Molecular basis of regulatory mechanism for cell growth and differentiation by proprotein convertases, Structure and function of plant proteases, Development of animal cellulases by protein engineering (protease, processing, 翻訳後修飾, 血清タンパク, enzyme replacement therapy)
Xiaomei Sun, Yuxin Ye, Naofumi Sakurai, Koji Kato, Keizo Yuasa, Akihiko Tsuji and Min Yao : Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing., Acta Crystallographica. Section F, Structural Biology Communications, 76, 1, 20-24, 2020.
Akihiko Tsuji and Keizo Yuasa : Identification and enzymatic characterization of clip domain serine protease in the digestive fluid of the sea hare, Aplysia kurodai., Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 237, 110322, 2019.
(Summary)
Clip domain serine proteases (CDSPs) participate in the extracellular signaling cascades of various biological processes such as innate immune responses in invertebrates. CDSP genes have been isolated from numerous invertebrates. Nevertheless, the enzymatic properties of mollusk CDSPs are poorly understood. In the present study, we demonstrated that the amino acid sequences of the trypsin-like serine protease purified from the digestive fluid of the sea hare, Aplysia kurodai resemble those of the unidentified CDSP-type protein (TPS3) of Aplysia californica predicted by genome analysis. The purified enzyme produced single 34 and 26.5 kDa bands on SDS-PAGE under non-reducing and reducing conditions, respectively. The 34-kDa band generated two amino-terminal sequences that were similar to the deduced sequences of the clip and catalytic domains of TPS3. The single amino-terminal sequence of the 26.5 kDa band showed a single sequence homologous to the catalytic domain. Thus, the purified enzyme consists of clip and catalytic domains bridged by disulfide linkage(s). The subsite specificity and inhibitor sensitivity of the purified enzyme were clearly distinct from those of horseshoe crab and silkworm CDSPs. A good substrate for the sea hare enzyme was pyroglutamyl-Arg-Thr-Lys-Arg-4-methyl-7-coumarylamide. The enzyme activity was strongly inhibited by aprotinin but not leupeptin. The physiological function of the enzyme in the digestive fluid remains to be determined.
Akihiko Tsuji, Keizo Yuasa and Chikako Asada : Cellulose-binding activity of a 21-kDa endo-ß-1,4-glucanase lacking cellulose-binding domain and its synergy with other cellulases in the digestive fluid of Aplysia kurodai, PLoS ONE, 13, 11, e0205915, 2018.
(Summary)
Endo-ß-1,4-glucanase AkEG21 belonging to glycosyl hydrolase family 45 (GHF45) is the most abundant cellulase in the digestive fluid of sea hare (Aplysia kurodai). The specific activity of this 21-kDa enzyme is considerably lower than those of other endo ß-1,4-glucanases in the digestive fluid of A. kurodai, therefore its role in whole cellulose hydrolysis by sea hare is still uncertain. Although AkEG21 has a catalytic domain without a cellulose binding domain, it demonstrated stable binding to cellulose fibers, similar to that of fungal cellobiohydrolase (CBH) 1 and CBH 2, which is strongly inhibited by cellohexaose, suggesting the involvement of the catalytic site in cellulose binding. Cellulose-bound AkEG21 hydrolyzed cellulose to cellobiose, cellotriose and cellotetraose, but could not digest an external substrate, azo-carboxymethyl cellulose. Cellulose hydrolysis was considerably stimulated by the synergistic action of cellulose-bound AkEG21 and AkEG45, another ß-1,4-endoglucanase present in the digestive fluid of sea hare; however no synergy in carboxymethylcellulose hydrolysis was observed. When AkEG21 was removed from the digestive fluid by immunoprecipitation, the cellulose hydrolyzing activity of the fluid was significantly reduced, indicating a critical role of AkEG21 in cellulose hydrolysis by A. kurodai. These findings suggest that AkEG21 is a processive endoglucanase functionally equivalent to the CBH, which provides a CBH-independent mechanism for the mollusk to digest seaweed cellulose to glucose.
Yuna Oue, Sara Murakami, Kinuka Isshiki, Akihiko Tsuji and Keizo Yuasa : Intracellular localization and binding partners of death associated protein kinase-related apoptosis-inducing protein kinase 1., Biochemical and Biophysical Research Communications, 496, 4, 1222-1228, 2018.
(Summary)
Death associated protein kinase (DAPK)-related apoptosis-inducing protein kinase (DRAK)-1 is a positive apoptosis regulator. However, the molecular mechanisms underlying the DRAK1-mediated apoptotic pathway remain unclear. In this study, we demonstrated the intracellular localization and binding partners of DRAK1. In human osteosarcoma cell line U2OS cells, DRAK1 was mainly localized in the nucleus and translocated outside the nucleus through Ser phosphorylation by protein kinase C. In the nucleus, DRAK1 associated with tumor suppressor p53 and positively regulated p53 transcriptional activity in response to DNA-damaging agent cisplatin. On the other hand, DRAK1 interacted with the mitochondrial inner-membrane protein, adenine nucleotide translocase (ANT)-2, an anti-apoptotic oncoprotein, outside the nucleus. These findings suggest that DRAK1 translocates in response to stimuli and induces apoptosis through its interaction with specific binding partners, p53 and/or ANT2.
Shinya Matsuda, Kohei Kawamoto, Kenji Miyamoto, Akihiko Tsuji and Keizo Yuasa : PCTK3/CDK18 regulates cell migration and adhesion by negatively modulating FAK activity, Scientific Reports, 7, 45545, 2017.
(Summary)
PCTAIRE kinase 3 (PCTK3) is a member of the cyclin dependent kinase family, but its physiologicalfunction remains unknown. We previously reported that PCTK3-knockdown HEK293T cells showedactin accumulation at the leading edge, suggesting that PCTK3 is involved in the regulation of actinreorganization. In this study, we investigated the physiological function and downstream signaltransduction molecules of PCTK3. PCTK3 knockdown in HEK293T cells increased cell motility and RhoA/Rho-associated kinase activity as compared with control cells. We also found that phosphorylationat residue Tyr-397 in focal adhesion kinase (FAK) was increased in PCTK3-knockdown cells. FAKphosphorylation at Tyr-397 was increased in response to fibronectin stimulation, whereas itsphosphorylation was suppressed by PCTK3. In addition, excessive expression of PCTK3 led to theformation of filopodia during the early stages of cell adhesion in HeLa cells. These results indicate thatPCTK3 controls actin cytoskeleton dynamics by negatively regulating the FAK/Rho signaling pathway.
Norio Kamemura, Sara Murakami, Hiroaki Komatsu, Masahiro Sawanoi, Kenji Miyamoto, Kazumi Ishidoh, Koji Kishimoto, Akihiko Tsuji and Keizo Yuasa : Type II cGMP-dependent protein kinase negatively regulates fibroblast growth factor signaling by phosphorylating Raf-1 at serine 43 in rat chondrosarcoma cells., Biochemical and Biophysical Research Communications, 483, 1, 82-87, 2017.
(Summary)
Although type II cGMP-dependent protein kinase (PKGII) is a major downstream effector of cGMP in chondrocytes and attenuates the FGF receptor 3/ERK signaling pathway, its direct target proteins have not been fully explored. In the present study, we attempted to identify PKGII-targeted proteins, which are associated with the inhibition of FGF-induced MAPK activation. Although FGF2 stimulation induced the phosphorylation of ERK1/2, MEK1/2, and Raf-1 at Ser-338 in rat chondrosarcoma cells, pretreatment with a cell-permeable cGMP analog strongly inhibited their phosphorylation. On the other hand, Ser-43 of Raf-1 was phosphorylated by cGMP in a dose-dependent manner. Therefore, we examined the direct phosphorylation of Raf-1 by PKGII. Wild-type PKGII phosphorylated Raf-1 at Ser-43 in a cGMP-dependent manner, but a PKGII D412A/R415A mutant, which has a low affinity for cGMP, did not. Finally, we found that a phospho-mimic mutant, Raf-1 S43D, suppressed FGF2-induced MAPK pathway. These results suggest that PKGII counters FGF-induced MEK/ERK activation through the phosphorylation of Raf-1 at Ser-43 in chondrocytes.
Akihiko Tsuji, Shuji Kuwamura, Akihiro Shirai and Keizo Yuasa : Identification and Characterization of a 25 kDa Protein That Is Indispensable for the Efficient Saccharification of Eisenia bicyclis in the Digestive Fluid of Aplysia kurodai, PLoS ONE, 12, 1, e0170669, 2017.
(Summary)
The digestive fluid of the sea hare Aplysia kurodai can liberate approximately 2.5 mg of glucose from 10 mg of dried Eisenia bicyclis powder. Although laminaran, a major storage polysaccharide in E. bicyclis, is easily digested to glucose by the synergistic action of the 110 and 210 kDa A. kurodai -glucosidases (BGLs), glucose is not liberated from E. bicyclis by direct incubation with these BGLs. To clarify this discrepancy, we searched for an Eisenia hydrolysis enhancing protein (EHEP) in the digestive fluid of A. kurodai. A novel 25 kDa protein that enhances E. bicyclis saccharification by -glucosidases was purified to a homogeneous state from the digestive fluid of A. kurodai, and its cDNA was cloned from total cDNAs reverse-transcribed from hepatopancreas total RNA. The E. bicyclis extract strongly inhibited BGLs, suggesting some compound within this brown alga functioned as a feeding deterrent. However, when E. bicyclis was incubated with BGLs in the presence of EHEP, glucose production was markedly increased. As E. bicyclis is rich in phlorotannin, which are only found in brown algae, our study suggested that these compounds are the main BGL inhibitors in E. bicyclis extract. EHEP protects BGLs from phlorotannin inhibition by binding to phlorotannins and forming an insoluble complex with phloroglucinol and phlorotannins. These findings indicated that EHEP plays a key role in the saccharification of brown seaweeds containing phlorotannins in the digestive fluid of A. kurodai. This is the first report of EHEP as a phlorotannin-binding protein that protects BGLs from inhibition.
Ichiro Yoshida, Chihiro Ito, Shinya Matsuda, Akihiko Tsuji, Noriyuki Yanaka and Keizo Yuasa : Alisol B, a triterpene from Alismatis rhizoma (dried rhizome of Alisma orientale), inhibits melanin production in murine B16 melanoma cells, Bioscience, Biotechnology, and Biochemistry, 81, 3, 534-540, 2017.
(Summary)
To develop new whitening agents from natural products, we screened 80 compounds derived from crude drugs in Kampo medicine in a melanin synthesis inhibition assay using murine B16 melanoma cells. The screen revealed that treatment with alisol B, a triterpene from Alismatis rhizoma, significantly decreased both melanin content and cellular tyrosinase activity in B16 cells. However, alisol B did not directly inhibit mushroom tyrosinase activity in vitro. Therefore, we investigated the mechanism underlying the inhibitory effect of alisol B on melanogenesis. Alisol B suppressed mRNA induction of tyrosinase and its transcription factor, microphthalmia-associated transcription factor (MITF). Furthermore, alisol B reduced the phosphorylation of CREB and maintained the activation of ERK1/2. These results suggest that the reduction in melanin production by alisol B is due to the downregulation of MITF through the suppression of CREB and activation of ERK and that alisol B may be useful as a new whitening agent.
Kohichi Kuwahara, Hiroshi Hirata, Kengo Ohbuchi, Kentaro Nishi, Akira Maeda, Akihiko Kuniyasu, Daisuke Yamada, Takehiko Maeda, Akihiko Tsuji, Makoto Sawada and Hitoshi Nakayama : The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin-like protease(s) in a subpopulation of microglia in neonatal rat brain, Glia, 64, 11, 1938-1961, 2016.
Kinuka Isshiki, Taishi Hirase, Shinya Matsuda, Kenji Miyamoto, Akihiko Tsuji and Keizo Yuasa : Death-associated protein kinase 2 mediates nocodazole-induced apoptosis through interaction with tubulin, Biochemical and Biophysical Research Communications, 468, 1-2, 113-118, 2015.
(Summary)
Death-associated protein kinase 2 (DAPK2) is a positive regulator of apoptosis. Although we recently reported that 14-3-3 proteins inhibit DAPK2 activity and its subsequent apoptotic effects via binding to DAPK2, the molecular mechanisms underlying the DAPK2-mediated apoptotic pathway remain unclear. Therefore, we attempted to further identify DAPK2-interacting proteins using pull-down assays and mass spectrometry. The microtubule β-tubulin was identified as a novel DAPK2-binding protein in HeLa cells. Pull-down assays revealed that DAPK2 interacted with the α/β-tubulin heterodimer, and that the C-terminal region of DAPK2, which differs from that of other DAPK family members, was sufficient for the association with β-tubulin. Although the microtubule-depolymerizing agent nocodazole induced apoptosis in HeLa cells, the level of apoptosis was significantly decreased in the DAPK2 knockdown cells. Furthermore, we found that treatment with nocodazole resulted in an increased binding of DAPK2 to β-tubulin. These findings indicate that DAPK2 mediates nocodazole-induced apoptosis via binding to tubulin.
Yuki Yoshikatsu, Yo-ichi Ishida, Haruka Sudo, Keizo Yuasa, Akihiko Tsuji and Masami Nagahama : NVL2, a nucleolar AAA-ATPase, is associated with the nuclear exosome and is involved in pre-rRNA processing, Biochemical and Biophysical Research Communications, 464, 3, 780-786, 2015.
(Summary)
Nuclear VCP-like 2 (NVL2) is a member of the chaperone-like AAA-ATPase family and is involved in the biosynthesis of 60S ribosomal subunits in mammalian cells. We previously showed the interaction of NVL2 with a DExD/H-box RNA helicase MTR4/DOB1, which is a known cofactor for an exoribonuclease complex, the exosome. This finding implicated NVL2 in RNA metabolic processes during ribosome biogenesis. In the present study, we found that a series of mutations within the ATPase domain of NVL2 causes a defect in pre-rRNA processing into mature 28S and 5.8S rRNAs. Co-immunoprecipitation analysis showed that NVL2 was associated with the nuclear exosome complex, which includes RRP6 as a nucleus-specific catalytic subunit. This interaction was prevented by depleting either MTR4 or RRP6, indicating their essential role in mediating this interaction with NVL2. Additionally, knockdown of MPP6, another cofactor for the nuclear exosome, also prevented the interaction by causing MTR4 to dissociate from the nuclear exosome. These results suggest that NVL2 is involved in pre-rRNA processing by associating with the nuclear exosome complex and that MPP6 is required for maintaining the integrity of this rRNA processing complex.
Keizo Yuasa, Reina Ota, Matsuda Shinya, Kinuka Isshiki, Masahiro Inoue and Akihiko Tsuji : Suppression of death-associated protein kinase 2 by interaction with 14-3-3 proteins, Biochemical and Biophysical Research Communications, 464, 1, 70-75, 2015.
(Summary)
Death-associated protein kinase 2 (DAPK2), a Ca(2+)/calmodulin-regulated serine/threonine kinase, induces apoptosis. However, the signaling mechanisms involved in this process are unknown. Using a proteomic approach, we identified 14-3-3 proteins as novel DAPK2-interacting proteins. The 14-3-3 family has the ability to bind to phosphorylated proteins via recognition of three conserved amino acid motifs (mode 1-3 motifs), and DAPK2 contains the mode 3 motif ((pS/pT)X1-2-COOH). The interaction of 14-3-3 proteins with DAPK2 was dependent on the phosphorylation of Thr(369), and effectively suppressed DAPK2 kinase activity and DAPK2-induced apoptosis. Furthermore, we revealed that the 14-3-3 binding site Thr(369) of DAPK2 was phosphorylated by the survival kinase Akt. Our findings suggest that DAPK2-induced apoptosis is negatively regulated by Akt and 14-3-3 proteins.
Kim Hyejin, Atsushi Tabata, Toshifumi Tomoyasu, Ueno Tomomi, Uchiyama Shigeto, Keizo Yuasa, Akihiko Tsuji and Hideaki Nagamune : Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells., Journal of Bone and Mineral Metabolism, 33, 1, 30-39, 2015.
(Summary)
Estrogenic compounds include endogenous estrogens such as estradiol as well as soybean isoflavones, such as daidzein and its metabolite equol, which are known phytoestrogens that prevent osteoporosis in postmenopausal women. Indeed, mineralization of MC3T3-E1 cells, a murine osteoblastic cell line, was significantly decreased in medium containing fetal bovine serum treated with charcoal-dextran to deplete endogenous estrogens, but estradiol and these soybean isoflavones dose-dependently restored the differentiation of MC3T3-E1 cells; equal was tenfold more effective than daidzein. These differentiation promoting effects were inhibited by the addition of fulvestrant, which is a selective downregulator of estrogen receptors. Analysis of the expression pattern of bone-related genes by reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qRT-PCR), which focused on responsiveness to the estrogen stimuli, revealed that the transcription of PACE4, a subtilisin-like proprotein convertase, was tightly linked with the differentiation of MC3T3-E1 cells induced by estrogen stimuli. Moreover, treatment with RNAi of PACE4 in MC3T3-E1 cells resulted in a drastic decrease of mineralization in the presence of estrogen stimuli. These results strongly suggest that PACE4 participates in bone formation at least in osteoblast differentiation, and estrogen receptor-mediated stimuli induce osteoblast differentiation through the upregulation of PACE4 expression.
