Protein disulfide isomerase does not control recombinant IgG4 productivity in mammalian cell lines.

Post-translational limitations in the endoplasmic reticulum during recombinant monoclonal antibody production are an important factor in lowering the capacity for synthesis and secretion of correctly folded proteins. Mammalian protein disulfide isomerase (PDI) has previously been shown to have a role in the formation of disulfide bonds in immunoglobulins. Several attempts have been made to improve the rate of recombinant protein production by overexpressing PDI but the results from these studies have been inconclusive. Here we examine the effect of (a) transiently silencing PDI mRNA and (b) increasing the intracellular levels of members of the PDI family (PDI, ERp72, and PDIp) on the mRNA levels, assembly and secretion of an IgG4 isotype. Although transiently silencing PDI in NS0/2N2 cells suggests that PDI is involved in disulfide bond formation of this subclass of antibody, our results show that PDI does not control the overall IgG4 productivity. Furthermore, overexpression of members of the PDI family in a Chinese hamster ovary (CHO) cell line does not improve productivity and hence we conclude that the catalysis of disulfide bond formation is not rate limiting for IgG4 production.

Interaction of the periplasmic peptidylprolyl cis-trans isomerase SurA with model peptides. The N-terminal region of SurA id essential and sufficient for peptide binding.

One of the rate-limiting steps in protein folding has been shown to be the cis-trans isomerization of proline residues, which is catalyzed by a range of peptidylprolyl cis-trans isomerases. To characterize the interaction between model peptides and the periplasmic peptidylprolyl cis-trans isomerase SurA from E. coli, we employed a chemical cross-linking strategy that has been used previously to elucidate the interaction of substrates with other folding catalysts. The interaction between purified SurA and model peptides was significant in that it showed saturation and was abolished by denaturation of SurA; however the interaction was independent of the presence of proline residues in the model peptides. From results obtained by limited proteolysis we conclude that an N-terminal fragment of SurA, comprising 150 amino acids that do not contain the active sites involved in the peptidylprolyl cis-trans isomerization, is essential for the binding of peptides by SurA. This was confirmed by probing the interaction of the model peptide with the recombinant N-terminal fragment, expressed in Escherichia coli. Hence we propose that, similar to protein disulfide isomerase and other folding catalysts, SurA exhibits a modular architecture composed of a substrate binding domain and distinct catalytically active domains.

Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding.

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways.

The pancreas-specific protein disulphide-isomerase PDIp interacts with a hydroxyaryl group in ligands.

Using a cross-linking approach, we have recently demonstrated that radiolabelled model peptides or misfolded proteins specifically interact in vitro with two members of the protein disulphide- isomerase family, namely PDI and PDIp, in a crude extract from sheep pancreas microsomes. In addition, we have shown that tyrosine and tryptophan residues within a peptide are the recognition motifs for the binding to PDIp. Here we examine non-peptide ligands and present evidence that a hydroxyaryl group is a structural motif for the binding to PDIp; simple constructs containing this group and certain xenobiotics and phytoestrogens, which contain an unmodified hydroxyaryl group, can all efficiently inhibit peptide binding to PDIp. To our knowledge this is the first time that the recognition motif of a molecular chaperone or folding catalyst has been specified as a simple chemical structure.

Domains b' and a' of protein disulfide isomerase fulfill the minimum requirement for function as a subunit of prolyl 4-hydroxylase. The N-terminal domains a and b enhances this function and can be substituted in part by those of ERp57.

