Selective translocation of the A chain of diphtheria toxin across the membrane of purified endosomes.

Translocation is a necessary and rate-limiting step for diphtheria toxin (DT) cytotoxicity. We have reconstituted DT translocation in a cell-free system using endosomes purified from lymphocytes and have demonstrated this using two different probe/cell systems, which provided identical results: 125I-DT/human CEM cells and 125I-transferrin-DT/mouse BW cells. The cell-free DT translocation process was found to be dependent on the presence of the pH gradient endosome (pH 5.3)/cytosol (pH 7). Among the pH equilibrating agents, nigericin (5 microM) was found to be the most effective, inhibiting DT translocation by 88%. An optimum pH value of 7 on the cytosolic side of the membrane (pH gradient approximately 1.7) was determined. ATP per se is not required for DT translocation. 125I-DT translocation was 3-fold more active from late than from early endosomes, probably because of their slightly more acidic pH. Only the A chain of the toxin was found to escape from either 125I-DT/CEM or 125I-transferrin-DT/BW endosomes. Translocation of control endosome labels (125I-transferrin and 125I-horseradish peroxidase) was never observed. We also show that DT receptors present on resistant (mouse) cells block the translocation of the toxin and are responsible for the resistance of these cells to DT.     

Studies on the binding of staphylococcal 125I-labeled alpha-toxin to rabbit erythrocytes.

Staphylococcal alpha-toxin, a hemolytic exotoxin, can be iodinated using the lactoperoxidase method. 125 I-Labeled alpha-toxin binds to rabbit erythrocytes in an apparently irreversible and highly specific manner. The binding of 125 I-labeled alpha-toxin to erythrocytes of rabbit and human reflects the species specificity of native alpha-toxin. Binding of 125I-labeled alpha-toxin is blocked by the presence of native alpha-toxin, 127I-labeled alpha-toxin, or anti-alpha-toxin antibody. Simultaneous assays of 125I-labeled alpha-toxin binding and leakage of intracellular 86Rb+ suggest that toxin binding and membrane damage are separate, sequential functions. Both the rate and extent of binding are temperature dependent. Rabbit erythrocytes possess 5 X 10(3) binding sites/cell, while human erythrocytes possess no detectable binding sites. Treatment of rabbit erythrocytes with 125I-labeled alpha-toxin appears to decrease the number of unoccupied binding sites. Chaotropic ions can inhibit 125I-labeled alpha-toxin binding and cause bound 125I-labeled alpha-toxin to dissociate from rabbit erythrocyte membranes. Treatment of intact rabbit erythrocytes with pronase reduces both the binding capacity of the cells for 125I-labeled alpha-toxin, and the cells' sensitivity to hemolysis by native alpha-toxin. It is proposed that the primary binding site for alpha-toxin in biomembranes is a surface membrane protein.     

Staphylococcus aureus alpha-toxin. Dual mechanism of binding to target cells

Staphylococcal alpha-toxin was radiolabeled to high specific radioactivity (1,500-3,000 Ci/mmol) under retention of its hemolytic activity. Binding studies with susceptible rabbit erythrocytes and highly resistant human erythrocytes revealed that binding of alpha-toxin to target cells can occur via two different mechanisms. Binding of alpha-toxin to rabbit erythrocytes initially involves specific binding sites and occurs at low concentrations, with half-maximal binding at 1-2 nM. In contrast, toxin binding to human erythrocytes is absorptive and nonspecific, in this case, significant binding as well as hemolysis occur only at alpha-toxin concentrations exceeding 1 microM. Autoradiographic analyses of membrane-associated alpha-toxin from either cell species proved that hemolysis was inevitably associated with the formation of toxin hexamers. Our data indicate that the high susceptibility of certain target cells toward alpha-toxin is caused by the presence of specific binding sites. However, membrane damage of both susceptible and nonsusceptible target cells occurs via a common mechanism involving toxin oligomerization and pore formation.     