Shinya Matsuda, Kyohei Kominato, Shizuyo Koide-Yoshida, Kenji Miyamoto, Kinuka Isshiki, Akihiko Tsuji and Keizo Yuasa : PCTAIRE Kinase 3/Cyclin-dependent Kinase 18 Is Activated through Association with Cyclin A and/or Phosphorylation by Protein Kinase A, The Journal of Biological Chemistry, 289, 26, 18387-18400, 2014.
(Summary)
PCTAIRE kinase 3 (PCTK3)/cyclin-dependent kinase 18 (CDK18) is an uncharacterized member of the CDK family because its activator(s) remains unidentified. Here we describe the mechanisms of catalytic activation of PCTK3 by cyclin A2 and cAMP-dependent protein kinase (PKA). Using a pulldown experiment with HEK293T cells, cyclin A2 and cyclin E1 were identified as proteins that interacted with PCTK3. An in vitro kinase assay using retinoblastoma protein as the substrate showed that PCTK3 was specifically activated by cyclin A2 but not by cyclin E1, although its activity was lower than that of CDK2. Furthermore, immunocytochemistry analysis showed that PCTK3 colocalized with cyclin A2 in the cytoplasm and regulated cyclin A2 stability. Amino acid sequence analysis revealed that PCTK3 contained four putative PKA phosphorylation sites. In vitro and in vivo kinase assays showed that PCTK3 was phosphorylated by PKA at Ser(12), Ser(66), and Ser(109) and that PCTK3 activity significantly increased via phosphorylation at Ser(12) by PKA even in the absence of cyclin A2. In the presence of cyclin A2, PCTK3 activity was comparable to CDK2 activity. We also found that PCTK3 knockdown in HEK293T cells induced polymerized actin accumulation in peripheral areas and cofilin phosphorylation. Taken together, our results provide the first evidence for the mechanisms of catalytic activation of PCTK3 by cyclin A2 and PKA and a physiological function of PCTK3.
Akihiko Tsuji, Nami Nishiyama, Miki Ohshima, Saori Maniwa, Shuji Kuwamura, Masataka Shiraishi and Keizo Yuasa : Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α-amylases and two α-glucosidases, FEBS Open Bio, 4, 560-570, 2014.
(Summary)
Sea lettuce (Ulva pertusa) is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the sea hare Aplysia kurodai, shows a clear feeding preference for sea lettuce. Compared with cellulose, sea lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on sea lettuce. A. kurodai contain two α-amylases (59 and 80 kDa) and two α-glucosidases (74 and 86 kDa). The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca(2+) or Cl(-). Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When sea lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, sea hares efficiently digest sea lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase-glucoamylase and sucrase-isomaltase complexes.
Junki Fukumoto, Mohd Nor Ismaliza Ismail, Masaki Kubo, Keita Kinoshita, Masahiro Inoue, Keizo Yuasa, Makoto Nishimoto, Hitoshi Matsuki and Akihiko Tsuji : Possible role of inter-domain salt bridges in oligopeptidase B from Trypanosoma brucei: critical role of Glu172 of non-catalytic -propeller domain in catalytic activity and Glu490 of catalytic domain in stability of OPB., The Journal of Biochemistry, 154, 5, 465-473, 2013.
(Summary)
Oligopeptidase B (OPB) is a member of the prolyl oligopeptidase (POP) family of serine proteases. OPB in trypanosomes is an important virulence factor and potential pharmaceutical target. Characteristic structural features of POP family members include lack of a propeptide and presence of a -propeller domain (PD), although the role of the -PD has yet to be fully understood. In this work, residues Glu(172), Glu(490), Glu(524) and Arg(689) in Trypanosoma brucei OPB (Tb OPB), which are predicted to form inter-domain salt bridges, were substituted for Gln and Ala, respectively. These mutants were evaluated in terms of catalytic properties and stability. A negative effect on kcat/Km was obtained following mutation of Glu(172) or Arg(689). In contrast, the E490Q mutant exhibited markedly decreased thermal stability, although this mutation had less effect on catalytic properties compared to the E172Q and R689A mutants. Trypsin digestion showed that the boundary regions between the -PD and catalytic domains (CDs) of the E490Q mutant are unfolded with heat treatment. These results indicated that Glu(490) in the CD plays a role in stabilization of Tb OPB, whereas Glu(172) in the -PD is critical for the catalytic activity of Tb OPB.
Akihiko Tsuji, Keiko Tominaga, Nami Nishiyama and Keizo Yuasa : Comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the sea hare aplysia kurodai. efficient glucose release from sea lettuce by synergistic action of 45 kDa endoglucanase and 210 kDa ß-glucosidase., PLoS ONE, 8, 6, 2013.
(Summary)
Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 ß-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU-ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.
Masahiro Inoue, Kouichi Yasuda, Haruki Uemura, Natsumi Yasaka, Achim Schnaufer, Mihiro Yano, Hiroshi Kido, Daisuke Kohda, Hirofumi Doi, Toshihide Fukuma, Akihiko Tsuji and Nobuo Horikoshi : Trypanosoma brucei 14-3-3I and II proteins predominantly form a heterodimer structure that acts as a potent cell cycle regulator in vivo., The Journal of Biochemistry, 153, 5, 431-439, 2013.
(Summary)
Hetero- and homodimerization of 14-3-3 proteins demonstrate distinctive functions in mammals and plants. Trypanosoma brucei 14-3-3I and II (Tb14-3-3I and II) play pivotal roles in motility, cytokinesis and the cell cycle; however, the significance and the mechanism of Tb14-3-3 dimerization are remained to be elucidated. We found that ectopically expressed epitope-tagged Tb14-3-3I and II proteins formed hetero- and homodimers with endogenous Tb14-3-3I and II proteins. However, we also found the ability to form hetero- or homodimers between Tb14-3-3I and II proteins was clearly affected by the sequence and location of the epitope tag used. We found a blue native polyacrylamide gel electrophoresis system followed by western blotting may distinguish monomer from dimer structure, and stable from unstable conformation of Tb14-3-3. Combined with co-immunoprecipitation results, we revealed that Tb14-3-3 proteins mainly existed as heterodimeric form. Furthermore, co-overexpression of Tb14-3-3I and II proteins in T. brucei induced aberrant numbers of organelles in cells, but overexpression of either isoform alone rarely produced such morphology. These results suggest that heterodimers play more significant roles than homodimers not only in the maintenance of steady-state levels of the 14-3-3 proteins but also in the regulation of cytokinesis.
Koichiro Hada, Kinuka Isshiki, Shinya Matsuda, Keizo Yuasa and Akihiko Tsuji : Engineering of α1-antitrypsin variants with improved specificity for the proprotein convertase furin using site-directed random mutagenesis, Protein Engineering, Design & Selection, 26, 2, 123-131, 2013.
(Summary)
Furin, PACE4, PC5/6 and PC7 are members of the subtilisin-like proprotein convertase (SPC) family. Although these enzymes are known to play critical roles in various physiological and pathological events including cell differentiation, tumor growth, virus replication and the activation of bacterial toxins, their distinct functions are yet to be fully delineated. α1-PDX is an engineered α1-antitrypsin variant carrying the RXXR consensus motif for furin within its reactive site loop. However, α1-PDX inhibits other SPCs in addition to furin. In this work, we prepared various rat α1-antitrypsin variants containing Arg at the P1 site within the reactive site loop, and examined their respective selectivity. The novel α1-antitrypsin variant AVNR (AVPM352/AVNR) was identified as a highly selective inhibitor of furin. This variant formed a sodium dodecyl sulfate- and heat-stable furin/α1-antitrypsin complex and inhibited furin activity ex vivo and in vitro. Other SPC members including PACE4, PC5/6 and PC7 were not inhibited by the AVNR variant. Furin-mediated maturation of bone morphogenetic protein-4 was completely inhibited by ectopic expression of the AVNR variant. The AVNR variant should prove to be a useful inhibitor in identifying the specific role of furin.
(Keyword)
Amino Acid Motifs / Amino Acid Substitution / Animals / Antithrombin III / Bone Morphogenetic Protein 4 / COS Cells / Catalytic Domain / Cercopithecus aethiops / Furin / HEK293 Cells / Humans / Mutagenesis, Site-Directed / Proprotein Convertase 5 / Proprotein Convertases / Protein Binding / Protein Precursors / Protein Processing, Post-Translational / Protein Stability / Proteolysis / Rats / Serine Endopeptidases / Substrate Specificity / alpha 1-Antitrypsin
Akihiko Tsuji, Kana Tsukamoto, Keiko Iwamoto, Yuka Ito and Keizo Yuasa : Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination., The Journal of Biochemistry, 153, 1, 73-83, 2013.
(Summary)
Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.
Akihiko Tsuji, Shiori Sato, Ayumi Kondo, Keiko Tominaga and Keizo Yuasa : Purification and characterization of cellulase from North Pacific krill (Euphausia pacifica). Analysis of cleavage specificity of the enzyme., Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 163, 3-4, 324-333, 2012.
(Summary)
Krill are filter feeders that consume algae, plankton and detritus, indicating that krill possess an adequate cellulose digesting system. However, less is known about the enzymatic properties of crustacean cellulases compared to termite cellulases. In the present study, 48 kDa-cellulase was highly purified from krill (Euphausia pacifica) in an effort to determine the cleavage specificity of the enzyme. The most notable characteristic of the enzyme was its high activity against both lichenan and carboxymethyl cellulose. The enzyme hydrolyzed internal β-1,4 glycosidic bonds within lichenan as well as carboxymethyl cellulose to release oligosaccharides and glucose. The effects of pH and temperature on the activity and stability of both enzyme activities were almost identical. Cello-oligosaccharides with a degree of polymerization of 4-6 were hydrolyzed by the enzyme, and the same endo-products, cellotriose, cellobiose and glucose, were produced from these oligosaccharides. Neither cellotriose nor cellobiose was hydrolyzed by the enzyme. The enzyme digested filter paper and sea lettuce to produce cellobiose, cellotriose and glucose as major products. Although amino acid sequence homology of the enzyme with termite cellulases and the presence of oligosaccharides in the enzyme suggested that the enzyme is produced by krill itself, further analysis is necessary.
Keizo Yuasa, Go Futamatsu, Tsuyoshi Kawano, Masaki Muroshita, Yoko Kageyama, Hiromi Taichi, Hiroshi Ishikawa, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : Subtilisin-like proprotein convertase paired basic amino acid-cleaving enzyme 4 is required for chondrogenic differentiation in ATDC5 cells, The FEBS Journal, 279, 21, 3997-4009, 2012.
(Summary)
Bone morphogenetic proteins (BMPs) have been implicated in the regulation of multiple stages of endochondral bone development. BMPs are synthesized as inactive precursors, and activated by removal of the propeptide. The subtilisin-like proprotein convertase (SPC) family comprises seven members [furin/SPC1, PC2/SPC2, PC1/PC3/SPC3, paired basic amino acid-cleaving enzyme 4 (PACE4)/SPC4, PC4/SPC5, PC6/PC5/SPC6, and PC8/PC7/LPC/SPC7], and activates various signaling molecules, including BMPs. In this study, we analyzed the role of this family in chondrogenic differentiation by using the mouse embryonal carcinoma-derived clonal cell line ATDC5. Both SPC-specific inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone and α1-antitrypsin Portland variant, suppressed chondrogenic differentiation. RT-PCR analysis revealed that PACE4 mRNA levels increased markedly during chondrogenic differentiation, whereas furin expression remained unchanged. Knockdown of PACE4 expression significantly reduced chondrogenic differentiation. Furthermore, proBMP6, which shows an expression pattern similar to that of PACE4, was efficiently processed into its mature form by PACE4, whereas furin could not process proBMP6. These results suggest that PACE4 may regulate the rate of hypertrophic conversion of ATDC5 cells through activation of proBMP6.
Kimihiko Mizutani, Sae Tsuchiya, Mayuko Toyoda, Yuko Nanbu, Keiko Tominaga, Keizo Yuasa, Nobuyuki Takahashi, Akihiko Tsuji and Bunzo Mikamia : Structure of β-1,4-mannanase from the common sea hare Aplysia kurodai at 1.05 Å resolution., Acta Crystallographica. Section F, Structural Biology and Crystallization Communications, 68, 10, 1164-1168, 2012.
(Summary)
β-1,4-Mannanase (EC 3.2.1.78) catalyzes the hydrolysis of β-1,4-glycosidic bonds within mannan, a major constituent group of the hemicelluloses. Bivalves and gastropods possess β-1,4-mannanase and may degrade mannan in seaweed and/or phytoplankton to obtain carbon and energy using the secreted enzymes in their digestive systems. In the present study, the crystal structure of AkMan, a gastropod β-1,4-mannanase prepared from the common sea hare Aplysia kurodai, was determined at 1.05 Å resolution. This is the first report of the three-dimensional structure of a gastropod β-1,4-mannanase. The structure was compared with bivalve β-1,4-mannanase and the roles of residues in the catalytic cleft were investigated. No obvious binding residue was found in subsite +1 and the substrate-binding site was exposed to the molecular surface, which may account for the enzymatic properties of mannanases that can digest complex substrates such as glucomannan and branched mannan.
Kinuka Isshiki, Shinya Matsuda, Akihiko Tsuji and Keizo Yuasa : cGMP-dependent protein kinase I promotes cell apoptosis through hyperactivation of death-associated protein kinase 2., Biochemical and Biophysical Research Communications, 422, 2, 280-284, 2012.
(Summary)
cGMP-dependent protein kinase-I (cGK-I) induces apoptosis in various cancer cell lines. However, the signaling mechanisms involved remain unknown. Using protein microarray technology, we identified a novel cGK substrate, death-associated protein kinase 2 (DAPK2), which is a Ca(2+)/calmodulin-regulated serine/threonine kinase. cGK-I phosphorylated DAPK2 at Ser(299), Ser(367) and Ser(368). Interestingly, a phospho-mimic mutant, DAPK2 S299D, significantly enhanced its kinase activity in the absence of Ca(2+)/calmodulin, while a S367D/S368D mutant did not. Overexpression of DAPK2 S299D also resulted in a twofold increase in apoptosis of human breast cancer MCF-7 cells as compared with wild-type DAPK2. These results suggest that DAPK2 is one of the targets of cGK-I in apoptosis induction.
Keizo Yuasa, Takeshi Nagame, Makoto Dohi, Yayoi Yanagita, Shin Yamagami, Masami Nagahama and Akihiko Tsuji : cGMP-dependent protein kinase I is involved in neurite outgrowth via a Rho effector, rhotekin, in Neuro2A neuroblastoma cells., Biochemical and Biophysical Research Communications, 421, 2, 239-244, 2012.
(Summary)
Although the cGMP/cGMP-dependent protein kinase (cGK) signaling is involved in the regulation of neurite outgrowth, its mechanism remains to be clarified. In this study, we identified a Rho effector, rhotekin, as a cGK-I-interacting protein. Rhotekin was also a substrate for cGK-Iα. In neurite-extended Neuro2A neuroblastoma cells, cGK-Iα and rhotekin were colocalized in the plasma membrane and extended neurites, while treatment with cGMP resulted in translocation of rhotekin to the cytoplasm. In addition, we found that cGK-Iα and rhotekin synergistically suppressed Rho-induced neurite retraction. Our findings suggest that cGK-Iα interacts with and phosphorylates rhotekin, thereby contributing to neurite outgrowth regulation.
Keizo Yuasa, Kaori Tada, Genki Harita, Tomomi Fujimoto, Masao Tsukayama and Akihiko Tsuji : Sudachitin, a polymethoxyflavone from Citrus sudachi, suppresses lipopolysaccharide-induced inflammatory responses in mouse macrophage-like RAW264 cells., Bioscience, Biotechnology, and Biochemistry, 76, 3, 598-600, 2012.
(Summary)
Although some polymethoxyflavones possess several important biological properties, including neuroprotective, anticancer, and anti-inflammatory ones, sudachitin, a polymethoxyflavone from <I>Citrus sudachi</I>, has been little studied. In this study, we found that sudachitin inhibited nitric oxide production by suppressing the expression of inducible nitric oxide synthase in lipopolysaccharide-stimulated macrophages, indicating that sudachitin has an anti-inflammatory effect.
