Natriuretic peptide receptor-C releases and activates guanine nucleotide-exchange factor H1 in a ligand-dependent manner.

Biochemical and Biophysical Research Communications


Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing.

Acta Crystallographica. Section F, Structural Biology Communications


Identification and enzymatic characterization of clip domain serine protease in the digestive fluid of the sea hare, Aplysia kurodai.

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.

Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology


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

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.


Citrus peel polymethoxyflavones, sudachitin and nobiletin, induce distinct cellular responses in human keratinocyte HaCaT cells

Bioscience, Biotechnology, and Biochemistry


Intracellular localization and binding partners of death associated protein kinase-related apoptosis-inducing protein kinase 1.

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.


PCTK3/CDK18 regulates cell migration and adhesion by negatively modulating FAK activity

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.


Type II cGMP-dependent protein kinase negatively regulates fibroblast growth factor signaling by phosphorylating Raf-1 at serine 43 in rat chondrosarcoma cells.

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.


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

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.


Alisol B, a triterpene from Alismatis rhizoma (dried rhizome of Alisma orientale), inhibits melanin production in murine B16 melanoma cells

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.


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


Death-associated protein kinase 2 mediates nocodazole-induced apoptosis through interaction with tubulin

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.

Biochemical and Biophysical Research Communications


NVL2, a nucleolar AAA-ATPase, is associated with the nuclear exosome and is involved in pre-rRNA processing

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.


Suppression of death-associated protein kinase 2 by interaction with 14-3-3 proteins

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.


Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells.

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.


PCTAIRE Kinase 3/Cyclin-dependent Kinase 18 Is Activated through Association with Cyclin A and/or Phosphorylation by Protein Kinase A

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.


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

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.


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.

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.


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.

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.


Trypanosoma brucei 14-3-3I and II proteins predominantly form a heterodimer structure that acts as a potent cell cycle regulator in vivo.

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.


Engineering of α1-antitrypsin variants with improved specificity for the proprotein convertase furin using site-directed random mutagenesis

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.


Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination.

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.


Purification and characterization of cellulase from North Pacific krill (Euphausia pacifica). Analysis of cleavage specificity of the enzyme.

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.

Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology


Subtilisin-like proprotein convertase paired basic amino acid-cleaving enzyme 4 is required for chondrogenic differentiation in ATDC5 cells

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.


Structure of β-1,4-mannanase from the common sea hare Aplysia kurodai at 1.05 Å resolution.

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.

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications


cGMP-dependent protein kinase I promotes cell apoptosis through hyperactivation of death-associated protein kinase 2.

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.

Biochemical and Biophysical Research Communications


cGMP-dependent protein kinase I is involved in neurite outgrowth via a Rho effector, rhotekin, in Neuro2A neuroblastoma cells.

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.


Sudachitin, a polymethoxyflavone from Citrus sudachi, suppresses lipopolysaccharide-induced inflammatory responses in mouse macrophage-like RAW264 cells.

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.

Bioscience, Biotechnology, and Biochemistry


Identification of a plant aminopeptidase with preference for aromatic amino acid residues as a novel member of the prolyl oligopeptidase family of serine proteases.

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.


Functional regulation of transient receptor potential canonical 7 by cGMP-dependent protein kinase Iα.

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.


A critical role for highly conserved Glu610 residue of oligopeptidase B Tryoanosoma brucei in thermal stability

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.


Transcriptional regulation of cGMP-dependent protein kinase II (cGK-II) in chondrocytes.

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.


Production of n-octanoyl-modified Ghrelin in Cultured Cells Requires Prohormone Proceeding Protease and Ghrelin O-acyltransferase, as well as n-octanoic Acid

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.


Subtilisin-like Proprotein Convertase PACE4 is Required for Skeletal Muscle Differentiation

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.


UBXD1 is a VCP-interacting protein that is involved in ER-associated degradation

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.

Biochemical and Biophysical Research Communications


Transcriptional Regulation of Fibrillin-2 Gene by E2F Family Members in Chondrocyte Differentiation

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.


Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativus

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.


Isolation and Biochemical Characterization of Two Forms of RD21 from Cotyledons of Daikon Radish (Raphanus sativus).

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.


Trafficking of cGMP-dependent protein kinase II via interaction with Rab11

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.


Structural and biochemical studies on Pompe disease and a "pseudodeficiency of acid α-glucosidase"

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.


