title: Candida albicans possesses Sap7 as a pepstatin A-insensitive secreted aspartic protease.
abstract: BACKGROUND
  Candida albicans, a commensal organism, is a part of the normal flora of healthy individuals. However, once the host immunity is compromised, C. albicans opportunistically causes recurrent superficial or fatal systemic candidiasis. Secreted aspartic proteases (Sap), encoded by 10 types of SAP genes, have been suggested to contribute to various virulence processes. Thus, it is important to elucidate their biochemical properties for better understanding of the molecular mechanisms that how Sap isozymes damage host tissues.
  
  
  RESULTS
  The SAP7 gene was cloned from C. albicans SC5314 and heterogeneously produced by Pichia pastoris. Measurement of Sap7 proteolytic activity using the FRETS-25Ala library showed that Sap7 was a pepstatin A-insensitive protease. To understand why Sap7 was insensitive to pepstatin A, alanine substitution mutants of Sap7 were constructed. We found that M242A and T467A mutants had normal proteolytic activity and sensitivity to pepstatin A. M242 and T467 were located in close proximity to the entrance to an active site, and alanine substitution at these positions widened the entrance. Our results suggest that this alteration might allow increased accessibility of pepstatin A to the active site. This inference was supported by the observation that the T467A mutant has stronger proteolytic activity than the wild type.
  
  
  CONCLUSIONS
  We found that Sap7 was a pepstatin A-insensitive protease, and that M242 and T467 restricted the accessibility of pepstatin A to the active site. This finding will lead to the development of a novel protease inhibitor beyond pepstatin A. Such a novel inhibitor will be an important research tool as well as pharmaceutical agent for patients suffering from candidiasis.
authors.affiliation: Japan Society for the Promotion of Science, Sakyo-ku, Kyoto, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Aoki
authors.forename: Nao
authors.initials: N
authors.lastname: Kitahara
authors.forename: Natsuko
authors.initials: N
authors.lastname: Miura
authors.forename: Hironobu
authors.initials: H
authors.lastname: Morisaka
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Kouichi
authors.initials: K
authors.lastname: Kuroda
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Ueda
mesh_terms: D000595:Amino Acid Sequence; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000378:metabolism*; D002176:Candida albicans / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010436:Pepstatins / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014774:Virulence
chemical_list: D010436:Pepstatins; D020033:Protein Isoforms; D057055:Aspartic Acid Proteases; C031375:pepstatin