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.     

A point mutation of proline 308 in diphtheria toxin B chain inhibits membrane translocation of toxin conjugates

Diphtheria toxin (DT) is a soluble protein that translocates across hydrophobic lipid bilayers in response to low pH. The translocation activity of DT has been localized to the 40-kDa toxin B chain and can be expressed independently of the C-terminal receptor binding site. Buried hydrophobic domains in DT are thought to participate in the membrane translocation process. We have identified a mutant form of DT, CRM 102, that has a point mutation at position 308 (Pro----Ser) within one of these hydrophobic domains. CRM 102 conjugated to a monoclonal antibody against the T cell receptor, the transferrin receptor, or transferrin itself is approximately 10-fold less toxic than native DT or a control DT mutant, CRM 103, linked to the same binding moieties. Direct measurement of membrane translocation activity by exposure of cells to low extracellular pH demonstrates that CRM 102 conjugates express only 10% of the translocation activity of the control toxin conjugates. However, when CRM 102 or 102 conjugates bind and kill cells via the DT receptor, no reduction in membrane translocation activity is observed. The defect in CRM 102 is not evident in the presence of 20 mM NH4Cl. The defect in translocation also has no effect on the ratio of the lag time before protein synthesis inhibition begins to the rate of protein synthesis inhibition. Thus, the proline-serine substitution at position 308 disrupts the membrane translocation process and distinguishes between two routes of DT entry: DT receptor-mediated entry and entry mediated by alternate receptors.     

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.     

Transgenic mice expressing a fully nontoxic diphtheria toxin mutant, not CRM197 mutant, acquire immune tolerance against diphtheria toxin

We previously developed a method termed "toxin receptor-mediated cell knockout" (TRECK). By the TRECK method, a single or repeated shot(s) of diphtheria toxin (DT) conditionally ablates a specific cell population from transgenic mice expressing the DT receptor transgene under the control of a cell type-specific promoter. In some cases of TRECK, frequent and high-dose administration of DT is required, raising the concern that these frequent injections of DT could cause production of anti-DT antibody, which would neutralize further DT administration. To solve this problem, we aimed to generate transgenic mice genetically expressing a nontoxic DT mutant, with the expectation that they may naturally acquire immune tolerance to DT. Unexpectedly, the G52E DT mutant, which is well known as the nontoxic DT variant cross reacting material 197 (CRM197), exhibited cytotoxicity in yeast and mammalian cells. Cytotoxicity of CRM197 was abrogated in cells mutated for elongation factor 2 (EF-2), indicating that CRM197 exerts its toxic effects through EF-2, similar to wild-type DT. On the other hand, the K51E/E148K DT mutant exhibited no detectable cytotoxicity. This led us to successfully obtain DT gene transgenic mice, which exhibited no histological abnormalities, and indeed acquired immune tolerance to DT.     

Molecular Cloning,Structural Modeling and the Production of Soluble Triple-Mutated Diphtheria Toxoid (K51E/G52E/E148K) Co-expressed with Molecular Chaperones in Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33 ± 17.47 μg/ml) under the optimal condition (induction with 0.1 mM IPTG at 20 °C for 24 h). Cells containing pG-Tf2, expressing trigger factor and GroEL-GroES, accumulated the highest amount of soluble CRM197EKTrxHis at 111.24 ± 10.40 μg/ml after induction for 24 h at 20 °C. The soluble CRM197EKTrxHis still possesses nuclease activity and completely digest λDNA at 25 and 37 °C with 8- and 4-h incubation, respectively. Molecular modeling of diphtheria toxin, CRM197 and CRM197EK indicated that substitutions of two amino acids (K51E/E148K) may cause poor NAD binding, consistent with the lack of toxicity. Therefore, CRM197EK might be used as a new potential carrier protein. However, further in vivo study is required to confirm its roles as functional carrier protein in conjugate vaccines.     