Akihiko Tsuji, Yoshinori Fujisawa, Takeru Mino and Keizo Yuasa : Identification of a plant aminopeptidase with preference for aromatic amino acid residues as a novel member of the prolyl oligopeptidase family of serine proteases., The Journal of Biochemistry, 150, 5, 525-534, 2011.
(Summary)
Genome analysis has indicated that plants, like animals, possess a variety of protease genes. However, bulk of putative proteases has not been characterized at the enzyme level. In this article, a novel enzyme that hydrolyses phenylalanyl-4-methylcoumaryl 7-amide (phenylalanyl-MCA) was purified from cotyledons of daikon radish by ammonium sulphate fractionation and successive chromatography with DEAE-cellulose, phenyl-Sepharose, Sephacryl S-200 and Mini-Q. The molecular mass of the enzyme was estimated to be 78 kDa by SDS-PAGE under reducing conditions and 74 kDa by gel filtration, indicating that the enzyme is a monomer. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that the enzyme is an orthologue of Arabidopsis unidentified protein, acylpeptide hydrolase-like protein (AHLP; UniProt ID: Q9FG66) belonging to the prolyl oligopeptidase (POP) family of a serine-type peptidase predicted from genetic information. Good substrates identified for the enzyme include phenylalanyl-MCA, tyrosyl-MCA and enkephalin. Neither acylamino acid-releasing activity nor endopeptidase activity was detected. The enzyme cleaved enkephalin (YGGFM, YGGFL), whereas, BAM-12 P (YGGFMRRVGRPE) and dynorphin A (YGGFLRRIRPKLK) were not digested. These results suggested that the enzyme possesses strict size selectivity of substrate. We propose the name 'tyrosyl aminopeptidase' for the uncharacterized protein AHLP.
Keizo Yuasa, Taito Matsuda and Akihiko Tsuji : Functional regulation of transient receptor potential canonical 7 by cGMP-dependent protein kinase Iα., Cellular Signalling, 23, 7, 1179-1187, 2011.
(Summary)
The cGMP/cGMP-dependent protein kinase (cGK) signaling pathway is implicated in the functional regulation of intracellular calcium levels. In the present study, we investigated the regulation of transient receptor potential canonical 7 (TRPC7) by the cGMP/cGK-I pathway. TRPC7 contains three putative cGK phosphorylation sites (Arg-Arg/Lys-Xaa-Ser/Thr). However, the role of cGK-I in the regulation of TRPC7 activity remains unclear. In vitro and in vivo kinase assays have revealed that cGK-Iα phosphorylates mouse TRPC7 but not mouse TRPC3. Site-directed mutagenesis analysis revealed that TRPC7 was phosphorylated by cGK-Iα at threonine 15. Phosphorylation of TRPC7 significantly suppressed carbachol-induced calcium influx and CREB phosphorylation. Furthermore, co-immunoprecipitation assay demonstrated that cGK-Iα interacted with the ankyrin repeat domain in the N terminus of TRPC7. cGK-Iβ also bound to TRPC7, while the type II regulatory subunit of cAMP-dependent protein kinase did not bind. These data indicate that cGK-Iα interacts with and phosphorylates TRPC7, contributing to the quick and accurate regulation of calcium influx and CREB phosphorylation.
Ismail Ismaliza Mohd Nor, Tsuyoshi Yuasa, Keizo Yuasa, Yuko Nambu, Makoto Nishimoto, Masaki GOTO, Hitoshi Matsuki, Masahiro Inoue, Masami Nagahama and Akihiko Tsuji : A critical role for highly conserved Glu610 residue of oligopeptidase B Tryoanosoma brucei in thermal stability, The Journal of Biochemistry, 147, 2, 201-211, 2010.
(Summary)
Oligopeptidase B from Trypanosoma brucei (Tb OPB) is a virulence factor and therapeutic target in African sleeping sickness. Three glutamic acid residues at positions 607, 609 and 610 of the catalytic domain are highly conserved in the OPB subfamily. In this study, the roles of Glu(607), Glu(609) and Glu(610) in Tb OPB were investigated by site-directed mutagenesis. A striking effect on k(cat)/K(m) was obtained following mutation of Glu(607) to glutamine. In contrast, the heat stability of Tb OPB decreased markedly following the single mutation of Glu(610) to glutamine, although this mutation had significantly less effect on catalytic properties compared with the Glu(607) mutation. Although no differences were found in the tertiary and secondary structures between wild-type (WT) OPB and the E610Q mutant prior to heat treatment, the E610Q mutant is inactivated more rapidly than WT OPB following heat treatment in a manner correlating with its attendant structural changes. Trypsin digestion showed that the boundary regions between the beta-propeller and catalytic domain of the E610Q mutant are unfolded with heat treatment. It is concluded that Glu(607) is essential for the catalytic activity of Tb OPB and that Glu(610) plays a critical role in stabilization rather than catalytic activity despite their close proximity.
Keizo Yuasa, Shotaro Uehara, Masami Nagahama and Akihiko Tsuji : Transcriptional regulation of cGMP-dependent protein kinase II (cGK-II) in chondrocytes., Bioscience, Biotechnology, and Biochemistry, 74, 1, 44-49, 2010.
(Summary)
The C-type natriuretic peptide/natriuretic peptide receptor-B/cGMP pathway plays an important role in the regulation of endochondral ossification. In chondrocytes, the physiological effect of cGMP is mediated primarily by the activation of cGMP-dependent protein kinase II (cGK-II). In this study, we investigated the transcriptional regulation of cGK-II in chondrocytes. The expression pattern of cGK-II transcripts was examined during chondrogenic differentiation of ATDC5 cells. cGK-II mRNA was not detectable in undifferentiated cells, but increased dramatically prior to differentiation to the hypertrophic stage. To analyze the transcriptional regulation of cGK-II, the 5'-flanking region of the mouse cGK-II gene was isolated and characterized. The promoter activity of the cGK-II gene decreased markedly following deletion and mutagenesis of the putative Nkx-binding site between nucleotide positions -292 and -286. These results suggest that the homeobox gene Nkx family is critical for the transcriptional regulation of cGK-II during chondrogenesis.
Tomoko Takahashi, Takanori Ida, Takahiro Sato, Yoshiki Nakashima, Yuki Nakamura, Akihiko Tsuji and Masayasu Kojima : Production of n-octanoyl-modified Ghrelin in Cultured Cells Requires Prohormone Proceeding Protease and Ghrelin O-acyltransferase, as well as n-octanoic Acid, The Journal of Biochemistry, 146, 5, 675-682, 2009.
(Summary)
Ghrelin was originally isolated from rat stomach as an endogenous ligand for the GH secretagogue receptor. The major active form of ghrelin is a 28-amino acid peptide modified by an n-octanoic acid on the serine 3 residue, and this lipid modification is essential for the biological activity of ghrelin. However, it is not clear whether prohormone convertase (PC) and ghrelin O-acyltransferase (GOAT) are the minimal requirements for synthesis of acyl-modified ghrelin in cultured cells. By using three cultured cell lines, TT, AtT20 and COS-7, in which the expression levels of processing proteases and GOAT vary, we examined the processing patterns of ghrelin precursor. We found that not only PC1/3 but also both PC2 and furin could process proghrelin to the 28-amino acid ghrelin. Moreover, the presence of PC and GOAT in the cells, as well as n-octanoic acid in the culture medium, was necessary to produce n-octanoyl ghrelin.
Keizo Yuasa, Tetsuya Masuda, Chihiro Yoshikawa, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : Subtilisin-like Proprotein Convertase PACE4 is Required for Skeletal Muscle Differentiation, The Journal of Biochemistry, 146, 3, 407-415, 2009.
(Summary)
Most growth factors stimulate myoblast proliferation and prevent differentiation, whereas insulin-like growth factors (IGFs) promote myoblast differentiation through the phosphatidylinositol 3-kinase (PI3K) pathway. Subtilisin-like proprotein convertases (SPCs) are involved in cell growth and differentiation via activation of pro-growth factors. However, the role of SPCs in myogenesis remains poorly understood. Here we show that PACE4, a member of the SPC family, plays a critical role in myogenic differentiation of C2C12 cells. PACE4 mRNA levels increased markedly during myogenesis, whereas the expression of other member of SPC family, furin and PC6, remained unchanged. The expression pattern of pro-IGF-II, which is processed extracellularly by SPCs, was similar to that of PACE4. The expression of shRNA targeting PACE4, but not furin, suppressed the expression of the muscle-specific myosin light chain (MLC). Interestingly, reduced expression of MLC was restored following treatment with recombinant mature IGF-II. Finally, we demonstrated that the PI3K inhibitor LY294002 blocked the induction of PACE4 mRNA, a result not observed when another myogenic differentiation inhibitor, SB203580 (p38 MAP kinase inhibitor), was employed, indicating the presence of a positive feedback loop regulating PACE4 expression. These results suggest that PACE4 plays an important role in myogenic differentiation through its association with the IGF-II pathway.
(Keyword)
Animals / Cell Differentiation / Cell Line / Feedback, Physiological / Gene Knockdown Techniques / Insulin-Like Growth Factor II / Matrix Metalloproteinase 11 / Mice / Muscle Development / MyoD Protein / Myoblasts, Skeletal / Myogenin / Myosin Light Chains / Phosphatidylinositol 3-Kinases / Proprotein Convertases / Protein Precursors / Signal Transduction / Up-Regulation / p38 Mitogen-Activated Protein Kinases
Masami Nagahama, Machi Ohnishi, Yumiko Kawate, Takayuki Matsui, Hitomi Miyake, Keizo Yuasa, Katsuko Tani, Mitsuo Tagaya and Akihiko Tsuji : UBXD1 is a VCP-interacting protein that is involved in ER-associated degradation, Biochemical and Biophysical Research Communications, 382, 2, 303-308, 2009.
(Summary)
AAA ATPase VCP and its yeast ortholog Cdc48, in a complex with the Ufd1-Npl4 heterodimer as an adaptor, play an essential role in endoplasmic reticulum-associated degradation (ERAD). Several UBX domain-containing proteins function to recruit ubiquitylated substrates to VCP/Cdc48 by binding both VCP/Cdc48 and other ERAD components such as ubiquitin ligases. Here we show that mammalian UBXD1 is an additional UBX domain-containing protein involved in the ERAD process. UBXD1 is a cytosolic protein that interacts with VCP and Derlin-1. Overexpression of UBXD1 in cells causes selective dissociation of Ufd1 from VCP, resulting in inhibition of mutant cystic fibrosis transmembrane conductance regulator (CFTR) degradation by ERAD. Additionally, depletion of endogenous UBXD1 protein by RNA interference also results in a defect in CFTR degradation. Collectively, these findings suggest that UBXD1 is a regulatory component of ERAD that may modulate the adaptor binding to VCP.
Takuya Yanagino, Keizo Yuasa, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : Transcriptional Regulation of Fibrillin-2 Gene by E2F Family Members in Chondrocyte Differentiation, Journal of Cellular Biochemistry, 106, 4, 580-588, 2009.
(Summary)
Mutation in fibrillin-2, a major structural component of extracellular microfibrils in connective tissue, results in the autosomal dominant disease congenital contractural arachnodactyly. This genetic disease is characterized by dolichostenomelia and arachnodactyly, in addition to contractures of the large joints and abnormal pinnae formation, thus indicating the significance of fibrillin-2 in chondrogenesis. In this study, we investigated the transcriptional regulation of fibrillin-2 in chondrogenic differentiation. Although mRNA expression of fibrillin-1, a highly homologous protein to fibrillin-2, remained almost unchanged during chondrogenesis of mouse ATDC5 cells, fibrillin-2 mRNA expression varied. Fibrillin-2 was highly expressed at the early stage and declined progressively during differentiation. The 5'-flanking region of the fibrillin-2 gene contains potential binding sites for E2F, Runx, AP-2, and Sox transcription factors. The promoter activity of fibrillin-2 decreased markedly following deletion and mutagenesis of the E2F binding site between -143 and -136 bp. Overexpression of E2F1 resulted in a marked increase in its promoter activity, whereas expression of other transcription factors including AP-2alpha and Runx2 had no effect. The increase in promoter activity by E2F1 was completely suppressed by the coexpression of E2F4. E2F2 and E2F3 had positive effects on the promoter activity. Although ATDC5 cells expressed transcripts for the E2F family genes at all stages of differentiation, the expression profiles differed. E2F1 expression remained almost unchanged, whereas E2F4 expression increased markedly at the late stage of differentiation. These results indicated that coordinated expression of the E2F family is critical for the transcriptional regulation of fibrillin-2 during chondrogenesis.
Akihiko Tsuji, Yayoi Kikuchi, Kentaro Ogawa, Hiroko Saika, Keizo Yuasa and Masami Nagahama : Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativus, The FEBS Journal, 275, 21, 5429-5443, 2008.
(Summary)
Plant cathepsin B-like cysteine protease (CBCP) plays a role in disease resistance and in protein remobilization during germination. The ability of animal cathepsin B to function as a dipeptidyl carboxypeptidase has been attributed to the presence of a dihistidine (His110-His111) motif in the occluding loop, which represents a unique structure of cathepsin B. However, a dihistidine motif is not present in the predicted sequence of the occluding loop of plant CBCP, as determined from cDNA sequence analysis, and the loop is shorter. In an effort to investigate the enzymatic properties of plant CBCP, which possesses the unusual occluding loop, we have purified CBCP from the cotyledons of daikon radish (Raphanus sativus) by chromatography through Sephacryl S-200, DEAE-cellulose, hydroxyapatite and organomercurial-Sepharose. The molecular mass of the enzyme was estimated to be 28 kDa by SDS/PAGE under reducing conditions. The best synthetic substrate for CBCP was t-butyloxycarbonyl Leu-Arg-Arg-4-methylcoumaryl 7-amide, as is the case with human cathepsin B. However, the endopeptidase activity of CBCP towards glucagon and adrenocorticotropic hormone showed broad cleavage specificity. Human cathepsin B preferentially cleaves model peptides via its dipeptidyl carboxypeptidase activity, whereas daikon CBCP displays both endopeptidase and exopeptidase activities. In addition, CBCP was found to display carboxymonopeptidase activity against the substrate o-aminobenzoyl-Phe-Arg-Phe(4-NO(2)). Daikon CBCP is less sensitive (1/7000) to CA-074 than human cathepsin B. Expression analysis of CBCP at the protein and RNA levels indicated that daikon CBCP activity in cotyledons is regulated by post-transcriptional events during germination.
Yayoi Kikuchi, Hiroko Saika, Keizo Yuasa, Masami Nagahama and Akihiko Tsuji : Isolation and Biochemical Characterization of Two Forms of RD21 from Cotyledons of Daikon Radish (Raphanus sativus)., The Journal of Biochemistry, 144, 6, 789-798, 2008.
(Summary)
RD21 (Responsive to desiccation-21) is an Arabidopsis cysteine protease which possesses a granulin-like domain at the C-terminus. Although two forms of RD21 have been identified, consisting of an intermediate form (iRD21) containing a granulin domain and a mature form (mRD21) lacking this domain, the enzymatic properties of these enzymes remain poorly understood. In this study, mRD21 orthologue was purified to homogeneity from the cotyledons of daikon radish (Raphanus sativus). RD21 preferentially cleaved peptide bond that had an aromatic or hydrophobic amino acid at the P2 position. Furthermore, the presence of a polar amino acid at the P1 position enhanced the cleavage susceptibility of the peptide bond, although the importance of the type of amino acid residue at the P1 and P1' positions was not as significant as the residue located at the P2 position. The iRD21 was also identified as an oligomeric form by gel filtration and sedimentation analyses. The expression of RD21 mRNA was initiated by imbibition and continued at almost constant levels during germination. On the other hand, the enzyme activity increased markedly 5 days after imbibition. These results indicate that this elevation of RD21 activity is generated post-transcriptionally.
(Keyword)
Amino Acid Sequence / Angiotensin II / Base Sequence / Chromatography, Gel / Cotyledon / Cysteine Endopeptidases / Dynorphins / Hydrolysis / Molecular Sequence Data / Plant Proteins / Protein Isoforms / Raphanus / Substrate Specificity
Keizo Yuasa, Shin Yamagami, Masami Nagahama and Akihiko Tsuji : Trafficking of cGMP-dependent protein kinase II via interaction with Rab11, Biochemical and Biophysical Research Communications, 374, 3, 522-526, 2008.
