Identification of an Entamoeba histolytica gene encoding a protein disulfide isomerase that functionally complements the dsbA mutation in Escherichia coli.

0 d In vivo, as in vitro, the tertiary structure of a protein is etermined by its amino acid sequence. Despite the ability f some isolated polypeptide to be denatured and refolded n vitro in the absence of other proteins, correct folding and ssembly of polypeptides in vivo involves the assistance of ther proteins, collectively named “foldases” [1]. Protein isulfide isomerase activity (PDI, EC 5.3.4.1), encoded by n eukaryotic family of foldase genes, is a protein-thiol xidoreductase that catalyzes the oxidation, reduction, and somerization of disulfide bonds in nascent polypeptides [2]. n the endoplasmic reticulum compartment, PDI exhibits xidative and reductive activities, while in reducing envionments (such as the cytosol, endosomes or cytoplasmic embrane) behaves just as a reductase [3]. In yeast and ammalian cells, the PDI subcellular localization and funcion suggest that it plays a key roll in the secretory pathway 4]. Structurally, PDI proteins share a region, of about 00 amino acid residues, that is highly homologous to the rokaryotic protein thioredoxin, thus known as “thioredoxinr Tx-domain”. The active site of Tx-domains contains the otif WCGHCK, in which the two cysteine residues cycle Amebiasis is a major cause of death in developing countries. Worldwide, it has been estimated that at least 40,000 people die every year by invasive infection of the etiological agent, the protozoan parasite Entamoeba histolytica [5,6]. In this pathogen the formation of accurate intraor inter-molecular disulfide bonds is an important biochemical step in favor of the correct folding status of proteins, such as the virulence factors amoebapores and Gal/GalNAc-binding lectin [7,8]. We have investigated whether E. histolytica encodes proteins with disulfide oxidoreductase activity, like PDI enzymes, particularly with an active in vivo oxidase activity. Our approach was based on the molecular isolation of actively expressed genes, followed by a functional complementation test. In an initial report, we described the isolation of a cDNA fragment coding for the C-terminal region of a putative EhPDI protein [9]. Here we extend that observation and report the complete characterization of an E. histolytica cDNA sequence encoding a polypeptide with high similarity to the PDI protein family, hence named EhPDI, as well as a phylogenetic inference analysis, and the functional evaluation of its oxidase activity (using an etween the dithiol and disulfide oxidation states [2,4].