Startseite The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase
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The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase

  • Jiří Dostál , Helena Dlouhá , Petr Maloň , Iva Pichová und Olga Hrušková-Heidingsfeldová
Veröffentlicht/Copyright: 6. September 2005
Biological Chemistry
Aus der Zeitschrift Band 386 Heft 8

Abstract

Opportunistic pathogens of the genus Candida produce secreted aspartic proteinases (Saps) that play an important role in virulence. Saps are synthesized as zymogens, but cell-free culture supernatants of Candida spp. contain only mature Saps. To study the zymogen conversion, the gene encoding a precursor of C. parapsilosis proteinase Sapp1p was cloned, expressed in E. coli and the product was purified. When placed in acidic conditions, the precursor was autocatalytically processed, yielding an active proteinase. The self-activation proceeded through an intermediate product and the resulting enzyme was one amino acid shorter than the authentic enzyme. This truncation did not cause changes in proteinase activity or secondary structure compared to the authentic Sapp1p. Accurate cleavage of the pro-mature junction, however, required a processing proteinase. A crude membrane fraction prepared from C. parapsilosis cells contained an enzyme with Kex2-like activity, which processed the Sapp1p precursor at the expected site. The pro-segment appeared to be indispensable for Sapp1p to attain an appropriate structure. When expressed without the pro-segment, the Sapp1p mature domain was not active and had a lower content of α-helical conformation, as measured by circular dichroism. A similar effect was observed when a His6-tag was linked to the C-terminus of Sapp1p or its precursor.

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References

Abad-Zapatero, C., Goldman, R., Muchmore, S.W., Hutchins, C., Stewart, K., Navaza, J., Payne, C.D., and Ray, T.L. (1996). Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: implications for the design of antifungal agents. Protein Sci.5, 640–652.Suche in Google Scholar

Achstetter, T. and Wolf, D.H. (1985). Hormone processing and membrane-bound proteinases in yeast. EMBO J.4, 173–177.10.1002/j.1460-2075.1985.tb02333.xSuche in Google Scholar

Achstetter, T., Emter, O., Ehmann, C., and Wolf, D.H. (1984). Proteolysis in eucaryotic cells. Identification of multiple proteolytic enzymes in yeast. J. Biol. Chem.259, 13334–13343.Suche in Google Scholar

Andreánsky, M. and Hunter, E. (1994). Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E. coli. Biotechniques16, 626–633.Suche in Google Scholar

Beggah, S., Léchenne, B., Reichard, U., Foundling, S., and Monod, M. (2000). Intra- and intermolecular events direct the propeptide-mediated maturation of the Candida albicans secreted aspartic proteinase Sap1p. Microbiology146, 2765–2773.10.1099/00221287-146-11-2765Suche in Google Scholar

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.72, 248–254.10.1016/0003-2697(76)90527-3Suche in Google Scholar

Cutfield, S.M., Dodson, E.J., Anderson, B.F., Moody, P.C.E., Marshall, C.J., Sullivan, P.A., and Cutfield, J.F. (1995). The crystal structure of a major secreted aspartic proteinase from Candida albicans in complexes with two inhibitors. Structure3, 1261–1271.10.1016/S0969-2126(01)00261-1Suche in Google Scholar

De Bernardis, F., Arancia, S., Morelli, L., Hube, B., Sanglard, D., Schafer, W., and Cassone, A. (1999a). Evidence that members of the secretory aspartyl proteinase gene family, in particular SAP2, are virulence factors for Candida vaginitis. J. Infect. Dis.179, 201–208.10.1086/314546Suche in Google Scholar PubMed

De Bernardis, F., Mondello, F., San Millan, R., Ponton, J., and Cassone, A. (1999b). Biotyping and virulence properties of skin isolates of Candida parapsilosis. J. Clin. Microbiol.37, 3481–3486.10.1128/JCM.37.11.3481-3486.1999Suche in Google Scholar PubMed PubMed Central