(Summary)
cGMP-dependent protein kinase II (cGK-II) is implicated in several physiological functions including intestinal secretion, bone growth, and learning and memory, but the detailed mechanisms are still unclear. To identify proteins that are involved in cGMP/cGK-II signaling, we performed yeast two-hybrid screening and identified Rab11b as a cGK-II-interacting protein that regulates the slow-recycling pathway. Interestingly, cGK-II interacted with the GDP-bound form of Rab11b (Rab11b S25N), but not the GTP-bound form, in mammalian cells. Immunofluorescence staining revealed that Rab11b S25N promoted the translocation of cGK-II from the plasma membrane to the cytoplasm and that the localization of cGK-II extensively overlapped with Rab11b. Furthermore, treatment with a membrane-permeable cGMP analog caused the rapid retranslocation of cGK-II and Rab11b S25N to the membrane. These data indicate that Rab11b is necessary for the trafficking of cGK-II and that the cGMP/cGK-II signaling pathway is closely related to Rab11b recycling pathway.
(Keyword)
Animals / Cell Line / Cell Membrane / Cyclic GMP-Dependent Protein Kinases / Guanine Nucleotides / Humans / Mice / Protein Transport / Signal Transduction / Two-Hybrid System Techniques / rab GTP-Binding Proteins
Youichi Tajima, Fumiko Matsuzawa, Sei-ichi Aikawa, Toshika Okumiya, Michiru Yoshimizu, Takahiro Tsukimura, Masahiko Ikekita, Seiichi Tsujino, Akihiko Tsuji, Tim Edmunds and Hitoshi Sakuraba : Structural and biochemical studies on Pompe disease and a "pseudodeficiency of acid α-glucosidase", Journal of Human Genetics, 52, 11, 898-906, 2007.
(Summary)
We constructed structural models of the catalytic domain and the surrounding region of human wild-type acid alpha-glucosidase and the enzyme with amino acid substitutions by means of homology modeling, and examined whether the amino acid replacements caused structural and biochemical changes in the enzyme proteins. Missense mutations including p.R600C, p.S619R and p.R437C are predicted to cause apparent structural changes. Nonsense mutation of p.C103X terminates the translation of acid alpha-glucosidase halfway through its biosynthesis and is deduced not to allow formation of the active site pocket. The mutant proteins resulting from these missense and nonsense mutations found in patients with Pompe disease are predictably unstable and degraded quickly in cells. The structural change caused by p.G576S is predicted to be small, and cells from a subject homozygous for this amino acid substitution exhibited 15 and 11% of the normal enzyme activity levels for an artificial substrate and glycogen, respectively, and corresponding amounts of the enzyme protein on Western blotting. No accumulation of glycogen was found in organs including skeletal muscle in the subject, and thus the residual enzyme activity could protect cells from glycogen storage. On the other hand, p.E689K, which is known as a neutral polymorphism, little affected the three-dimensional structure of acid alpha-glucosidase. Structural study on a mutant acid alpha-glucosidase in silico combined with biochemical investigation is useful for understanding the molecular pathology of Pompe disease.
(Keyword)
Amino Acid Sequence / Blotting, Western / Cells, Cultured / Glycogen Storage Disease Type II / Humans / Molecular Sequence Data / Sequence Homology, Amino Acid / alpha-Glucosidases
Keizo Yuasa, Kaori Suzue, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : Transcriptional regulation of subtilisin-like proprotein convertase PACE4 by E2F: Possible role of E2F-mediated upregulation of PACE4 in tumor progression, Gene, 402, 1-2, 103-110, 2007.
(Summary)
PACE4, a member of the subtilisin-like proprotein convertase (SPC) family, is expressed at high levels in certain tumor cells and plays a role in metastatic progression through activation of matrix metalloproteinases. The mechanism leading to overexpression of PACE4 in tumor cells remains unclear. In this study, we show that the E2F1 transcription factor, which is implicated in carcinoma invasiveness, upregulates the expression of PACE4. HT1080 (highly tumorigenic and invasive) cells expressed much higher levels of PACE4 and E2F family (E2F1 and E2F2) transcripts than IMR90 (normal fibroblast) cells. Expression levels of other SPCs (furin and PC6) remained unchanged in these cells. Promoter analysis indicated that two E2F consensus binding sites (-117/-110 and -86/-79) in the 5'-flanking region of the human PACE4 gene function as positive regulatory elements. Mutation of these sites abolished PACE4 promoter response to E2F1 as well as binding of E2F1 in electrophoretic mobility-shift assays. Other E2F members, E2F2 and E2F3, also activated PACE4 expression, as in the case of E2F1. These results indicate a novel mechanism for E2F family-mediated promotion of carcinoma invasiveness through PACE4.
Akihiko Tsuji, Hiroki Kanie, Hirotaka Makise, Keizo Yuasa, Masami Nagahama and Yoshiko Matsuda : Engineering of α1-antitrypsin cariants selective for subtilisin-like proprotein convertases PACE4 and PC6: Importance of the P2' residue in stable complex formation of the serpin with proprotein convertase, Protein Engineering, Design & Selection, 20, 4, 163-170, 2007.
(Summary)
Furin and PACE4, members of the subtilisin-like proprotein convertase (SPC) family, have been implicated in the metastatic progression of certain tumors in addition to the activation of viral coat proteins and bacterial toxins, indicating that these enzymes are potential targets for therapeutic agents. Alpha1-Antitrypsin Portland is an engineered alpha1-antitrypsin designed as a furin-specific inhibitor and has been used as a tool in the functional analysis of furin. In this work, we engineered rat alpha1-antitrypsin to create a PACE4-specific inhibitor. Substituting Arg-Arg-Arg-Arg for Ala-Val-Pro-Met(352) at P4-P1 and Ala for Leu(354) at P2' created a potent PACE4- and PC6-specific inhibitor. This variant (RRRRSA) formed an SDS- and heat-stable serpin/proteinase complex with PACE4 or PC6 and inhibited both enzyme activities. The RRRRSA variant was efficiently cleaved by furin without formation of the stable complex. This is the first report of a highly selective protein-based inhibitor of PACE4 and PC6. This inhibitor will be useful in delineating the roles of PACE4 and PC6 localized in the extracellular matrix.
Masami Nagahama, Takeshi Yamazoe, Yoshimitsu Hara, Katsuko Tani, Akihiko Tsuji and Mitsuo Tagaya : The AAA-ATPase NVL2 is a component of pre-ribosomal particles that interacts with the DexD/H-box RNA helicase DOB1, Biochemical and Biophysical Research Communications, 346, 3, 1075-1082, 2006.
(Summary)
Nuclear VCP/p97-like protein 2 (NVL2) is a member of the chaperone-like AAA-ATPase family with two conserved ATP-binding modules. Our previous studies have shown that NVL2 is localized to the nucleolus by interacting with ribosomal protein L5 and may participate in ribosome synthesis, a process involving various non-ribosomal factors including chaperones and RNA helicases. Here, we show that NVL2 is associated with pre-ribosomal particles in the nucleus. Moreover, we used yeast two-hybrid and co-immunoprecipitation assays to identify an NVL2-interacting protein that could yield insights into NVL2 function in ribosome biogenesis. We found that NVL2 interacts with DOB1, a DExD/H-box RNA helicase, whose yeast homologue functions in a late stage of the 60S subunit synthesis. DOB1 can interact with a second ATP-binding module mutant of NVL2, which shows a dominant negative effect on ribosome synthesis. In contrast, it cannot interact with a first ATP-binding module mutant, which does not show the dominant negative effect. When the dominant negative mutant of NVL2 was overexpressed in cells, DOB1 appeared to remain associated with nuclear pre-ribosomal particles. Such accumulation was not observed upon overexpression of wild-type NVL2 or a nondominant-negative mutant. Taken together, our results suggest that NVL2 might regulate the association/dissociation reaction of DOB1 with pre-ribosomal particles by acting as a molecular chaperone.
(Keyword)
Adenosine Triphosphatases / Cell Line / Cell Nucleus / Humans / Mutation / Protein Binding / RNA Helicases / Ribosomes
Akihiko Tsuji, Yayoi Kikuchi, Yukimi Sato, Shizuyo Koide, Keizo Yuasa, Masami Nagahama and Yoshiko Matsuda : A proteomic approach reveals transient association of reticulocalbin-3, a novel member of the CREC family, with the precursor of subtilisin-like proprotein convertase, PACE4, The Biochemical Journal, 396, 1, 51-59, 2006.
(Summary)
SPCs (subtilisin-like proprotein convertases) are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins. In an effort to examine the substrate protein for PACE4 (paired basic amino-acid-cleaving enzyme-4), an SPC, a potent protein inhibitor of PACE4, an alpha1-antitrypsin RVRR (Arg-Val-Arg-Arg) variant, was expressed in GH4C1 cells. Ectopic expression of the RVRR variant caused accumulation of the 48 kDa protein in cells. Sequence analysis indicates that the 48 kDa protein is a putative Ca2+-binding protein, RCN-3 (reticulocalbin-3), which had previously been predicted by bioinformatic analysis of cDNA from the human hypothalamus. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand Ca2+-binding proteins localized to the secretory pathway. The most interesting feature of the RCN-3 sequence is the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of SPCs. Biosynthetic studies showed that RCN-3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4-RCN-3 complex in cells. Furthermore, autoactivation and secretion of PACE4 was increased upon co-expression with RCN-3. Our findings suggest that selective and transient association of RCN-3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4.
Akihiko Tsuji, Tadashi Yoshimoto, Keizo Yuasa and Yoshiko Matsuda : Pratamine: a unique and patent inhibitor of oligopeptidase B, Journal of Peptide Science, 12, 1, 65-71, 2006.
(Summary)
Oligopeptidase B is a serine endopeptidase found in prokaryotes, unicellular eukaryotes and higher plants. The enzyme has been shown recently to play a central role in the pathogenesis of several parasitic diseases such as African trypanosomiasis, and to be a potential therapeutic target. This study reports that protamine, a basic peptide rich in arginine, is a potent inhibitor at the nanomolar level of oligopeptidase B from E. coli and wheat. Protamines 1B, 2C, 3A and TP17 displayed similar inhibitory activities and were capable of binding strongly to oligopeptidase B without proteolytic cleavage. The concentration of protamine needed for 50% inhibition (IC50) of oligopeptidase B was 10(4)-fold lower than the IC50 of trypsin. Oligopeptidase B was highly sensitive to inhibition by protamines even in the presence of serum (IC50, 1 microM). These data indicate that protamines might provide information useful for the design of more specific synthetic oligopeptidase B inhibitors.
Akihiko Tsuji, Keizo Yuasa and Yoshiko Matsuda : Identification of Oligopeptidase B in Higher Plants. Purification and Characterization of Oligopeptidase B from Quiescent Wheat Embryo, Triticum aestivum, The Journal of Biochemistry, 136, 5, 673-681, 2004.
Shizuyo Koide, Ichiro Yoshida, Akihiko Tsuji and Yoshiko Matsuda : The expression of proprotein convertase PACE4 is highly regulated by Hash-2 in placenta: Possible role of placenta-specific basic helix-loop-helix transcription factor, human Achaete-Scute homologue-2, The Journal of Biochemistry, 134, 3, 433-440, 2003.
Akihiko Tsuji, Kensuke Sakurai, Emi Kiyokage, Takahito Yamazaki, Shizuyo Koide, Kazunori Toida, Kazunori Ishimura and Yoshiko Matsuda : Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix. Potential role of PACE4 in the activation of proproteins in the extracellular matrix, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1645, 1, 95-104, 2003.
Miwa Bando, Atsushi Matsuoka, Akihiko Tsuji and Yoshiko Matsuda : The proprotein convertase PACE4 is upregulated by PDGF-BB in megakaryocytes: gene expression of PACE4 and furin is regulated differently in Dami cells, The Journal of Biochemistry, 132, 1, 127-134, 2002.
(Summary)
The differentiation of megakaryocytes into platelets is highly regulated by many cytokines and growth factors. PACE4 and furin are Ca(2+)-dependent serine endoproteases belonging to the subtilisin-like proprotein convertase (SPC) family. These enzymes are involved in the proteolytic activation of proteins that play essential roles in cell growth and differentiation. In this study, we examined the expression of PACE4 and furin during the differentiation of megakaryoblastic cell lines, Dami and HEL cells, induced by phorbol 12-myristate 13-acetate (PMA). PMA stimulates not only the expression of platelet-derived growth factor-B (PDGF-B) mRNA, but also PACE4 mRNA in these cell lines. The expression of PACE4 transcripts (both the PACE4A and PACE4C/CS isoforms) was upregulated more than 4-fold by PMA. Moreover, direct treatment with PDGF-BB also resulted in an increase in the level of PACE4 mRNA. Further, the effect of PDGF-BB on PACE4 expression was confirmed by promoter assay of the PACE4 gene. Although the furin mRNA level was increased by TGF-beta1 in Dami cells, it was not affected by PDGF-BB. These results indicate for the first time that PACE4 expression is specifically upregulated by PDGF-BB in differentiated megakaryoblasts, suggesting a unique role for PACE4 in platelet production.
K. Utsumi, Akihiko Tsuji, R. Kase, A. Tanaka, T. Tanaka, E. Uyama, T. Ozawa, H. Sakuraba, Y. Komaba, M. Kawabe, Y. Iino and Y. Katayama : Western blotting analysis of the beta-hexosaminidase alpha and beta-subunits in cultured fibroblasts frim cases of various forms of GM2 gangliosidosis, Acta Neurologica Scandinavica, 105, 6, 427-430, 2002.
Akihiko Tsuji, Takayuki Ikoma, Emi Hashimoto and Yoshiko Matsuda : Development of selectivity of α1-antitrypsin variant by mutagenesis in its reactive site loop against proprotein convertase. A crutial role of the P4 arginine in PACE4 inhibition, Protein Engineering, 15, 2, 123-130, 2002.
Takazumi Taniguchi, Rieko Kuroda, Kensuke Sakurai, Masami Nagahama, Ikuo Wada, Akihiko Tsuji and Yoshiko Matsuda : A Critical Role for the Carboxy Terminal Region of the Proprotein Convertase, PACE4A, in the Regulation of Its Autocatalytic Activation Coupled with Secretion, Biochemical and Biophysical Research Communications, 290, 2, 878-884, 2002.
Kenji Mori, Akiyoshi Imamaki, Kaya Nagata, Yasuo Yonetomi, Ryoko Kiyokage-Yoshimoto, John T Martin, Matthew T Gillespie, Masami Nagahama, Akihiko Tsuji and Yoshiko Matsuda : Subtilisin-Like Proprotein Convertase, PACE4 and PC8, as Well as Furin, are Endogenous Proalbumin Convertases in HepG2 Cells, The Journal of Biochemistry, 125, 3, 627-633, 1999.
(Summary)
Serum albumin is synthesized as a larger precursor form, proalbumin, which undergoes proteolytic processing at a dibasic site by a hepatic proprotein convertase within the secretory pathway to generate the mature form. Although furin, a member of the subtilisin-like proprotein convertase (SPC) family, was thought to be the only candidate hepatic convertase for proalbumin, SPC family members other than furin were recently suggested to also be involved in proalbumin processing. This study was designed to identify the endogenous proprotein convertases involved in proalbumin processing. Since human hepatoma HepG2 cells are highly differentiated and produce major plasma proteins, this cell line was used as a model for hepatocytes. Northern blot analysis revealed that PACE4, furin and PC8 of the SPC family were expressed in HepG2 cells as well as in the liver. Ribonuclease protection assay showed that PACE4A-II mRNA is the major transcript in HepG2 cells among the PACE4 isoforms. The coexpression studies showed that furin, PACE4A-II and PC8 were all able to convert proalbumin to albumin correctly. To elucidate the roles of these endogenous SPC family members in proalbumin processing, the antisense RNA for PACE4, furin and PC8 was stably expressed in HepG2 cells, respectively. The expression of each antisense RNA resulted in approximately 30% inhibition of endogenous proalbumin processing. We therefore concluded that PACE4 and PC8, as well as furin, are involved in the processing of proalbumin in HepG2 cells, and that these SPC family members are functionally redundant in this processing.
Akihiko Tsuji, Emi Hashimoto, Takayuki Ikoma, Takazumi Taniguchi, Kenji Mori, Masami Nagahama and Yoshiko Matsuda : Inactivation of Proprotein Convertase, PACE4 by α1-Antitrypsin Portland (α1-PDX) which is a Blocker of Proteolytic Activation of Bone Morphogenetic Protein during Embryogenesis., The Journal of Biochemistry, 126, 3, 591-603, 1999.