Identification of diphtheria toxin receptor and a nonproteinous diphtheria toxin-binding molecule in Vero cell membrane

Two substances possessing the ability to bind to diphtheria toxin (DT) were found to be present in a membrane fraction from DT-sensitive Vero cells. One of these substances was found on the basis of its ability to bind DT and inhibit its cytotoxic effect. This inhibitory substance competitively inhibited the binding of DT to Vero cells. However this inhibitor could not bind to CRM197, the product of a missense mutation in the DT gene, and did not inhibit the binding of CRM197 to Vero cells. Moreover, similar levels of the inhibitory activity were observed in membrane fractions from DT-insensitive mouse cells, suggesting the inhibitor is not the DT receptor which is specifically present in DT-sensitive cells. The second DT-binding substance was found in the same Vero cell membrane preparation by assaying the binding of 125I-labeled CRM197. Such DT-binding activity could not be observed in membrane preparation from mouse L cells. From competition studies using labeled DT and CRM proteins, we conclude that this binding activity is due to the surface receptor for DT. Treatment of these substances with several enzymes revealed that the inhibitor was sensitive to certain RNases but resistant to proteases, whereas the DT receptor was resistant to RNase but sensitive to proteases. The receptor was solubilized and partially purified by chromatography on CM-Sepharose column. Immunoprecipitation and Western blotting analysis of the partially purified receptor revealed that a 14.5-kD protein is the DT receptor, or at least a component of it.     

Differential effects of abrin on normal and tumor cells

The effects of the plant toxin abrin on normal mouse embryonic fibroblasts (MEF), an untransformed mouse cell line (NIH 3T3), and two mouse tumor cell lines (LMTK- and S-180) were studied. Measurements of cell growth and colony formation showed that MEF and S-180 cells were more sensitive to abrin intoxication than NIH 3T3 and LMTK- cells. Also, the effects of abrin on the inhibition of [3H]leucine and [3H]thymidine incorporation were more evident in MEF and S-180 cells. The basis for these varying responses to abrin by the four different cells was examined. The number of abrin binding sites per cell was determined from [125I]abrin binding studies: NIH 3T3 and LMTK- cells had significantly fewer abrin binding sites than MEF and S-180 cells. The fate of the [125I]abrin after internalization was examined by gel electrophoresis and autoradiography. A pattern of time-dependent degradation was observed, degradation being more rapid in NIH 3T3 and S-180 cells than in LMTK- and MEF cells. We conclude that the varying responses of different cells to the toxin abrin may be due to several factors, including the relative number of abrin binding sites on the cell surface and the rate of degradation of the toxin once internalized. The results also show that the sensitivities of the cells to abrin do not necessarily correlate with their normal or neoplastic state.     

Matrix assembly sites for exogenous fibronectin are decreased on human fibroblasts after treatment with agents which increase intracellular cAMP

Studies with cultured fibroblasts have shown that plasma as well as cellular fibronectin can be organized into fibrillar structures and that this organization is mediated by sites at the cell surface. Treatment of human skin fibroblasts with cholera toxin resulted in a prompt decrease in the number of binding sites for 125I-labeled plasma fibronectin and a 125I-labeled 70-kDa amino-terminal fragment of fibronectin. This decrease was accompanied by less incorporation of labeled fibronectin into deoxycholate-insoluble extracellular matrix. Binding of 125I-fibronectin was also decreased in cultures treated with epinephrine, isoproterenol, or forskolin. These results, therefore, indicate that G proteins and the adenylate cyclase system are involved in regulation of fibronectin matrix assembly sites may be one mechanism whereby hormones or growth factors can modify extracellular matrix characteristics.     

Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells.

We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell strains fall into two distinct complementation groups. The Dipr group encompasses Chinese hamster strains that are resistant only to diphtheria toxin, as well as mouse LM cells. These strains possess a normal complement of high-affinity binding sites for diphtheria toxin, but these receptors are unable to deliver active toxin fragment A to the cytosol. Cells of the DPVr group have a broader spectrum of resistance, including Pseudomonas exotoxin A and several enveloped viruses as well as diphtheria toxin. In these studies, which investigate the resistance of these cells to diphtheria toxin, we demonstrate that they possess a reduced number of specific binding sites for this toxin and behave, phenotypically, like cells treated with the proton ionophore monensin. Their resistance is related to a defect in a mechanism required for release of active toxin from the endocytic vesicle.     

Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 6.4, 31 and 24 micrograms/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 X 10(4), 1.5 X 10(4) and 2.5 X 10(5). The binding of 125I-LDL was competitively inhibited by LDL greater than VLDL greater than total HDL; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL greater than total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the Kd values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.     

