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. 1994 Nov;62(11):4938–4947. doi: 10.1128/iai.62.11.4938-4947.1994

An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides.

P L Mäkinen 1, K K Mäkinen 1, S A Syed 1
PMCID: PMC303210  PMID: 7523301

Abstract

An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides.

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Selected References

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  1. Barrett A. J., Rawlings N. D. Oligopeptidases, and the emergence of the prolyl oligopeptidase family. Biol Chem Hoppe Seyler. 1992 Jul;373(7):353–360. doi: 10.1515/bchm3.1992.373.2.353. [DOI] [PubMed] [Google Scholar]
  2. Blumberg S., Teichberg V. I., Charli J. L., Hersh L. B., McKelvy J. F. Cleavage of substance P to an N-terminal tetrapeptide and a C-terminal heptapeptide by a post-proline Cleaving enzyme from bovine brain. Brain Res. 1980 Jun 23;192(2):477–486. doi: 10.1016/0006-8993(80)90898-7. [DOI] [PubMed] [Google Scholar]
  3. Boehringer H., Taichman N. S., Shenker B. J. Suppression of fibroblast proliferation by oral spirochetes. Infect Immun. 1984 Jul;45(1):155–159. doi: 10.1128/iai.45.1.155-159.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cockayne A., Sanger R., Ivic A., Strugnell R. A., MacDougall J. H., Russell R. R., Penn C. W. Antigenic and structural analysis of Treponema denticola. J Gen Microbiol. 1989 Dec;135(12):3209–3218. doi: 10.1099/00221287-135-12-3209. [DOI] [PubMed] [Google Scholar]
  5. Cornish-Bowden A. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem J. 1974 Jan;137(1):143–144. doi: 10.1042/bj1370143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dawson J. R., Ellen R. P. Tip-oriented adherence of Treponema denticola to fibronectin. Infect Immun. 1990 Dec;58(12):3924–3928. doi: 10.1128/iai.58.12.3924-3928.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ferris G., Grow T. E., Low S. B., Ferris R. T. Measurement of gingival crevicular fluid conductivity, in vivo. J Periodontol. 1987 Jan;58(1):46–50. doi: 10.1902/jop.1987.58.1.46. [DOI] [PubMed] [Google Scholar]
  8. Grenier D. Nutritional interactions between two suspected periodontopathogens, Treponema denticola and Porphyromonas gingivalis. Infect Immun. 1992 Dec;60(12):5298–5301. doi: 10.1128/iai.60.12.5298-5301.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grenier D., Uitto V. J., McBride B. C. Cellular location of a Treponema denticola chymotrypsinlike protease and importance of the protease in migration through the basement membrane. Infect Immun. 1990 Feb;58(2):347–351. doi: 10.1128/iai.58.2.347-351.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haapasalo M., Singh U., McBride B. C., Uitto V. J. Sulfhydryl-dependent attachment of Treponema denticola to laminin and other proteins. Infect Immun. 1991 Nov;59(11):4230–4237. doi: 10.1128/iai.59.11.4230-4237.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hersh L. B. Immunological, physical, and chemical evidence for the identity of brain and kidney post-proline cleaving-enzyme. J Neurochem. 1981 Jul;37(1):172–178. doi: 10.1111/j.1471-4159.1981.tb05305.x. [DOI] [PubMed] [Google Scholar]
  12. Heylings R. T. Electron microscopy of acute ulcerative gingivitis (Vincent's type). Demonstration of the fusospirochaetal complex of bacteria within pre-necrotic gingival epithelium. Br Dent J. 1967 Jan 17;122(2):51–56. [PubMed] [Google Scholar]
  13. Holt S. C., Bramanti T. E. Factors in virulence expression and their role in periodontal disease pathogenesis. Crit Rev Oral Biol Med. 1991;2(2):177–281. doi: 10.1177/10454411910020020301. [DOI] [PubMed] [Google Scholar]
  14. Kalwant S., Porter A. G. Purification and characterization of human brain prolyl endopeptidase. Biochem J. 1991 May 15;276(Pt 1):237–244. doi: 10.1042/bj2760237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kanatani A., Yoshimoto T., Kitazono A., Kokubo T., Tsuru D. Prolyl endopeptidase from Aeromonas hydrophila: cloning, sequencing, and expression of the enzyme gene, and characterization of the expressed enzyme. J Biochem. 1993 Jun;113(6):790–796. doi: 10.1093/oxfordjournals.jbchem.a124120. [DOI] [PubMed] [Google Scholar]
  16. Kumar S. Selective modification and immune evasion: a hypothesis. Immunol Cell Biol. 1993 Apr;71(Pt 2):141–143. doi: 10.1038/icb.1993.15. [DOI] [PubMed] [Google Scholar]