(Summary)
PACE4 (SPC4), a member of the subtilisin-like proprotein convertase (SPC) family of proteases that cleave at paired basic amino acids, exhibits a dynamic expression pattern during embryogenesis and colocalizes with bone morphogenetic proteins (BMPs). Recently Cui et al. reported that the ectopic expression of alpha1-antitrypsin variant Portland (alpha1-PDX), an engineered serpin that contains the minimal SPC consensus motif in its reactive loop, blocks the proteolytic activation of BMP4, leading to abnormal embryogenic development [Cui, Y. et al. (1998) EMBO J. 17, 4735-4743]. TGFbeta-related factors such as BMPs are synthesized as inactive precursors and activated by limited proteolysis at multibasic amino acids. Therefore, an alpha1-PDX-inhibitable protease is thought to participate in BMP activation. However, conflicting properties, including sensitivity to alpha1-PDX, have been reported for PACE4. In this study, we examined whether alpha1-PDX is responsible for the inhibition of PACE4 by measuring the protease/inhibitor complex directly. Here we show that alpha1-PDX has the ability to form an SDS-stable acyl-intermediate (180 kDa) with PACE4 in vivo and in vitro. Further, we characterized the PACE4 secreted into the culture medium from Cos-1 cells by a specific immunological assay. An alpha1-PDX-insensitive and decanoyl-RVKR-chloromethylketone-sensitive 60-kDa protease(s) is greatly activated in conditioned medium by PACE4 overexpression, suggesting that the activation of an unknown protease(s) other than PACE4 is the cause of the variation in the properties of PACE4. PACE4 is a Ca(2+)-dependent protease with an optimal Ca(2+) requirement of 2 mM, and shows its highest activity at weakly basic pH. PACE4 activity is completely inhibited by EDTA and EGTA, but not by leupeptin. These results show that PACE4 activity can be inhibited by alpha1-PDX as well as furin (SPC1) and suggest that the inhibition of PACE4-mediated activation of factors such as BMPs by alpha1-PDX causes abnormal embryogenic development.
(Keyword)
Animals / Antibodies, Monoclonal / Base Sequence / Bone Morphogenetic Protein 4 / Bone Morphogenetic Proteins / COS Cells / calcium / Chromatography, Affinity / Culture Media, Conditioned / DNA Primers / Embryonic and Fetal Development / Hydrolysis / Proprotein Convertases / Protease Inhibitors / Rats / Serine Endopeptidases / Sodium Dodecyl Sulfate / Substrate Specificity / alpha 1-Antitrypsin
Akihiko Tsuji, Shigeru Yoshida, Shinichi Hasegawa, Miwa Bando, Ichiro Yoshida, Shizuyo Koide, Kenji Mori and Yoshiko Matsuda : Human Subtilisin-Like Proprotein Convertase, PACE4 (SPC4) Gene Expression Is Highly Regulated through E-Box Elements in HepG2 and GH4C1 Cells, The Journal of Biochemistry, 126, 3, 494-502, 1999.
(Summary)
PACE4 (SPC4) is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which participates in maturation of precursor proteins. PACE4 is expressed at high levels in the anterior pituitary, central nervous system, the developing olfactory bulb, heart, and liver. Recently, we determined the gene structure of human PACE4. [Tsuji et al. (1997) J. Biochem. 122, 438-452]. The 5'-flanking region of PACE4 gene contains 12 E-boxes (E1 to E12) within 1 kb upstream of the transcription initiation site. To examine the function of these E-box elements in the regulation of PACE4 expression, deletion and mutation constructs of the 5'-flanking region were ligated to the luciferase gene and analyzed for promoter activity in HepG2 and GH4C1 cells, which express PACE4 at high level. Some differences were observed in the activity of each promoter construct between HepG2 and GH4C1 cells, although the overall profiles of activity for the promoter fragment series were similar regardless of cell type. We showed that the basal promoter activity of the PACE4 gene is first determined by sequences lying between -315 and -1 bp and further regulated by positive and negative elements in the upstream region. Site-directed mutagenesis of E-boxes in these regulatory elements showed that the E10 E-box act as positive regulator, whereas an E-box cluster (E4-E9) acts as a negative regulator in both cells. E2 E-box acts as a positive regulator only in HepG2 cells. Other E-boxes (E1, E3, and E12) had no effect on the promoter activity. These results indicate that E-box elements play a critical role in controlling PACE4 expression in HepG2 and GH4C1 cells and that PACE4 expression is regulated by a mechanism distinct from that of other SPC family proteases.
(Keyword)
Base Sequence / Cell Line / DNA Primers / Gene Expression Regulation, Enzymologic / Humans / Mutagenesis, Site-Directed / Promoter Regions, Genetic / Proprotein Convertases / Serine Endopeptidases
SPC4 (PACE4), a member of the eukaryotic family of subtilisin-like proprotein convertases, is synthesized as a proenzyme (proSPC4) which undergoes proteolytic removal of N-terminal propeptide during transit through the secretory pathway. As this propeptide processing seems to be a key event in the functional expression of SPC4, we have investigated its mechanism and the intracellular site where it occurs. In transfected fibroblast cells, the 110-kDa proSPC4 undergoes slow cleavage to generate a 103-kDa mature enzyme in the endoplasmic reticulum (ER). Site-directed mutagenesis studies demonstrate that the proteolytic activation of SPC4 occurs mainly through a unimolecular autocatalytic process and propeptide cleavage is a prerequisite for its export from the ER. Sedimentation velocity and chemical cross-linking analysis demonstrate that the precursor protein in the cells exists as both a monomer and a dimer-sized complex whereas mature SPC4 exists only as a monomer. These results suggest that the cleavage of the N-terminal propeptide of SPC4 plays a regulatory role in its activation and secretion through the change in its oligomeric state.
(Keyword)
Animals / Cell Line / Humans / Isoenzymes / Proprotein Convertases / Protein Conformation / Protein Processing, Post-Translational / Serine Endopeptidases
Hideaki Nagamune, Hiroki Kourai, Ayako Katsuura, Yasuki Taoka, Kenzo Fushitani, Robert A. Whiley, Kikuji Yamashita, Akihiko Tsuji, Yoshiko Matsuda, Hiroki Kourai and Seiichiro Kitamura : Intermedilysin A cytolytic toxin specific for human cells of a Streptococcus intermedius isolated from human liver abscess in "Streptococci and The Host", Advances in Experimental Medicine and Biology, 418, 773-775, 1997.
(Summary)
S. intermedius肝膿瘍分離株から単離されたヒト細胞特異的な細胞溶解毒素インターメディリシン(ILY)の性質を検討した．これまで知られていたチオール基修飾試薬への非感受性やコレステロールへの低感受性に加え，電子顕微鏡観察からILYは直径30-50nmの膜孔を形成し細胞を破壊することが示された．またILYの部分遺伝子構造解析の結果からILYはチオール活性化毒素群と同じ分子祖先から進化したものであることが示唆された．
Akihiko Tsuji, Chiemi Hine, Yasuhiro Tamai, Kayoko Yonemoto, Kazushige Mori, Shigeru Yoshida, Miwa Bando, Eri Sakai, Kenji Mori, Tetsuya Akamatsu and Yoshiko Matsuda : Genomic organization and alternative splicing of human PACE4 (SPC4), Kexin-like processing endoprotease., The Journal of Biochemistry, 122, 2, 438-452, 1997.
(Summary)
PACE4 (paired basic amino acid cleaving enzyme) is a member of a family of the mammalian kexin-like proprotein convertases containing a subtilisin-like catalytic domain. Previously we reported seven isoform mRNAs of PACE4 that vary in size and 3'-coding sequence [A. Tsuji et al. (1994) Biochem. Biophys. Res. Commun. 200, 943-950; K. Mori et al. (1997) J. Biochem. 121, 941-948]. To determine the origin of these isoforms, the entire human PACE4 gene has been isolated as a set of overlapping genomic DNA fragments, and analyzed by restriction enzyme digestion and nucleotide sequence determination. The human PACE4 gene spans at least 250 kb and is distributed over 25 exons that range in size from 39 to 1,422 base pairs. Human PACE4 gene is the largest kexin-like proprotein convertase gene reported to date. The most striking feature of its genomic structure is the size of the introns and the number of exons, although the general organization of signal peptide, propeptide, and catalytic domains, which are conserved in this family, is very similar to that reported for other kexin-like protease genes. The structural analysis of PACE4 genomic DNA indicates that multiple PACE4 transcripts are produced as a consequence of alternative RNA splicing events, including exon skipping, and differences in the usage of the inner 5'-splicing donor and polyadenylation sites. A major transcriptional start site was detected 314 bp upstream from the ATG translational start site by primer extension analysis. Sequence analysis of the 5'-flanking region revealed that PACE4 gene lacks TATA and CCAAT boxes in the proximal upstream region of the start site, although potential binding sites for several transcription factors including SP1, AP1, AP2, PEA3, Ets-1, GHF (growth hormone factor)-1, CREB (cyclic AMP response element binding protein), and basic helix-loop-helix proteins, were present. An unusual sequence of six tandem repeats of a nonadecamer (GGCCTGGGGGTTCACCTGC) containing an E box is found in the 5'-flanking region. These results suggest that PACE4 is not a constitutive gene product and its expression is regulated by various transcription factors.
(Keyword)
Processing protease / PACE4 / Gene structure / Alternative splicing
Tetsuya Akamatsu, Shigeo Daikoku, Hideaki Nagamune, Shigeru Yoshida, Kenji Mori, Akihiko Tsuji and Yoshiko Matsuda : Developmental expression of a novel Kexin family protease, PACE4E, in the rat olfactory system., Histochemistry and Cell Biology, 108, 2, 95-103, 1997.
(Summary)
PACE4 is a mammalian Kexin family protease that is involved in the maturation of precursor proteins. Four PACE4 isoforms have been identified. We identified a novel PACE4 isoform, PACE4E, from a human cerebellum cDNA library, which possesses a hydrophobic cluster in its C-terminus participating in membrane association. The size of PACE4E mRNA from adult rat brain was estimated by Northern blotting to be 4.4 kb. In situ hybridization histochemistry revealed that the highest level of PACE4E mRNA was expressed in the mitral cells of the adult rat olfactory bulb (OB). The OB is a unique sensory organ in that it has a lifelong regenerating capacity and it affects brain development. We further analyzed the expression of PACE4E mRNA in the developing olfactory system. On day 13.5 of gestation, PACE4E mRNA was expressed at high levels in the neuroepithelium of the forebrain vesicle (FV), olfactory epithelium, and cells in the fiber bundles projecting to the FV. As development proceeded, PACE4E mRNA was expressed in developing mitral cells but decreased in the olfactory epithelium. In the newborn, its expression was confined to the mitral cells in both the main and accessory OB and in some periglomerular cells, as shown in adult rats. The spatio-temporal expression of PACE4E suggests that it plays a role in the establishment and maintenance of the olfactory receptor system.
(Keyword)
Processing protease / Olfactory system / Mitral cell
Kenji Mori, Sachiko Kii, Akihiko Tsuji, Masami Nagahama, Akiyoshi Imamaki, Keiko Hayashi, Tetsuya Akamatsu, Hideaki Nagamune and Yoshiko Matsuda : A Novel Human PACE4 Isoform, PACE4E Is an Active Processing Protease Containing a Hydrophobic Cluster at the Carboxy Terminus, The Journal of Biochemistry, 121, 5, 941-948, 1997.
(Summary)
PACE4 is a processing protease which processes the precursor protein to the mature protein. Currently, four PACE4 isoforms have been reported [Tsuji, A. et al. (1994) Biochem. Biophys. Res. Commun. 200, 943 950]. In this study, we have cloned cDNA encoding a novel isoform, PACE4E, by screening the human brain cerebellum cDNA library and reverse transcriptase polymerase chain reaction analysis of total RNA from human hepatoma HepG2 cells. The PACE4E cDNA encoded an amino acid sequence of 975 residues. The sequence from the amino terminus to Arg900 of PACE4E was identical to the corresponding sequence of PACE4A, but the carboxy terminal sequence (75 residues) was unique and contained a hydrophobic cluster (Leu952-Gly968). PACE4E cDNA was transiently transfected in COS-1 cells, and the expressed proteins were a 112-kDa precursor form and a 105-kDa mature form. They were secreted into the culture medium, but their secretion was retarded compared with that of PACE4A. The expression of a mutant of PACE4E truncated up to the hydrophobic cluster from the carboxy terminus resulted in a remarkable increase in secretion level, suggesting that PACE4E tends to be retained intracellularly due to interaction with the membrane through the hydrophobic cluster. On the contrary, the transient expression experiment of PACE4C showed that only 68-kDa protein (precursor form) was detected in the cell and not secreted into the medium. In addition, coexpression experiment revealed that PACE4E was able to process the precursor form of von Willebrand factor to the mature form, but PACE4C did not process it.
(Keyword)
Amino Acid Sequence / Animals / Cell Line / Cloning, Molecular / DNA Mutational Analysis / DNA, Complementary / Gene Deletion / Humans / Isoenzymes / Molecular Sequence Data / Proprotein Convertases / Protein Conformation / Protein Precursors / Protein Processing, Post-Translational / Serine Endopeptidases
Hideaki Nagamune, Chikako Ohnishi, Ayako Katsuura, Kenzo Fushitani, Robert A. Whiley, Akihiko Tsuji and Yoshiko Matsuda : Intermedilysin, a novel cytotoxin specific for human cells, secreted by Streptococcus intermedius UNS46 isolated from a human liver abscess, Infection and Immunity, 64, 8, 3093-3100, 1996.
(Summary)
A novel cytotoxin (intermedilysin) specific for human cells was identified as a cytolytic factor of Streptococcus intermedius UNS46 isolated from a human liver abscess. Intermedilysin caused human cell death with membrane blebs. Intermedilysin was purified from UNS46 culture medium by means of gel filtration and hydrophobic chromatography. The purified toxin was resolved into major and minor bands of 54 and 53 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins reacted with an antibody against intermedilysin. Five internal peptide fragments of intermedilysin were sequenced and found to have 42 to 71% homology with the thiol-activated cytotoxin pneumolysin. However, the action of intermedilysin differed from that of thiol-activated cytotoxins, especially in terms of a lack of activation by dithiothreitol and resistance to treatments with N-ethylmaleimide and 5,5'-dithio-bis-(2-nitrobenzoic acid), although cholesterol inhibited the toxin activity. Intermedilysin was potently hemolytic on human erythrocytes but was 100-fold less effective on chimpanzee and cynomolgus monkey erythrocytes. Intermedilysin was not hemolytic in nine other animal species tested. Since human erythrocytes treated with trypsin were far less sensitive to intermedilysin than were the intact cells, a cell membrane protein(s) may participate in the intermedilysin action. These data demonstrated that intermedilysin is distinguishable from all known bacterial cytolysins.
Akihiko Tsuji, Keisuke Edazawa, Koji Sakiyama, Kaya Nagata, Yuka Sasaki, Hideaki Nagamune and Yoshiko Matsuda : Purification and characterization of a novel serine proteinase from the microsomal fraction of bovine pancreas., The Journal of Biochemistry, 119, 1, 100-105, 1996.
(Summary)
牛膵臓膜画分より，CHAPSによる可溶化，ゲルろ過，トリプシンインヒビターセファロース，アルギニンーセファロースによって，新規のトリプシン様セリンプロテアーゼを単一に精製した．この酵素は分子量29500で，グルカゴンを用いて切断特異性を調べると，塩基性アミノ酸とロイシンのC末側を切断した．N末のアミノ酸配列は，エラスターゼIV, II, IIIに類似していた．
新本 美智枝, Akihiko Tsuji, 楊 瑞成, 鈴木 義之, 野村 房子 and 瀬川 昌也 : 制限酵素断片長多型を利用した連鎖解析によるX染色体劣性遺伝病の遺伝子診断, The Journal of the Japan Pediatric Society, 91, 3440-3447, 1996.
Hideaki Nagamune, Kazuaki Muramatsu, Tetsuya Akamatsu, Yasuhiro Tamai, Keisuke Izumi, Akihiko Tsuji and Yoshiko Matsuda : Distribution of the kexin family proteases in pancreatic islets., --- PACE4C is specifically expressed in B cells of pancreatic islets ---, Endocrinology, 136, 1, 357-360, 1995.
(Summary)
The distribution of Kexin family proteases in adult rat pancreatic islets was investigated by immunohistochemical means using a series of specific antibodies specific for PC1, PC2, PC6, Furin, PACE4A and a recently identified member of the Kexin family, PACE4C. PACE4C expression was limited to B cells of the pancreatic islets. PC2 was found in A and in some D cells more than in B cells and PC1 was evident only in B cells. Furin and PC6 were weakly and evenly expressed in the entire islet. PACE4A was hardly found in the islets. These findings indicated that individual Kexin family proteases are uniquely distributed in the islets and suggested that these proteases share roles in these cells as follows: PC2 is involved in the peptide hormone precursor processing in A cells and in D cells, and PACE4C, PC1 and PC2 (mainly PACE4C and PC1) are responsible for the processing event(s) specific to B cells.
