuoles formed more extensively in hyphae that were not in contact with nutrients, and vacuoles that were interconnected by tubules in the mature mycelial region displayed traces of microautophagy-mediated degradation of cytoplasm. The aim of this addendum is to discuss the possible involvement of vacuoles in degrading, transporting, and recycling nutrients from the mature mycelial region to hyphal tips, to support the continuous tip growth.
[Show abstract][Hide abstract] ABSTRACT: Endocytosis is an important process for cellular activities. However, in filamentous fungi, the existence of endocytosis has been so far elusive. In this study, we used AoUapC-EGFP, the fusion protein of a putative uric acid-xanthine permease with enhanced green fluorescent protein (EGFP) in Aspergillus oryzae, to examine whether the endocytic process occurs or not. Upon the addition of ammonium into the medium the fusion protein was internalized from the plasma membrane. The internalization of AoUapC-EGFP was completely blocked by sodium azide, cold, and cytochalasin A treatments, suggesting that the internalization possesses the general features of endocytosis. These results demonstrate the occurrence of endocytosis in filamentous fungi. Moreover, we discovered that the endosomal compartments appeared upon the induction of endocytosis and moved in a microtubule-dependent manner.
Biochemical and Biophysical Research Communications 03/2006; 340(3):784-91. DOI:10.1016/j.bbrc.2005.12.077 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra- and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both Deltavam3 and Deltapep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.