Conformational changes in diphtheria toxoids. Analysis with monoclonal antibodies

Monoclonal antibodies (Mab) were raised against CRM197, a non-toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRM1001, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mab11.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as 'closed' in contrast with an 'open' conformation of CRM197, CRM176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.     

Cloned fragment of diphtheria toxin linked to T cell-specific antibody identifies regions of B chain active in cell entry

The role of discrete domains of diphtheria toxin (DT) B chain in cytosol entry and cytotoxicity was investigated by linking a monoclonal antibody recognizing the human T cell-specific antigen T3 (UCHT1) to diphtheria toxin (UCHT1-DT), DT A subunit (UCHT1-DTA), or to a genetically engineered form of DT (UCHT1-MspSA) lacking the C-terminal 17-kDa portion of the B subunit. The N-terminal 21-kDa region of DT B chain increased toxicity of UCHT1-DTA 100-fold (UCHT1-MspSA) while addition of the C-terminal 17-kDa region (UCHT1-DT) increased toxicity 100-fold more. The cytotoxicity was dependent upon antibody binding as demonstrated by blocking toxicity with excess UCHT1. The differences in toxicity between these reagents were not due to differences in ADP-ribosylation activity of DT A chain, binding activity of the antibody moiety, extent of DT nicking, or the cross-linking method, so we conclude that the large differences in toxicity were due to the presence of different B chain domains. The large increase in toxicity by the C-terminal region of DT B did not appear to be caused by DT receptor binding. The lysosomotropic agent NH4Cl blocked the cytotoxic effect of DT, UCHT1-DT, and UCHT1-MspSA but not UCHT1-DTA.     

CRM197 (nontoxic diphtheria toxin): effects on advanced cancer patients

Purpose: Many years ago, diphtheria toxin (DT) showed antitumor activity in mice and in humans, but it was unclear whether this depended on the toxicity of the molecule only or on its strong inflammatory-immunological property as well. To deal with this open question, we planned to treat a group of cancer patients with cross-reacting material 197 (CRM197). CRM197 is a nontoxic mutant of DT that shares the immunological properties of the native molecule and its ability to bind to heparin-binding epidermal growth factor (HB-EGF), the specific cell-membrane receptor for DT that is often overexpressed in cancer. Methods: 25 outpatients with various advanced tumors who were refractory to standard therapies (23 subjects) or had refused, in whole or in part, conventional therapies (2 subjects) were treated with CRM197 injected subcutaneously in the abdominal wall, on alternate days, for 6 days. Three different dosages (1.7, 2.6, or 3.5 mg/day) were used according to the patients degree of immunological reactivity to DT/CRM197 (none, moderate, or high). Results: After the first administration of CRM197, a significant increase in the number of circulating neutrophils and in the serum level of TNF- was detected. Toxicities were minimal. Only patients with delayed-type hypersensitivity to DT/CRM197 had irritating skin reactions in the injection sites and a flu-like syndrome with fever. Pharmacokinetics showed a mean peak concentration (12.7 ng/ml) 12 h after the first injection and a mean half-life of 18.1 h. There were two complete and one partial responses (metastatic breast carcinoma, neuroblastoma, and metastatic breast carcinoma) lasting 4, 45+, and 15 months, respectively. Six cases of stable disease, lasting from 1 to 15 months, were also recorded. Conclusions: CRM197 injected subcutaneously elicited an inflammatory-immunological reaction, caused tolerable toxicities, was absorbed to a good extent into the circulatory system, and exerted some degree of biological antitumor activity. A possible role of neutrophils and TNF- in the mode of action of the molecule is hypothesized.     

