An important virulence factor of the opportunistic human pathogen is elastase, a secreted thermolysin-like neutral zinc-metalloprotease (TNP). Elastase is synthesized as a larger precursor with an amino-terminal 18 kDa propeptide, and was the first TNP shown to require its propeptide as an intramolecular chaperone (IMC) for activity and secretion. This paper reports the analysis of the elastase propeptide to identify residues conserved among other TNP precursors that may be critical for its IMC function. The prosequences of TNP precursors from both Gram-negative ( species and species) and Gram-positive ( species) bacteria were found to show homology to the elastase propeptide. Two regions of conserved residues were observed: a hydrophilic region (ProM) found in the middle of the elastase propeptide and a more hydrophobic region (ProC) located proximal to the propeptide-processing site. To test whether such conserved motifs were important to function, single residue substitutions at eight conserved amino acids were introduced within the full-length pre-proelastase precursor by site-specific mutagenesis of , the gene encoding elastase. Mutant alleles were expressed from plasmids within a -deleted strain, FRD740, and the effects of these propeptide alterations on elastase enzyme activity, processing, stability and accumulation inside and outside of the cell were examined. Within the ProM region, substitution at Arg74 resulted in a dramatic accumulation of proelastase in the cell, suggesting a secretion defect, and a dramatic reduction in supernatant elastolytic activity. Substitution at Asn68 adversely affected the amount of elastase in the culture supernatant, apparently as a result of the reduced stability of the mutated proelastase in the cell. Within the ProC region, mutations at Ile181 and Ala183 abolished the accumulation of a stable elastase molecule in the supernatant. Most mutations produced a phenotype consistent with a defect in protein folding and stability. Overall, the data from this preliminary study show that conserved residues within the elastase propeptide are essential for its function and begin to define the mechanisms of action of IMCs in the TNP family.


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  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.(1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef] [Google Scholar]
  2. Baker, D., Sohl, J. L. & Agard, D. A.(1992). A protein-folding reaction under kinetic control. Nature 356, 263–265.[CrossRef] [Google Scholar]
  3. Braun, P., Tommassen, J. & Filloux, A.(1996). Role of the propeptide in folding and secretion of elastase of Pseudomonas aeruginosa. Mol Microbiol 19, 297–306.[CrossRef] [Google Scholar]
  4. Braun, P., Bitter, W. & Tommassen, J.(2000). Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide-enzyme complex. Microbiology 146, 2565–2572. [Google Scholar]
  5. Bryan, P., Wang, L., Hoskins, J., Ruvinov, S., Strausberg, S., Alexander, P., Almog, O., Gilliland, G. & Gallagher, T.(1995). Catalysis of a protein folding reaction: mechanistic implications of the 2·0 Å structure of the subtilisin-prodomain complex. Biochemistry 34, 10310–10318.[CrossRef] [Google Scholar]
  6. Eder, J., Rheinnecker, M. & Fersht, A.(1993). Folding of subtilisin BPN′: characterization of a folding intermediate. Biochemistry 32, 18–26.[CrossRef] [Google Scholar]
  7. Filloux, A., Michel, G. & Bally, M.(1998). GSP-dependent protein secretion in gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa. FEMS Microbiol Rev 22, 177–198.[CrossRef] [Google Scholar]
  8. Fujishige, A., Smith, K. R., Silen, J. L. & Agard, D. A.(1992). Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli. J Cell Biol 118, 33–42.[CrossRef] [Google Scholar]
  9. Goldberg, J. B. & Ohman, D. E.(1984). Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J Bacteriol 158, 1115–1121. [Google Scholar]
  10. Gustin, J. K., Kessler, E. & Ohman, D. E.(1996). A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion. J Bacteriol 178, 6608–6617. [Google Scholar]
  11. Hase, C. C. & Finkelstein, R. A.(1990). Comparison of the Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase. Infect Immun 58, 4011–4015. [Google Scholar]
  12. Ikemura, H., Takagi, H. & Inouye, M.(1987). Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. J Biol Chem 262, 7859–7864. [Google Scholar]
  13. Inouye, M.(1991). Intermolecular chaperone: the role of the pro-peptide in protein folding. Enzyme 45, 314–321. [Google Scholar]
  14. Janknecht, R., de Martynoff, G., Lou, J., Hipskind, R. A., Nordheim, A. & Stunnenberg, H. G.(1991). Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A 88, 8972–8976.[CrossRef] [Google Scholar]
  15. Kessler, E. & Ohman, D. E.(1998). Pseudolysin (Pseudomonas aeruginosa elastase). In Handbook of Proteolytic Enzymes, pp. 1058–1064. Edited by A. J. Barrett, N. D. Rawlings & J. F. Woessner. San Diego: Academic Press.
