Protein disulfide isomerase assisted protein folding in malaria parasites.

In eukaryotes, the formation of protein disulfide bonds among cysteine residues is mediated by protein disulfide isomerases and occurs in the highly oxidised environment of the endoplasmic reticulum. This process is poorly understood in malaria parasites. In this paper, we report the gene isolation, sequence and phylogenetic comparisons, protein structure and thioredoxin-domain analyses of nine protein disulfide isomerases-like molecules from five species of malaria parasites including Plasmodium falciparum and Plasmodium vivax (human), Plasmodium knowlesi (simian) and Plasmodium berghei and Plasmodium yoelii (murine). Four of the studied protein disulfide isomerases belong to P. falciparum malaria and have been named PfPDI-8, PfPDI-9, PfPDI-11 and PfPDI-14, based on their chromosomal location. Among these, PfPDI-8 bears the closest similarity to a prototype PDI molecule with two thioredoxin domains (containing CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL. PfPDI-8 is expressed during all stages of parasite life cycle and is highly conserved (82-96% identity at amino acid level) in the other four Plasmodium species studied. Detailed biochemical analysis of PfPDI-8 revealed that this molecule is a potent oxido-reductase enzyme that facilitated the disulfide-dependent conformational folding of EBA-175, a leading malaria vaccine candidate. These studies open the avenues to understand the process of protein folding and secretory pathway in malaria parasites that in turn might aid in the production of superior recombinant vaccines and provide novel drug targets.

Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum.

Protein disulfide isomerase (PDI) is a protein-thiol oxidoreductase that catalyzes the oxidation, reduction and isomerization of protein disulfides. In the endoplasmic reticulum PDI catalyzes both the oxidation and isomerization of disulfides on nascent polypeptides. Under the reducing condition of the cytoplasm, endosomes and cell surface. PDI catalyzes the reduction of protein disulfides. At those locations, PDI has been demonstrated to participate in the regulation of reception function, cell-cell interaction, gene expression, and actin filament polymerization. These activities of PDI will be discussed, as well as its activity as a chaperone and subunit of prolyl 4-hydroxylase and microsomal triglyceride transfer protein.

Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.

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 protein-disulfide 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.

Secretion, surface localization, turnover, and steady state expression of protein disulfide isomerase in rat hepatocytes.

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.

The role of the thiol/disulfide centers and peptide binding site in the chaperone and anti-chaperone activities of protein disulfide isomerase.

The complexity of protein folding is often aggravated by the low solubility of the denatured state. The inefficiency of the oxidative refolding of reduced, denatured lysozyme results from a kinetic partitioning of the unfolded protein between pathways leading to aggregation and pathways leading to the native structure. Protein disulfide isomerase (PDI), a resident foldase of the endoplasmic reticulum, catalyzes the in vitro oxidative refolding of reduced, disulfide-containing proteins, including denatured lysozyme. Depending on the concentrations of foldase and denatured substrate and the order in which they are added to initiate folding, PDI can exhibit either a chaperone activity or an anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem 269, 7764-7771). PDI's chaperone activity leads to quantitative recovery of native lysozyme. Its anti-chaperone activity diverts substrate away from productive folding and facilitates disulfide cross-linking of lysozyme into large, inactive aggregates that specifically incorporate PDI. A mutant PDI (NmCm-PDI), in which both the N- and C-terminal active site cysteines have been changed to serines, loses all chaperone activity and behaves as an anti-chaperone at all substrate and PDI concentrations tested. The dithiol/disulfide sites of PDI are essential for the chaperone activity observed at high PDI concentrations, but they are not required for the anti-chaperone activity found at low PDI concentrations. Inactivation of PDI's peptide/protein binding site by a specific photoaffinity label (Noiva, R., Freedman, R. B., and Lennarz, W. J. (1993) J. Biol. Chem. 268, 19210-19217) inhibits the disulfide isomerase and chaperone activity, but the protein still retains its anti-chaperone activity. In a glutathione redox buffer, lysozyme-PDI aggregates are disulfide cross-linked; however, disulfide cross-linking is not required for aggregate formation or for the incorporation of PDI into the aggregates. Although both the peptide binding site and the catalytic active sites of PDI are required for chaperone and disulfide isomerase activity, neither of these sites are involved in PDI's anti-chaperone activity. PDI's anti-chaperone activity could serve as a quality control device by providing an efficient mechanism to retain misfolded proteins in the endoplasmic reticulum (Marquardt, T., and Helenius, A. (1992) J. Cell. Biol. 117, 505-513).