Transcriptional regulation of subtilisin-like proprotein convertase PACE4 by E2F: Possible role of E2F-mediated upregulation of PACE4 in tumor progression

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.


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

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.


The AAA-ATPase NVL2 is a component of pre-ribosomal particles that interacts with the DexD/H-box RNA helicase DOB1

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.

Biochemical and Biophysical Research Communications


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

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.


Pratamine: a unique and patent inhibitor of oligopeptidase B

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.


Identification of Oligopeptidase B in Higher Plants. Purification and Characterization of Oligopeptidase B from Quiescent Wheat Embryo, Triticum aestivum

小麦発芽過程において，著明に活性が上昇するセリンプロテアーゼを見出し，小麦胚芽より各種クロマトグラフィーによって単一に精製した．精製酵素はオリゴぺプチダーゼbに類似した酵素学的性質を有するとともに，その部分アミノ酸配列も大腸菌やトリパノゾーマオリゴぺプチダーゼbの配列と極めて相同性が高かった．以上の結果より，今までバクテリアやトリパノゾーマのみ存在すると考えられていたオリゴペプチダーゼbは高等植物にも存在することが蛋白レベルで証明された．


Molecular cloning and expression analysis of novel wheat cysteine protease

小麦発芽過程で著明に発現量が増加する新規のシステインプロテアーゼのクローニングを行った．得られたcDNAから推定されるアミノ酸配列はこのプロテアーゼがパパインファミリーに属する新規のシステインプロテアーゼであることを示した．このプロテアーゼは小麦，大麦に存在し，米，シロイヌナズナには存在しなかった．抗体を作成し，その蛋白レベルにおける発現を調べると，発芽2日目以降に著明に増加することが判明し，小麦の発芽過程における細胞の増殖，分化に寄与している可能性が高い．

Biochimica et Biophysica Acta (BBA) - General Subjects


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

培養細胞Bewoは絨毛癌に由来する培養細胞で，トロホブラスト細胞のモデル細胞として使われている．Bewo細胞にはPACE4が発現し，その発現はE-boxに結合するbHLH転写因子で制御されることを明らかにした．また，ヒト胎盤の免疫染色によってPACE4は胎盤トロホブラストの分化過程で発現量が変動することを見出した．


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

PACE4のヘパリンセファロースゲルに対する結合能を調べた．PACE4はヘパリンカラムに強く結合し，塩化ナトリウム1モル以上の濃度で溶出された．また，結合したPACE4はヘパリンで溶出されたが，コンドロイチン硫酸では全く溶出されなかった．PACE4のC末に塩基性アミノ酸に富んだヘパリン結合部位を同定した．さらに，培養細胞を用いてPACE4は細胞外マトリックスに存在するプロテオグリカン分子のヘパラン硫酸糖鎖に結合していることを証明した．

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics


The proprotein convertase PACE4 is upregulated by PDGF-BB in megakaryocytes: gene expression of PACE4 and furin is regulated differently in Dami cells

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.

血小板の分化過程において，巨核芽球から血小板への分化は多くのサイトカインや分化因子によって高度に制御されている．本研究では，巨核芽球のモデル細胞であるDami, HEL細胞を用いて，分化因子前駆体を活性化するプロプロテインコンベルターゼ(SPC)の発現制御について解析した．SPCの中で，特にPACE4は細胞自身から分泌されるPDGFによって著明に発現量が増加することが判明し，PACE4は血小板の分化において重要な役割を持つことが示された


Western blotting analysis of the beta-hexosaminidase alpha and beta-subunits in cultured fibroblasts frim cases of various forms of GM2 gangliosidosis

GM2ガングリオシドーシス患者皮膚培養繊維芽細胞のヘキソサミニダーゼ発現量や分子量をウエスタンブロット法で解析する方法を確立し，この方法は，簡便で正確なGM2ガングリオシドーシス診断法であることを証明した．


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

ズブチリシンプロプロテインコンベルターゼ(SPC)は，哺乳類では7種類が同定されているが，これらの酵素は類似した基質特異性を持ち，さらに複数の酵素がひとつの細胞で発現しているため，その生理機能解析は困難である．本研究では，これらSPCの機能解析のツールとして使用できる選択的阻害特性を有する阻害タンパクを作成することを目的として，アンチトリプシンのタンパク工学的改変を行い，その阻害特異性について検討した．SPCの中でPACE4とFurinを識別可能な改変体を作成することができた．


