Faculty Profiles - OKAMOTO Kuniaki

Papers - OKAMOTO Kuniaki

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Role of N-glycosylation in cathepsin E. A comparative study of cathepsin E with distinct N-linked oligosaccharides and its nonglycosylated mutant Reviewed

Yoshiyuki Yasuda, Shinobu Ikeda, Hideaki Sakai, Takayuki Tsukuba, Kuniaki Okamoto, Kazuhisa Nishishita, Akifumi Akamine, Yuzo Kato, Kenji Yamamoto

European Journal of Biochemistry 266 ( 2 ) 383 - 391 1999.12

Language：English Publishing type：Research paper (scientific journal)

Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N- glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N- glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex- type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high- mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its- proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.

DOI： 10.1046/j.1432-1327.1999.00863.x

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Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis : construction of mutants with rgpA, rgpB, kgp, and hagA.

Shi Y, Ratnayake DB, Okamoto K, Abe N, Yamamoto K, Nakayama K

Journal of Biological Chemistry 274 ( 25 ) 17955 - 17960 1999

カテプシンEにおける糖鎖の生理的意義について

安田善之, 岡元邦彰, 池田仁子, 西下一久, 坂井英昭, 加藤有三, 山本健二

生化学 70 ( 8 ) 927 - 927 1998.8

Biochemical and functional properties of lysine-specific cysteine proteinase (Lys-Gingipain) as a virulence factor of Porphyromonas gingivalis in periodontal disease Reviewed

Naoko Abe, Tomoko Kadowaki, Kuniaki Okamoto, Koji Nakayama, Masamichi Ohishi, Kenji Yamamoto

Journal of Biochemistry 123 ( 2 ) 305 - 312 1998

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press

The oral anaerobic bacterium Porphyromonas gingivalis has been implicated as a major etiologic agent of progressive periodontal disease. A novel lysine-specific cysteine proteinase, termed 'Lys-gingipain,' was purified from the culture supernatant of the Arg-gingipain-deficient mutant of P. gingivalis (KDP112) by a simple method including immunoaffinity chromatography. The purified enzyme was found to be composed of a single polypeptide of M(r) = 51,000. Analysis of the enzymatic properties revealed several distinctive features of this enzyme. The proteolytic activity was remarkably activated by thiol-reducing agents and inhibited by idoacetamide, idoacetic acid, and leupeptin. The enzyme was also inhibited by the chloromethyl ketones of tosyl-L-lysine and tosyl-L-phenylalanine. However, internal protease inhibitors, such as cystatins and α1-antichymotrypsin, had no effect on the activity, suggesting its resistance to normal host defense systems in vivo. Despite its narrow specificity for synthetic substrates containing Lys in the P1 site, the enzyme extensively degraded human type I collagen and immunoglobulins G and A (both serum and secretory types). Most important, the enzyme was able to disrupt the functions of polymorphonuclear leukocytes, as shown by its inhibitory effect on the generation of active oxygen species from the activated cells. These results suggest that Lys-gingipain, like Arg-gingipain, plays a crucial role as a virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction of periodontal tissue components and the disruption of normal host defense mechanisms.

DOI： 10.1093/oxfordjournals.jbchem.a021937

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Arg-gingipain acts as a major processing enzyme for various cell surface proteins in Porphyromonas gingivalis. Reviewed

Kadowaki T, Nakayama K, Yoshimura F, Okamoto K, Abe N, Yamamoto K

J. Biol. Chem. 273 29072 - 29076 1998

Involvement of a Lysine-specific cysteine Proteinase in Hemoglobin Adsorption and Heme Accumulation by Porphyromonas gingivalis. Reviewed

Okamoto K, Nakayama K, Kadowaki T, Abe N, Ratnayake D.B, Yamamoto K

J. Biol. Chem. 273 21225 - 21231 1998

Possible roles of N-linked oligosaccharides on cathepsin E Reviewed

H Sakai, S Ikeda, K Nishishita, Y Kato, K Okamoto, T Tsukuba, K Yamamoto

PROTEOLYSIS IN CELL FUNCTIONS 13 55 - 60 1997

Language：English Publishing type：Research paper (international conference proceedings) Publisher：I O S PRESS