分子伴侣对白喉毒素无毒突变体CRM197重组蛋白在大肠杆菌中可溶性表达的促进作用

为了获得有活性的白喉毒素突变体蛋白 (Cross-reacting material 197,CRM197),本研究利用分子伴侣pG-KJE8与重组质粒pET28a-CRM197在大肠杆菌原核表达系统中进行共表达,来促进目的蛋白的正确折叠,进而提高CRM197蛋白的可溶性表达。将质粒转化至大肠杆菌后并诱导其表达目的蛋白,再通过SDS-PAGE胶染色、Western blotting等技术对所得蛋白进行检测分析。结果发现：利用体外重组技术成功得到了pET28a-CRM197重组蛋白原核表达质粒,且CRM197重组蛋白在原核表达系统中主要以包涵体形式表达;通过探索和优化,确定了诱导蛋白的最佳浓度和温度,当加入终浓度为1.0 mmol/L IPTG、0.5 mg/mL L-阿拉伯糖、5.0 ng/mL四环素,在20 ℃条件下诱导16 h时,目的蛋白的可溶性表达得到显著提高;可溶性表达的CRM197重组蛋白可以与CRM197一抗发生特异性结合,免疫反应性良好。因此,研究发现分子伴侣pG-KJE8可以促进CRM197重组蛋白在大肠杆菌中以可溶性形式表达,且能很好地与CRM197一抗发生特异性结合,证实CRM197重组蛋白具有良好的免疫反应性,为CRM197蛋白的工业化生产及应用奠定了一定的基础。     

The cytotoxic effect of diphtheria toxin on the actin cytoskeleton

Diphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin. The toxin entry rate into cells (HUVEC) was determined by measuring the ADP-ribosyltransferase activity. DT uptake was nearly 80% after 30 min. The efficiency was determined as K_m = 2.2 nM; V_max = 0.25 pmol.min⁻¹. The nuclease activity was tested with hyperchromicity experiments, and it was concluded that G-actin has an inhibitory effect on DT nuclease activity. In thepresence of DT and mutant of diphtheria toxin (CRM197), F-actin depolymerisation was determined with gel filtration, WB and fluorescence techniques. In the presence of DT and CRM197, 60–65% F-actin depolymerisation was observed. An in vitro FA-actin interaction and F-actin depolymerisation were reported in our previous paper. The present study thus confirms the depolymerisation of actin cytoskeleton in vivo.     

Characterization of the diphtheria toxin receptor-binding domain

The carboxyl-terminal region of diphtheria toxin (DT) has been analysed in order to determine regions of receptor recognition. Biochemical cleavage of the toxin with hydroxylamine (HA) was used to generate the peptides HA9DT (residues 454–535), HA6DT (residues 482–535), and HA3DT (residues 454–461). Characterization of HA6DT demonstrated that the final 54 amino acids of DT are sufficient to constitute the receptor-binding domain of the toxin. Within HA9DT, the region encompassing HA3DT and containing the highly cationic polyphosphate-binding site did not contribute to the binding ability of HA6DT. Consistent with this observation, HA3DT itself did not compete for binding of radiolabelled DT to Vero cells. A 30-amino acid synthetic peptide composed of residues 506–535 did not block receptor binding of DT, indicating that residues toward the amino-terminus of HA6DT, or the entire HA6DT region, are required for receptor recognition.     

A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity

The role of diphtheria toxin (DT) B-chain subdomains in DT cytotoxicity and immunotoxin mechanism of action has been investigated. OKT3 (mAb to the CD3 surface Ag of human T lymphocytes) was conjugated to DT or the DT mutant CRM 1001, which has a cys----tyr substitution at position 471 of the B chain. OKT3-CRM 1001 immunotoxin was about 1400-fold less cytotoxic for CD3 Jurkat cells than OKT3-DT and had a 12-fold slower kinetics of protein synthesis inactivation, CRM 1001 killed DT-sensitive Vero cells at a 5000-fold higher concentration than DT. Its cell surface-binding activity was comparable to DT. Based on kinetics of cell inactivation, toxicity determination at low extracellular pH and Triton X-114 distribution, it was concluded that CRM 1001 is defective in at least one crucial step of toxin penetration and is unable to cross cell membranes as efficiently as DT. The substituted cysteine appears to be important for DT translocating functions. Data on the function of DT B-chain subdomains are relevant for the study of whole toxin conjugates and their mechanism of action.     

