Characterization of a secreted aspartyl protease of the fungal pathogen Paracoccidioides brasiliensis.
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.
Tacco
Bruno Aluisio Coutinho de Assis
BA
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, 74001-970, Goiânia, Goiás, Brazil.
Parente
Juliana Alves
JA
Barbosa
Mônica Santiago
MS
Báo
Sônia Nairs
SN
Gsóes
Tércio de Souza
Tde S
Pereira
Maristela
M
Soares
Célia Maria de Almeida
CM
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
D018076:DNA, Complementary; D004792:Enzyme Precursors; D005656:Fungal Proteins; D011994:Recombinant Proteins; D005780:Gelatin; D057055:Aspartic Acid Proteases