Enzymatic catalysis of disulfide formation.

The formation of disulfide bridges in membrane and secretory proteins occurs during the protein folding process in the endoplasmic reticulum of eukaryotic cells and the periplasm of prokaryotes. The formation of disulfide bridges is facilitated by the thiol-disulfide oxidoreductases, protein disulfide isomerase (PDI) in eukaryotes and dsbA in prokaryotes. Structure-function analysis of these soluble proteins demonstrates that their active sites are sequences with similarity to the active site of the redox protein thioredoxin. Although these active sites share homology with thioredoxin, it is evident that other structural determinants change the redox characteristics of these proteins to enable them to facilitate the formation of correct disulfide bridging in the nascent protein. The analysis of structure-function relationships of PDI and dsbA have indicated that these thiol-disulfide oxidoreductases act as protein oxidants to facilitate the formation of disulfides during the folding process. The ability of PDI and dsbA to catalyze the formation and/or isomerization of disulfide bridging establishes their usefulness in facilitating the in vitro and in vivo folding of proteins. Protocols for the purification and assay of PDI activity have been described. Systems for the expression of PDI in Escherichia coli and Spodoptera frugiperda cells have been developed which may prove useful in the expression of recombinant proteins.

Peptide binding to protein disulfide isomerase occurs at a site distinct from the active sites.

Protein disulfide isomerase (PDI) is a multifunctional protein resident in the lumen of the rough endoplasmic reticulum that facilitates protein folding via disulfide bond isomerization. Previously we determined that PDI binds a variety of peptides that can be covalently attached to this protein via a photoreactive cross-linker. We have now investigated the relationship between the peptide binding site and the ability of PDI to catalyze disulfide bond isomerization. PDI has two identical sequences, -WCGHCK-, that have been demonstrated to be important in PDI-catalyzed disulfide isomerization. We have found that other proteins containing these thioredoxin-like active site sequences do not bind the photoreactive peptide probes. Moreover, although chemical modification of the 2 cysteines within the thioredoxin-like active site regions completely inhibits PDI-catalyzed disulfide isomerization, these modifications do not affect peptide binding by PDI. Both of these observations suggest that peptide binding occurs at a site other than the putative PDI active sites. To localize the site in PDI at which binding occurs, we used a radiolabeled peptide photoaffinity probe. Peptide fragments generated by cleavage of 125I-peptide-labeled PDI with cyanogen bromide yielded a single 8-kDa polypeptide fragment containing the 125I-labeled peptide site, but neither of the putative catalytic sites of PDI. An 125I-labeled tryptic peptide was generated from this cyanogen bromide fragment and determined by microsequencing to contain residues 451-476 of PDI; this 26-residue peptide is noteworthy because of its extremely high content of acidic amino acids. Based on these findings we conclude that the peptide binding site is located in the COOH-terminal domain of the protein, and it is distinct from the two active sites for PDI-catalyzed disulfide isomerization and from the region of PDI that has estrogen receptor sequence similarity.

Peptide binding by protein disulfide isomerase, a resident protein of the endoplasmic reticulum lumen.

Previously we had demonstrated by photoaffinity labeling that a 57-kDa protein of the endoplasmic reticulum can bind and become covalently linked to glycosylatable photoreactive peptides containing the sequence-Asn-Xaa-Ser/Thr-. Subsequently, it was found that this protein, called glycosylation site-binding protein, was a multifunctional protein, i.e. it was identical to protein disulfide isomerase (PDI), the beta-subunit of prolyl hydroxylase and thyroid hormone-binding protein. In this study, the peptide specificity for binding to this 57-kDa protein, hereafter called PDI, has been investigated in more detail using photoaffinity probes. The results reveal that although N-glycosylation by oligosaccharyl transferase in the endoplasmic reticulum has an absolute requirement for an hydroxyamino acid in the third amino acid residue of the glycosylation site sequence, no such specificity is observed in the binding of such peptides to PDI. In addition to the lack of specificity for an hydroxyamino acid in the third residue position, no specificity was observed for the asparagine residue in the first position. Thus, binding is not restricted to peptides containing N-glycosylation sites. We have investigated the discrepancy between this apparent lack of sequence specificity and earlier results indicating that binding of peptides to PDI was specific for N-glycosylation site sequences. We now demonstrate that PDI in the lumen of microsomes is more efficiently labeled by peptides containing photoreactive-Asn-Xaa-Ser/Thr- sequences than by nonacceptor site sequences because the former become glycosylated. This increased labeling does not occur because the glycosylated form of the probes are preferentially recognized by PDI. Rather, it appears that increased polarity of the affinity probe after attachment of the oligosaccharide chain prevents its exit from the sealed microsomes, in effect concentrating it within the lumen of the microsome. These results, coupled with other studies on the multifunctional nature of PDI, suggest that the observed peptide binding may be a manifestation of the ability of PDI to recognize the backbone of polypeptides in the lumen of the endoplasmic reticulum.

