Peptides fused to the amino-terminal end of diphtheria toxin are translocated to the cytosol.

Diphtheria toxin belongs to a group of toxic proteins that enter the cytosol of animal cells. We have here investigated the effect of NH2-terminal extensions of diphtheria toxin on its ability to become translocated to the cytosol. DNA fragments encoding peptides of 12-30 amino acids were fused by recombinant DNA technology to the 5'-end of the gene for a mutant toxin. The resulting DNA constructs were transcribed and translated in vitro. The translation products were bound to cells and then exposed to low pH to induce translocation across the cell membrane. Under these conditions all of the oligopeptides tested, including three viral peptides and the leader peptide of diphtheria toxin, were translocated to the cytosol along with the enzymatic part (A-fragment) of the toxin. Neither hydrophobic nor highly charged sequences blocked translocation. The results are compatible with a model in which the COOH-terminus of the A-fragment first crosses the membrane, whereas the NH2-terminal region follows behind. The possibility of using nontoxic variants of diphtheria toxin as vectors to introduce peptides into the cytosol to elicit MHC class I-restricted immune response and clonal expansion of the relevant CD8+ cytotoxic T lymphocytes is discussed.

TCF11/Nrf1 overexpression increases the intracellular glutathione level and can transactivate the gamma-glutamylcysteine synthetase (GCS) heavy subunit promoter.

Gamma-glutamylcysteinylglycine or glutathione (GSH) performs important protective functions in the cell through maintenance of the intracellular redox balance and elimination of xenobiotics and free radicals. The production of GSH involves a number of enzymes and enzyme subunits offering multiple opportunities for regulation. Two members of the CNC subfamily of bZIP transcription factors (TCF11/Nrf1 and Nrf2) have been implicated in the regulation of detoxification enzymes and the oxidative stress response. Here we investigate the potential role of one of these factors, TCF11/Nrf1, in the regulation of GSH levels in the cell and particularly its influence on the expression of one of the enzymatic components necessary for the synthesis of GSH, the heavy subunit of gamma-glutamylcysteine synthetase (GCS(h)). Using overexpression of the transcription factor in COS-1 cells we show that TCF11/Nrf1 stimulates GSH accumulation. Using co-transfection with reporter constructs where reporter expression is driven through the GCS(h) promoter we show that this increase may be mediated in part by induced expression of the GCS(h) gene by TCF11/Nrf1. We further show that a distal portion of the promoter including two antioxidant-response elements (AREs) predominantly mediates the TCF11/Nrf1 transactivation and an electromobility shift assay showed that just one of these AREs specifically binds TCF11/Nrf1 as heterodimers with small Maf proteins. We suggest that TCF11/Nrf1 can operate through a subset of AREs to modulate the expression of GCS(h) together with other components of the pathway and in this way play a role in regulating cellular glutathione levels.

Role of the C-termini of human and chicken plasma retinol-binding proteins.

The most prominent differences between mammalian and non-mammalian vertebrate retinol-binding proteins (RBP) are in the C-terminal sequences. We have cloned and sequenced the cDNA for chicken RBP. Transfected COS cells that transiently expressed mammalian (human) or non-mammalian (chicken) RBP were used to demonstrate that both proteins were able to bind retinol and human transthyretin. However, we observed an increased retinol-independent secretion in cells expressing chicken RBP and reduced ligand-dependent secretion compared to the human protein. It can therefore be concluded that the C-terminal amino acid tail which is missing in chicken RBP compared to human RBP might play a role in retention and ligand-induced secretion.

Retinol-induced secretion of human retinol-binding protein in yeast.

Retinol-binding protein (RBP) functions as a transporter for retinol (vitamin A) in plasma in higher eukaryotes. We have successfully expressed human RBP in Saccharomyces cerevisiae, and its secretion was found to be induced by retinol also in this lower eukaryote. Reduced induction of secretion by retinol in a temperature-sensitive sec18-1 mutant that is blocked in secretion at the restricted temperature suggests that as in mammalian cells, RBP can be released from the endoplasmic reticulum upon addition of retinol. Thus, the molecular mechanism involved in retinol-dependent secretion of RBP appears to be conserved in yeast, and this points to yeast as a putative model system for studying retinol-regulated secretion of RBP. RBP purified from yeast was found to be indistinguishable from RBP purified from human plasma in several functional assays.

