title: Cloning, characterization and expression of a calnexin homologue from the pathogenic fungus Paracoccidioides brasiliensis.
abstract: We report the cloning of a Paracoccidioides brasiliensis cDNA, here named PbCnx, 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 PbCnx 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 PbCnx is encoded by a single, or a low number of, gene copies. PbCnx contains the hallmark KPEDWD motifs that are found in all members of the calnexin/calreticulin family proteins. A cDNA-encoding PbCnx was overexpressed as recombinant protein in Escherichia coli. The purified recombinant PbCnx 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-PbCnx mouse serum against yeast forms, a cytoplasmic staining pattern was observed.
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia da Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil.
authors.forename: Luciano
authors.initials: L
authors.lastname: dos Santos Feitosa
authors.forename: Célia Maria
authors.initials: CM
authors.lastname: de Almeida Soares
authors.forename: Márcia Regina Machado
authors.initials: MR
authors.lastname: Dos Santos
authors.forename: Alexandre Melo
authors.initials: AM
authors.lastname: Bailão
authors.forename: Patrícia
authors.initials: P
authors.lastname: Xander
authors.forename: Renato Arruda
authors.initials: RA
authors.lastname: Mortara
authors.forename: José Daniel
authors.initials: JD
authors.lastname: Lopes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D037281:Calnexin / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010228:Paracoccidioides / Q000235:genetics* / Q000378:metabolism*; D010229:Paracoccidioidomycosis / Q000097:blood / Q000276:immunology; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D017385:Sequence Homology
chemical_list: D018076:DNA, Complementary; D004271:DNA, Fungal; D000939:Epitopes; D012331:RNA, Fungal; D011994:Recombinant Proteins; D037281:Calnexin