The role of the diphtheria toxin receptor in cytosol translocation

The role of the receptor in the transport of diphtheria toxin (DT) to the cytosol was examined. A point-mutant form of DT, CRM 107 (CRM represents cross-reacting material), that has an 8,000-fold lower affinity for the DT receptor than native toxin was conjugated to transferrin and monoclonal antibodies specific for the cell-surface receptors T3 and Thy1. Conjugating the binding site-inactivated CRM 107 to new binding moieties reconstituted full toxicity, indistinguishable from native DT linked to the same ligand, indicating that the entry activity of the DT B chain can be fully separated from the receptor binding function. Like DT, the toxin conjugates exhibited a dose-dependent lag period before first-order inactivation of protein synthesis. Inactivation of the binding site of the toxin portion of the conjugate was found to have no effect on the kinetics of protein synthesis inactivation. The receptor used by the toxin determined the length of the lag period relative to the killing rate. Comparing the potency of CRM 107 conjugates with native DT, standardized for receptor occupancy, shows that new receptors can be as or more efficient than the DT receptor in transporting DT to the cytosol. The transferrin-CRM 107 conjugate, unlike native DT, was highly toxic to murine cells. All the data presented are consistent with a model that the DT receptor, other than initiating rapid internalization of the toxin to low pH compartments, is unnecessary for transport of the toxin to the cytosol and that membrane translocation activity is expressed by the DT B subunit independent of the receptor-binding site.     

Hints of nonhierarchical folding of acidic fibroblast growth factor

We have analyzed by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) the helical propensity of the all-beta protein acidic fibroblast growth factor (aFGF) and two peptides corresponding to beta-strand 8 (beta8 peptide, amino acids 95-107) and the beta-strand 8/turn/beta-strand 9 hairpin (beta8/9 peptide, amino acids 95-114), which has been involved in receptor binding. A secondary structure prediction of aFGF carried out by several procedures labels the 95-104 sequence as predominantly alpha-helical. A titration of aFGF with 2,2,2-trifluoroethanol (TFE) induces a change in the far-UV CD spectrum of the protein giving rise to a prominent alpha-helical shape (22% alpha-helix). The cooperativity of the transition and the moderate TFE concentrations used (midpoint at 24%) suggest that the effect of TFE is specific. Moreover, a titration performed at pH 2 yields a higher amount of alpha-helix (55%) at a smaller TFE concentration. Synthetic peptides containing the beta8 and beta8/9 sequences display a random coil conformation at pH 7 but acquire alpha-helical structure in the presence of TFE, methanol, and SDS micelles. At pH below 3.0 a significant amount (20-30%) of alpha-helical conformation is present in both the beta8 and beta8/9 peptides even in the absence of other solvent additives. The secondary structure of the peptides was determined by proton nuclear magnetic resonance (1H NMR). These results suggest that the 95-114 sequence of aFGF has helical propensity and that the protein may fold nonhierarchically in the early steps of folding, acquiring its final beta-structure by a later interaction with the rest of the polypeptide.     

Extension of juxtamembrane domain of diphtheria toxin receptor arrests translocation of diphtheria toxin fragment A into cytosol

Diphtheria toxin (DT) binds to the EGF-like domain of the DT receptor (DTR), followed by internalization and translocation of the enzymatically active fragment A into the cytosol. The juxtamembrane domain (JM) of the DTR is the linker domain connecting the transmembrane and EGF-like domains. We constructed mutants of DTRs with altered JMs and studied their abilities for DT intoxication. Although DTR mutants with extended JMs showed normal DT binding activity, the cells expressing the mutants showed both reduced translocation of DT fragment A into the cytosol and reduced sensitivity to DT, when compared with cells expressing wild-type DTR. These results indicate that the JM contributes to DT intoxication by providing a space appropriate for the interaction of DT with the cell membrane. The present study also indicates that consideration of epitopes of an immunotoxins would be an important factor in the design of potent immunotoxins.     