A Critical Role for the Carboxy Terminal Region of the Proprotein Convertase, PACE4A, in the Regulation of Its Autocatalytic Activation Coupled with Secretion

ズブチリン様プロプロテインコンベルターゼファミリーであるPACE4は，自己活性化されたのち分泌されるが，培養細胞で過剰発現しても，その自己活性化速度が非常に遅いため，分泌される活性型酵素の量が少ない．本研究では，PACE4のC末端側に自己活性化を制御する領域があることを証明した．

Biochemical and Biophysical Research Communications


Proprotein convertase PACE4 is down-regulated by the basic helix-loop-helix transcription factor hASH-1 and MASH-1

Mash-1は，神経分化に必須のbHLH型転写因子であるが，その標的遺伝子は不明である．本研究では，種々のMash-1発現量の異なるヒト神経芽細胞腫を用いて，Mash-1のズブチリシン様プロプロテインコンベルターゼであるPACE4発現に及ぼす影響について検討し，Mash-1はPACE4遺伝子上流のE-boxクラスタに結合し，そのプロモーター活性をネガティブに制御する転写因子であることを証明した．


Subtilisin-Like Proprotein Convertase, PACE4 and PC8, as Well as Furin, are Endogenous Proalbumin Convertases in HepG2 Cells

血清アルブミンはプロ体として肝臓で合成されたのち，細胞内でプロペプチドがプロセスされ血液中に分泌される．しかし このプロセシング機構についてはまだ不明な点が多い．本研究では，アンチセンスRNA法を用いて，ズブチリシン様プロプロテインコンベルターゼであるFurin, PACE4, PC8のアルブミンプロセシングにおける役割を解析し，Furinのみならず，PACE4, PC8もアルブミンプロセシングに関わっていることを証明した．


Inactivation of Proprotein Convertase, PACE4 by α1-Antitrypsin Portland (α1-PDX) which is a Blocker of Proteolytic Activation of Bone Morphogenetic Protein during Embryogenesis.

PACE4はSPCファミリーに属するプロテアーゼで，特に細胞分化過程でその発現量が高度に制御されているが，その機能に関しては不明な点が多い．本研究では，BMPの活性化を阻害し，細胞分化を抑制するアンチトリプシン改変体が，PACE4に対して強い阻害作用を持つことを証明し，PACE4がBMPプロセシングプロテアーゼとして機能していることを示唆した．またPACE4の酵素学的性質についても検討した．


Human Subtilisin-Like Proprotein Convertase, PACE4 (SPC4) Gene Expression Is Highly Regulated through E-Box Elements in HepG2 and GH4C1 Cells

ヒトPACE4の発現制御機構を明らかにするため，その遺伝子5'上流域の構造，機能解析を行った．ヒトPACE4遺伝子上流域には多くの転写因子結合サイトが認められたが，特に上流1kbにE-box配列が12個存在した．リポータージーンアッセイにより，E4-E9のE-boxクラスタが強力な負のエンハンサーとして機能していることを明らかにした．E-boxに結合するbHLH型転写因子は細胞分化を制御するものが多く，PACE4と細胞分化の関わりを示唆した．


Biosynthetic processing and quoternary interactions of proprotein convertase SPC4 (PACE4).

ズブチリン様プロプロテインコンベルターゼファミリーに属するPACE4の活性化機構について解析した．プロPACE4のプロペプチドの切断はPACE4の活性基を変異した不活性体では起こらず，また不活性体と活性体を共発現しても不活性体のプロセシングがなかったことから，このプロセシングは分子内自己活性化であることを証明した．また自己活性化はPACE4の分泌に必須であり，PACE4の活性化と細胞内移行は共役していることを明らかにした．


Biosynthetic processing and quaternary interactions of proprotein convertase SPC4 (PACE4).


Intermedilysin A cytolytic toxin specific for human cells of a Streptococcus intermedius isolated from human liver abscess in "Streptococci and The Host"

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．

Advances in Experimental Medicine and Biology


Genomic organization and alternative splicing of human PACE4 (SPC4), Kexin-like processing endoprotease.

