Protein disulfide isomerase plays a key role in catalyzing the folding of secretory proteins. It features two catalytically inactive thioredoxin domains inserted between two catalytically active thioredoxin domains and an acidic C-terminal tail. The crystal structure of yeast PDI reveals that the four thioredoxin domains are arranged in the shape of a twisted "U" with the active sites facing each other across the long sides of the "U." The inside surface of the "U" is enriched in hydrophobic residues, thereby facilitating interactions with misfolded proteins. The domain arrangement, active site location, and surface features strikingly resemble the Escherichia coli DsbC and DsbG protein disulfide isomerases. Biochemical studies demonstrate that all domains of PDI, including the C-terminal tail, are required for full catalytic activity. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.

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Protein disulfide isomerase in isolated rat hepatocytes was present at a concentration of 7 micrograms/mg cell protein, representing a approximately 2-fold enrichment compared to isolated hepatic non-parenchymal cells. Though localized mainly in microsomal fractions of hepatocytes, direct immunofluorescence and cell surface radioiodination followed by immunoprecipitation revealed the presence of M(r) 57,000 disulfide isomerase at the cell surface. Electrostatic interaction of the protein with the cell surface was suggested by susceptibility to carbonate washing. Metabolic radiolabeling and immunoprecipitation studies also indicated that some of the newly synthesized M(r) 57,000 disulfide isomerase was secreted. Treatment of cells with colchicine markedly reduced the recovery of disulfide isomerase from the media, indicating microtubular-directed secretion of the protein. Partial staphlococcal V8 proteolytic digestion of the secreted protein revealed a peptide pattern similar to that of the cellular protein. Immunoprecipitation with antibody specific to the -KDEL peptide retention sequence confirmed the presence of this sequence in the secreted protein. Studies of the turnover of disulfide isomerase revealed a half-life of approximately 96 h. Treatment of cells with tunicamycin or heat shock resulted in an increased recovery of newly synthesized disulfide isomerase from cell lysates but diminished recovery from the media. The secretion and cell surface distribution of disulfide isomerase in hepatocytes may be important for the pathogenesis of immune mediated liver injury.

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic proteindisulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca 2؉ -dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.Many secreted proteins have disulfide bonds that are crucial for their structure or function. It has long been known that although the necessary information for folding is dictated by the primary structure of a protein, this process can be exceedingly slow (1). In vivo, the actions of specialized enzymes located in specific compartments of both bacterial and eukaryotic cells catalyze the formation and isomerization of disulfide bonds. In Gram-negative bacteria, the crucial function of disulfide bond formation is sequestered within the periplasmic space between the inner and outer cell membranes and is catalyzed by a group of enzymes belonging to the Dsb (disulfide bond) family. These proteins have a single thioredoxin-like active site consisting of a pair of cysteines in a CXXC motif. In eukaryotic cells, the endoplasmic reticulum (ER) 1 is the only cellular compartment that is sufficiently oxidizing for disulfide bond formation. The ER has a high concentration of the enzymes and molecular chaperones involved in the folding and assembly of proteins (2). The ER-resident enzyme, proteindisulfide isomerase (PDI; EC 5.3.4.1), catalyzes thiol-disulfide exchange reactions. Like Dsb proteins, PDI is a member of the thioredoxin superfamily, but has two thioredoxin-like active sites (CGHC) that are involved in disulfide bond formation and rearrangement reactions (3).In the case of the protozoan parasite Giardia lamblia, it is likely that PDI activity plays a key role in the folding of outer surface proteins that are central to avoidance of the host immune system and survival of this parasite in the external environment (4). Specifically, the plasmalemma of the trophozoite i...

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