  17. LEVY H. M., LEBER P. D., RYAN E. M. INACTIVATION OF MYOSIN BY 2,4-DINITROPHENOL AND PROTECTION BY ADENOSINE TRIPHOSPHATE AND OTHER PHOSPHATE COMPOUNDS. J Biol Chem. 1963 Nov;238:3654–3659. [PubMed] [Google Scholar]
  18. Listgarten M. A., Helldén L. Relative distribution of bacteria at clinically healthy and periodontally diseased sites in humans. J Clin Periodontol. 1978 May;5(2):115–132. doi: 10.1111/j.1600-051x.1978.tb01913.x. [DOI] [PubMed] [Google Scholar]
  19. Loesche W. J., Syed S. A., Schmidt E., Morrison E. C. Bacterial profiles of subgingival plaques in periodontitis. J Periodontol. 1985 Aug;56(8):447–456. doi: 10.1902/jop.1985.56.8.447. [DOI] [PubMed] [Google Scholar]
  20. Loesche W. J., Syed S. A., Stoll J. Trypsin-like activity in subgingival plaque. A diagnostic marker for spirochetes and periodontal disease? J Periodontol. 1987 Apr;58(4):266–273. doi: 10.1902/jop.1987.58.4.266. [DOI] [PubMed] [Google Scholar]
  21. Maltha J. C., Mikx F. H., Kuijpers F. J. Necrotizing ulcerative gingivitis in beagle dogs. III. Distribution of spirochetes in interdental gingival tissue. J Periodontal Res. 1985 Sep;20(5):522–531. doi: 10.1111/j.1600-0765.1985.tb00836.x. [DOI] [PubMed] [Google Scholar]
  22. Mentlein R. Proline residues in the maturation and degradation of peptide hormones and neuropeptides. FEBS Lett. 1988 Jul 18;234(2):251–256. doi: 10.1016/0014-5793(88)80092-9. [DOI] [PubMed] [Google Scholar]
  23. Mikx F. H., Maltha J. C., van Campen G. J. Spirochetes in early lesions of necrotizing ulcerative gingivitis experimentally induced in beagles. Oral Microbiol Immunol. 1990 Apr;5(2):86–89. doi: 10.1111/j.1399-302x.1990.tb00233.x. [DOI] [PubMed] [Google Scholar]
  24. Mikx F. H., Ngassapa D. N., Reijntjens F. M., Maltha J. C. Effect of splint placement on black-pigmented Bacteroides and spirochetes in the dental plaque of beagle dogs. J Dent Res. 1984 Nov;63(11):1284–1288. doi: 10.1177/00220345840630110601. [DOI] [PubMed] [Google Scholar]
  25. Mikx F. H., de Jong M. H. Keratinolytic activity of cutaneous and oral bacteria. Infect Immun. 1987 Mar;55(3):621–625. doi: 10.1128/iai.55.3.621-625.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Miles E. W. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol. 1977;47:431–442. doi: 10.1016/0076-6879(77)47043-5. [DOI] [PubMed] [Google Scholar]
  27. Moore W. E., Holdeman L. V., Cato E. P., Smibert R. M., Burmeister J. A., Palcanis K. G., Ranney R. R. Comparative bacteriology of juvenile periodontitis. Infect Immun. 1985 May;48(2):507–519. doi: 10.1128/iai.48.2.507-519.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mäkinen K. K., Mäkinen P. L., Syed S. A. Purification and substrate specificity of an endopeptidase from the human oral spirochete Treponema denticola ATCC 35405, active on furylacryloyl-Leu-Gly-Pro-Ala and bradykinin. J Biol Chem. 1992 Jul 15;267(20):14285–14293. [PubMed] [Google Scholar]
  29. Ohta K., Makinen K. K., Loesche W. J. Purification and characterization of an enzyme produced by Treponema denticola capable of hydrolyzing synthetic trypsin substrates. Infect Immun. 1986 Jul;53(1):213–220. doi: 10.1128/iai.53.1.213-220.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Olsen I. Attachment of Treponema denticola to cultured human epithelial cells. Scand J Dent Res. 1984 Feb;92(1):55–63. doi: 10.1111/j.1600-0722.1984.tb00860.x. [DOI] [PubMed] [Google Scholar]
  31. Polgar L. pH-dependent mechanism in the catalysis of prolyl endopeptidase from pig muscle. Eur J Biochem. 1991 Apr 23;197(2):441–447. doi: 10.1111/j.1432-1033.1991.tb15930.x. [DOI] [PubMed] [Google Scholar]
  32. Pougeois R., Satre M., Vignais P. V. N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, a new inhibitor of the mitochondrial F1-ATPase. Biochemistry. 1978 Jul 25;17(15):3018–3023. doi: 10.1021/bi00608a013. [DOI] [PubMed] [Google Scholar]
  33. Rawlings N. D., Barrett A. J. Evolutionary families of peptidases. Biochem J. 1993 Feb 15;290(Pt 1):205–218. doi: 10.1042/bj2900205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rawlings N. D., Polgar L., Barrett A. J. A new family of serine-type peptidases related to prolyl oligopeptidase. Biochem J. 1991 Nov 1;279(Pt 3):907–908. doi: 10.1042/bj2790907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Saccomani G., Barcellona M. L., Sachs G. Reactivity of gastric (H+ + K+)-ATPase to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. J Biol Chem. 1981 Dec 10;256(23):12405–12410. [PubMed] [Google Scholar]