Glycosylation site-binding protein is not required for N-linked glycoprotein synthesis.

In prior studies we identified a 57-kDa protein in the lumen of the endoplasmic reticulum that, in addition to having both protein disulfide isomerase and thyroid hormone-binding protein activities, bound a photoaffinity probe containing the N-glycosylation-site sequence Asn-Xaa-Ser/Thr. It was hypothesized that this multifunctional protein, called glycosylation site-binding protein (GSBP), participated in the process of N-glycosylation of proteins. To test this hypothesis we have employed various conditions to deplete the lumen of GSBP and then assess the level of N-glycosylation catalyzed by oligosaccharyltransferase (OTase). Although most conditions leading to depletion resulted in partial loss of OTase activity, this loss was independent of the extent of GSBP depletion. Indeed, virtually complete loss (greater than 99%) of GSBP with partial retention of OTase activity was frequently observed. Moreover, repletion of the microsomal lumen with GSBP did not restore OTase activity to control levels. Thus, no correlation between GSBP content and OTase activity before or after reconstitution was found. These results suggest that this multifunctional 57-kDa protein is not an essential component of the enzymatic reaction in which oligosaccharide chains are transferred from dolichyl-P-P-GlcNAc2Man9Glc3 to nascent polypeptides or to synthetic tripeptide acceptors.

Thyroid hormone binding protein contains glycosylation site binding protein activity.

Several lines of evidence provided by other workers indicate that within the same species thyroid hormone binding protein, the beta-subunit of prolyl hydroxylase, and protein disulfide isomerase are the same protein. We sought to determine if glycosylation site binding protein, a lumenal protein of the endoplasmic reticulum, also has the same primary structure. To accomplish this the level of glycosylation site binding protein (GSBP) activity, measured by photolabeling with a glycosylation site peptide probe, was carried out in preparations of 3T3 cells and in E. coli transformed with human thyroid hormone binding protein cDNA. The results strongly support the idea that GSBP is identical to these other lumenal proteins of the endoplasmic reticulum.

Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER.

A 57 kd component of oligosaccharyl transferase, termed glycosylation site binding protein, specifically recognizes a photoaffinity probe containing the N-glycosylation site sequence Asn-Lys-Thr. It is present in the lumen of the ER (endoplasmic reticulum) and its release from this compartment results in a loss of N-glycosylation. Antibodies against this protein were used to identify cDNA clones from a lambda gt11 expression library. Analysis of its cDNA sequence reveals high sequence similarity to three other 57 kd luminal endoplasmic reticulum proteins: protein disulfide isomerase, the beta-subunit of prolyl hydroxylase, and thyroid hormone binding protein. This finding suggests that the capacity to recognize multiple polypeptide domains may reside in a single luminal protein that participates in co- and/or posttranslational modifications of newly synthesized proteins.

Bovine serum hemopexin: properties of the protein from a single animal.

1. Hemopexin was isolated from bovine serum of a single animal in a yield of 0.5 mg/ml. 2. Bovine hemopexin was found to exist in two isoforms of mol. wt 68,000 and 65,000. 3. Treatment of hemopexin with glycopeptidase F yields a single band corresponding to a mol. wt of 51,000. 4. The protein binds heme on an equimolar ratio and shows a single component in reverse-phase high performance liquid chromatography. 5. The amino acid composition of bovine hemopexin compares with that of hemopexin isolated form other animals.

Selenium concentrations in plasma of patients with arteriographically defined coronary atherosclerosis.

A prospective epidemiological study (Lancet ii: 175-179, 1982) implicates low concentrations of selenium in plasma in coronary atherogenesis. We examined this relationship more directly by fluorometry of selenium in the plasma of 91 hospitalized patients who were being examined by coronary arteriography for clinical evaluation of chest pain. We observed a significant, inverse correlation between the plasma selenium and severity of coronary atherosclerosis. These results confirm those of the epidemiological studies, but the role, if any, of selenium in atherogenesis still is unclear. Its concentration in plasma is decreased by ethanol and cigarette use; possibly this is the mechanism of its relation to hypertension and atherosclerosis.