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Cloning, characterization and expression of a calnexin homologue from the pathogenic fungus Paracoccidioides brasiliensis
Author(s) -
dos Santos Feitosa Luciano,
de Almeida Soares Célia Maria,
dos Santos Márcia Regina Machado,
Bailão Alexandre Melo,
Xander Patrícia,
Mortara Renato Arruda,
Lopes José Daniel
Publication year - 2007
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1438
Subject(s) - calnexin , calreticulin , biology , paracoccidioides brasiliensis , endoplasmic reticulum , complementary dna , microbiology and biotechnology , chaperone (clinical) , kdel , molecular cloning , recombinant dna , open reading frame , peptide sequence , gene , biochemistry , golgi apparatus , medicine , pathology
We report the cloning of a Paracoccidioides brasiliensis cDNA, here named Pb Cnx, encoding the homologue of the endoplasmic reticulum molecular chaperone calnexin. Calnexin specifically recognizes monoglucosylated glycoproteins in the endoplasmic reticulum, thus being an essential component of the complex that interacts with the folded state of nascent secreted glycoproteins. The Pb Cnx open reading frame was found in a 1701 base pair (bp) fragment that encodes a 567 amino acid protein with an estimated mass of 62 680 Da. Northern and Southern blot hybridizations showed that Pb Cnx is encoded by a single, or a low number of, gene copies. Pb Cnx contains the hallmark KPEDWD motifs that are found in all members of the calnexin/calreticulin family proteins. A cDNA‐encoding Pb Cnx was overexpressed as recombinant protein in Escherichia coli. The purified recombinant Pb Cnx was recognized by 6 out of 10 sera from PCM patients, a result that rules out its possible consideration for further use in diagnosis. Using confocal microscopy with anti‐ Pb Cnx mouse serum against yeast forms, a cytoplasmic staining pattern was observed. Copyright © 2006 John Wiley & Sons, Ltd.