本研究ではプロセシング酵素Kexin familyの1つPACE4のヒトゲノムを単離し, その構造を解析し, ファミリーの中でも最も大きく複雑な構造であること, 多種類存在するアイソフォームが全て同一遺伝子から選択的スプライシングにより生じること, およびプロモーター領域がユニークな構造であること等を初めて明らかにした.


Developmental expression of a novel Kexin family protease, PACE4E, in the rat olfactory system.

本研究では新規Kexin family proteaseであるPACE4Eがラット脳発生過程において時期·空間特異的に発現することを明らかにし, 特に嗅球においては僧帽細胞に極めて特異的に発現し, 僧帽細胞の分化成熟と密接に関わることを見い出した.


A Novel Human PACE4 Isoform, PACE4E Is an Active Processing Protease Containing a Hydrophobic Cluster at the Carboxy Terminus

ズブチリシン様プロプロテインコンベルターゼファミリーに属するPACE4の新規アイソフォームPACE4E-I, PACE4E-IIをヒト脳cDNAライブラリーよりクローニングした．PACE4Eは，PACE4Aと比べ，C末に75残基からなる，疎水性アミノ酸のクラスタを含むユニークな配列を持っていた．この部分を欠失したPACE4Eは分泌速度が上昇した．共発現実験によって，PACE4E-1, E-IIは，PACE4Aと同様にプロセシング活性を持っていることが証明された．


Cyclic AMP-inducible procalcitonin processing in thyroidal parafollicular cells is regulated by the kexin family protease, PC1

甲状腺におけるカルシトニンのプロセシングに関与するズブチリシン様プロプロテインコンベルターゼを同定するため，特異抗体による免疫染色とモデル細胞TT細胞におけるcAMPの影響について調べた．カルシトニン産生細胞で発現していたのは，主にPC1とPC2であったが，カルシトニンの産生を刺激するcAMPによって発現が増加したのは，PC1であった．これらの結果は，PC1がプロカルシトニンの活性化酵素であることを示している．


Intermedilysin, a novel cytotoxin specific for human cells, secreted by Streptococcus intermedius UNS46 isolated from a human liver abscess

S. intermediusのヒト肝膿瘍分離株が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)を分泌することを発見した． ILYは約54KDaの蛋白で，その部分一次構造はニューモリシンと高い相同性を示した． しかしSH修飾試薬により失活せず，コレステロール対しても感受性が低かった． またトリプシン処理したヒト赤血球はILYに対して抵抗性を示すようになることから，ILY作用には宿主細胞膜の蛋白質が関与している事が示された．


Purification and characterization of a novel serine proteinase from the microsomal fraction of bovine pancreas.

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．


Novel members of mammalian kexin family proteases, PACE4C, PACE4D, PC7A and PC7B.


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

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

X染色体劣性遺伝病である筋ジストロフィーとオルニチントランスカルバミラーゼ欠損症について，その患者と家系のRFLP解析を行った．白人のRFLP解析で有用なプローブは，日本人においても有効であり，人種による差は認められなかった．この方法は，キャリアー診断においても有用であることが判明した．


Distribution of the kexin family proteases in pancreatic islets. --- PACE4C is specifically expressed in B cells of pancreatic islets

Kexin Family Proteasesはペプチドホルモン等のプロセシング酵素として同定され, 血糖調節ホルモンのインスリン等のプロセシングにも関与することが示唆されていたが, 本研究では我々が新規にクローニングしたPACE4CをはじめとするKexin Family Proteasesとインスリン等との膵島細胞での局在, 共存関係を初めて明らかにし, その成果は雑誌表紙にも掲載された.


A Novel Member, PC7, of The Mammalian Kexin-Like Protease Family --- Homology to PACE4A, Its Brainspecific Expression and Identification of Isoforms

哺乳類には7種類のKexin様プロプロテインコンベルターゼが存在する．哺乳類Kexin様プロテアーゼ間で保存された触媒領域のプライマーを用い，ラット脳下垂体からcDNA断片をPCR増幅し，これをプローブにしてラット脳下垂体cDNAライブラリーからPC7をクローニングした．PC7(PC7AとPC7B)はRXKRの活性化サイトを持ち，触媒，HomoB及びシステインリッチ領域を持っており，PACE4Aと非常に高いホモロジーを示した．PC7は脳で強く発現し，肝，腎，心，脾では検出されなかった．