De Viragh, P.A., Sanglard, D., Togni, G., Falchetto, R., and Monod, M. (1993). Cloning and sequencing of two Candida parapsilosis genes encoding acid proteases. J. Gen. Microbiol.139, 335–342.10.1099/00221287-139-2-335Suche in Google Scholar PubMed

Deléage, G. and Geourjon, C. (1993). An interactive graphic program for calculating the secondary structures content of proteins from circular dichroism spectrum. Comp. Appl. Biosci.9, 197–199.10.1093/bioinformatics/9.2.197Suche in Google Scholar PubMed

Eder, J. and Fersht, A.R. (1995). Pro-sequence-assisted protein folding. Mol. Microbiol.16, 609–614.10.1111/j.1365-2958.1995.tb02423.xSuche in Google Scholar PubMed

Felk, A., Kretschmar, M., Albrecht, A., Schaller, M., Beinhauer, S., Nichterlein, T., Sanglard, D., Korting, H.C., Schafer, W., and Hube, B. (2002). Candida albicans hyphal formation and the expression of the Efg1-regulated proteinases Sap4 to Sap6 are required for the invasion of parenchymal organs. Infect. Immun.70, 3689–3700.10.1128/IAI.70.7.3689-3700.2002Suche in Google Scholar PubMed PubMed Central

Fukuda, R., Horiuchi, H., Ohta, A., and Takagi, M. (1994). The prosequence of Rhizopus niveus aspartic proteinase-I supports correct folding and secretion of its mature part in Saccharomyces cerevisiae. J. Biol. Chem.269, 9556–9561.10.1016/S0021-9258(17)36917-XSuche in Google Scholar

Fuller, R.S., Brake, A., and Thorner, J. (1989). Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease. Proc. Natl. Acad. Sci. USA86, 1434–1438.10.1073/pnas.86.5.1434Suche in Google Scholar

Fusek, M. and Větvička, V. (1995). Aspartic Proteases: Physiology and Pathology (New York, USA: CRC Press), pp. 32–36.Suche in Google Scholar

Fusek, M., Smith, E.A., Monod, M., Dunn, B., and Foundling, S.I. (1994). Extracellular aspartic proteinases from Candida albicans, Candida tropicalis and Candida parapsilosis yeast differ substantially in their specificities. Biochemistry33, 9791–9799.10.1021/bi00198a051Suche in Google Scholar

Haynes, K. (2001). Virulence in Candida species. Trends Microbiol.9, 591–596.10.1016/S0966-842X(01)02237-5Suche in Google Scholar

Hoffman, C.S. and Winston, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene57, 267–272.10.1016/0378-1119(87)90131-4Suche in Google Scholar

Hrušková-Heidingsfeldová, O., Dostál, J., Hamal, P., Pazlarová, J., Ruml, T., and Pichová, I. (2001). Enzymological characterization of secreted proteinases from Candida parapsilosis and Candida lusitaniae. Collect. Czech. Chem. Commun.66, 1707–1719.10.1135/cccc20011707Suche in Google Scholar

Hube, B. and Naglik, J. (2001). Candida albicans proteinases: resolving the mystery of a gene family. Microbiology147, 1997–2005.10.1099/00221287-147-8-1997Suche in Google Scholar

Khan, A.R., Khazanovich-Bernstein, N., Bergmann, E.M., and James, M.N.G. (1999). Structural aspects of activation pathways of aspartic proteinase zymogens and viral 3C protease precursors. Proc. Natl. Acad. Sci. USA96, 10968–10975.10.1073/pnas.96.20.10968Suche in Google Scholar

Koelsch, G., Mareš, M., Metcalf, P., and Fusek, M. (1994). Multiple functions of pro-parts of aspartic proteinase zymogens. FEBS Lett.343, 6–10.10.1016/0014-5793(94)80596-2Suche in Google Scholar

Koelsch, G., Tang, J., Loy, J.A., Monod, M., Jackson, K., Foundling, S.I., and Lin, X. (2000). Enzymic characteristics of secreted aspartic proteases of Candida albicans. Biochim. Biophys. Acta1480, 117–131.10.1016/S0167-4838(00)00068-6Suche in Google Scholar