Quantal entry of diphtheria toxin to the cytosol

The rate-limiting step in diphtheria toxin (DT) intoxication of Vero cells has been determined utilizing cycloheximide as an inhibitor of the intoxication process. Cycloheximide is shown to inhibit the toxin catalyzed ADP-ribosylation of elongation factor 2 (EF-2). The inhibition is blocked by puromycin thus establishing the ribosome as the location of cycloheximide protection. Washing cells free of cycloheximide rapidly reverses the protective effect. The initial rates of protein synthesis inhibition observed after removal of cycloheximide from DT-intoxicated cells are 5 to 12-fold greater than rates observed in unprotected cells and are shown to reflect ADP-ribosylation of EF-2 by cytosolic DT. Ten to thirty minutes after cycloheximide removal, the rate of protein synthesis inhibition abruptly changes to values identical to those of unprotected cells. Both the initial rates and extent of the initial rapid inactivation are directly related to toxin concentration and time of incubation with DT in the presence of cycloheximide. We concluded that: the rate-limiting step in protein synthesis inhibition by DT is not the ADP-ribosylation of EF-2 by cytosolic toxin but rather the earlier entry step of DT into the cytosol. DT enters the cytosol as a bolus of sufficient size to rapidly inactivate all EF-2 in that cell. It is inferred from 1 and 2 that the first order inactivation rate exhibited by DT is the result of the probability of the release of a bolus of toxin to the cytosol of any cell in the population per unit time. Autoradiographic analysis of intoxicated cell populations support this two-population state model. The size of a single bolus or quantum of DT is calculated from data over the range of 10(-11) to 10(-9) M DT and is found to remain constant. We suggest that the cytosolic entry mechanism of DT results from a unique ability of the internalized toxin molecules to destabilize the vesicular membrane resulting in a random release of a bolus of toxin into the cytosol. Because the bolus size remains constant over a 50-fold change in receptor occupancy the possibility is raised that DT undergoes a post-receptor packaging process, package size remaining a constant and package number increasing with receptor occupancy.     

Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo

A detailed proteolysis study of internalized diphtheria toxin (DT) within rat liver endosomes was undertaken to determine whether DT-resistant species exhibit defects in toxin endocytosis, toxin activation by cellular enzymes or toxin translocation to its cytosolic target. Following administration of a saturating dose of wild-type DT or nontoxic mutant DT (mDT) to rats, rapid endocytosis of the intact 62-kDa toxin was observed coincident with the endosomal association of DT-A (low association) and DT-B (high association) subunits. Assessment of the subsequent post-endosomal fate of internalized mDT revealed a sustained endo-lysosomal transfer of the mDT-B subunit accompanied by a net decrease in intact mDT and mDT-A subunit throughout the endo-lysosomal apparatus. In vitro proteolysis of DT, using an endosomal lysate, was observed at both neutral and acidic pH, with the subsequent generation of DT-A and DT-B subunits (pH 7) or DT fragments with low ADP-ribosyltransferase activity (pH 4). Biochemical characterization revealed that the neutral endosomal DT-degrading activity was due to a novel luminal 70-kDa furin enzyme, whereas the aspartic acid protease cathepsin D (EC 3.4.23.5) was identified as being responsible for toxin degradation at acidic pH. Moreover, an absence of in vivo association of the DT-A subunit with cytosolic fractions was identified, as well as an absence of in vitro translocation of the DT-A subunit from cell-free endosomes into the external milieu. Based on these findings, we propose that, in rat, resistance to DT may originate from two different mechanisms: the ability of free DT-A subunits to be rapidly proteolyzed by acidic cathepsin D within the endosomal lumen, and/or the absence of DT translocation across the endosomal membrane, which may arise from the absence of a functional cytosolic translocation factor previously reported to participate in the export of DT from human endosomes.     

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.     

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.     

Possible activity of acidic fibroblast growth factor as a progression factor rather than a transforming factor.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are mitogens for mesoderm- and neuroectoderm-derived cells. The facts that FGF-related proteins are oncogenic and that FGFs are expressed in a variety of tumor cell lines or tumor tissues suggest the transforming activities of FGFs. To examine such an activity of aFGF, we introduced a human aFGF expression vector into two populations of Rat-1 cells; one was non-transformed (nRat-1), the other was partially-transformed (tRat-1). tRat-1 cells transfected with aFGF cDNA formed larger colonies in soft agar and produced larger and more malignant tumors in nude mice than those of parental cells. In contrast, nRat-1 cells transfected with aFGF cDNA neither formed colonies in soft agar nor produced tumors in nude mice. Our results suggest that high expression of aFGF may enhance a tumorigenic potential of Rat-1 cells rather than confer such a potential de novo.     