Biochemical and Biophysical Research Communications


The tissue distribution of mRNAs for the PACE4 isoforms, kexin-like processing protease: PACE4C and PACE4D mRNAs are major transcripts among PACE4 isoforms

新たに見いだされたPACE4アイソフォーム(PACE4CとPACE4D)の組織分布をRT-PCR法を用いて解析した． 両アイソフォームのmRNAともに，PACE4Aと同様に多くの組織で見いだされたが，それぞれの発現パターンは異なっていた．また，PACE4C及びPACE4DのmRNAはPACE4Aのそれより遙かに多く，PACE4 mRNAの主要な部分はPACE4CとPACE4DのmRNAである事が示唆された．


Lysosomal enzyme replacement using α2-macroglobulin as a transport vehicle

α2-マクログロブリン(α2M)を運搬担体に用いたリソソーム酵素欠損性遺伝病の酵素補充療法システムを開発した． α2Mはレセプターを介してリソソームに取り込まれることから，豚肝臓から精製した α-グルコシダーゼ或いはα-ガラクトシダーゼにα2Mを運搬担体として二価架橋試薬で連結し，リソソーム指向性の酵素補充療法剤を構築した． これを用いてin vitro実験を行い，ポンペ病及びファブリー病細胞の細胞病態が改善されることを証明した．


Identification of Novel cDNAs Encording Human Kexin-like Protease, PACE4 Isoforms

ヒト胎盤cDNAライブラリーより，新規のPACE4アイソフォーム，PACE4CとPACE4Dをクローニングし，塩基配列を決定した．PACE4Cは，PACE4AのC末に存在するシステインリッチ領域を持たず，ホモBドメインのC末にC-特異的配列を持っていた．PACE4Dはシグナルペプチド，プロペプチドおよびシステインリッチ領域を欠失していた．


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.

血清蛋白質であるα-マクログロブリン(αM)は種々の酵素に対するインヒビターとして機能すると考えられていたが, 生体内で標的となる酵素は不明であった. 本研究ではαMが酵素と複合体形成後, リソゾームに運ばれ分解されることに着目し, リソゾーム内の分解を抑制したラット肝臓リソゾームよりαM-酵素複合体を単離し, 肥満細胞トリプターゼが生体内でαMの標的酵素の1つであることを初めて明らかにした.


Hepsin, a putative cell-surface serine protease, is required for mammalian cell growth

細胞膜結合セリンプロテアセーゼ，ヘプシンの生理機能解析のため，抗体による阻害，またはアンチセンスRNAによる発現阻害の細胞に及ぼす影響を調べた．ヘプシンの活性を現象させると，著名な細胞増殖の抑制が見られ，ヘプシンは細胞増殖を制御するプロテアーゼであることが明らかになった．

Proceedings of the National Academy of Sciences of the United States of America


Hepsin, a cell membrane-associated protease --- characterization, tissue distribution and gene localization

ヘプシンはトリプシン様セリンプロテアーゼの活性セリン周辺の保存された配列から設計されたオリゴヌクレオチドプローブによって，肝臓からクローニングされたが，その機能に関しては全く不明である．本研究では，染色体局在，ノーザンブロットによる組織分布，ペプチド抗体によるヘプシンタンパクの同定を行った．ヘプシン遺伝子は第19染色体に局在し，特に肝臓で強く発現していた．免疫染色によって，ヘプシンは膜結合酵素として細胞膜に発現していることが判明した．


The major component of a large, intracellular proteinase accumulated by inhibitors is a complex of alpha-2 macroglobulin and thrombin

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.

プロテアーゼ阻害剤存在下で培養した細胞に蓄積する高分子量のプロテアーゼは，α2マクログロブリンとスロンビンの複合体であることを証明した．

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology


Biosynthesis of human alpha-N-acetylgalactosaminidase: Defective phosphorylation and maturation in infantile alpha-NAGA deficiency

正常者およびα-N-アセチルガラクトサミニダーゼ欠損症患者繊維芽細胞におけるα-N-アセチルガラクトサミニダーゼの生合成を解析した．正常者では分子量52,000の前駆体として合成された後，リン酸化された49,000の成熟体に変換したが，患者細胞では前駆体が合成されたが，リン酸化されず，細胞内で分解された．

Biochemical and Biophysical Research Communications


Abnormal protein spots revealed by two-dimensional electrophoresis in mycoplasma-infected human fibroblasts

