Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI.     

Post-translational processing of chicken bone phosphoproteins. Identification of the bone phosphoproteins of embryonic tibia.

After periodate oxidation and incubation with a dihydrazide, cross-linking of the two heavy chains of immunoglobulins G from several species proceeds specifically through their oligosaccharides. We have used malonic acid dihydrazide, adipic acid dihydrazide and dithiodipropionic acid dihydrazide. The last compound is introduced in this work as a cleavable-carbohydrate-specific cross-linker. It was found that in rabbit and human immunoglobulins the degree of cross-linking was strongly dependent on the oxidation conditions but only very weakly dependent on the concentration and size of the dihydrazides. Papain cleavage of the cross-linked rabbit IgG indicated that the cross-linking occurred predominantly, if not exclusively, in the Fc region, probably through the two glycans linked to Asn-297 in the CH2 domain of each of the two heavy chains. The immunoglobulins from sheep, pig, goat and guinea pig show a comparable cross-linking pattern, indicating that the sugar chains from these immunoglobulins have a spatial structure closely related to that of rabbit and human IgG. When dithiodipropionic acid dihydrazide was used as the cross-linker, the cross-link could be cleaved by mercaptoethanol.     

Interchain disulfide bonds promote protein cross-linking during protein folding

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.     

Molecular cloning and expression of Caenorhabditis elegans ERp57-homologue with transglutaminase activity.

Formation of cross-linking between proteins via a gamma-glutamyl-epsilon-lysine residue is an important process in many biological phenomena including apoptosis. Formation of this linkage is catalyzed by the enzyme transglutaminase, which is widely distributed from bacteria to the animal kingdom. The simple multi-cellular organism Caenorhabditis elegans also possesses transglutaminase activity associated with apoptosis [Madi, A. et al. (1998) Eur. J. Biochem. 253, 583-590], but no gene with significant homology to vertebrate or bacterial transglutaminases has been found in the C. elegans genome sequence database. On the other hand, protein disulfide isomerases were recently recognized as a new family of transglutaminases [Chandrashekar, R. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 531-536]. To identify the molecule with transglutaminase activity in C. elegans, we isolated from C. elegans a gene homologous to ERp57, which encodes a protein disulfide isomerase, expressed it in recombinant form, and characterized the transglutaminase and protein disulfide isomerase activities of the resultant protein. The C. elegans ERp57 protein had both enzyme activities, and the transglutaminase activity had similar characteristics to the activity in lysate of the whole worm. These results suggested that the ERp57 homologue was one of the substances with transglutaminase activity in C. elegans.     

Oxidative Folding of Conopeptides Modified by <Emphasis Type="Italic">Conus</Emphasis> Protein Disulfide Isomerase

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis–trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis–trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.     

Cell-free production of aggregation-prone proteins in soluble and active forms

We have developed an efficient cell-free protein synthesis system for the production of soluble and active eukaryotic proteins that are predominantly produced as inclusion bodies in bacteria. S30 extracts (indicating the supernatant of cell homogenate when centrifuged at 30,000g) for cell-free protein synthesis were prepared from Escherichia coli that was modified to overexpress a set of chaperones (GroEL/ES or DnaK/J-GrpE) and disulfide isomerase (leader sequence-free mature DsbC expressed in the cytoplasm). The solubility and biological activity concentration (biological activity per unit volume of cell-free protein synthesis reaction mixture) of the protein synthesized by the new cell-free protein synthesis system showed a dramatic improvement. Solubility enhancement was most dramatic with the existence of DnaK/J-GrpE. It shows that the co-translational interaction with DnaK/J-GrpE prior to folding trial is important in maintenance of the aggregation-prone protein in a folding-competent soluble state. For maximizing the biological activity concentration of the expressed protein, the additional presence of GroEL/ES and DsbC was required. When human erythropoietin was expressed in the developed cell-free protein synthesis system including endogenously overexpressed chaperones and/or DsbC, the biological activity concentration of erythropoietin was enhanced by 700%. It implies that the post-translational folding and disulfide bond reshuffling as well as co-translational folding are important in acquiring functionally active protein from cell-free expression system. This is the first report of using S30 extracts including endogenously overexpressed chaperones and/or disulfide isomerase for the efficient production of soluble and active proteins in cell-free protein synthesis. This new cell-free protein synthesis system was capable of introducing much larger amounts of chaperones and disulfide isomerase compared to a conventional method that supplements them separately. The developed cell-free protein synthesis system supported efficient expression of the eukaryotic proteins in soluble and active forms without the need of any exogenous addition or coexpression of folding effectors.     

