title: Characterization of a secreted aspartyl protease of the fungal pathogen Paracoccidioides brasiliensis.
abstract: Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis, a human systemic disease prevalent in Latin America. Proteases have been described as playing an important role in the host invasion process in many pathogenic microorganisms. Here we describe the identification and characterization of a secreted aspartyl protease (PbSAP), isolated from a cDNA library constructed with RNAs of mycelia transitioning to yeast cells. Recombinant PbSAP was produced in Escherichia coli, and the purified protein was used to develop a polyclonal antibody that was able to detect a 66 kDa protein in the P. brasiliensis proteome. PbSAP was detected in culture supernatants of P. brasiliensis and this data strongly suggest that it is a secreted molecule. The protein was located in the yeast cell wall, as determined by immunoelectron microscopy. In vitro deglycosylation assays with endoglycosidase H, and in vivo inhibition of the glycosylation by tunicamycin demonstrated N-glycosylation of the PbSAP molecule. Zymogram assays indicated the presence of aspartyl protease gelatinolytic activity in yeast cells and culture supernatant.
authors.affiliation: Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, 74001-970, Goiânia, Goiás, Brazil.
authors.forename: Bruno Aluisio Coutinho de Assis
authors.initials: BA
authors.lastname: Tacco
authors.forename: Juliana Alves
authors.initials: JA
authors.lastname: Parente
authors.forename: Mônica Santiago
authors.initials: MS
authors.lastname: Barbosa
authors.forename: Sônia Nairs
authors.initials: SN
authors.lastname: Báo
authors.forename: Tércio de Souza
authors.initials: Tde S
authors.lastname: Gsóes
authors.forename: Maristela
authors.initials: M
authors.lastname: Pereira
authors.forename: Célia Maria de Almeida
authors.initials: CM
authors.lastname: Soares
mesh_terms: D000595:Amino Acid Sequence; D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010228:Paracoccidioides / Q000201:enzymology* / Q000235:genetics; D010229:Paracoccidioidomycosis / Q000382:microbiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D004792:Enzyme Precursors; D005656:Fungal Proteins; D011994:Recombinant Proteins; D005780:Gelatin; D057055:Aspartic Acid Proteases