  16. Kessler, E. & Safrin, M.(1988). Synthesis, processing, and transport of Pseudomonas aeruginosa elastase. J Bacteriol 170, 5241–5247. [Google Scholar]
  17. Kessler, E. & Safrin, M.(1994). The propeptide of Pseudomonas aeruginosa elastase acts as an elastase inhibitor. J Biol Chem 269, 22726–22731. [Google Scholar]
  18. Kessler, E., Safrin, M., Gustin, J. K. & Ohman, D. E.(1998). Elastase and LasA protease of Pseudomonas aeruginosa are secreted with their propeptides. J Biol Chem 273, 30225–30231.[CrossRef] [Google Scholar]
  19. Kobayashi, T. & Inouye, M.(1992). Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule. J Mol Biol 226, 931–933.[CrossRef] [Google Scholar]
  20. Lerner, C. G., Kobayashi, T. & Inouye, M.(1990). Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis. J Biol Chem 265, 20085–20086. [Google Scholar]
  21. Maniatis, T., Fritsch, E. F. & Sambrook, J.(1982).Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  22. Marie-Claire, C., Roques, B. P. & Beaumont, A.(1998). Intramolecular processing of prothermolysin. J Biol Chem 273, 5697–5701.[CrossRef] [Google Scholar]
  23. McIver, K. S., Kessler, E. & Ohman, D. E.(1991). Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase. J Bacteriol 173, 7781–7789. [Google Scholar]
  24. McIver, K. S., Kessler, E., Olson, J. C. & Ohman, D. E.(1995). The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol 18, 877–889.[CrossRef] [Google Scholar]
  25. O'Donohue, M. J. & Beaumont, A.(1996). The roles of the prosequence of thermolysin in enzyme inhibition and folding in vitro. J Biol Chem 271, 26477–26481.[CrossRef] [Google Scholar]
  26. Ohman, D. E., Cryz, S. J. & Iglewski, B. H.(1980). Isolation and characterization of a Pseudomonas aeruginosa PAO mutant that produces altered elastase. J Bacteriol 142, 836–842. [Google Scholar]
  27. Shinde, U. & Inouye, M.(1993). Intramolecular chaperones and protein folding. Trends Biochem Sci 18, 442–446.[CrossRef] [Google Scholar]
  28. Shinde, U. P. & Inouye, M.(1994). The structural and functional organization of intramolecular chaperones: the N-terminal propeptides which mediate protein folding. J Biochem 115, 629–636. [Google Scholar]
  29. Shinde, U. & Inouye, M.(1996). Propeptide-mediated folding in subtilisin: the intramolecular chaperone concept. Adv Exp Med Biol 379, 147–154. [Google Scholar]
  30. Shinde, U. & Inouye, M.(2000). Intramolecular chaperones: polypeptide extensions that modulate protein folding. Semin Cell Dev Biol 11, 35–44.[CrossRef] [Google Scholar]
  31. Silen, J. L. & Agard, D. A.(1989). The α-lytic protease pro-region does not require a physical linkage to activate the protease domain in vivo. Nature 341, 462–464.[CrossRef] [Google Scholar]
  32. Silen, J. L., Frank, D., Fujishige, A., Bone, R. & Agard, D. A.(1989). Analysis of prepro-α-lytic protease expression in Escherichia coli reveals that the pro region is required for activity. J Bacteriol 171, 1320–1325. [Google Scholar]
  33. Wandersman, C.(1989). Secretion, processing and activation of bacterial extracellular proteases. Mol Microbiol 3, 1825–1831.[CrossRef] [Google Scholar]
  34. Wetmore, R. D., Wong, S.-L. & Roche, R. S.(1992). The role of the pro-sequence in the processing and secretion of the thermolysin-like neutral protease from Bacillus cereus. Mol Microbiol 6, 1593–1604.[CrossRef] [Google Scholar]
  35. Zhu, X., Ohta, Y., Jordan, F. & Inouye, M.(1989). Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature 339, 483–484.[CrossRef] [Google Scholar]

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