Cathepsin E (CE) is a glycosylated intracellular aspartic proteinase. In this study, the role of N-glycosylation in intracellular trafficking and stability of CE was investigated in human gastric adenocarcinoma cells expressing endogenous CE (AGS-1) and recombinant normal rat kidney (NRK) cells transfected with rat CE cDNA. When these cells were treated with tunicamycin, a patent inhibitor for N-glycosylation, the initially synthesized unglycosylated proCE with an apparent molecular mass of 43 kDa was rapidly disappeared from the cells. Since secretion into the medium was not observed, the unglycosylated CE was suggested to be degraded within these cells. The amino acid sequence predicted from the cDNA revealed that rat CE (RCE) contained two potential N-glycosylation sites in the NH2-terminal (Asn(73)-Phe-Thr(75)) and COOH-terminal (Asn(305)-Val-Tnr(307)) portions. To further elucidate the significance of N-glycosylation on the stability of CE, we constructed the N-glycosylation-deficient mutants of RCE by site-directed mutagenesis (Thr(75)-->Ala(75) or Asn(305)-->Gln(305)) and expressed in NRK cells. In contrast to unglycosylated RCE generated by tunicamycin treatment, each mutant was stably retained in the cells and converted into a fully catalytically active form by a brief acid treatment in a similar manner as the wild-type CE. Pulse-chase experiments with S-35.methionine also revealed that there was no significant difference in the half-lives between each mutant and the wild-type CE in the cells. The results suggest that the deficiency of either potential N-glycosylation site alone does not affect the intracellular stability of RCE.

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Biosynthesis and trafficking of cathepsin E Reviewed

K Yamamoto, H Nakanishi, T Tsukuba, K Okamoto, H Sakai, K Nishishita, Y Kato

PROTEOLYSIS IN CELL FUNCTIONS 13 215 - 222 1997

Language：English Publishing type：Research paper (international conference proceedings) Publisher：I O S PRESS

Cathepsin E (CE) is an intracellular aspartic proteinase that exists mainly as a homodimer with a molecular mass of about 82 kDa and partly as a monomer of 42 kDa. In contrast to the lysosomal aspartic proteinase cathepsin D (CD), CE has a limited distribution in some animal species and is normally located in non-lysosomal compartments such as the plasma membrane, the endocytic organelles, and the endoplasmic reticulum (ER). The intracellular localization appears to be dependent on cell types expressing this enzyme. Our recent studies with pulse-chase kinetic analysis in Various cell types revealed that CE was initially synthesized as a larger precursor and underwent different proteolytic and glycolytic processing depending on cell types. This paper describes three different processing systems of CE in certain cell types. The biosynthesis and processing were studied by pulse-chase analysis with S-35-methionone and immunocytochemistry using specific antibodies to CE. In primary cultures of rat peritoneal macrophages and brain microglial cells, CE was first synthesized as a proenzyme with high-mannose-type oligosaccharide chains and subsequently processed to the mature form bearing complex-type oligosaccharides via the intermediate form within a 4 h chase period. The maturation of proCE in these cells was completely blocked by bafilomycin A1, a potent inhibitor of a vacuolar type of H+-ATPase, indicating that the maturation of CE precursor occurs in cellular acidic compartments. In rat thymocytes and mouse Friend erythroleukemia cells, the initially synthesized proCE was processed to the intermediate form within a 6 h chase period, but scarcely processed to the mature form even after a 48 h chase. However, the CE precursors in these cells became resistant to digestion by endo-beta-N-acetylglucosaminidase (Endo H) within several hours of synthesis. Interestingly, CE in thymocytes from dexamethasone-treated rats was found to be greatly processed to the mature form. In recombinant CHO and NRK cells expressing exogenous CE, the initially synthesized proenzyme was processed to the intermediate form, but not to the mature form, which did not undergo glycolytic processing.

