Cloning, characterization and expression of a calnexin homologue from the pathogenic fungus Paracoccidioides brasiliensis.
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.
dos Santos Feitosa
Luciano
L
Departamento de Microbiologia, Imunologia e Parasitologia da Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil.
de Almeida Soares
Célia Maria
CM
Dos Santos
Márcia Regina Machado
MR
Bailão
Alexandre Melo
AM
Xander
Patrícia
P
Mortara
Renato Arruda
RA
Lopes
José Daniel
JD
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
D018076:DNA, Complementary; D004271:DNA, Fungal; D000939:Epitopes; D012331:RNA, Fungal; D011994:Recombinant Proteins; D037281:Calnexin