Pseudomonas toxin binds triton X-114 at low pH.

Pseudomonas toxin was found to bind Triton X-114 at pH values below pH 5.0, whereas much less binding was observed at higher pH values. The toxin bound Triton X-114 at a higher pH value in the presence of 0.14 M-NaCl, -KCl or -NaNO3 than at low salt concentrations. The results suggest that low pH in an intracellular compartment facilitates the transport of Pseudomonas toxin across the membrane and into the cytosol by inducing a conformational change in the molecule.

Insertion of diphtheria toxin B-fragment into the plasma membrane at low pH. Characterization and topology of inserted regions.

When the enzymatically active A-fragment of diphtheria toxin is translocated to the cytosol, the B-fragment inserts into the membrane in such a way that a 25-kDa polypeptide becomes shielded from proteases added to the external medium. We have attempted to determine the boundaries of this polypeptide within the toxin B-fragment as well as the topology of the B-fragment in the membrane. Chemical cleavage of the 25-kDa polypeptide with hydroxylamine and o-iodosobenzoic acid yielded fragments of sizes indicating that the 25-kDa polypeptide starts at residue approximately 300 and extends to the COOH-terminal end. Experiments where the toxin was labeled with [35S]cysteine at distinct positions of the B-fragment supported this conclusion. Treatment of cells with inserted B-fragment with L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin and with V8 protease from Staphylococcus aureus yielded protected 27- and 30-kDa fragments in addition to 25 kDa, indicating that the region 240-264 is also at the outside. The topology of the inserted B-fragment is discussed.

Rapid cellular removal of a membrane-inserted foreign polypeptide.

We have developed a system that makes it possible to study the fate of a foreign polypeptide that is inserted in the plasma membrane. Diphtheria toxin is a bacterial protein toxin that, upon acidification, has the ability to insert into the plasma membrane from the outside of eukaryotic cells. We present results that indicate endocytic uptake and degradation of the diphtheria toxin B-fragment after insertion into the membrane of Vero cells. The degradation rate of the fragment was found to be very high (t1/2 = 6 min) and dependent on cleavage of the extracellular part of the polypeptide with protease. Degradation was strongly inhibited in ATP-depleted cells, as well as at temperatures below 18 degrees C, and it was partially inhibited when the cytosol was acidified to block endocytosis from clathrin-coated pits. Degradation was also reduced in the presence of NH4Cl. The results indicate that the inserted and cleaved B-fragment is degraded by a process requiring endocytosis and transport to late endosomes or to lysosomes.

Cell-mediated reduction of the interfragment disulfide in nicked diphtheria toxin. A new system to study toxin entry at low pH.

When 125I-labeled nicked diphtheria toxin bound to Vero cells was exposed to pH less than 5.0, a small fraction was reduced to yield A- and B-fragments. The pH required for reduction correlates well with that required to induce intoxication, and the amount of A-fragment released was of the same order as that required to intoxicate the cells. Conditions that protect cells against intoxication, such as acidification of the cytosol, treatment with anion transport inhibitors, or treatment with anti-diphtheria toxin antibodies, prevented the reduction of the interfragment disulfide in cell-bound toxin. In vitro, thioredoxin reduced nicked diphtheria toxin only at pH 5.0 and lower, and the reduction was inhibited by anti-toxin antibodies. This indicates that a conformational change in the toxin, necessary for reduction by the thioredoxin system, is prevented by the antibodies. Reduction by glutathione and cysteine was most efficient at neutral pH and was not inhibited by anti-toxin. The results are consistent with the possibility that cell-mediated reduction of the interfragment disulfide is a measure of the entry of fragment A into the cytosol.

Cell-populations involved in the humoral leukocyte adherence inhibition reaction. The T8-suppressor/cytotoxic cells are both effectors and responders.