Akihiko Tsuji, Chiemi Hine, Kenji Mori, Yasuhiro Tamai, Kazuya Higashine, Hideaki Nagamune and Yoshiko Matsuda : A Novel Member, PC7, of The Mammalian Kexin-Like Protease Family, --- Homology to PACE4A, Its Brainspecific Expression and Identification of Isoforms ---, Biochemical and Biophysical Research Communications, 202, 3, 1452-1459, 1994.
(Summary)
By polymerase chain reaction (PCR) with primers corresponding to the sequences of catalytic domain conserved among the mammalian kexin-like protease family, a cDNA fragment encoding a novel member of the family was obtained from rat pituitary. A cDNA for the novel protease was obtained from three overlapping clones isolated from a rat pituitary cDNA library using the PCR product as a screening probe. The protein, designated as PC7, encoded by this cDNA contained a putative activation site with the sequence RXKR, subtilisin-like catalytic domain and a homo B region which are typical of mammalian kexin-like proteases, and short cysteine-rich region at the carboxyl-terminal end. It exhibited surprising sequence similarity to PACE4A. The PC7 transcript was expressed at high levels in the brain. However it was undetectable in the liver, kidney, heart, and spleen. The presence of PC7 isoforms (PC7A and PC7B) was also shown.
Akihiko Tsuji, Kenji Mori, Chiemi Hine, Yasuhiro Tamai, Hideaki Nagamune and Yoshiko Matsuda : The tissue distribution of mRNAs for the PACE4 isoforms, kexin-like processing protease: PACE4C and PACE4D mRNAs are major transcripts among PACE4 isoforms, Biochemical and Biophysical Research Communications, 202, 3, 1215-1221, 1994.
Akihiko Tsuji, Ryoichi Oda, Kohji Sakiyama, Hideaki Nagamune, Kouji Itou, Ryoichi Kase, Hitoshi Sakuraba, Yoshiyuki Suzuki and Yoshiko Matsuda : Lysosomal enzyme replacement using α2-macroglobulin as a transport vehicle, The Journal of Biochemistry, 115, 5, 937-944, 1994.
(Summary)
Improvement of the delivery of exogenous enzymes is essential to achieve effective enzyme replacement therapy in lysosomal storage diseases. To test whether alpha 2-macroglobulin, an endogenous plasma protein, could serve as a transport vehicle of therapeutic agents to cells, alpha 2-macroglobulin and acid alpha-glucosidase or alpha-galactosidase A were coupled using two heterobifunctional cross-linking reagents. The alpha-glucosidase-alpha 2-macroglobulin conjugate was internalized and transported into lysosomes of acid alpha-glucosidase-deficient fibroblasts. The enzyme activity was stable after being taken up by the cells. Uptake of the conjugate resulted in the degradation of glycogen accumulated in lysosomes. The alpha-galactosidase A-alpha 2-macroglobulin conjugate was also internalized into the lysosomes of alpha-galactosidase A-deficient fibroblasts. Internalized alpha-galactosidase A-conjugate degraded globotriaosylceramide accumulated in lysosomes. The endocytosis of both conjugate was inhibited by alpha 2-macroglobulin-trypsin complex, indicating that the conjugates were endocytosed by an alpha 2-macroglobulin receptor system. These results showed the usefulness of alpha 2-macroglobulin as a transport vehicle of lysosomal enzymes for effective enzyme replacement.
Akihiko Tsuji, Kazuya Higashine, Chiemi Hine, Kenji Mori, Yasuhiro Tamai, Hideaki Nagamune and Yoshiko Matsuda : Identification of Novel cDNAs Encording Human Kexin-like Protease, PACE4 Isoforms, Biochemical and Biophysical Research Communications, 200, 2, 943-950, 1994.
(Summary)
PACE4 has been identified as a second human subtilisin-like protease by Keifer et al. [DNA and Cell Biology (1991) 10, 757-769]. In this study, we isolated two novel cDNAs coding for PACE4 isoforms (PACE4C and PACE4D) from a human placenta cDNA library. The deduced PACE4C protein sequence (652 amino acids) lacks the cysteine-rich region located at the carboxy terminus of PACE4. The DNA sequence of the 3'-untranslated region (1 kb) of PACE4C is not homologous to the PACE4 sequence. The protein (497 amino acids) encoded by PACE4D cDNA lacks a signal peptide, a propeptide and the cysteine-rich region. These isoform cDNAs were also isolated from rat pituitary cDNA library.
Akihiko Tsuji, Tetsuya Akamatsu, Hideaki Nagamune and Yoshiko Matsuda : Identification of targeting proteinase for rat α1-macroglobulin in vivo., --- Mast-cell tryptase is a major component of the α1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes. ---, The Biochemical Journal, 298, 1, 79-85, 1994.
(Summary)
The alpha 1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes was purified by a series of column chromatographic steps on concanavalin A-Sepharose, Sephacryl S-300, DEAE-cellulose and TSK gel DEAE-5PW columns. The complex contained no detectable alpha 2-macroglobulin. Studies on the substrate specificity indicated that the complex had tryptase-like activities towards various synthetic substrates, but no elastase, chymotrypsin, cathepsin-B and cathepsin-L activities. The proteinase activity was completely inhibited by di-isopropyl fluorophosphate, leupeptin and antipain, indicating that the proteinase bound to alpha 1-macroglobulin is a serine proteinase. Two protein bands (62 and 59 kDa) of the complex were labelled with [3H]diisopropyl fluorophosphate and both bands cross-reacted with anti-(mast-cell tryptase)antibody. These results suggest that mast-cell tryptase is a major targeting proteinase for alpha 1-macroglobulin in vivo. The main alpha-macroglobulin-proteinase complex in the adjuvant-treated rats was also the alpha 1-macroglobulin-tryptase complex, even though the plasma level of alpha 2-macroglobulin was elevated.
Torres-Rosado Adrian, O'Shea K.Sue, Akihiko Tsuji, Chou Shan-Ho and Kurachi Kotoku : Hepsin, a putative cell-surface serine protease, is required for mammalian cell growth, Proceedings of the National Academy of Sciences of the United States of America, 90, 15, 7181-7185, 1993.
(Summary)
Hepsin was previously identified as a putative cell-surface serine protease. When hepatoma cells were treated with anti-hepsin antibodies, their growth was substantially arrested, suggesting the requirement of hepsin molecules present at the cell surface for normal cell growth. This was further supported by a gross inhibition of cell growth with hepsin-specific antisense oligonucleotides. Upon treatment of cells with antisense oligonucleotides, rapid reduction in cellular hepsin was observed. This reduction in cellular hepsin levels was accompanied by drastic morphological changes. Various tissues in the developing mouse embryo showed greatly elevated hepsin levels in regions of active proliferation. These results indicate that hepsin plays an essential role in cell growth and maintenance of cell morphology.
Akihiko Tsuji, Adrian Torres-Rosado, Toshiro Arai, Michelle M. LeBeau, Richard S. Lemons, Shan-Ho Chou and Kotoku Kurachi : Hepsin, a cell membrane-associated protease, --- characterization, tissue distribution and gene localization ---, The Journal of Biological Chemistry, 266, 25, 16948-16953, 1991.
(Summary)
Hepsin, a putative membrane-bound serine protease, was originally identified as a human liver cDNA clone (Leytus, S.P., Loeb, K.R., Hagen, F.S., Kurachi, K., and Davie, E.W. (1988) Biochemistry 27, 1067-1074). In the present study the human hepsin gene was localized to chromosome 19 at q11-13.2. The messenger RNA of hepsin is 1.85 kilobases in size and present in most tissues, with the highest level in liver. Hepsin is synthesized as a single polypeptide chain, and its mature form of 51 kDa was found in various mammalian cells including HepG2 cells and baby hamster kidney cells. It is present in the plasma-membrane in a molecular orientation of type II membrane-associated proteins, with its catalytic subunit (carboxyl-terminal half) at the cell surface, and its amino terminus facing the cytosol. Hepsin is found neither in cytosol nor in culture media. The results obtained suggest that hepsin has an important role(s) in cell growth and function.
Akihiko Tsuji, Tishiro Arai, P. S. Furcinitti, J. P. Langmore and Kotoku Kurachi : The major component of a large, intracellular proteinase accumulated by inhibitors is a complex of alpha-2 macroglobulin and thrombin, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1078, 1, 85-93, 1991.
(Summary)
A large, intracellular proteinase accumulated by inhibitors (PABI) was found in cultured mammalian cells as a large, multicatalytic proteinase with a greatly elevated concentration in the presence of small peptide proteinase inhibitors (Tsuji and Kurachi (1989) J. Biol. Chem. 264, 16093). Electron microscopic analysis showed that the tertiary structure of PABI highly resembled that of alpha 2-macroglobulin complexed with a proteinase(s). Isolation of the anti-PABI cross-reacting material from calf serum added to the culture media of baby hamster kidney cells further supported that the primary component of PABI was alpha 2-macroglobulin. Immunoblot analyses and the substrate specificity of PABI indicated that the major proteinase component contained in PABI was thrombin. When alpha 2-macroglobulin was added to the PABI-depleted serum, a significant accumulation or a degradation of the intracellular alpha 2-macroglobulin was observed in the presence or absence of leupeptin, respectively. Similarly, when thrombin was added to the PABI-depleted fetal calf serum supplemented with fresh alpha 2-macroglobulin, a significant amount of intracellular thrombin was found only in the presence of leupeptin. These results indicate that the major component of the intracellular PABI molecules is a complex of alpha 2-macroglobulin with thrombin which is internalized from the culture media. Intracellular accumulation of PABI, therefore, is a phenomenon primarily relevant to the culture cells. Whether or not PABI is also generated in certain physiological or pathological conditions requires further study.
P. Hu, A J J Reuser, H. C. Janse, W. J. Kleijer, D. Schindler, H. Sakuraba, Akihiko Tsuji, Y. Suzuki and O. van P. Diggelen : Biosynthesis of human alpha-N-acetylgalactosaminidase: Defective phosphorylation and maturation in infantile alpha-NAGA deficiency, Biochemical and Biophysical Research Communications, 175, 3, 1097-1103, 1991.
(Summary)
The biosynthesis of human alpha-N-acetylgalactosaminidase (alpha-NAGA) was studied in normal fibroblasts and in cells from patients with infantile alpha-NAGA deficiency. Normal alpha-NAGA is synthesized as a 52 kDa precursor which matures to a 49 kDa species through phosphorylation and carbohydrate triming. Fibroblasts from the patients synthesize normal amounts of a 52 kDa precursor, however phosphorylation does not occur and this precursor is subsequently degraded intracellularly.
Yang Rei-Cheng, Akihiko Tsuji and Suzuki Yoshiyuki : Abnormal protein spots revealed by two-dimensional electrophoresis in mycoplasma-infected human fibroblasts, Electrophoresis, 11, 4, 344-346, 1990.
(Summary)
Six new protein spots were detected by two-dimensional electrophoresis in cultured human fibroblasts infected with mycoplasma. No changes were observed in other protein spots present in normal and pathological cells. After treatment with an antimycoplasma drug for a week, the new spots disappeared and the cells became negative for mycoplasma stain.
Rei-Cheng Yang, Yoshiyuki Suzuki and Akihiko Tsuji : Two-dimentional electrophoresis aided personal computer analusis for screening of mutant proteins in inherited diseases, Electrophoresis, 10, 11, 785-792, 1989.
(Summary)
A rapid and reproducible method of two-dimensional electrophoresis was developed for screening of abnormal proteins expressed in fibroblasts from patients with inherited diseases. After silver staining, the electrophoresis gel was subjected to semiautomatic digitizer-personal computer analysis: scanning with an image sensor video camera connected to a digitizer, followed by quantitative determination and statistical analysis with a personal computer. The protein spots analyzed by this method showed quantitative variations of various degrees, particularly in 2 of 247 spots examined. Seven spots were not always detected in control and pathological cells in this study. Slight variations in molecular weight were observed in 3 different spots.
(Keyword)
Adolescent / adult / Age Factors / Cells, Cultured / children / Child, Preschool / Electronic Data Processing / Electrophoresis, Gel, Two-Dimensional / Electrophoresis, Polyacrylamide Gel / female / Genetic Diseases, Inborn / Humans / infant / male / Mutation / Proteins / Sex Factors
Akihiko Tsuji and Kotoku Kurachi : Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors, The Journal of Biological Chemistry, 264, 27, 16093-16099, 1989.
(Summary)
We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.
Akihiko Tsuji and Kotoku Kurachi : Isolation and characterization of a novel large protease accumulated mammalian cells in the presence of inhibitors, The Journal of Biological Chemistry, 264, 27, 16093-16099, 1989.
81.
Yuji Iwasaki, Akihiko Tsuji, Kiyoshi Omura and Yoshiyuki Suzuki : Purification and characterization of beta-mannosidase from human placenta, The Journal of Biochemistry, 106, 2, 331-335, 1989.
Akihiro Ohshima, Akihiko Tsuji, Yoshiro Nagao, Hitoshi Sakuraba and Yoshiyuki Suzuki : Cloning, sequencing, and expression of cDNA for human beta-galactosidase, Biochemical and Biophysical Research Communications, 157, 1, 238-244, 1988.
(Summary)
We cloned and sequenced the full-length cDNA for human placental beta-galactosidase. The 2379-nucleotide sequence contains 2031 nucleotides which encode a protein of 677 amino acids. The amino acid sequence includes a putative signal sequence of 23 amino acids and 7 potential asparagine-linked glycosylation sites. The cDNA in the expression vector pSVL was used to transfect COS cells. Expression of the cDNA in transfected COS cells produced immunoprecipitable proteins and led to an increase in beta-galactosidase activity.
Shinmoto Michie, Akihiko Tsuji and Suzuki Yoshiyuki : Restriction fragment length polymorphism on the short arm of X-chromosome among the Japanese population, The Japanese Journal of Human Genetics, 33, 333-338, 1988.
Akihiko Tsuji and Yoshiyuki Suzuki : Solubilization of Membrane-Bound Acid α-Glucosidase by Proteolysis, Biochemical and Biophysical Research Communications, 151, 3, 1358-1363, 1988.
Akihiko Tsuji, Kiyoshi Omura and Yoshiyuki Suzuki : Intracelluler Transport of Acid α-Glucosidase in Human Fibroblasts: Evidence for Involvement Phosphomannosyl Receptor-Independent System, The Journal of Biochemistry, 104, 2, 276-278, 1988.
(Summary)
Intracellular transport of two lysosomal enzymes, acid alpha-glucosidase and beta-hexosaminidase, was analyzed in human fibroblasts. The precursors of beta-hexosaminidase in normal fibroblasts were released from the membrane fraction by treatment with mannose 6-phosphate, but the precursor of alpha-glucosidase was not. Percoll density gradient centrifugation revealed a normal amount of acid alpha-glucosidase activity in heavy lysosomes in I-cell disease fibroblasts despite impaired maturation and defective phosphorylation, and beta-hexosaminidase activity was markedly reduced in lysosomes. It was concluded that the membrane-bound precursor of acid alpha-glucosidase is transported to lysosomes by a phosphomannosyl receptor-independent system although the enzyme lacks the recognition marker for the phosphomannosyl receptor and processing of an intermediate form to mature forms does not occur in this disease.
Yoshiyuki Suzuki, Akihiko Tsuji, Kiyoshi Omura, Gen Nakamura, Shoichi Awa, Mrian Kroos and Arnold J.J. Reuser : Km mutant of acid α-glucosidase in a case of cardiomyopathy without signs of skeletal muscle involvement, Clinical Genetics, 33, 376-385, 1988.
Eiji Nanba, Akihiko Tsuji, Kiyoshi Omura and Yoshiyuki Suzuki : Galactosialidosis: molecular heterogeneity in biosynthesis and processing of peotective protein for β-galactosidase, Human Genetics, 80, 4, 329-332, 1988.
(Summary)
Biosynthesis and processing of the protective protein for beta-galactosidase in normal and galactosialidosis fibroblasts were investigated using specific antiserum preparations. A 45-kd precursor was processed to a mature 30-kd protein in normal fibroblasts. The mature protective protein was not detected in any of the twelve galactosialidosis fibroblast strains examined in this study. The precursor was not detected in two cases and in the others was of heterogeneous molecular weight, i.e., normal, abnormally low, or abnormally high. These molecular abnormalities were not correlated with clinical manifestations of the patients.
Michie Shinmoto, Akihiko Tsuji, Rei-Cheng Yang, Yoriko Nomura, Masaya Segawa and Yosiyuki Suzuki : DNA deletion and recombination in the gene locus of X-linked muscular dystrophies, Journal of Inherited Metabolic Disease, 11, 324-328, 1988.