Lin, X., Tang, J., Koelsch, G., Monod, M., and Foundling, S. (1993). Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. J. Biol. Chem.268, 20143–20147.10.1016/S0021-9258(20)80705-4Suche in Google Scholar

McIver, K.S., Kessler, E., Olson, J.C., and Ohman, D.E. (1995). The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol. Microbiol.18, 877–889.10.1111/j.1365-2958.1995.18050877.xSuche in Google Scholar PubMed

Monod, M., Togni, G., Hube, B., and Sanglard, D. (1994). Multiplicity of genes encoding secreted aspartic proteinases in Candida species. Mol. Microbiol.13, 357–368.10.1111/j.1365-2958.1994.tb00429.xSuche in Google Scholar PubMed

Naglik, J.R., Challacombe, S.J., and Hube, B. (2003). Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiol. Mol. Biol. Rev.67, 400–428.10.1128/MMBR.67.3.400-428.2003Suche in Google Scholar PubMed PubMed Central

Newport, G. and Agabian, N. (1997). KEX2 influences Candida albicans proteinase secretion and hyphal formation. J. Biol. Chem.272, 28954–28961.10.1074/jbc.272.46.28954Suche in Google Scholar PubMed

Newport, G., Kuo, A., Flattery, A., Gill, C., Blake, J.J., Kurtz, M.B., Abruzzo, G.K., and Agabian, N. (2003). Inactivation of Kex2p diminishes the virulence of Candida albicans. J. Biol. Chem.278, 1713–1720.10.1074/jbc.M209713200Suche in Google Scholar PubMed

Pichová, I., Pavlíčková, L., Dostál, J., Dolejší, E., Hrušková-Heidingsfeldová, O., Weber, J., Ruml, T., and Souček, M. (2001). Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae: inhibition with peptidomimetic inhibitors. Eur. J. Biochem.268, 2669–2677.10.1046/j.1432-1327.2001.02152.xSuche in Google Scholar PubMed

Pohl, J. and Dunn, B.M. (1988). Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site. Biochemistry27, 4827–4834.10.1021/bi00413a037Suche in Google Scholar PubMed

Richter, C., Tanaka, T., and Yada, R.Y. (1998). Mechanism of activation of the gastric proteinases: pepsinogen, progastricsin and prochymosin. Biochem. J.335, 481–490.10.1042/bj3350481Suche in Google Scholar PubMed PubMed Central

Rockwell, N.C., Krysan, D.J., Komiyama, T., and Fuller, R.S. (2002). Precursor processing by kex2/furin proteases. Chem. Rev.102, 4525–4548.10.1021/cr010168iSuche in Google Scholar PubMed

Ruissen, A.L., Groenink, J., Krijtenberg, P., Walgreen-Weterings, E., van't Hof, W., Veerman, E.C., and Amerongen, A.V.N. (2003). Internalisation and degradation of histatin 5 by Candida albicans. Biol. Chem.384, 183–190.10.1515/BC.2003.020Suche in Google Scholar

Sanglard, D., Hube, B., Monod, M., Odds, F.C., and Gow, N.A. (1997). A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect. Immun.65, 3539–3546.10.1128/iai.65.9.3539-3546.1997Suche in Google Scholar

Stewart, K. and Abad-Zapatero, C. (2001). Candida proteases and their inhibition: prospects for antifungal therapy. Curr. Med. Chem.8, 941–948.10.2174/0929867013372698Suche in Google Scholar

Symersky, J., Monod, M., and Foundling, S.I. (1997). High-resolution structure of the extracellular aspartic proteinase from Candida tropicalis yeast. Biochemistry36, 12700–12710.10.1021/bi970613xSuche in Google Scholar

van den Hazel, H.B., Kielland-Brandt, M.C., and Winther, J.R. (1993). The propeptide is required for in vivo formation of stable active yeast proteinase A and can function even when not covalently linked to the mature region. J. Biol. Chem.268, 18002–18007.10.1016/S0021-9258(17)46804-9Suche in Google Scholar

Published Online: 2005-09-06
Published in Print: 2005-08-01

©2005 by Walter de Gruyter Berlin New York

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