CLONAGEM, CARACTERIZAÇÃO E EXPRESSÃO HETERÓLOGA DE UMA CALNEXINA HOMÓLOGA DO FUNGO PATOGÊNICO Paracoccidioides brasiliensis / Cloning, characterization and heterologue expression of a calnexin homologue from the pathogenic fungus Paracoccidioides brasiliensis. / CLONAGEM, CARACTERIZAÇÃO E EXPRESSÃO HETERÓLOGA DE UMA CALNEXINA HOMÓLOGA DO FUNGO PATOGÊNICO Paracoccidioides brasiliensis / Cloning, characterization and heterologue expression of a calnexin homologue from the pathogenic fungus Paracoccidioides brasiliensis.

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

We report the cloning of a Paracoccidioides brasiliensis cDNA, here named PbCnx, encoding the homologue of the endoplasmic reticulum calnexin. This chaperone specifically recognizes monoglucosylated glycoproteins in the endoplasmic reticulum. Thus, it is an essential component of the folding process of nascent secreted glycoproteins. PbCnx open reading frame was found in a 1,701-base pair fragment that encodes a 567-residue amino acids protein with estimated mass of 62,680 Da. The deduced amino acid sequence shares identity with the described sequences of calnexin from Aspergillus fumigatus and Aspergillus niger. It also shows twenty potential phosphorylation sites and two potential N-glycosylation sites. Moreover, this amino acid sequence showed a remarkable degree of conservation, especially in the central portion named highly conserved central domain (hcd) that contains, as a hallmark, KPEDWD-repeats. These repeats represent a high-affinity calcium-binding site and are found in all members of the calnexin/calreticulin family proteins. Northern and Southern blots hybridizations analysis showed that PbCnx is encoded by a single or low number of copies genes. A cDNA encoding PbCnx was overexpressed as recombinant protein in Escherichia coli. The purified recombinant PbCnx was recognized by sera of Paracoccidiomycosis patients (chronic form). The monoespecific polyclonal serum generated against the recombinant calnexin allowed its cellular localization by confocal microscopy. The staining pattern observed by immunofluorescence showed this protein intensely distributed in the cytoplasmic region. Furthermore, these polyclonal serum specifically recognized native calnexin on P. brasiliensis yeast form cellular extract by immunoblotting.

ASSUNTO(S)

1. chaperone. 2. calnexina. 3. proteína recombinante. 4.p. brasiliensis. microbiologia

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