Papers by Takayuki Nemoto
Science, Aug 9, 1985
Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and ... more Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. Comparison of the amino acid sequence data for seven different subunits of arthropod hemocyanins from crustaceans and chelicerates shows many highly conserved residues and extensive regions of near identity. This correspondence can be matched closely with the three domain structure established by x-ray crystallography for spiny lobster hemocyanin. The degree of identity is particularly striking in the second domain of the subunit that contains the six histidines which ligate the two oxygen-binding copper atoms. The polypeptide architecture of spiny lobster hemocyanin appears to be the same in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, about 540 to 600 million years ago.
Journal of Biochemistry, Aug 1, 1996
The 94-kDa glucose-regulated protein (GRP94) is a member of the 90-kDa heat-shock protein (HSP90)... more The 94-kDa glucose-regulated protein (GRP94) is a member of the 90-kDa heat-shock protein (HSP90) family. In this study, we expressed the barley (Hordeum vulgare L.) GRP94 and the a isoform of human HSP90 (HSP90a) in Escherichia coli and compared their dimer-forming abilities. Native polyacrylamide gel electrophoresis revealed that GRP94 (amino acids 69-809) and the full-length form of HSP90a existed in the dimeric state. The C-terminal 326 amino acids of GRP94 or the C-terminal 200 amino acids of HSP90a were sufficient for the dimerization. Limited proteolysis of the C-terminal half of GRP94 with thrombin revealed a 16-kDa fragment, which was derived from the C-terminus of GRP94 through the cleavage of either the Arg710-ffis711 or the Arg735-Leu736 bond. These cleavage sites were nearly, if not completely, equivalent to the proteolyzed region of HSP90a. Their structural similarity prompted us to investigate, by use of a coexpression system, the possibility that the two proteins form a heterodimeric complex. A two-step affinity chromatography that specifically trapped only the complex revealed that the C-terminal 200 amino acids of HSP90a and the C-terminal 326 amino acids of GRP94 associated with HSP90a and GRP94, respectively. However, the C-terminal 326 amino acids of GRP94 failed to form a complex with HSP90a. In conclusion, these results indicate the similarity of the general dimeric conformation of the two HSP90 family member proteins, but show that the similarity is not sufficient to allow heterodimer formation.
Analytical Biochemistry, 1998
reaction volume is to be kept to a minimum and the alkaline digestion is from either a membrane b... more reaction volume is to be kept to a minimum and the alkaline digestion is from either a membrane blot or in solution, then the use of a mineral oil overlay should be considered to prevent concentration of the alkali and hydrolysis of the phosphate moiety.
Nihon Shishubyo Gakkai Kaishi (Journal of the Japanese Society of Periodontology)
Cell Biochemistry and Function, 2019
There are two types of bisphosphonates (BPs), nitrogen-containing (N-BPs) and those free from nit... more There are two types of bisphosphonates (BPs), nitrogen-containing (N-BPs) and those free from nitrogen (non-N-BPs). Although N-BPs show greater inhibition of bone resorption than non-N-BPs, their effects are likely accompanied with inflammation, which non-N-BPs mitigate. We examined the competitive effects of zoledronate (ZOL), an N-BP, and etidronate (ETI), a non-N-BP, in osteoblasts. ZOL, but not ETI, markedly reduced alkaline phosphatase activity and cell viability in osteoblastic MC3T3-E1 and Saos2 cells, while that inhibition was relieved by simultaneous administration of ETI, possibly because of competition with ZOL for cellular uptake. However, phosphonoformate, an inhibitor of the phosphonate transporters SLC20A and SLC34A, did not mitigate the reducing effects of ZOL, suggesting that those transporters are not involved in BP uptake in osteoblastic cells. Additionally, ZOL reduced fibroblastic NIH3T3 and C3H10T1/2 cell viability, which was relieved by administration of both ETI and phosphonoformate. Transporter gene expression levels were significantly lower in osteoblasts as compared to fibroblasts, which may account for the distinct effects of phosphonoformate with different cell types. Together, our results suggest existence of a common uptake route of N-BPs and non-N-BPs into osteoblastic cells that is unrelated to the SLC20A and SLC34A families.
