Novel Protein-disulfide Isomerases from the Early-diverging Protist Giardia lamblia

Abstract

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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