Protein disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b', and a', plus an acidic C-terminal extension, c. PDI carries out multiple functions, acting as the beta subunit in the animal prolyl 4-hydroxylases and in the microsomal triglyceride transfer protein and independently acting as a protein folding catalyst. We report here that the minimum sequence requirement for the assembly of an active prolyl 4-hydroxylase alpha(2)beta(2) tetramer in insect cell coexpression experiments is fulfilled by the PDI domain construct b'a' but that the sequential addition of the b and a domains greatly increases the level of enzyme activity obtained. In the assembly of active prolyl 4-hydroxylase tetramers, the a and b domains of PDI, but not b' and a', can in part be substituted by the corresponding domains of ERp57, a PDI isoform that functions naturally in association with the lectins calnexin and calreticulin. The a' domain of PDI could not be substituted by the PDI a domain, suggesting that both b' and a' domains contain regions critical for prolyl 4-hydroxylase assembly. All PDI domain constructs and PDI/ERp57 hybrids that contain the b' domain can bind the 14-amino acid peptide Delta-somatostatin, as measured by cross-linking; however, binding of the misfolded protein "scrambled" RNase required the addition of domains ab or a' of PDI. The human prolyl 4-hydroxylase alpha subunit has at least two isoforms, alpha(I) and alpha(II), which form with the PDI polypeptide the (alpha(I))(2)beta(2) and (alpha(II))(2)beta(2) tetramers. We report here that all the PDI domain constructs and PDI/ERp57 hybrid polypeptides tested were more effectively associated with the alpha(II) subunit than the alpha(I) subunit.

Specificity in substrate binding by protein folding catalysts: tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp.

Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding.

Mutations that destabilize the a' domain of human protein-disulfide isomerase indirectly affect peptide binding.

Protein-disulfide isomerase (PDI) is a catalyst of folding of disulfide-bonded proteins and also a multifunctional polypeptide that acts as the beta-subunit in the prolyl 4-hydroxylase alpha(2)beta(2)-tetramer (P4H) and the microsomal triglyceride transfer protein alphabeta-dimer. The principal peptide-binding site of PDI is located in the b' domain, but all domains contribute to the binding of misfolded proteins. Mutations in the C-terminal part of the a' domain have significant effects on the assembly of the P4H tetramer and other functions of PDI. In this study we have addressed the question of whether these mutations in the C-terminal part of the a' domain, which affect P4H assembly, also affect peptide binding to PDI. We observed a strong correlation between P4H assembly competence and peptide binding; mutants of PDI that failed to form a functional P4H tetramer were also inactive in peptide binding. However, there was also a correlation between inactivity in these assays and indicators of conformational disruption, such as protease sensitivity. Peptide binding activity could be restored in inactive, protease-sensitive mutants by selective proteolytic removal of the mutated a' domain. Hence we propose that structural changes in the a' domain indirectly affect peptide binding to the b' domain.

Oxidative stress: Protein folding with a novel redox switch.

A novel cellular response to oxidative stress has been discovered, in which the activity of a molecular chaperone, Hsp33, is modulated by the environmental redox potential. This provides a rapid first defence mechanism against the potentially very harmful toxic effects of oxidative stress.

A pancreas-specific glycosylated protein disulphide-isomerase binds to misfolded proteins and peptides with an interaction inhibited by oestrogens.

Using a cross-linking approach, we have demonstrated that radiolabeled model peptides or misfolded proteins specifically interact in vitro with two different luminal proteins in a crude extract from sheep pancreas microsomes. One of the proteins was identified as protein disulphide-isomerase (PDI), the other one was a related protein (PDIp). We have shown that PDIp was expressed exclusively in the pancreas. Interspecies conservation of PDIp was confirmed and, unlike other members of the PDI family, PDIp from various sources was found to be a glycoprotein. PDIp interacted with peptides and also a misfolded protein, but not with native proteins, suggesting that it might act as a molecular chaperone. The initial binding process was independent of the presence of Cys residues in the probed peptides. Certain oestrogens strongly inhibited the interaction between peptides and PDIp, with 17beta-oestradiol being the most potent inhibitor.

The b' domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins.

Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide-bonded proteins. A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding. We recently demonstrated by cross-linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate-binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coli, with different radiolabelled model peptides. We observed that the b' domain of human PDI is essential and sufficient for the binding of small peptides. In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b' domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required. Hence we propose that the different domains of PDI all contribute to the binding site, with the b' domain forming the essential core.

Interactions between protein disulphide isomerase and peptides.

There is growing evidence that protein disulphide isomerase (PDI) has a common chaperone function in the endoplasmic reticulum. To characterise this function, we investigated the interaction of purified PDI with radiolabelled model peptides, somatostatin and mastoparan, by cross-linking. The interaction between the peptides and PDI was specific, for it showed saturation and was abolished by denaturation of PDI. The interaction between a hydrophobic peptide without cysteine residues was much more sensitive to Triton X-100 than the interaction between PDI and a more hydrophilic peptide with or without cysteine residues. We therefore propose that hydrophobic interactions between protein disulphide isomerase and peptides play an important role in the binding process. The interaction between PDI and the bound peptide therefore is enhanced by the formation of mixed disulphide bonds.

