The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein.
Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.
Borges
Clayton L
CL
Laboratório de Biologia Molecular, ICBII, Universidade Federal de Goiás, 74001-970 Goiânia, Goiás, Brazil.
Pereira
Maristela
M
Felipe
Maria S S
MS
de Faria
Fabrícia P
FP
Gomez
Francisco J
FJ
Deepe
George S
GS
Soares
Célia M A
CM
D000581:Amidohydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000946:Antigens, Fungal / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D000939:Epitopes; D004926:Escherichia coli / Q000378:metabolism; D005559:Formamides / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010228:Paracoccidioides / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000946:Antigens, Fungal; D018076:DNA, Complementary; D000939:Epitopes; D005559:Formamides; D011994:Recombinant Proteins; C031066:formamide; D000581:Amidohydrolases; C101846:formamidase