培養細胞は病因蛋白質の解析に使われるが，マイコプラズマに感染した細胞では，その解析が困難になる．本研究では，マイコプラズマ感染によって特異的に発現が誘導される蛋白6種類を同定した．


Isolation and characterization of a novel large protease accumulated mammalian cells in the presence of inhibitors


Purification and characterization of beta-mannosidase from human placenta

ヒト胎盤から約10,000倍にβマンノシダーゼを精製した．分子量は110,000で，等電点は4.7であった．主に高マンノース型糖鎖うぃ持っていた．至適pHは，pH 4.5であった．


Two-dimentional electrophoresis aided personal computer analusis for screening of mutant proteins in inherited diseases

2次元電気泳動を用い，正常者と先天代謝異常症患者皮膚繊維芽細胞の発現蛋白質の違いを検討した．泳動パターンをコンピューターに取込み，移動度が異なる蛋白，発現量に差がある蛋白を敏速に検出できる方法を開発した．


Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors

トリプシン様セリンプロテアーゼおよびシステインプロテアーゼ阻害剤であるロイペプチン存在下で培養したBHK細胞に分子量の大きなプロテアーゼが蓄積することを見いだし，単一に精製し，その酵素学的性質について検討した．分子量約700,000で，トリプシン様活性を持ち，阻害剤を培養液から除くと敏速に細胞内で分解された．


Cloning, sequencing, and expression of cDNA for human beta-galactosidase

完全長ヒトβガラクトシダーゼcDNAをヒト胎盤cDNAライブラリーより，抗体を用いたスクリーニング法でクローニングした．677アミノ酸からなる配列が推定された．7か所のN型糖鎖結合サイトが存在した．また培養細胞で，このcDNAを発現させ，βガラクトシダーゼ蛋白の発現と活性の上昇を確認した．このcDNAによって，GM1-ガングリオシドーシス症の遺伝学的研究が可能となった．


Solubilization of Membrane-Bound Acid α-Glucosidase by Proteolysis


Intracelluler Transport of Acid α-Glucosidase in Human Fibroblasts: Evidence for Involvement Phosphomannosyl Receptor-Independent System

本論文は，正常Ⅰ-cell病皮膚繊維芽細胞におけるα-グルコシダーゼ及びβ-ヘキソサミニダーゼの活性，細胞内局在性を比較したものである．Ⅰ-cell病患者は，多くのリソゾーム酵素の活性が低下する．しかし酸性α-グルコシダーゼの活性，細胞内局在性はⅠ-cell病細胞においても異常が認められず，本酵素のリソゾーム転送機構は，従来報告されているホスホマンノシルレセプター系とは異なる可能性が示唆された．


Km mutant of acid α-glucosidase in a case of cardiomyopathy without signs of skeletal muscle involvement

本論文は，16歳時に不整脈と診断され，17歳時に急性心不全で死亡した患者の酸性α-グルコシダーゼを生検試料，皮膚線維芽細胞を用いて研究したものである．その結果，患者の酸性α-グルコシダーゼはグリコーゲンに対するKm値が，正常酵素に比べ異常に高いことが判明し，この症例は今まで報告されていない新しいタイプのKm変異酸性α-グルコシダーゼによる糖原病Ⅱ型であることがわかった．


Galactosialidosis: molecular heterogeneity in biosynthesis and processing of peotective protein for β-galactosidase

本論文は，ガラクトシアリドーシス患者皮膚線維芽細胞におけるβ-ガラクトシダーゼの保護タンパク(プロテクティブプロテイン)の生合成，プロセッシングについて詳細に検討したものである．


Restriction fragment length polymorphism on the short arm of X-chromosome among the Japanese population

日本人におけるX染色体短腕DNAプローブ(13種類)の制限酵素断片長多型性について，白人の多型性と比較した．プローブ782，754は，得られる断片は白人の結果より長く，またプローブC7, pERT87-8, XJ5.1は断片の比率が白人の場合と全く異なっていた．これらの結果は，RFLP解析で使用されるプローブは人種の違いを考慮する必要があることを示した．


I-cell disease: evidence for a mannose 6-phospahte independent pathway for translocation of lysosomal enzymes in lymphoblastoid cells