Akihiko Tsuji and Yoshiyuki Suzuki : Biosynthesis of two components of human acid α-glucosidase, Archives of Biochemistry and Biophysics, 259, 2, 234-240, 1987.
(Summary)
Two acid alpha-glucosidase components of different molecular sizes (80 and 71 kDa) were separated from human placenta by DEAE-cellulose chromatography. Their catalytic properties were similar, and they showed almost the same molecular structure with regard to immunological properties and carboxy-terminal sequences, although the amino acid composition, the total hexose content, and the circular dichroism spectra were different. The pulse-chase labeling acid alpha-glucosidase with [3H]leucine revealed a processing pathway from a 110 kDa precursor to a 95 kDa intermediate form, then finally to 80 and 71 kDa mature forms. However, the sequence of appearance was different between the two mature enzymes. The 80 kDa component appeared first after chase for 5 h, and then the 71 kDa component followed. Their amounts became equal at 2 to 4 days. When ammonium chloride or leupeptin was added to the culture medium after chase for 5 h, the 71 kDa component failed to appear and the 80 kDa component was not converted to 71 kDa. It is concluded that probably only a part of the 80 kDa component is processed to form the 71 kDa component, although another possibility that cannot be excluded is that these two components are converted independently from the common intermediate 95 kDa protein.
Akihiko Tsuji and Yoshiyuki Suzuki : The precursor of acid α-glucosidase is synthesized as a membrane bound enzyme, Biochemistry International, 15, 5, 945-952, 1987.
(Summary)
A pulse-chase study in human skin fibroblasts showed that a 110 kDa precursor of acid alpha-glucosidase was synthesized as a membrane-bound protein, which was solubilized in vitro not by mannose 6-phosphate or 1M KCl but by Triton X-100 or trypsin. This 110 kDa precursor form bound to the membrane was detected in control fibroblasts treated with tunicamycin and in I-cell disease fibroblasts as well. The precursors in human placenta were found also in the membrane fraction. It was concluded that the newly synthesized acid alpha-glucosidase precursor is located on the surface of the membrane, and the phosphomannosyl receptor does not participate in the enzyme-membrane binding.
Akihiko Tsuji and Yoshiyuki Suzuki : Purification of human placental acid α-mannosidase by an immunological method, Biochemistry International, 15, 3, 483-489, 1987.
(Summary)
An immunoaffinity column was used for the purification of alpha-mannosidase from human placenta. The enzyme was purified to homogeneity by extraction in the presence of various protease inhibitors, immunoaffinity chromatography, Ultrogel AcA-34 gel filtration and hydroxyapatite chromatography. Two subunits were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their molecular weights were 65 kDa and 27 kDa. Heterogeneity of the molecular weight of the large subunit was not observed in our preparation. This method is relatively simple and rapid for obtaining the purified enzyme which is structurally not modified during purification procedures.
(Keyword)
Amino Acids / Centrifugation, Density Gradient / Chromatography, Affinity / Chromatography, Gel / Electrophoresis, Polyacrylamide Gel / female / Humans / Immune Sera / Kinetics / Mannosidases / Placenta / alpha-Mannosidase
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 3426623
Eiji Nanba, Akihiko Tsuji, Kiyoshi Omura and Yoshiyuki Suzuki : Galactosialidosis: A direct evidence that 49-kilodalton protein restores deficient enzyme activities in fibroablast, Biochemical and Biophysical Research Communications, 144, 1, 138-142, 1987.
Akihiko Tsuji, Rei-Cheng Yang, Kiyoshi Omura, Tomoko Imabayashi and Yoshiyuki Suzuki : A simple differential immunoprecipitation assey of urinary asid and neutral α-glucosidase for glucogenosisII, Clinica Chimica Acta, 167, 3, 313-320, 1987.
(Summary)
A specific assay for acid alpha-glucosidase in urine was developed to facilitate the diagnosis of glycogenosis II. This enzyme activity was calculated as a difference between the alpha-glucosidase activities before and after immunoprecipitation with antiserum to acid alpha-glucosidase. Acid alpha-glucosidase accounted for 86% of the total activity in control urine. All the cases of various clinical types of glycogenosis II showed either a marked decrease or a complete deficiency of this enzyme activity. A marked decrease of acid alpha-glucosidase was demonstrated by immunoblotting of the urine from patients with late-onset forms of this disease. These results indicate that assays of urinary acid alpha-glucosidase by this immunological method are useful for detection of the various types of glycogenosis II.
(Keyword)
Adult / children / Diagnosis, Differential / female / Glycogen Storage Disease / Glycogen Storage Disease Type II / Humans / Hydrogen-Ion Concentration / Immunoassay / male / Pregnancy / alpha-Glucosidases
Eiji Nanba, Akihiko Tsuji, Kiyoshi Omura and Yoshiyuki Suzuki : Galactosialidosis: Studies on residual enzymes in early and late onset clinical phenotypes, Journal of Clinical Biochemistry and Nutrition, 3, 2, 149-157, 1987.
Matsuko Moriyasu, Kichiko Koike, Yoshishige Urata, Akihiko Tsuji and Masahiko Koike : Rapid and simple isolation procedure for three component enzyme of pig heart 2-oxoglutarate dehydrogenase complex, Journal of Nutritional Science and Vitaminology, 32, 1, 33-40, 1986.
(Summary)
A novel procedure was developed for rapid separation of the three component enzymes of pig heart 2-oxoglutarate dehydrogenase complex by high performance liquid chromatography on a gel filtration column. The complex was dissociated and separated into two fractions of the first dihydrolipoamide succinyltransferase and a second yellow fraction within 1 h by chromatography on a preparative TSK-GEL G4000SW column equilibrated with 0.05 M potassium phosphate buffer (pH 7.0) containing 0.7 M guanidine hydrochloride, 0.05% Triton X-100 and 2 mM dithiothreitol at 10 degrees C. The dihydrolipoamide succinyltransferase fraction was further purified by incubation with 0.5% sodium deoxycholate and subsequent ammonium sulfate fractionation. The other two component enzymes, 2-oxoglutarate dehydrogenase and lipoamide dehydrogenase were separated from the second yellow fraction by chromatography on a calcium phosphate gel-cellulose column. The TSK-GEL column permitted very rapid dissociation and separation of the three component enzymes accompanied by good preservation of their activities and high overall yields.
Katsuko Yamashita, Yoko Tachibana, Akira Hitoi, Yoshiko Matsuda, Akihiko Tsuji, Nobuhiko Katunuma and Akira Kobata : Differences in the sugar chains of two subunits and isozymic forms of rat kidney γ-glutamyltranspeptidase, Archives of Biochemistry and Biophysics, 227, 1, 225-232, 1983.
(Summary)
A comparative study by gel-permeation chromatographic analysis of oligosaccharides released from the heavy and the light subunits of rat kidney gamma-glutamyltranspeptidase has revealed that high-mannose-type sugar chains are found only in the heavy subunit, and the nonsialylated and nonfucosylated biantennary complex-type sugar chains are included only in the light subunit. By the same analysis of the oligosaccharide fractions obtained from four isozymic forms of rat kidney gamma-glutamyltranspeptidase, it was found that all these enzymes contain 2 mol of neutral sugar chains but different numbers of acidic sugar chains. The total numbers of sialic acid residues showed a reciprocal relationship to the isoelectric point of each isozymic form, and an increase of 1 mol of sialic acid residue corresponds to a decrease of 0.5 in the value of the isoelectric point.
Yoshiko Matsuda, Akihiko Tsuji, Nobuhiko Katunuma and N. Katunuma : Biosynthesis and degradation of gamma-glutamyltranspeptidase of rat kidney, The Journal of Biochemistry, 94, 3, 755-765, 1983.
Katsuko Yamashita, Akira Hitoi, Yoshiko Matsuda, Akihiko Tsuji, Nobuhiko Katunuma and Akira Kobata : Structural studies of the carbohydrate moieties of rat kidney γ-glutamyltranspeptidase, The Journal of Biological Chemistry, 258, 2, 1098-1107, 1983.
Yoshiko Matsuda, Akihiko Tsuji and Nobuhiko Katunuma : Studies on the structure of γ-glutamyltranspeptidase. III. Evidence that the amino terminus of the heavy subunit is membrane binding segment, The Journal of Biochemistry, 93, 5, 1427-1433, 1983.
Tsutomu Miura, Yoshiko Matsuda, Akihiko Tsuji and Nobuhiko Katunuma : Immunological cross reactivity of γ-glutamyltranspeptidase from human and rat kidney, liver and bile, The Journal of Biochemistry, 89, 1, 217-222, 1981.
Yoshiko Matsuda, Akihiko Tsuji and Nobuhiko Katunuma : Studies on the structure of gamma-glutamyltranspeptidase. I. Correlation between sialylation and isozymic forms., The Journal of Biochemistry, 87, 4, 1243-1248, 1980.
(Summary)
Papain solubilized gamma-glutamyltranspeptidase was purified by DEAE-cellulose column chromatography. Four fractions were obtained as single proteins, each with a single isoelectric point, and fifth and sixth fractions were obtained as mixtures of several components. The molecular seights of the subunits of each form and their amino acid compositions and carboxyl-terminals were determined and found to be identical. The isoelectric points of the four purified enzymes and the main bands of the fifth fraction were pH 8.3, 8.0, 7.5, 6.7, and 6.0, respectively. The sixth fraction gave five bands with isoelectric points of pH 5.6, 5.3, 5.1, 4.6, and 4.4. An asialo-form of the enzyme, obtained by treatment with neuraminidase, had an isoelectric point of pH 8.6. A linear relationship was found between the isoelectric points of the enzymes and their contents of sialic acid, indicating that the heterogeneity of papain-solubilized gamma-glutamyltranspeptidase is mostly due to differences in the extends of sialylation of the enzymes. The distribution of the activity in rat kidney as follows: 29% in a poorly sialylated fraction (isoelectric point (Ip) 8.3 to 6.7), 24% in a moderately sialylated fraction (Ip 6.6 to 6.0), and 45% in a highly sialylated fraction (Ip 5.6 to 4.4).
Akihiko Tsuji, Yoshiko Matsuda and Nobuhiko Katunuma : Studies on the structure of g-glutamyltranspeptidase. II. Location of the segment anchoring γ-glutamyltranspeptidase to the membrane, The Journal of Biochemistry, 87, 6, 1567-1571, 1980.
Akihiko Tsuji, Yoshiko Matsuda and Nobuhiko Katunuma : Purification and characterization of γ-glutamyltranspeptidase from human bile, Biomedical Research, 1, 5, 410-416, 1980.
Yoshiko Matsuda, Akihiko Tsuji, Nobuhiko Katunuma, Masanori Hayashi and Yoshiyata Takahashi : Studies on liver argininosuccinate synthetase in a patient with citrullinemia and in normal subjects, The Journal of Biochemistry, 85, No 1, 192-195, 1979.
Yoshiko Matsuda, Hitoshi Hori, Akihiko Tsuji, Hideko Nagasawa, Yoshihiro Uto, Rieko Kuroda, 葛西 宗江, Miwa Bando, 吉田 一郎, 上田 祐司 and サハルディン B. モハマッド : 新医薬品の開発をめざした脳神経特異的発現遺伝子産物のプロテオミクス解析, Bulletin of Faculty of Engineering, The University of Tokushima, 46, 49-56, 2001.
Masami Nagahama, Ichiro Yoshida, Keiichi Uyemura, Akihiko Tsuji and Yoshiko Matsuda : Proteolytic Cleavage of the Neural Cell Adhesion Molecule L1 by Subtilisin-Like Proprotein Convertases, Keio University Symposia for Life Science and Medicine, 2, 261-266, 1999.
Suzuki Yoshiyuki, Omura Kiyoshi, Nanba Eiji, Akihiko Tsuji, Yang Rei-Cheng, Kato Goro, Sakuraba Hitoshi and Igarashi Takashi : Lysosomopathies: Analysis of intracellular abnormalities of functional proteins and a survey of new therapeutic approach, Child Neurology and Developmental Disabilities, 21-27, 1988.
T. Izumi, Y. Fukuyama, Akihiko Tsuji, T. Yamanaka, Y. Hirabayashi and Y. Suzuki : GM2-Gangliosidosis: B1 variant with thermostable beta-hexosaminidase A and molecular analysis of the mutany enzyme, Lipid Storage Disorders, 237-245, 1988.
Yoshiko Matsuda, Akihiko Tsuji and Nobuhiko Katunuma : Quantitative abnormality of argininosuccinate synthetase in a patient with citrullinemia, Advancesin Experimental Medicine and Biology, Urea Cycle Disease, 153, 77-82, 1984.
Shogo Abe, Saki Hirose, Mami Nishitani, Akihiko Tsuji and Keizo Yuasa : Citrus peel polymethoxyflavones, sudachitin and nobiletin, induce distinct cellular responses in human keratinocyte HaCaT cells via the MAPK pathways, The 7th International Conference on Food Factors (ICoFF2019)/The 12th International Conference and Exhibition on Nutraceuticals and Functional Foods (ISNFF2019), Kobe, Japan, Dec. 2019.
2.
Matsuda Shinya, Akihiko Tsuji and Keizo Yuasa : PCTK3/CDK18 regulates cell migration by negatively modulating the FAK1 activity, 16th International Conference of Biochemistry and Molecular Biology: Signalling Pathways in Development, Disease and Aging, Vancouver, BC, Canada, Jun. 2016.
3.
Shinya Matsuda, Kyohei Kominato, Akihiko Tsuji and Keizo Yuasa : PCTAIRE kinase 3/cyclin dependent kinase 18 is activated through association with cyclin A and/or phosphorylation by protein kinase A, Experimental Biology 2015, Boston, Massachusetts, Mar. 2015.
Akihiko Tsuji : Characterization of proteases expressed in cotyledons of daikon radish during germination., 6th General Meeting of the International Proteolysis Society, Gold Coast, Oct. 2009.
5.
Keizo Yuasa, Masami Nagahama and Akihiko Tsuji : The role of subutilisin-like proproteinn connvertase PACE4 in myogenesis, 5th General Meeting of the International Proteolysis Society, Patras, Greece, Oct. 2007.
6.
Kentaro Ogawa, Tsuyoshi Yuasa, Keizo Yuasa, Masami Nagahama and Akihiko Tsuji : Characterization of proteases expressed in the embryo of germinating wheat seed, International Symposium on Medical and Biological Perspectives in Proteases and Their Inhibitors. Satellite meeting of the 20th IUBMB International congress and 11th FAOBMB Congress, Hyogo, Jun. 2006.
7.
Keizo Yuasa, Kaori Suzue, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : Subtilisin-like proprotein convertase PACE4 is transcriptionally regulated by E2F/Rb, International Symposium on Medical and Biological Perspectives in Proteases and Their Inhibitors. Satellite meeting of the 20th IUBMB International congress and 11th FAOBMB Congress, Hyogo, Jun. 2006.
8.
Hiroki Kanie, Keizo Yuasa, Masami Nagahama and Akihiko Tsuji : Development of selectivity of 1-antitrypsin variant by mutagenesis in reactive site loop against proprotein convertase, International Symposium on Medical and Biological Perspectives in Proteases and Their Inhibitors. Satellite meeting of the 20th IUBMB International congress and 11th FAOBMB Congress, Hyogo, Jun. 2006.
9.
Tetsuya Masuda, Keizo Yuasa, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : Transcriptional Activation of processing protease PACE4 during myogenic differentiation., International Symposium on Medical and Biological Perspectives in Proteases and Their Inhibitors. Satellite meeting of the 20th IUBMB International congress and 11th FAOBMB Congress, Hyogo, Jun. 2006.
10.
Akihiko Tsuji : A novel protein which regulates autoactivation and secretion of subtilisin-like proprotein convertase. International Symposium on Medical and Biological Perspectives in Proteases and Their Inhibitors, Satellite meeting of the 20th IUBMB International congress and 11th FAOBMB Congress, Hyogo, Jun. 2006.
11.
Yayoi Kikuchi, Keizo Yuasa, Masami Nagahama and Akihiko Tsuji : RCN3 facilitates the secretion and activation of PACE4, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jun. 2006.
12.
Keizo Yuasa, Shin Yamagami, Shotaro Uehara, Masami Nagahama and Akihiko Tsuji : cGMP-dependent protein kinase II is required for chondrogenic differentiation, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jun. 2006.
13.
Takuya Yanagino, Keizo Yuasa, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : Transcriptional Regulation of Fibrillin-2 Gene during Chondrogenrsis, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jun. 2006.
14.
Akihiko Tsuji, Keizo Yuasa, Masami Nagahama and Yoshiko Matsuda : Role of PACE4 and furin in cell differentiation, International Conference on Serine-Carboxyl Peptidases 2005, Kyoto, Nov. 2005.
15.