Journal of Biological Chemistry, 1997
Domain structures of the 90-kDa heat-shock protein (HSP90) have been investigated with a library ... more Domain structures of the 90-kDa heat-shock protein (HSP90) have been investigated with a library of anti-HSP90 monoclonal antibodies (mAbs) and by limited proteolysis with trypsin and chymotrypsin. Thirty-three mAbs were obtained by immunization with bacterially expressed human HSP90␣ and HSP90 isoforms. Among them, ten and three mAbs reacted specifically with HSP90␣ and HSP90, respectively. Immunoblotting and enzyme-linked immunosorbent analyses revealed that major immunogenic domains were located at two restricted regions of HSP90␣, i.e. amino acids 227-310 (designated Region I) and 702-716 (Region II), corresponding to a highly charged region and a region near the C terminus, respectively. Taken together with the characteristics of the amino acid sequences, these two immunogenic regions appeared to be exposed at the outer surface of HSP90. We further investigated the domain structures of HSP90 by limited proteolysis in combination with N-terminal sequencing and immunoblotting analyses. Tryptic cleavages of HSP90␣ at low concentrations revealed the existence of major susceptible sites at Arg 400-Glu 401 , Lys 615-Ala 616 , and Arg 620-Asp 621. Proteolysis at higher trypsin concentrations caused successive cleavages only toward the N-terminal direction from these sites, and Region I was included in the region selectively deleted during this process, thereby further suggesting its surface location. From these results, we propose three domain structures of HSP90 consisting of amino acids 1-400, 401-615, and 621-732. Differences in the protease sensitivity and immunogenicity further suggest that every domain is composed of two subdomains. This is the first study describing the domain structures and the immunogenic regions of HSP90.
Journal of Biochemistry, 2004
FEMS Microbiology Letters
Multiple dipeptidyl-peptidases (DPPs) are present in the periplasmic space of Porphyromonas gingi... more Multiple dipeptidyl-peptidases (DPPs) are present in the periplasmic space of Porphyromonas gingivalis, an asaccharolytic periodontopathic bacterium. Dipeptides produced by DPPs are presumed to be transported into the bacterial cells and metabolized to generate energy and cellular components. The present study aimed to identify a transporter responsible for dipeptide uptake in the bacterium. A real-time metabolic analysis demonstrated that P. gingivalis preferentially incorporated Gly–Xaa dipeptides, and then, single amino acids, tripeptides and longer oligopeptides to lesser extents. Heterologous expression of the P. gingivalis serine/threonine transporter (SstT; PGN_1460), oligopeptide transporter (Opt; PGN_1518) and proton-dependent oligopeptide transporter (Pot; PGN_0135) genes demonstrated that Escherichia coli expressing Pot exclusively incorporated Gly–Gly, while SstT managed Ser uptake and Opt was responsible for Gly–Gly–Gly uptake. Dipeptide uptake was significantly decreas...
Japanese Journal of Oral Biology, 1985
In the submandibular gland of intact mice , there is a sex diffecence in cytosol androgen recepto... more In the submandibular gland of intact mice , there is a sex diffecence in cytosol androgen receptor level, being higher in females. The exchange assay using mersalyl and monothioglycerol revealed that cytosol androgen receptor in both sexes is mostly unoccupied. To understand the action of androgen via androgen receptor in the submandibular gland , cytosol androgen receptor level and androgen-dependent N-tosyl-L-arginine methyl ester esterase (TAMEase) activity, being higher in males were determined under endocrine manipulations. In males, castration elevated unoccupied cytosol androgen receptor level and reduced the TAMEase activity. Injection of testosterone decreased unoccupied cytosol androgen receptor level and increased TAMEase activity in females, whereas injection of estradiol exerted no effects on cytosol androgen receptor level and TAMEase activity in males.