Pancreas specific protein disulfide isomerase, PDIp, is in transient contact with secretory proteins during late stages of translocation.

Protein disulfide isomerase (PDI) and an additional lumenal protein of dog pancreas microsomes were previously observed to be in transient contact with secretory proteins during late stages of their co- or posttranslational translocation into these mammalian microsomes. The second protein was characterized as a 57 kDa glycoprotein. Here we identified this glycoprotein as the canine equivalent of human PDIp, a protein which was recently described as a new protein disulfide isomerase which is highly expressed in human pancreas. Canine PDIp is also a very abundant protein, its concentration in pancreatic microsomes approaches the concentration of PDI and of the major microsomal molecular chaperones. Apparently, PDIp shares with PDI not just the enzymatic but also the polypeptide binding or chaperoning activity. Furthermore, we suggest that PDIp, too, can be involved in completion of cotranslational as well as posttranslational translocation of proteins into mammalian microsomes.

Cyclosporin A inhibits the degradation of signal sequences after processing of presecretory proteins by signal peptidase.

Targeting of presecretory proteins to, and insertion into, the microsomal membrane are mediated by signal sequences. These signal sequences are removed from presecretory proteins by signal peptidase. We demonstrate that the signal sequence of preprolactin, after translocation into microsomes and cleavage by signal peptidase, is converted to an intermediate form. This intermediate was found outside the microsomes, where it was degraded in the presence of cytosol. Degradation of the signal sequence of another presecretory protein, preprocecropinA, occurred even in the absence of cytosol. The immunosuppressant cyclosporin A inhibited trimming of the preprolactin signal sequence and degradation of the preprocecropinA signal sequence. We observed by cross-linking studies that cleaved signal sequences are bound to two microsomal proteins prior to degradation.

Protein disulphide isomerase and a lumenal cyclophilin-type peptidyl prolyl cis-trans isomerase are in transient contact with secretory proteins during late stages of translocation.

The transport of a presecretory protein into the mammalian endoplasmic reticulum can be divided into early translocation events which include specific targeting of the presecretory protein to and insertion into the endoplasmic reticulum membrane and late translocation events, comprising signal sequence cleavage, completion of translocation and folding of the secretory protein into a functional conformation. The microsomal membrane proteins Sec61 alpha p and translocating-chain-associating membrane protein were previously identified as being in close contact with a nascent presecretory protein at an early step of translocation. Here, we investigated whether additional microsomal proteins are in contact with translocating chains during or immediately after transit. This was addressed by crosslinking after release of the nascent chain from Sec61 alpha p. We observed two additional membrane proteins interacting with the nascent precursor in the early stages of translocation and three lumenal proteins interacting with the processed polypeptide chain in the late stages of translocation. One of the lumenal proteins was identified as protein disulphide isomerase by immunoprecipitation. Another of the lumenal proteins was suggested to be a lumenal cyclophilin-type peptidyl prolyl cis-trans isomerase by the effect of cyclosporin A. We propose that molecular chaperones, such as protein disulphide isomerase and cyclophilin may represent two of the lumenal proteins which are involved in completion of translocation.

The membrane proteins TRAMp and sec61 alpha p may be involved in post-translational transport of presecretory proteins into mammalian microsomes.

The presecretory protein ppcecDHFR, a hybrid between preprocecropinA and dihydrofolate reductase, is transported into mammalian microsomes post-translationally, i.e. independent of ribosome and signal recognition particle. Here, the involvement of microsomal proteins in ribonucleoparticle-independent transport of ppcecDHFR was analyzed by transport into trypsin-pretreated microsomes and by transport of a truncated version of ppcecDHFR and subsequent chemical cross-linking. We observed that post-translational transport of ppcecDHFR can occur into microsomes which had been pretreated with trypsin (final concentration, 100 micrograms/ml) and that of the known transport components only TRAMp and sec61 alpha p are still present under these conditions. Furthermore, we found that the truncated ppcecDHFR, ppcecDHFR-98mer', can be cross-linked to 36 kDa microsomal membrane proteins during post-translational transport. Therefore, the two microsomal membrane proteins with molecular masses of about 36 kDa, TRAMp and sec61 alpha p, appear to be involved in the post-translational transport of ppcecDHFR and ppcecDHFR-98mer.