Akihiko Tsuji, Yayoi Kikuchi and Yoshiko Matsuda : Reticulocalbin-3: A novel protein which regulates biosynthesis of subtilisin-like proprotein convertase, The 1st Pacific-Rim International Conference on Protein Science, 129, Yokohama, Apr. 2004.
Go Futamatsu, Tomonori Shimada, Akihiko Tsuji and Yoshiko Matsuda : Functional analysis of the processing protease PACE4 during chondrogenic differentiation of ATDC5 cells, International Symposium on Medical and Biological Perspectives in Proteases and Their Inhibitors., 36, Hyogo, Nov. 2003.
17.
Akihiko Tsuji, Shizuyo Koide, Keizo Yuasa and Yoshiko Matsuda : Involvement of PACE4 in activation of TGF beta-related factors in the ECM, International Symposium on Medical and Biological Perspectives in Proteases and Their Inhibitors., 20, Hyogo, Nov. 2003.
18.
Akihiko Tsuji, Yayoi Kikuchi, Yukimi Sato, Shizuyo Koide and Yoshiko Matsuda : Reticulocalbin-like protein (RCN3): A novel protein which regulates biosynthesis of subtilisin-like proprotein convertase (SPC), 3rd General Meeting of the International Proteolysis Society, 187, Nagoya, Nov. 2003.
19.
Akihiko Tsuji and Yoshiko Matsuda : Enhancement of selectivity of α1-antitrypsin variant against subtilisin-like proprotein convertase by mutagenesis in RSL and application for functional analysis, Second General Meeting of International Proteolysis Society, 59, Germany, Oct. 2001.
Akihiko Tsuji : Biosynthesis of PACE4, Hormonal and neural peptide biosynthesis Gordon Research Conference, New Hampshire, Jul. 2000.
21.
Akihiko Tsuji and Yoshiko Matsuda : Unique Mechanism Governing Cell-Specific Expression and Biosynthetic Processing of PACE4, International Proteolysis Society First Gemeral Meeting, 38, Michigan, USA, Sep. 1999.
22.
Akihiko Tsuji and Yoshiko Matsuda : Regulation of PACE4 expression under dynamic bHLH network and its physiological importance, 6th CGGH Symposium "Roles of Proteolysis in Health and Disease", 3, Tokushima, May 1999.
23.
Kenji Mori, Akihiko Tsuji and Yoshiko Matsuda : Subtilisin-like Proprotein Convertase, PACE4, participates in the Processing of Plasma protein precursors in human hepatoma HepG2 cells, 6th CGGH Symposium "Roles of Proteolysis in Health and Disease", 80, Tokushima, May 1999.
24.
Shin-ichi Hasegawa, Ichiro Yoshida, Shizuyo Koide, Akihiko Tsuji and Yoshiko Matsuda : Transcriptional Regulation of the Human PACE4 Gene Expression in HepG2 and GH4C1 cells, 6th CGGH Symposium "Roles of Proteolysis in Health and Disease", 83, Tokushima, May 1999.
25.
Emi Hashimoto, Takayuki Ikoma, Akihiko Tsuji and Yoshiko Matsuda : Proprotein Convertase PACE4 is inhibited by α1-Antitrypsin cariant Portland, 6th CGGH Symposium "Roles of Proteolysis in Health and Disease", 82, Tokushima, May 1999.
26.
Miwa Bando, Atsushi Matsuoka, Akihiko Tsuji and Yoshiko Matsuda : Physiological Function of PACE4 on megakaryoblastic differentiation, 6th CGGH Symposium "Roles of Proteolysis in Health and Disease", 84, Tokushima, May 1999.
27.
Ichiro Yoshida, Shizuyo Koide, Akira Nakagawara, Akihiko Tsuji and Yoshiko Matsuda : Regulation of PACE4 Expression by bHLH factor in neuroblastoma cells, 6th CGGH Symposium "Roles of Proteolysis in Health and Disease", 85, Tokushima, May 1999.
28.
Akihiko Tsuji : Enzymatic characterization of human SPC4, Hormonal and neural peptide biosynthesis Gordon Research Conference, New Hampshire, Aug. 1998.
29.
Hideaki Nagamune, Chikako Ohnishi, Ayako Katsuura, Yasunori Taoka, Kenzo Fushitani, Robert A. Whiley, Kikuji Yamashita, Akihiko Tsuji, Yoshiko Matsuda, Hiroki Kourai and Seiichiro Kitamura : Intermedilysin A cytolytic toxin specific for human cells of a Streptococcus intermedius isolated from human liver abscess, XIII Lancefield International Symposium on Streptococci and Streptococcal Diseases; In "Streptococci and The Host"(T. Horaud, A. Bouvet, R. Leclercq, H. de Montclos, M. Sicard, eds.): Advances in Experimental Medicine and Biology, 418, 773-775, Paris, Sep. 1997.
(Summary)
S. intermedius肝膿瘍分離株から単離されたヒト細胞特異的な細胞溶解毒素インターメディリシン(ILY)の性質を検討した．これまで知られていたチオール基修飾試薬への非感受性やコレステロールへの低感受性に加え，電子顕微鏡観察からILYは直径30-50nmの膜孔を形成し細胞を破壊することが示された．またILYの部分遺伝子構造解析の結果からILYはチオール活性化毒素群と同じ分子祖先から進化したものであることが示唆された．
Kenji Mori, 今牧 明義, Tetsuya Akamatsu, Akihiko Tsuji, Masami Nagahama and Yoshiko Matsuda : A novel human PACE4 isoform, PACE4E, is an active protease containing a hydrophobic cluster at the carboxy terminus, 17th International Congress of Biochemistry and Molecular Biology, San Francisco, Aug. 1997.
31.
Akihiko Tsuji, Chiemi Hine, Shigeru Yoshida, Miwa Bando, Kenji Mori, Tetsuya Akamatsu and Yoshiko Matsuda : Gene organization and Alternative Splicing of Human PACE4, Subtilisin-Like Proprotein Convertase, 17th International Congress of Biochemistry and Molecular Biology, FASEB.J., 11, 9, 1224, San Francisco, Aug. 1997.
32.
Hideaki Nagamune, Kazuaki Muramatsu, Masakatsu Higashine, Tetsuya Akamatsu, Yasuhiro Tamai, Yasuo Yonetomi, Akihiko Tsuji, Keisuke Izumi, Takemasa Sakaguchi, Tetsuya Yoshida and Yoshiko Matsuda : Cyclic AMP-inducible procalcitonin processing in thyroidal parafollicular cells is regulated by the Kexin family protease, PC1., International Symposium on Medical Aspects of Protease Inhibitors; In "Medical Aspects of Proteases and Protease Inhibitors": Biomedical and Health Research, 15, 22-33, Amsterdam, May 1997.
Tetsuya Akamatsu, 吉田 茂, 大黒 成夫, Hideaki Nagamune, 長濱 正巳, Akihiko Tsuji and Yoshiko Matsuda : Expression of a novel PACE4 isoform, PACE4E, in developmental rat brain, The 12th International Symposium of Federation of Asian and Oceanian Biochemists and Molecular Biologists, Tokushima, Jun. 1996.
34.
Akihiko Tsuji, Adrian Torres-Rosado, Toshihiro Arai, Shan-Ho Chou and Kotoku Kurachi : Characterization of hepsin, a membrane bound protease, Biomedica Biochimica Acta, 50, 4-6, 791-793, 1991.
(Summary)
Hepsin is a membrane bound protease of 51 kDa present in mammalian cells. It contains a stretch of hydrophobic sequence of 27 amino acid residues in its N-terminal region. By employing fluorescent immunostaining of cells and western blot analysis of the various cell subfractions, the catalytic subunit (carboxyl terminal half) of hepsin is found at the cell surface. The hepsin gene is expressed in most tissues of a young adult baboon, particularly in the liver at high levels.
Sun Xiaomei, Ye Yuxin, Sakurai Naofumi, Kato Koji, Yu Jian, Keizo Yuasa, Akihiko Tsuji and Yao Min : Characterization and ligand-binding manner of EHEP and BGL for producing biofuel from brown algae, 日本結晶学会 令和2年(2020年)度年会, Nov. 2020.
2.
Mika Nishida, Maki Shimada, Kenji Miyamoto, Akihiko Tsuji and Keizo Yuasa : ナトリウム利尿ペプチド受容体NPR-Cのグアニンヌクレオチド交換因子GEF-H1を介した新たなシグナル伝達機構, 日本農芸化学会2020年度中四国支部大会(第57回講演会), Sep. 2020.
3.
Mika Nishida, Kenji Miyamoto, Yuki Shimizu, Akihiko Tsuji and Keizo Yuasa : ナトリウム利尿ペプチド受容体NPR-Cの新たなシグナル伝達機構, 日本農芸化学会中四国支部第54回講演会, Jun. 2019.
Koji Kishimoto, Takashi Hraguchi, Kenji Shimizu, Aiko Yamaguchi, Toshitada Yoshihara, Mikiko Kishi, Munenori Ide, Tetsunari Oyama, Yoshito Tsushima, Seishi Yobita, Akihiko Tsuji and Takashi Izumi : A GPCR, G2A, regulates stemness of cancer stem cells, 第56回 日本生化学会 中国四国支部例会(口頭), 16, May 2015.
43.
宮本 賢治, 清水 友紀, 松田 真弥, Akihiko Tsuji and Keizo Yuasa : ナトリウム利尿ペプチド受容体Cの新規結合タンパク質同定, 日本農芸化学会2015年度大会, Mar. 2015.
44.
一色 衣香, 平瀬 大志, Akihiko Tsuji and Keizo Yuasa : Death-associated protein kinase-2 (DAPK2)と tubulinの相互作用解析及びアポトーシスへの関連性, 日本農芸化学会2015年度大会, Mar. 2015.
45.
Teruaki Ito, Yasuhiko Kawamura, Akihiko Tsuji, Masaki Hashizume and Toshihiro Moriga : Academic collaboration towards manufacturing system globalization, 日本機械学会生産システム部門研究発表講演会2015・講演論文集, 15, 8, 45-46, Mar. 2015.
(Summary)
TokushimaU-UTeM Academic Center (TMAC) has been successfully established in 2014 to promote the joint collaboration between the two institutions in terms of both education and research. One of the critical factors of this success was a good relationship between the two institutions based on the academic collaboration with alumni network in the past one decade. Presenting the overview of TMAC with its history and some of the critical on-going activities, this paper shows the feasibility of manufacturing system globalization through academic collaboration, and emphasized the importance of global engineer education.
松田 真弥, 小湊 恭平, 小出(吉田) 静代, 宮本 賢治, Akihiko Tsuji and Keizo Yuasa : PCTK3はcyclin A及びPKAによって活性調節を受け，アクチン動態を制御する, 日本農芸化学会2014年度大会, Mar. 2014.
56.
伊藤 千尋, 小出(吉田) 静代, Reger Albert, Akihiko Tsuji, Kim Choel and Keizo Yuasa : cGMP-dependent protein kinase II と小胞輸送制御因子 Rab11B との相互作用の解析, 日本農芸化学会2014年度大会, Mar. 2014.
Junki Fukumoto, Ismaliza Ismail Nor Mohd, 雅広 Inoue, Keizo Yuasa and Akihiko Tsuji : Oligopeptidase Bにおけるβ-プロペラドメインと触媒ドメインの相互作用に重要な塩橋の同定, 第85回日本生化学大会, Dec. 2012.
74.
Hyejin Kim, Atsushi Tabata, Toshifumi Tomoyasu, Tomomi Ueno, Naruhito Uchiyama, Keizo Yuasa, Akihiko Tsuji and Hideaki Nagamune : A study on the mechanism of osteoblast differentiation induced by estrogen-stimuli, 日本生化学会, Dec. 2012.
Akihiko Tsuji, Hiroshi Ishikawa and Keizo Yuasa : BMP活性化におけるPACE4とFurinの機能分担, 第16回日本病態プロテアーゼ学会, Aug. 2011.
95.
Junki Fukumoto, NOR MOHD BINTI ISMALIZA ISMAIL, Keizo Yuasa and Akihiko Tsuji : トリパノソーマ症の病原因子であるOligopeptidase Bのドメイン間相互作用の安定化機構, 第11回 日本蛋白質科学会年会, Jun. 2011.
96.
Genki Harita, Keizo Yuasa, 津嘉山 正夫 and Akihiko Tsuji : スダチ由来ポリフェノールsudachitinによる神経保護作用, 日本農芸化学会中四国支部 第30回講演会, May 2011.
97.
土肥 真, Keizo Yuasa, 長目 健 and Akihiko Tsuji : cGMP依存性プロテインキナーゼとRhoエフェクターrhotekinの相互作用部位および細胞内局在の解析, 第52回日本生化学会中国・四国支部例会, May 2011.
98.
Keiko Tominaga, Chika Ikeda, Ayumi Kondo, 佐藤 しおり, Keizo Yuasa and Akihiko Tsuji : アメフラシのセルロース消化システムの解析, 第52回日本生化学会中国・四国支部例会, May 2011.
99.
Takeru Mino, Yoshinori Fujisawa, Keizo Yuasa and Akihiko Tsuji : カイワレダイコンフェニルアラニルアミノぺプチダーゼの酵素特性解析及びクローニング, 第52回日本生化学会中国・四国支部例会, May 2011.
100.
Genki Harita, Keizo Yuasa, 津嘉山 正夫 and Akihiko Tsuji : Effect of the polymethoxyflavone in citrus sudachi on neuroprotection, 日本農芸化学会2011年度大会, Mar. 2011.
101.
Keizo Yuasa, 松田 泰斗 and Akihiko Tsuji : Functional regulation of transient receptor potential channel TRPC7 by cGMP-dependent protein kinase, 日本農芸化学会2011年度大会, Mar. 2011.
Takuya Yanagino, Keizo Yuasa, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : Transcriptional regulation of fibrillin-2 during chondrogenesis, 第78回日本生化学会大会, Oct. 2005.
153.
Tetsuya Masuda, Keizo Yuasa, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : Transcriptional activation of proprotein convertase PACE4 during skeletal muscle differentiation, 第78回日本生化学会大会, Oct. 2005.
154.
Akihiko Tsuji, Kentaro Ogawa, Tsuyoshi Yuasa, Keizo Yuasa, Masami Nagahama and Tadashi Yoshimoto : Protamine: a unique and potent inhibitor of oligopeptidase B, 第78回日本生化学会大会, Oct. 2005.
155.
Kentaro Ogawa, Keizo Yuasa, Masami Nagahama and Akihiko Tsuji : Expression of cysteine proteases and oligopeptidase B in early seedling growth of wheat, 第78回日本生化学会大会, Oct. 2005.
156.
Yayoi Kikuchi, Keizo Yuasa, Masami Nagahama, Yoshiko Matsuda and Akihiko Tsuji : The role of Reticulocalbin-3 in regulation of PACE4 activation and secretion, 第78回日本生化学会大会, Oct. 2005.
Keizo Yuasa, Kaori Suzue, Akihiko Tsuji and Yoshiko Matsuda : Transcriptional activation of proprotein convertase PACE4 by E2F1, 第77回日本生化学会大会, Oct. 2004.
162.
Akihiko Tsuji, Keizo Yuasa and Yoshiko Matsuda : Purification and characterization of oligopeptidase B from wheat germ. First identification of oligopeptidase B at the protein level in higer plant, 第77回日本生化学会大会, Oct. 2004.
163.
Yoshiko Matsuda, Akihiko Tsuji and Keizo Yuasa : Subtilisin like proprotein convertase familyの多様性と役割分担:ECM局在PACE4の細胞分化における役割, 第51回マトリックス研究会大会, 20-21, Apr. 2004.
Akihiko Tsuji, 菊池 弥生 and Yoshiko Matsuda : ズブチリシン様プロプロテインコンベルターゼ生合成におけるEF-Hand Ca結合タンパク，Reticulocalbin-like Protein (RLP)の機能同定, 第26回日本分子生物学会, Dec. 2003.
166.
Yayoi Kikuchi, Yukimi Sato, Akihiko Tsuji and Yoshiko Matsuda : Reticulocabin: A novel protein which regulates biosynthesis of subtilisin-like proprotein convertase, 第76回日本生化学会大会, Oct. 2003.
167.
Yoshiko Matsuda and Akihiko Tsuji : A unique 26-kDa cysteine protease induced in wheat embryo during germination, 第76回日本生化学会大会, Oct. 2003.
168.
Akihiko Tsuji, Hirotaka Makise and Yoshiko Matsuda : Antitrypsin mutants: Tool for functional analysis of serine-endopeptidases, 第76回日本生化学会大会, Oct. 2003.
169.
Akihiko Tsuji : 蛋白質の分析技術, 第40回分析化学講習会, 49-54, Aug. 2003.