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Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-Gingipain) in porphyromonas gingivalis: Structural relationship with the arginine-specific cysteine proteinase (Arg-Gingipain) Reviewed

Kuniaki Okamoto, Tomoko Kadowaki, Koji Nakayama, Kenji Yamamoto

Journal of Biochemistry 120 ( 2 ) 398 - 406 1996

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press

Lys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5,169-bp open reading frame encoding 1,723 amino acids with a calculated molecular mass of 218 kDa. As the extracellular mature enzyme had an apparent molecular mass of 51 kDa in gels, the precursor of KGP was found to comprise at least four domains, the signal peptide, the NH2-terminal prodomain, the mature proteinase domain, and the COOH-terminal hemagglutinin domain, and to be proteolytically processed during its transport. Importantly, the COOH-terminal region contained three direct repeats of two different amino acid sequences, LKWD(or E)AP and YTYTVYRDGTKI, and the subdomains located between the two repeats exhibited strong similarity to those of Arg-gingipain (RGP), another major cysteine proteinase produced by the organism and having cleavage specificity for arginine residues, although the arrangement of the subdomains was not necessarily identical in the two enzymes. Since the I(GP activity was greatly decreased in RGP-deficient mutants and since the most probable site of the propeptide cleavage was present in the homologous sequence highly susceptible to proteolysis by RGP, the precursor of KGP is likely to be processed by RGP to form the mature enzyme.

DOI： 10.1093/oxfordjournals.jbchem.a021426

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Structure and function of a novel arginine-specific cysteine proteinase (argingipain) as a major periodontal pathogenic factor from Porphyromonas gingivalis Reviewed

Kenji Yamamoto, Tomoko Kadowaki, Kuniaki Okamoto, Masahiro Yoneda, Koji Nakayama, Yoshio Misumi, Yukio Ikehara

Advances in Experimental Medicine and Biology 389 33 - 42 1996

Isolation and sequencing of two cDNA clones encoding rat spleen cathepsin E and analysis of the activation of purified procathepsin E

Kuniaki Okamoto, Hao Yu, Yoshio Misumi, Yukio Ikehara, Kenji Yamamoto

Archives of Biochemistry and Biophysics 322 ( 1 ) 103 - 111 1995.9

Language：English Publishing type：Research paper (scientific journal)

Cathepsin E (CE) is an intracellular, nonlysosomal aspartic proteinase consisting of two identical subunits with a molecular mass of approximately 42 kDa and has a unique subcellular distribution in various rat tissues. In this study, we determined the complete amino acid sequence of rat spleen CE and examined the activation mechanism of the proenzyme purified from this tissue. Two cDNA clones encoding rat CE, termed pTNO5 and pTN1, were isolated. Comparison of the amino acid sequences predicted from the respective cDNA sequences revealed that they were essentially identical, except that pTN1 lacked a sequence corresponding to residues Tyr293-Pro325 of the longer cDNA clone (pTN05). Based on the structural analysis of purified enzyme forms, the CE precursor was found to comprise a signal peptide, a prosequence, and a mature protein region of 19, 39, and 337 (pTN05) or 304 (pTN1) residues, respectively. Despite a high degree of similarity in the overall structure of CE between rat and other mammalian species, the first 11 residues in the NH2-terminal sequence of rat mature enzyme were significantly different from those of other species. The purified pro-CE was analyzed for its conversion to the mature form. The results indicated that the maximal conversion occurred at pH 3.0-4.0 in a temperature- and time-dependent manner by autocatalytic cleavage at the site between Phe39-Ser40. This conversion was highly dependent on the protein concentrations of pro-CE and delayed by the presence of exogenous substrates, suggesting the predominance of intermolecular reaction for its conversion to the mature form. © 1995 Academic Press.

DOI： 10.1006/abbi.1995.1441

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Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis. Reviewed

Okamoto K, Misumi Y, Kadowaki T, Yoneda M, Yamamoto K

Arch. Biochem. Biophys. 316 917 - 925 1995

Construction and characterization of arginine-specific cysteine proteinase(Arg-ginigpain)-deficient mutants of Porphyromonas gingivalis : evidence for singificant contribution of Arg-gingipain to virulence.

Nakayama K, Kadowaki T, Okamoto K, Yamamoto K

Journal of Biological Chemistry 270 ( 40 ) 23619 - 23626 1995

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