The role of molecular chaperones in protein transport into the endoplasmic reticulum.

In eukaryotic cells export of the vast majority of newly synthesized secretory proteins is initiated at the level of the membrane of the endoplasmic reticulum (microsomal membrane). The precursors of secretory proteins are not transported across the microsomal membrane in their native state. Typically, signal peptides in the precursor proteins are involved in preserving the transport-competent state. Furthermore, there are two alternatively acting mechanisms involved in preserving transport competence in the cytosol. The first mechanism involves two ribonucleoparticles (ribosome and signal recognition particle) and their receptors on the microsomal surface and requires the hydrolysis of GTP. The second mechanism does not involve ribonucleoparticles and their receptors but depends on the hydrolysis of ATP and on cis-acting molecular chaperones, such as heat shock cognate protein 70 (hsc 70). In both mechanisms a translocase in the microsomal membrane mediates protein translocation. This translocase includes a signal peptide receptor on the cis-side of the microsomal membrane and a component that also depends on the hydrolysis of ATP. At least in certain cases, an additional nucleoside triphosphate-requiring step is involved which is related to the trans-acting molecular chaperone BiP.

A microsomal protein is involved in ATP-dependent transport of presecretory proteins into mammalian microsomes.

Ribonucleoparticle (i.e. ribosome and SRP)-independent transport of proteins into mammalian microsomes is stimulated by a cytosolic ATPase which involves proteins belonging to the hsp70 family. Here we addressed the question of whether there are additional nucleoside triphosphate requirements involved in this transport mechanism. We employed a purified presecretory protein which upon solubilization in dimethyl sulfoxide and subsequent dilution into an aqueous buffer was processed by and transported into mammalian microsomes in the absence of the cytosolic ATPase. Membrane insertion of this precursor protein was found to depend on the hydrolysis of ATP and to involve a microsomal protein which can be photoaffinity inactivated with azido-ATP. Furthermore, a microsomal protein with a similar sensitivity towards photoaffinity modification with azido-ATP was observed to be involved in ribonucleoparticle-dependent transport. We suggest that a novel microsomal protein which depends on ATP hydrolysis is involved in membrane insertion of both ribonucleoparticle-dependent and -independent precursor proteins.

Photoaffinity labeling of dog pancreas microsomes with 8-azido-ATP inhibits association of nascent preprolactin with the signal sequence receptor complex.

Transport of bovine preprolactin into dog pancreas microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with azido-ATP and which is distinct from the ATP-binding protein, immunoglobulin heavy chain binding protein. Here we addressed the question of what stage of preprolactin transport is affected. Thus a nascent presecretory protein which is related to preprolactin, termed ppl-86mer, was employed. Here we show that the nascent preprolactin did not become associated with the alpha-subunit of the signal sequence receptor complex after photoaffinity labeling of microsomes with azido-ATP. Therefore, we conclude that the microsomal protein which is sensitive to photoaffinity labeling with azido-ATP acts prior to the signal sequence receptor complex.

Protein export in prokaryotes and eukaryotes. Theme with variations.

Protein export in prokaryotes as well as in eukaryotes can be defined as protein transport across the plasma membrane. In both types of organisms there are various apparently ATP-dependent transport mechanisms which can be distinguished from one another and which show similarities when the prokaryotic mechanism is compared with the respective eukaryotic mechanism. First, one can distinguish between transport mechanisms which involve so-called signal or leader peptides and those which do not. The latter mechanisms seem to employ ATP-dependent transport systems which belong to the family of oligopeptide permeases and multiple drug resistance proteins. Second, in signal or leader peptide-dependent transport one can distinguish between transport mechanisms which involve ribonucleoparticles and those which employ molecular chaperones. Both mechanisms appear to converge at the level of ATP-dependent translocases.