Surface distribution of monosialoganglioside GM1 on human blood cells and the effect of exogenous GM1 and neuraminidase on cholera toxin surface labeling. A quantitative immunocytochemical study

The cholera toxin-colloidal gold-labeled IgG-F(ab')2 anticholera toxin ultrastructural immunocytochemical procedure was used for the localization of GM1 monosialoganglioside on the surface of human blood cells. The number of gold particles per micron of cell surface was counted and the data subjected to statistical analysis. Cholera toxin (CT) binding characteristics assessed in several subjects showed consistent labeling patterns for the various hemic cells, although some quantitative differences were noted in surface labeling densities between subjects. Neutrophils were invariably the most heavily labeled of the hemic cells, while lymphocytes, erythrocytes, and platelets exhibited only limited CT labeling. Exposure of hemic cells to neuraminidase induced a major increase in surface CT labeling that proved to be directly related to cell type and differed in many respects with the CT labeling pattern noted in nonenzyme treated cells. Newly exposed CT binding sites attributed to "masked" GM1 and/or to neuraminidase-transformed GD1a or GT1 gangliosides, showed that the number of new binding sites were nearly twice as abundant on platelet and monocyte sufaces as on the surfaces of neutrophil, lymphocyte, and erythrocyte populations. However, ratios of new CT binding sites to those normally available for CT binding were approximately 10:1 for erythrocytes, approximately 3--7:1 for lymphocytes, monocytes, and platelets, and approximately 1:1 for the neutrophil group. Exogenous GM1 was incorporated into the cell surface of the hemic cells in a differential manner. Platelets showed a dramatic increase in surface CT labeling, viz. approximately 12- to 20-fold, compared to that of other hemic cells; however, neutrophil and erythrocyte GM1 uptake was limited. Our studies have demonstrated that distinct differences exist in the extent of surface CT labeling of the various types of blood cells. They further indicated that the ability of the cell surface to incorporate exogenous GM1 may represent a differential expression of the physiochemical properties of the surface of the individual cell types.     

Modulation of the susceptibility of human erythrocytes to snake venom myotoxic phospholipases A(2): role of negatively charged phospholipids as potential membrane binding sites.

Cerrophidion (Bothrops) godmani myotoxins I (CGMT-I) and II (CGMT-II), Asp-49 and Lys-49 phospholipases A(2) (PLA2s), which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS- and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.     

Occurrence of hemolytic activity in the skin secretion of the caecilian siphonops paulensis.

The skin secretion of the caecilian Siphonops paulensis (SpSS) induces a time-and dose-dependent hemolytic response on red blood cells (RBC). When RBC from various animals species were subjected to the action of SpSS, a range of sensitivities was evident, sheep erythrocytes being the most susceptible, human, mouse and rabbit having moderate susceptibility, cow, snake and toad erythrocytes being more resistant, while S. paulensis RBC were entirely resistant. The hemolytic activity of SpSS was inhibited at temperatures higher than 60 degrees C. Both trypsin- and chymotrypsin-treated SpSS were ineffective in inducing RBC lysis. The treatment of SpSS with sheep RBC ghosts reduced its activity. There is no phospholipase activity in the SpSS.     

Effects of Ca2+-channel antagonists on nucleoside and nucleobase transport in human erythrocytes and cultured mammalian cells

Lidoflazine strongly inhibited the equilibrium exchange of uridine in human erythrocytes (Ki approximately 16 nM). Uridine zero-trans influx was similarly inhibited by lidoflazine in cultured HeLa cells (IC50 approximately to 80 nM), whereas P388 mouse leukemia and Novikoff rat hepatoma cells were three orders of magnitude more resistant (IC50 greater than 50 microM). Uridine transport was also inhibited by nifedipine, verapamil, diltiazem, prenylamine and trifluoperazine, but only at similarly high concentrations in both human erythrocytes and the cell lines. IC50 values ranged from about 10 microM for nifedipine and about 20 microM for verapamil to more than 100 microM for diltiazem, prenylamine and trifluoperazine. The concentrations required for inhibition of nucleoside transport are several orders higher than those blocking Ca2+ channels. Lidoflazine competitively inhibited the binding of nitrobenzylthioinosine to high-affinity sites in human erythrocytes, but did not inhibit the dissociation of nitrobenzylthioinosine from these sites on the transporter as is observed with dipyridamole and dilazep.     

Cell selective cytotoxicity of a peptide toxin from the cyanobacterium Microcystis aeruginosa

The effects of a cyclic peptide toxin, isolated from the cyanobacterium Microcystis aeruginosa, on cell morphology and ion transport in human erythrocytes, isolated rat hepatocytes and mouse fibroblasts (3T3) were studied. Neither in erythrocytes nor in fibroblasts did the toxin cause morphological alterations. In hepatocytes the toxin induced marked morphological alterations at a concentration of about 50 nM. In erythrocytes and fibroblasts no effects on ion transport were observed. In hepatocytes the toxin induced a significant increase in both phosphate and potassium efflux at concentrations far below the concentration causing morphological alterations (0.1 and 1 nM, respectively). It is suggested that the cytotoxicity of the toxin is not due to a non-specific interaction with the plasma membrane and that the effects of the toxin in hepatocytes are probably due to an interaction of the toxin with cytoskeletal elements.     