I-cell病患者と正常者のリンパ芽球細胞を培養し，リソソーム酵素の動態について解析，検討した．I-cell病患者皮膚繊維芽細胞ではほとんどのリソソーム酵素活性は著明に低下するが，リンパ芽球細胞ではガラクトシダーゼ以外の酵素活性はほぼ正常であり，細胞内局在性やプロセシングも正常細胞と差は見られず，マンノース6リン酸経路以外のリソソーム酵素局在化機構の存在が示唆された．


DNA deletion and recombination in the gene locus of X-linked muscular dystrophies

日本人のドゥシェンヌ型，ベッカー型筋ジストロフィー患者のRFLP解析結果をまとめた．日本人も白人と同様の比率で，遺伝子欠損または交差がみられた．また 一つの家系では，ジストロフィーのプローブでは異常はないが，その近傍で遺伝子欠損が認められた．


Biosynthesis of two components of human acid α-glucosidase

本論文は，酸性α-グルコシダーゼの生合成過程を詳細に検討するために分子量の異なる2種類の本酵素(分子量8万と7万1千)を分離精製し，両タンパクの物理化学的，タンパク化学的，免疫学的性質を比較したものである．更に，ヒト線維芽細胞の標識実験により，80K，71K分子の合成過程も検討した．その結果，両酵素は同一前駆体からプロセスされるものの，アミノ末端部分に差がみられることがわかった．


The precursor of acid α-glucosidase is synthesized as a membrane bound enzyme

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.

本論文は，ヒト皮膚線維芽細胞を[3H]ロイシンで標識後，経時的に細胞分画を行い，次に特異抗体を用いて酸性α-グルコシダーゼを分離し，本酵素の生合成過程における分子量の変化，細胞内局在の変化を調べたものである．その結果酸性α-グルコシダーゼは，膜結合酵素としてまず先駆体が合成され，以後のプロセシングにより可溶化され成熟体に変換されることがわかった．


Purification of human placental acid α-mannosidase by an immunological method

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.

本研究は，プロテアーゼ阻害剤共存下，抗体カラムを用いて敏速に酸性α-マンノシダーゼを精製したものである．この研究によって，従来の論文で報告されているα-マンノシダーゼのDEAE-カラムクロマトにおける多様性は，精製過程におけるプロテアーゼによる分解の結果によることが明らかとなった．


Galactosialidosis: A direct evidence that 49-kilodalton protein restores deficient enzyme activities in fibroablast

本研究は，ガラクトシアリドーシス患者で，欠損する分子量49kDaのタンパク質を，正常繊維芽細胞培養液より分離後，患者細胞に加え，患者細胞中のβ-ガラクトシダーゼ，シアリダーゼ活性の変化を調べたものであり，49kDaタンパク質はβ-ガラクトシダーゼ，シアリダーゼの安定化に寄与していることが分かった．

Biochemical and Biophysical Research Communications


A simple differential immunoprecipitation assey of urinary asid and neutral α-glucosidase for glucogenosisII

本論文は，糖源病Ⅱ型の診断法として，酸性α-グルコシダーゼ特異抗体を用いた尿中の酸性及び中性グルコシダーゼ活性測定法を開発したものである．本法によって，他の方法で糖源病Ⅱ型と診断されている患者尿と正常者の酸性α-グルコシダーゼ活性を測定すると，患者尿では活性がみられず，本法がこの遺伝病の診断に有効であることが判明した．


Galactosialidosis: Studies on residual enzymes in early and late onset clinical phenotypes

本論文は，β-ガラクトシダーゼ，シアリダーゼの欠損するガラクトシアリドーシスの乳児型，成人型患者皮膚繊維芽細胞の残存β-ガラクトシダーゼ，シアリダーゼの性質を比較したものである．乳児型，成人型とも著明に両酵素活性が低下しているものの，有意に乳児型に比べ成人型で両活性が高かった．

Journal of Clinical Biochemistry and Nutrition


Rapid and simple isolation procedure for three component enzyme of pig heart 2-oxoglutarate dehydrogenase complex

本研究は，高速液体クロマトグラフィーを用いて敏速かつ簡単に，ブタ心筋より2-オキソグルタル酸脱水素酵素複合体の3つの成分酵素を精製する方法を開発したもので，この方法により，従来の方法より高収量で，3つの各成分酵素を分離，精製することが可能となった．

Journal of Nutritional Science and Vitaminology


Differences in the sugar chains of two subunits and isozymic forms of rat kidney γ-glutamyltranspeptidase