In an attempt to identify the cell subpopulation involved in the humoral leukocyte adherence inhibition (H-LAI) reaction, mononuclear leukocytes were fractionated and the response with the different cell populations was measured in the H-LAI assay using sera from lung cancer patients and a lung cancer associated antigen. The monocytes were found to exhibit a suppressive effect on the H-LAI response. The B-lymphocytes seemed not to take an active part in the reaction. Depletion of the T-lymphocytes abolished the reaction. Subsequent studies on the T-lymphocyte subpopulations revealed that removal of the T4-cells had little effect while removal of the T8-cells resulted in complete abrogation of the H-LAI response. Direct blocking by monoclonal antibodies against the T4 and T8 surface determinants gave additional support for the T8-cell dependency of the H-LAI assay. The cells that specifically were induced to loose adherence in the H-LAI assay upon incubation with serum from a lung cancer patient and lung cancer associated antigen were also analyzed. Compared to the total mononuclear leukocyte population, the relative content of monocytes and B-cells were lower in the non-adherent population. An increase was found in the relative number of T-cells. While the relative number of T4-cells decreased, the content of T8-cells was significantly increased. Furthermore, a direct correlation was found between the loss of adherence of the T8-subpopulation and the index of the H-LAI response. The present study indicates the T8-subpopulation of the T-lymphocytes to be involved both in the effector and responder side of the H-LAI reaction and gives implications for an immunological reaction behind the response of the H-LAI assay.

Translocation of diphtheria toxin to the cytosol and formation of cation selective channels.

A number of protein toxins act by translocating an enzymatically active polypeptide to the cytosol. The translocation process is best understood in the case of diphtheria toxin which binds to cell surface receptors, is then taken up by endocytosis and is subsequently translocated to the cytosol, where it inactivates elongation factor 2. The translocation of the enzymatically active part of the toxin can be induced at the level of the plasma membrane upon exposure to low pH of cells with surface-bound toxin. Receptor molecules appear to be involved in the translocation process, which also requires an inward directed H(+)-gradient and permeant anions. Cation-selective channels are formed in the membrane upon toxin entry. The B-fragment alone is much more efficient in inducing channels than the whole toxin. The current model of the translocation process is discussed.

Low pH-induced release of diphtheria toxin A-fragment in Vero cells. Biochemical evidence for transfer to the cytosol.

When Vero cells with surface-bound 125I-labeled, nicked diphtheria toxin were exposed to pH 4.5, two polypeptides of Mr 20,000 and 25,000 became protected against externally applied Pronase E. The 20-kDa polypeptide appears to be the toxin A-fragment, whereas the 25-kDa polypeptide must be derived from the B-fragment. Permeabilization of the cells with saponin allowed efflux of the 20-kDa fragment to occur, whereas most of the 25-kDa polypeptide remained associated with the cells. A number of compounds and conditions which protect cells against diphtheria toxin prevented the protection against Pronase E. Protection of the 25-kDa polypeptide occurred even when the transmembrane proton gradient (delta pH) was dissipated by acidification of the cytosol, whereas protection and release of the A-fragment were prevented under these conditions. Electrical depolarization and ATP depletion of the cells did not inhibit protection and release of the A-fragment. The data indicate that delta pH is required for the transfer of the A-fragment to the cytosol, whereas the insertion of part of the B-fragment into the membrane occurs at low pH, even in the absence of a delta pH.

Role of anions in low pH-induced translocation of diphtheria toxin.

Previous work has shown that when Vero cells with surface-bound diphtheria toxin are exposed to low pH, toxin entry across the plasma membrane is induced and that this entry involves two steps, insertion of the B-fragment of the toxin into the membrane and translocation of the enzymatically active A-fragment to the cytosol. Here we have studied the role of permeant anions in this process. It was found that when the B-fragment was inserted into the membrane, part of it, a 25-kDa polypeptide, was shielded from externally added Pronase. This insertion did not require permeant anions. The translocation of the A-fragment was monitored by measuring either its ability to inhibit protein synthesis in the cells or the appearance of radioactively labeled 21-kDa fragment after treatment of the cells with externally applied Pronase. The translocation of the A-fragment was dependent on the presence of permeant anions in the medium. However, when the cells were depleted of Cl- by incubation in Cl- free buffer at high pH, translocation of the A-fragment did not require permeant anions in the medium. The possibility that translocation of the A-fragment is inhibited by an outward directed chloride gradient rather than by the absence of chloride is discussed.

Translocation of diphtheria toxin A-fragment to the cytosol. Role of the site of interfragment cleavage.