Energetics of heparin binding to human acidic fibroblast growth factor

The binding of low-molecular-weight heparin to an amino-terminal-truncated, 132-amino-acid, human acidic fibroblast growth factor form has been studied by isothermal titration calorimetry. This technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change are also calculated. Experiments in different buffers and pH values show that the protonic balance during the reaction is negligible. Experiments made at pH 7.0 with NaCl concentrations ranging from 0.20 to 0.60 M revealed changes in enthalpy and Gibbs energy in the range of -30- -17 and -27- -24 kJ x mol(-1), respectively. Isothermal titration calorimetry was also performed at different temperatures to obtain a value for the heat-capacity change at pH 7.0 and 0.4 M NaCl concentration of -96 J K- x mol(-1). A change in the length of heparin brought about no change in the thermodynamic parameters at 25 degrees C under the same experimental conditions. Changes upon ligand binding in the range of -50- -200 A2 in both polar and non-polar solvent-accessible surface areas were calculated from thermodynamic data by using different parametric equations taken from the literature. These values suggest a negligible overall conformational change in the protein when it binds to heparin and no formation of any protein-protein interface.     

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.     

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.     

Establishment of monoclonal antibodies against human acidic fibroblast growth factor.

Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibroblast growth factor (haFGF) were established using recombinant haFGF as an immunogen. The recognition sites of four MAbs designated AF1-52, 81, 114 and 1C10 for the haFGF molecule were examined by binding studies with synthetic polypeptides and with amino-terminal truncated forms of haFGF. These experiments suggested that AF1-52, 114, and 1C10 MAbs recognize epitopes within the 1-5, 44-132 and 6-43 amino acid sequences, respectively. However, the epitope recognized by the AF1-81 MAb could not be determined. The sandwich EIA method constructed with these MAbs was sensitive to 1.5 pg/well of haFGF and had no cross-reactivity with human basic FGF, bovine aFGF or the hst-1 gene product.     

Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life

The mechanism(s) by which heparin influences the biological activities of acidic and basic fibroblast growth factors (aFGF and bFGF) is not completely understood. One mechanism by which heparin could alter the biological activities of aFGF and bFGF is by altering their biological half-lives. We investigated the possibility that heparin potentiates aFGF-induced neurite outgrowth from PC12 cells by prolonging its biological half-life. Under conditions where heparin potentiated aFGF-induced neurite outgrowth, we observed that heparin increased the biological half-life of aFGF from 7 to 39 hr. We determined that greater than 25 hr of exposure to active aFGF was required for induction of neurite outgrowth. If aFGF activity was maintained for greater than 25 hr by periodic readdition of factor, heparin no longer potentiated aFGF-induced neurite outgrowth. These observations strongly suggest that heparin potentiates the activity of aFGF by prolonging its biological half-life. The protease inhibitors hirudin, leupeptin, and pepstatin A did not potentiate aFGF-induced neurite outgrowth, indicating that proteases inhibited by these inhibitors are not responsible for the loss of aFGF activity that we observed. However, aprotinin potentiated aFGF neurite-promoting activity approximately sevenfold, indicating that proteases that are inhibited by aprotinin are at least partially responsible for aFGF inactivation. These observations suggest that heparin regulates the activity of aFGF by regulating its proteolytic degradation, thereby regulating its biological half-life.     

Molecular characterization of recombinant human acidic fibroblast growth factor produced in E. coli: comparative studies with human basic fibroblast growth factor.

Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.     

Myogenic differentiation triggered by antisense acidic fibroblast growth factor RNA.

Acidic fibroblast growth factor (FGF) and related family members regulate differentiation in organisms as diverse as Xenopus laevis and mammals. We utilized a well-characterized model of myogenic development to directly assess the importance of endogenously produced FGF in controlling differentiation. A role for endogenous FGF is suggested by the previous finding that acidic and basic FGF abundance in cultured myocytes decreases during differentiation. In this study we inhibited the endogenous production of FGF in murine Sol 8 myoblasts by using antisense RNA and observed precocious myogenic differentiation. Exogenously supplied acidic FGF rescues this phenotype. Further results suggest that the effect of FGF on myogenic differentiation is mediated in part through inhibition of myogenin expression. These results demonstrate a direct role for endogenously synthesized growth factors in regulating myogenesis and provide support for a general role for related proteins in mammalian development.     

Effect of polyanions on the refolding of human acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.     

Amino acid sequence of human acidic fibroblast growth factor

The complete amino acid sequence of human brain acidic fibroblast growth factor (aFGF) has been established. Human aFGF consists of 140 amino acids and is highly homologous to bovine aFGF (11 amino acid replacements). Results from experiments involving alkylation of cysteine residues are compatible with the possibilities that in aFGF all three cysteines exist as free sulfhydryls, or alternatively, that a disulfide bridge is present but cannot be identified due to disulfide scrambling caused by the SH group of the remaining cysteine. A potential glycosylation site Asn114-Gly115-Ser116 is present in aFGF but the mitogen does not bind to lectins suggesting that it may not be glycosylated.