Journal of Biological Chemistry, 2022
Dipeptide production from extracellular proteins is crucial for Porphyromonas gingivalis, a patho... more Dipeptide production from extracellular proteins is crucial for Porphyromonas gingivalis, a pathogen related to chronic periodontitis, because its energy production is entirely dependent on the metabolism of amino acids predominantly incorporated as dipeptides. These dipeptides are produced by periplasmic dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11. Although the substrate specificities of these four DPPs cover most amino acids at the penultimate position from the N-terminus (P1), no DPP is known to cleave penultimate Gly, Ser, Thr, or His. Here we report an expanded substrate preference of bacterial DPP7 that covers those residues. MALDI-TOF MS analysis demonstrated that DPP7 efficiently degraded incretins and other gastrointestinal peptides, which were successively cleaved at every second residue, including Ala, Gly, Ser, and Gln, as well as authentic hydrophobic residues. Intravenous injection of DPP7 into mice orally administered glucose caused declines in plasma GLP-1 and insulin, accompanied by increased blood glucose levels. A newly developed coupled enzyme reaction system that uses synthetic fluorogenic peptides revealed that the P1' and P2' residues of substrates significantly elevated kcat values, providing an expanded substrate preference. This activity enhancement was most effective toward the substrates with non-favorable but non-repulsive P1 residues in DPP7. Enhancement of kcat by prime-side residues was also observed in DPP11, but not DPP4 and DPP5. Based on this expanded substrate specificity, we demonstrate that a combination of DPPs enables proteolytic liberation of all types of N-terminal dipeptides, and ensures P. gingivalis growth and pathogenicity.
Running title, 2 roles of the C-terminal domain of HSP90
Title Determination of three amino acids causing alteration of proteolytic activities of staphylo... more Title Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases. Author(s) Nemoto, Takayuki K; Ono, Toshio; Shimoyama, Yu; Kimura, Shigenobu; Ohara-Nemoto, Yuko Citation Biological chemistry, 390(3), pp.277-285; 2009 Issue Date 2009-03 URL http://hdl.handle.net/10069/23192 Right Walter de Gruyter. NAOSITE: Nagasaki University's Academic Output SITE
Porphyromonas gingivalis, a pathogen of chronic periodontitis, is an asaccharolytic microorganism... more Porphyromonas gingivalis, a pathogen of chronic periodontitis, is an asaccharolytic microorganism that solely utilizes nutritional amino acids as its energy source and cellular constituents. The bacterium is considered to incorporate proteinaceous nutrients mainly as dipeptides, thus exopeptidases that produce dipeptides from polypeptides are critical for survival and proliferation. We present here an overview of dipeptide production by P. gingivalis mediated by dipeptidyl‐peptidases (DPPs), e.g., DPP4, DPP5, DPP7, and DPP11, serine exopeptidases localized in periplasm, which release dipeptides from the N‐terminus of polypeptides. Additionally, two other exopeptidases, acylpeptidyl‐oligopeptidase (AOP) and prolyl tripeptidyl‐peptidase A (PTP‐A), which liberate N‐terminal acylated di‐/tri‐peptides and tripeptides with Pro at the third position, respectively, provide polypeptides in an acceptable form for DPPs. Hence, a large fraction of dipeptides is produced from nutritional polypep...
Analytical biochemistry, 2018
Bacterial dipeptidyl-peptidase (DPP) 7 liberates a dipeptide with a preference for aliphatic and ... more Bacterial dipeptidyl-peptidase (DPP) 7 liberates a dipeptide with a preference for aliphatic and aromatic penultimate residues from the N-terminus. Although synthetic substrates are useful for activity measurements, those currently used are problematic, because they are more efficiently degraded by DPP5. We here aimed to develop a potent and specific substrate and found that the k/K value for Phe-Met-methylcoumaryl-7-amide (MCA) (41.40 ± 0.83 μM s) was highest compared to Met-Leu-, Leu-Leu-, and Phe-Leu-MCA (1.06-3.77 μM s). Its hydrolyzing activity was abrogated in a Porphyromonas gingivalis dpp7-knockout strain. Conclusively, we propose Phe-Met-MCA as an ideal synthetic substrate for DPP7.