Biological activity of sea anemone proteins: II. Cytolysis and cell line toxicity

Potent cytolytic activity was exhibited by proteins extracted from three sea anemones viz. Heteractis magnifica, Stichodactyla haddoni and Paracodylactis sinensis by affecting the red blood corpuscles (RBC) and the mouse fibroblast cell line (L929) and leukemia cell line (P388). Crude toxin of all the three anemone species induced spontaneous hemolysis of chicken, goat and human erythrocytes. The crude toxin of H. magnifica (0.98 mg/ml) elicited hemolysis at levels of 4096, 512 and 4096 HU (hemolytic unit) in chicken, goat and human erythrocytes respectively. Subsequently, the crude toxin of S. haddoni (0.82 mg/ml) exhibited a hemolytic activity of 256, 128 and 512 HU and that of P. sinensis (0.60 mg/ml) had a hemolytic activity of 128, 4096 and 512 HU. Most of the partially purified proteins of these anemones also exhibited the activity against the three different erythrocytes. The viability of L929 and P388 was adversely affected on adding the crude toxins. The symptoms of toxicity shown by the cells were rounding, lysis and detachment from the substratum. These effects were the least in S. haddoni, as compared to those the crude toxins of the other two species. Inhibition of growth of L929 exhibited by the toxin of the three species ranged between 61.08 and 93.38%. Similarly, inhibition of the growth of P388 ranged between 51.32 and 86.16%. The present investigation reveal the cytotoxic nature of anemone toxins.     

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin- sensitive cells

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.     

Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.

A monkey cDNA (pDTS) encoding a diphtheria toxin (DT) sensitivity determinant was isolated by expression cloning in mouse L-M cells. Mouse cells are naturally resistant to DT, because they lack functional cell surface receptors for the toxin. Unlike wild-type L-M cells, pDTS-transfected mouse cells are extremely toxin sensitive and specifically bind radioiodinated DT. Intoxication of the transfected cells requires receptor-mediated endocytosis of the bound toxin. The cDNA is predicted to encode an integral membrane protein that is identical to the precursor of a heparin-binding EGF-like growth factor. The DT sensitivity protein is thus a growth factor precursor that DT exploits as a receptor.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

The binding of fluorescein-labelled stapbylococcal alpha toxoid to erythrocytes

Erythrocytes of different animal species have variable hemolytic sensitivity to staphylococcal alpha toxin. Specific and non-specific binding of toxin was measured using fluorescein-labelled toxoid. These studies indicate that toxoid binding to erythrocytes increases with concentration for all species tested. Scatchard plot analyses of 35 animals representing seven species indicate that rabbit, pig, cow, and chicken erythrocytes possess 125 980, 103 920, 82 500, and 41 200 receptors per cell, respectively. The number of receptors remains constant over a period of at least 10 days. No detectable receptors were found for human, rat, and guinea pig erythrocytes. A correlation coefficient of 0.992 exists between receptor number and hemolytic sensitivity for those species having receptors. Variation in hemolytic sensitivity is governed by receptor number and not by variation in the dissociation constant. A threshold sensitivity of 37 000 receptors per cell has been calculated. Since species lacking detectable receptors have considerable sensitivity to hemolysis, it is proposed that two binding mechanisms, specific and non-specific, exist which prepare erythrocytes for destruction.     

Familial insulin-resistant diabetes secondary to an affinity defect of the insulin receptor

We describe three siblings with a mild diabetes mellitus in combination with acanthosis nigricans and multiple minor physical abnormalities. Fasting plasma insulin was elevated up to 100-fold as compared with normal values, and the diabetes was classified as insulin resistant. Insulin-binding studies on erythrocytes, monocytes, and cultured fibroblasts disclosed an abnormally reduced binding capacity, as compared with that of healthy controls, which was most prominent at low concentrations of insulin. Scatchard analysis on erythrocytes of the three patients revealed a normal number of total insulin-binding sites per cell, but a complete lack of insulin binding to the high-affinity receptor component. The findings are consistent with the assumption of two genetically distinct types of insulin receptors.