Diphtheria toxin contains a trypsin-sensitive region with 3 closely spaced arginines in the sequence (Asn189, Arg190, Val191, Arg192, Arg193, Ser194). Cleavage of the toxin to yield A- and B-fragments ("nicking") appears to occur in a stochastic manner after either of these arginine residues. Isoelectric focusing of A-fragment prepared in vitro showed four bands of varying intensity with pI between 4.5 and 5.0, three of which could be accounted for by the three different cleavage sites. Exposure of cells with surface-bound toxin to pH less than 5.3 induces translocation of A-fragment to a position where it is shielded from external Pronase, presumably in the cytosol. A-fragment translocated in this manner had the same pI as the most acidic A-fragments, indicating that only A-fragments lacking both Arg192 and Arg193 are translocation-competent. This was confirmed by amino acid sequencing. Treatment of A-fragment with carboxypeptidase B eliminated the two bands with the highest pI while there was a concomitant increase in the bands corresponding to the two most acidic A-fragments. Such treatment of nicked diphtheria toxin increased the amount of translocated A-fragment and the ability of toxin to form cation-selective pores in the cell membrane. The site of trypsin cleavage therefore appears to be one of the factors limiting toxin entry to the cytosol.

The C-terminal RNLL sequence of the plasma retinol-binding protein is not responsible for its intracellular retention.

An in vitro model system using COS cells that transiently express human plasma retinol binding protein has been set up in which we are able to mimic the retinol dependent secretion of this protein observed in hepatocytes. In the absence of its ligand, plasma retinol binding protein is retained in the endoplasmic reticulum. It contains a C-terminal sequence, RNLL, that could function as a cryptic KDEL motif and thus be responsible for its retention in the endoplasmic reticulum. The model system has been used to test a mutant lacking these four last amino acids for retention and retinol induced secretion. The results obtained show that although plasma retinol binding protein is retained in the endoplasmic reticulum, the RNLL sequence does not seem to be responsible for its retention.

Membrane translocation of diphtheria toxin A-fragment: role of carboxy-terminal region.

The C-terminal end of diphtheria toxin A-fragment was altered and the consequences for toxicity and translocation of the A-fragment to the cytosol were studied. Mutations and deletions in the protease-sensitive, disulfide-bridged region linking the two functional parts of the toxin, the A- and B-fragments, reduced the toxicity of the protein as such, but when the mutant toxins were cleaved ("nicked") by trypsin before being added to cells, the toxicity was restored. Prevention of disulfide formation by removal of Cys186 resulted in complete loss of toxicity. To circumvent the nicking step, toxin was formed by reconstitution from separate A- and B-fragments where the A-fragments varied in the C-terminal sequences. The amino acids C-terminal to Cys186 were found not to be required for translocation. Furthermore, both charged and uncharged residues near the C-terminal end were compatible with translocation. The data indicate that the C-terminal amino acid sequence is not decisive for translocation of diphtheria toxin A-fragment to the cytosol.

Secretion of N-(4-hydroxyphenyl) retinamide-retinol-binding protein from liver parenchymal cells: evidence for reduced affinity of the complex for transthyretin.

The synthetic retinoid 4-HPR has been shown to markedly lower the plasma concentration of both retinol and RBP in rats and humans. We have studied the effect of 4-HPR on the secretion of retinol-RBP from liver cells in vivo and in vitro. In rats maintained with a normal diet, a vitamin A-deficient diet or a normal diet supplemented with 4-HPR, chylomicrons [3H]retinyl esters were rapidly cleared from the plasma. The secretion of chylomicron-derived [3H]retinol from tissues to the circulation, however, was different. In control rats, the lymph-derived [3H]retinol peaked after about 2 hr, whereas 4-HPR treatment effectively reduced this peak of [3H]retinol. Our results suggest that 4-HPR inhibits secretion of retinol-RBP from the liver. Therefore, we decided to study the effect of 4-HPR on the secretion of RBP using the human hepatoma cell line HepG2. Retinol and 4-HPR were found to induce the secretion of RBP. The medium from cells treated with 4-HPR was immunoprecipitated with antibodies against human RBP. HPLC analysis of the precipitated RBP revealed the presence of 4-HPR. When the medium from cells incubated with either 4-HPR or retinol was applied to a TTR affinity column, we found that RBP from cells incubated with 4-HPR had a considerably reduced affinity for TTR. We conclude that 4-HPR binds RBP and thereby induces secretion of RBP in HepG2 cells, and that the secreted 4-HPR-RBP complex has a reduced affinity for TTR. This observation may explain the 4-HPR-induced reduction of plasma retinol and RBP observed in in vivo studies.