  36. Shah H. N., Gharbia S. E., Zhang M. I. Measurement of electrical bioimpedance for studying utilization of amino acids and peptides by Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola. Clin Infect Dis. 1993 Jun;16 (Suppl 4):S404–S407. doi: 10.1093/clinids/16.supplement_4.s404. [DOI] [PubMed] [Google Scholar]
  37. Simonson L. G., Goodman C. H., Bial J. J., Morton H. E. Quantitative relationship of Treponema denticola to severity of periodontal disease. Infect Immun. 1988 Apr;56(4):726–728. doi: 10.1128/iai.56.4.726-728.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sorsa T., Ingman T., Suomalainen K., Haapasalo M., Konttinen Y. T., Lindy O., Saari H., Uitto V. J. Identification of proteases from periodontopathogenic bacteria as activators of latent human neutrophil and fibroblast-type interstitial collagenases. Infect Immun. 1992 Nov;60(11):4491–4495. doi: 10.1128/iai.60.11.4491-4495.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Szwajcer-Dey E., Rasmussen J., Meldal M., Breddam K. Proline-specific endopeptidases from microbial sources: isolation of an enzyme from a Xanthomonas sp. J Bacteriol. 1992 Apr;174(8):2454–2459. doi: 10.1128/jb.174.8.2454-2459.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Uitto V. J., Haapasalo M., Laakso T., Salo T. Degradation of basement membrane collagen by proteases from some anaerobic oral micro-organisms. Oral Microbiol Immunol. 1988 Sep;3(3):97–102. doi: 10.1111/j.1399-302x.1988.tb00092.x. [DOI] [PubMed] [Google Scholar]
  41. Walter R., Simmons W. H., Yoshimoto T. Proline specific endo- and exopeptidases. Mol Cell Biochem. 1980 Apr 18;30(2):111–127. doi: 10.1007/BF00227927. [DOI] [PubMed] [Google Scholar]
  42. Weinberg A., Holt S. C. Interaction of Treponema denticola TD-4, GM-1, and MS25 with human gingival fibroblasts. Infect Immun. 1990 Jun;58(6):1720–1729. doi: 10.1128/iai.58.6.1720-1729.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Welches W. R., Brosnihan K. B., Ferrario C. M. A comparison of the properties and enzymatic activities of three angiotensin processing enzymes: angiotensin converting enzyme, prolyl endopeptidase and neutral endopeptidase 24.11. Life Sci. 1993;52(18):1461–1480. doi: 10.1016/0024-3205(93)90108-f. [DOI] [PubMed] [Google Scholar]
  44. Welches W. R., Santos R. A., Chappell M. C., Brosnihan K. B., Greene L. J., Ferrario C. M. Evidence that prolyl endopeptidase participates in the processing of brain angiotensin. J Hypertens. 1991 Jul;9(7):631–638. doi: 10.1097/00004872-199107000-00008. [DOI] [PubMed] [Google Scholar]
  45. Wilk S. Prolyl endopeptidase. Life Sci. 1983 Nov 28;33(22):2149–2157. doi: 10.1016/0024-3205(83)90285-0. [DOI] [PubMed] [Google Scholar]
  46. Wyss C. Aspartame as a source of essential phenylalanine for the growth of oral anaerobes. FEMS Microbiol Lett. 1993 Apr 15;108(3):255–258. doi: 10.1111/j.1574-6968.1993.tb06111.x. [DOI] [PubMed] [Google Scholar]
  47. Yaron A., Naider F. Proline-dependent structural and biological properties of peptides and proteins. Crit Rev Biochem Mol Biol. 1993;28(1):31–81. doi: 10.3109/10409239309082572. [DOI] [PubMed] [Google Scholar]
  48. Yoshimoto T., Kado K., Matsubara F., Koriyama N., Kaneto H., Tsura D. Specific inhibitors for prolyl endopeptidase and their anti-amnesic effect. J Pharmacobiodyn. 1987 Dec;10(12):730–735. doi: 10.1248/bpb1978.10.730. [DOI] [PubMed] [Google Scholar]
  49. Yoshimoto T., Kanatani A., Shimoda T., Inaoka T., Kokubo T., Tsuru D. Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene. J Biochem. 1991 Dec;110(6):873–878. doi: 10.1093/oxfordjournals.jbchem.a123682. [DOI] [PubMed] [Google Scholar]
  50. Yoshimoto T., Simmons W. H., Kita T., Tsuru D. Post-proline cleaving enzyme from lamb brain. J Biochem. 1981 Aug;90(2):325–334. doi: 10.1093/oxfordjournals.jbchem.a133477. [DOI] [PubMed] [Google Scholar]
  51. Zolfaghari R., Baker C. R., Canizaro P. C., Feola M., Amirgholami A., Behal F. J. Human lung post-proline endopeptidase: purification and action on vasoactive peptides. Enzyme. 1986;36(3):165–178. doi: 10.1159/000469289. [DOI] [PubMed] [Google Scholar]

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