本研究は，ラット腎γ-グルタミルトランスぺプチダーゼを構成する2種のサブユニットの糖鎖構造を各々明らかにしたものである．


Biosynthesis and degradation of gamma-glutamyltranspeptidase of rat kidney

ラット腎臓γ-glutamyltranspepidaseの生合成，分解について調べた．ラットに放射性アミノ酸を投与し，蛋白質を標識後，抗体によって腎臓のγ-glutamyltranspepidaseを分離し，生合成について解析した．その結果，本酵素は一本鎖で合成された後，重鎖と軽鎖にプロセスされ，細胞膜に局在化することが明らかになった．


Structural studies of the carbohydrate moieties of rat kidney γ-glutamyltranspeptidase

本研究は，ラット腎γ-グルタミルトランスペプチダーゼの糖鎖構造を調べたもので，極めて多様性に富む糖鎖構造の概要が明らかにされた．


Studies on the structure of γ-glutamyltranspeptidase. III. Evidence that the amino terminus of the heavy subunit is membrane binding segment

γ-グルタミルトランスペプチダーゼの膜結合様式について詳細に検討し，ヘビーサブユニットのアミノ末端付近の疎水性アミノ酸に富む部分が膜との結合に関与していることを明らかにした．


Immunological cross reactivity of γ-glutamyltranspeptidase from human and rat kidney, liver and bile

本研究は，ヒト腎及びラット腎よりγ-グルタミルトランスペプチダーゼを精製し，各々特異抗体を調製したのち，ラット腎，肝及びヒト腎，胆汁中のγ-グルタミルトランスペプチダーゼの免疫学的性質を比較検討した．


Studies on the structure of γ-glutamyltranspeptidase. I. Correlation between sialylation and isozymic

本研究は，γ-グルタミルトランスペプチダーゼの等電点多様性とシアル酸含量の関係を調べたもので，等電点の多様性は，タンパク部分ではなく糖鎖中のシアル酸含量の違いによることを明らかにした．


Studies on the structure of g-glutamyltranspeptidase. II. Location of the segment anchoring γ-glutamyltranspeptidase to the membrane

本研究は，γ-グルタミルトランスペプチダーゼの膜結合様式について調べたもので，ヘビーサブユニットのアミノ末端付近に膜結合部位があることを明らかにした．


Characterization of human serum γ-glutamyltranspeptidase

本研究は，ヒト血清，胆汁中のγ-グルタミルトランスペプチダーゼの構造比較を行い，本酵素の血液·胆汁中への遊出機序について検討した．


Purification and characterization of γ-glutamyltranspeptidase from human bile

本研究は，ヒト胆汁よりγ-グルタミルトランスペプチダーゼを単一に精製し，その性質を調べた．


Studies on liver argininosuccinate synthetase in a patient with citrullinemia and in normal subjects

本研究は，肝生検試料を用いてシトルリン血症患者のアルギノコハク酸合成酵素の性質を正常者酵素と比較したもので，患者酵素の動力学的性質に異常を認め，臨床症状と酵素異常の関係を論じた．


本研究は，実験的肝炎，脂肪肝のウサギ血清オルニチントランスカルバミルトランスフェラーゼ活性を調べ，血清オルニチントランスカルバミルトランスフェラーゼ活性値は，脂肪肝の指標になることを示した．


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

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

本論文では，新薬開発における新規標的分子の同定のための脳神経特異的遺伝子産物のプロテオミクス解析に関する成果を報告した．

Bulletin of Faculty of Engineering, The University of Tokushima


Proteolytic Cleavage of the Neural Cell Adhesion Molecule L1 by Subtilisin-Like Proprotein Convertases

L1蛋白質は神経の分化，機能と関係していると考えられている接着蛋白である．L1蛋白質はプロセス部位にSubtilisin-like proprotein convertaseの切断配列である塩基性アミノ酸対を持っているが，そのプロセシング機構は不明である．本研究では，L1蛋白質のプロセシングに関与するプロテアーゼについて，特にSPCについて検討した．培養細胞でL1蛋白質前駆体とSPC (furin, PACE4, PC6, PC1, PC4, PC8)を共発現すると，furin, PACE4, PC6によってプロセシングされることが判明した．

Keio University Symposia for Life Science and Medicine