Evidence for an essential role of megalin in transepithelial transport of retinol.

Transepithelial transport of retinol is linked to retinol-binding protein (RBP), which is taken up and also synthesized in a number of epithelia. By immunocytochemistry of human, rat, and mouse renal proximal tubules, a strong staining in apical endocytic vacuoles, lysosomes, endoplasmic reticulum, Golgi, and basal vesicles was observed, in accordance with luminal endocytic uptake as well as a constitutive synthesis and basal secretion of RBP. Analysis of mice with target disruption of the gene for the major endocytic receptor of proximal tubules, megalin, revealed no RBP in proximal tubules of these mice. Western blotting and HPLC of the urine of the megalin-deficient mice instead revealed a highly increased urinary excretion of RBP and retinol, demonstrating that glomerular filtered RBP-retinol of megalin-deficient mice escapes uptake by proximal tubules. A direct megalin-mediated uptake of purified RBP-retinol was indicated by surface plasmon resonance analysis and uptake in immortalized rat yolk sac cells. Uptake was partially inhibited by a polyclonal megalin antibody and the receptor-associated protein. The present data show that the absence of RBP-binding megalin causes a significantly increased loss of RBP and retinol in the urine, demonstrating a crucial role of megalin in vitamin A homeostasis.

Megalin knockout mice as an animal model of low molecular weight proteinuria.

Megalin is an endocytic receptor expressed on the luminal surface of the renal proximal tubules. The receptor is believed to play an important role in the tubular uptake of macromolecules filtered through the glomerulus. To elucidate the role of megalin in vivo and to identify its endogenous ligands, we analyzed the proximal tubular function in mice genetically deficient for the receptor. We demonstrate that megalin-deficient mice exhibit a tubular resorption deficiency and excrete low molecular weight plasma proteins in the urine (low molecular weight proteinuria). Proteins excreted include small plasma proteins that carry lipophilic compounds including vitamin D-binding protein, retinol-binding protein, alpha(1)-microglobulin and odorant-binding protein. Megalin binds these proteins and mediates their cellular uptake. Urinary loss of carrier proteins in megalin-deficient mice results in concomitant loss of lipophilic vitamins bound to the carriers. Similar to megalin knockout mice, patients with low molecular weight proteinuria as in Fanconi syndrome are also shown to excrete vitamin/carrier complexes. Thus, these results identify a crucial role of the proximal tubule in retrieval of filtered vitamin/carrier complexes and the central role played by megalin in this process.

Nuclear deviation in hepatic parenchymal cells on sinusoidal surfaces in Arctic animals.

In normal rat and human, most of the nuclei of hepatic parenchymal cells are centrally located in the cytoplasm. However, it is reported that the nuclei of hepatic parenchymal cells are situated at a deviated position on sinusoidal surfaces under pathological situations such as chronic hepatitis, hepatocellular carcinoma, adenomatous hyperplasia, or regeneration. During a study on the mechanism of extreme vitamin A-accumulation in hepatic stellate cells of arctic animals including polar bears, arctic foxes, bearded seals, and glaucous gulls, we noticed that these arctic animals displayed the nuclear deviation in hepatic parenchymal cells on sinusoidal surfaces. In this study, we assessed the frequency of hepatic parenchymal cells showing the nuclear deviation on the sinusoidal surfaces in arctic animals. A significantly higher frequency of the nuclear deviation in hepatic parenchymal cells was seen in polar bears (89.8+/-3.4%), arctic foxes (68.6+/-10.5%), bearded seals (63.6+/-8.4%), and glaucous gulls (24.2+/-5.8%), as compared to that of control rat liver (9.8+/-3.5%). However, no pathological abnormality such as fibrosis or necrosis was observed in hepatic parenchymal and nonparenchymal cells of arctic animals, and there were no differences in the intralobular distribution of parenchymal cells displaying the nuclear deviation in the livers from either arctic animals and control rats. The hepatic sinusoidal littoral cells such as stellate cells or extracellular matrix components in the perisinusoidal spaces may influence the nuclear positioning and hence the polarity and intrinsic physiological function of parenchymal cells.