Biochimie, Jan 25, 2017
Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which... more Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which liberate dipeptides from the N-termini of polypeptides along with the penultimate hydrophobic and acidic residues, respectively. Their specificities are primarily defined by a single amino acid residue, Gly(673) in DPP7 and Arg(673) in DPP11 (numbering for Porphyromonas gingivalis DPP11). Bacterial species in the phyla Proteobacteria and Bacteroidetes generally possess one gene for each, while Bacteroides species exceptionally possess three genes, one gene as DPP7 and two genes as DPP11, annotated based on the full-length similarities. In the present study, we aimed to characterize the above-mentioned Bacteroides S46 DPPs. A recombinant protein of the putative DPP11 gene BF9343_2924 from Bacteroides fragilis harboring Gly(673) exhibited DPP7 activity by hydrolyzing Leu-Leu-4-methylcoumaryl-7-amide (MCA). Another gene, BF9343_2925, as well as the Bacteroides vulgatus gene (BVU_2252) with...
Journal of Dental Sciences
Background/purpose: Porphyromonas gingivalis is a major causative agent of chronic periodontitis,... more Background/purpose: Porphyromonas gingivalis is a major causative agent of chronic periodontitis, whilst circumstances for acquisition of the bacterium remain to be elucidated. To examine prevalence of the bacterium harboring distinct fimA types in dental plaque of children, we established PCR procedures that are applicable for specimens with limited amounts. By this method, all six fimA types including type I and Ib were directly identified, and prevalence of fimA types and their frequency of guardian-child transmission in Japanese children were assessed. Materials and methods: Genomic DNA was purified from dental plaque specimens of 132 periodontally healthy children (2e12 years old, 4.8 AE 0.2 years) and 19 mothers of resultant P. gingivalis-positive child subjects. PCR-based fimA genotyping was performed, and untypeable strains in the first PCR analysis were determined by a nested PCR. Results: P. gingivalis was found in 15.2% of the subjects (2e10 years old, 5.1 AE 0.6 years), and the most prevalent types were I and IV (37.0% each), followed by Ib and III (11.1% each), and then II (7.4%). Seven (35.0%) of the 20 P. gingivalis-positive subjects had combined colonization of type I with other fimA types. In most cases, bacterial prevalence and fimA types in the children were distinct from those of their mothers, indicating that its maternal transmission was not significant.
Japanese Dental Science Review, 2015
Porphyromonas gingivalis, an asaccharolytic bacterium, utilizes amino acids as energy and carbon ... more Porphyromonas gingivalis, an asaccharolytic bacterium, utilizes amino acids as energy and carbon sources. Since amino acids are incorporated into the bacterial cells mainly as diand tri-peptides, exopeptidases including dipeptidyl-peptidase (DPP) and tripeptidylpeptidase are considered to be prerequisite components for their metabolism. We recently discovered DPP11, DPP5, and acylpeptidyl oligopeptidase in addition to previously reported DPP4, DPP7, and prolyl tripeptidyl peptidase A. DPP11 is a novel enzyme specific for acidic P1 residues (Asp and Glu) and distributed ubiquitously in eubacteria, while DPP5 is preferential for the hydrophobic P1 residue and the first entity identified in prokaryotes. Recently, acylpeptidyl oligopeptidase with a preference for hydrophobic P1 residues was found to release N-terminally blocked di-and tri-peptides. Furthermore, we also demonstrated that gingipains R and K contribute to P1-basic dipeptide production. These observations implicate that most, if not all, combinations of di-and tri-peptides are produced from extracellular oligopeptides even with an N-terminal modification. Here, we review P. gingivalis exopeptidases mainly in regard to their enzymatic characteristics. These exopeptidases with various substrate specificities benefit P. gingivalis for obtaining energy and carbon sources from the nutritionally limited subgingival environment.
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Papers by Takayuki Nemoto