An atypical protein disulfide isomerase from the protozoan parasite Leishmania containing a single thioredoxin-like domain

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Carbohydrates Facilitate Correct Disulfide Bond Formation and Folding of Rotavirus VP7

It is well established that glycosylation is essential for assembly of enveloped viruses, but no information is yet available as to the function of carbohydrates on the nonenveloped but glycosylated rotavirus. We show that tunicamycin and, more pronouncedly, a combination of tunicamycin and brefeldin A treatment caused misfolding of the luminal VP7 protein, leading to interdisulfide bond aggregation. While formation of VP7 aggregates could be prevented under reducing conditions, they reoccurred in less than 30 min after a shift to an oxidizing milieu. Furthermore, while glycosylated VP7 interacted during maturation with protein disulfide isomerase, nonglycosylated VP7 did not, suggesting that glycosylation is a prerequisite for protein disulfide isomerase interaction. While native NSP4, which does not possess S-S bonds, was not dependent on N-linked glycosylation or on protein disulfide isomerase assistance for maturation, nonglycosylated NSP4 was surprisingly found to interact with protein disulfide isomerase, further suggesting that protein disulfide isomerase can act both as an enzyme and as a chaperone. In conclusion, our data suggest that the major function of carbohydrates on VP7 is to facilitate correct disulfide bond formation and protein folding.     

Synthesis of the bis-cystinyl-fragment 225-232/225'-232' of the human IgGl hinge region

In human IgGl the two heavy chains are crosslinked in the central portion of the molecule by two disulfide bridges forming a double chain bis-cystinyl cyclic peptide in parallel alignment. For our synthetic studies we have chosen the sequence portion 225-232/225'-232', i.e. [H-Thr-C1ys-Pro-Pro-C1ys-Pro-Ala-Pro-OH]2. By the use of a combination of the S-tert.-butylthio and the S-acetamidomethyl groups selective cysteine pairings in two successive steps produced the hinge hexadecapeptide in parallel and antiparallel alignment as homogeneous and well characterized compounds. Thiol disulfide interchange experiments on the antiparallel dimer led to over 90% conversion to the parallel isomer. Similarly random air-oxidation was found to generate again mainly the parallel dimer, thus strongly suggesting that this sequence portion contains sufficient structural information for a correct assembly of the two heavy chains of immunoglobulins without decisive contribution of a protein disulfide isomerase.     

Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus

The oxidative folding of disulfide-rich conotoxins is essential for their biological functions. In vivo, disulfide bond formation is mainly catalyzed by protein disulfide isomerase. To elucidate the physiologic roles of protein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus. Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase. Enzymatic analyses of recombinant C. marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase. Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C. marmoreus protein disulfide isomerase and found that it was much more efficient than glutathione in catalyzing oxidative folding and disulfide isomerization of conotoxins. We further demonstrated that macromolecular crowding had little effect on the protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization of conotoxins. On the basis of these data, we propose that the C. marmoreus protein disulfide isomerase plays a key role during in vivo folding of conotoxins.     

Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli.

The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5. The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.     

Isolation, purification, and characterization of a rat liver mitochondrial protein disulfide isomerase

The isolation and purification to electrophoretical homogeneity and characterization of a protein disulfide isomerase from rat liver mitochondria is reported. The purified enzyme exhibits a single band on sodium dodecylsulfatepolyacrylamide gel electrophoresis with an apparent molecular weight of approximately 54 kDa. Comparatively, the microsomal form shows an apparent molecular weight of 57 kDa indicating that the two forms are slightly different. The antibody raised against the microsomal isoform does not recognize the mitochondrial enzyme. To characterize the enzyme, different classical methodologies utilized for protein disulfide isomerase estimation have been adopted. The isolated enzyme is active with all of them, indicating that it comprises all the features of a typical protein disulfide isomerase. At the mitochondrial level the enzyme appears mostly localized at the membrane level. Its potential involvement in mitochondrial membrane permeability control is also discussed.     

A Novel Reaction of Peroxiredoxin 4 towards Substrates in Oxidative Protein Folding

Peroxiredoxin 4 (Prx4) is the only endoplasmic reticulum localized peroxiredoxin. It functions not only to eliminate peroxide but also to promote oxidative protein folding via oxidizing protein disulfide isomerase (PDI). In Prx4-mediated oxidative protein folding we discovered a new reaction that the sulfenic acid form of Prx4 can directly react with thiols in folding substrates, resulting in non-native disulfide cross-linking and aggregation. We also found that PDI can inhibit this reaction by exerting its reductase and chaperone activities. This discovery discloses an off-pathway reaction in the Prx4-mediated oxidative protein folding and the quality control role of PDI.     

Functional properties of PDIA from Aspergillus niger in renaturation of proteins

Functional properties of protein disulfide isomerase A (PDIA) from Aspergillus niger were investigated using ribonuclease A, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and prochymosin as substrates. PDIA was shown to function as an isomerase catalyzing the refolding of denatured and reduced ribonuclease A. PDIA also exhibited trx-independent chaperone activity preventing the aggregation of reduced, denatured GAPDH, an enzyme lacking disulfide bonds. Both isomerase activity and chaperone function of PDIA were essential for the efficient refolding of the reduced, denatured prochymosin.     

MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, <Emphasis Type="Italic">Methanothermobacter thermoautotrophicum</Emphasis> involving in stress response

MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50°C) and high (70°C) growth temperatures than under the optimal growth temperature for the organism (65°C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4°C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0°C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.     

Enhancing functional expression of β-glucosidase in Pichia pastoris by co-expressing protein disulfide isomerase

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Protein folding and disulfide bond formation were identified as rate-limited steps in recombinant protein secretion in yeast cells. For the production of β-glucosidase in Pichia pastoris, final β-glucosidase activity reached 1,749 U/mL after fermentation optimization in a 3 L bioreactor, while the specific activity decreased from 620 to 467 U/mg, indicating a potential protein misfolding. To solve this problem, protein disulfide isomerase, a chaperone protein which may effectively regulate disulfide bond formation and protein folding, was co-expressed with β-glucosidase. In the co-expression system, a β-glucosidase production level of 2,553 U/mL was achieved and the specific activity of the enzyme reached 721 U/mg, which is 1.54 fold that of the control.     

The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids.

To study the environment of a preprotein as it crosses the plasma membrane of Escherichia coli, unique cysteinyl residues were introduced into proOmpA and the genes for these mutant preproteins were fused to the gene of dihydrofolate reductase (Dhfr). A photoactivable, radiolabeled and reducible cross-linker was then attached to the unique cysteinyl residue of each purified protein. Partially translocated polypeptides were generated and arrested in their membrane transit by the folded structure of the dihydrofolate reductase domain. After photolysis to label their nearest neighbors and reduction of the disulfide bond between proOmpA-Dhfr and the cross-linker, radiolabeled cross-linker was selectively recovered with the SecA and SecY subunits of preprotein translocase. Strikingly, neither the SecE nor Band 1 subunits were cross-linked to any of the constructs and the membrane phospholipids were almost entirely shielded from cross-linking. The fact that SecY and SecA are the only membrane proteins cross-linked to the translocating chains suggests that they may form an entirely proteinaceous pathway through which secreted proteins pass during membrane transit.     

Protein engineering of a disulfide bond in a beta/alpha-barrel protein.

A disulfide bond has been introduced in the beta/alpha-barrel enzyme N-(5'-phosphoribosyl)anthranilate isomerase from Saccharomyces cerevisiae. The design of this disulfide bond was based on a model structure of this enzyme, built from the high-resolution crystal structure of the N-(5'-phosphoribosyl)anthranilate isomerase domain from Escherichia coli. The disulfide cross-link is spontaneously formed in vitro between residues 27 and 212, located in the structurally adjacent alpha-helices 1 and 8 of the outer helical ring of the beta/alpha-barrel. It creates a loop of 184 residues that account for 83% of the sequence of this enzyme, thus forming a quasi circular protein. The cross-linked mutant enzyme displays wild-type steady-state kinetic parameters. Measurements of the equilibrium constant for the reduction of this disulfide bond by 1,4-dithiothreitol show that its bond strength is comparable to that of other engineered protein disulfide bonds. The oxidized, cross-linked N-(5'-phosphoribosyl)anthranilate isomerase mutant is about 1.0 kcal/mol more stable than the wild-type enzyme, as estimated from its equilibrium unfolding transitions by guanidine hydrochloride.