Construction of an immunotoxin by linking a monoclonal antibody against the human epidermal growth factor receptor and a hemolytic toxin

Hybrid molecules obtained through conjugation of monoclonal antibodies and toxins constitute an approach under exploration to generate potential agents for the treatment of cancer and other diseases. A frequently employed toxic component in the construction of such immunotoxins is ricin, a plant toxin which inhibits protein synthesis at ribosomal level and so requires to be internalized by the cell. A hemolytic toxin isolated from the sea anemone Stichodactyla helianthus, which is active at the cell membrane level, was linked through a disulfide bond to the anti-epidermal growth factor receptor monoclonal antibody ior egf/r3. The resulting immunotoxin did not exhibit hemolytic activity except under reducing conditions. It was toxic for H125 cells that express the human epidermal growth factor receptor, but non-toxic for U1906 cells that do not express this receptor.     

Roles of Dppa2 in the regulation of the present status and future of pluripotent stem cells

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored protein, known as proHB-EGF. ProHB-EGF is cleaved by metalloproteases through a process referred to as 'ectodomain shedding', resulting in the formation of soluble HB-EGF. Both proHB-EGF and soluble HB-EGF are biologically active; the former acts on neighbouring cells through juxtacrine signalling, whereas the latter can move to distant locations. Elevated HB-EGF expression has been observed in ovarian and some other cancers. CRM197, a diphtheria toxin (DT) mutant, binds directly to the epidermal growth factor (EGF)-like domain and represses the mitogenic activity of HB-EGF. Recently, monoclonal antibodies (mAbs) specific for human HB-EGF were generated by immunizing HB-EGF-deficient mice with human HB-EGF (Hamaoka et al. (2010) J. Biochem. 148, 55-69). Most of the mAbs can bind to the EGF-like domain of HB-EGF, but fail to inhibit the mitogenic activity of soluble HB-EGF. However, some mAbs prevented the ectodomain shedding of proHB-EGF and inhibited the proliferation of EGF receptor-expressing cells stimulated by proHB-EGF-expressing cells. Hamaoka et al. showed that CRM197 prevents the ectodomain shedding of proHB-EGF. Thus, these mAbs function as specific inhibitors for the ectodomain shedding of HB-EGF and may be useful for treating cancers exhibiting elevated levels of HB-EGF.     

An IL 2-secreting T cell hybridoma that responds to a self class I histocompatibility antigen in the H-2D region

A self-reactive T cell hybridoma that secretes IL-2 in response to H-2d haplotype cells resulted from a fusion of BALB/cBy lymph node cells with the AKR thymoma BW5147. The lymph node cells used had been enriched for cells reactive to (TG)-A--L, but neither this antigen nor fetal calf serum were required for stimulation of the hybridoma designated 3DT52.5. The gene product responsible for stimulation mapped to the H-2D region. Allogeneic cells of the b, f, k, q, and s haplotypes failed to stimulate. Not all H-2d haplotype cells were effective stimulators of 3DT52.5. Peritoneal cells and splenic B cells were much more stimulatory than splenic T cells. Most tumor cell lines of H-2d derivation and of B cell or macrophage/monocyte lineage were stimulatory, whereas H-2d T cell lines were not. The capacity to stimulate 3DT52.5 did not correlate with the ability to stimulate I region-restricted hybridomas, or with the ability to be induced to stimulate such hybridomas. Stimulatory cell lines did not apparently produce a soluble factor required for stimulation, and negative cell lines were not inhibitory. The monoclonal antibody 27-11-13, which reacts with H-2D of the b, d, and q haplotypes, inhibited stimulation of 3DT52.5 but did not inhibit stimulation of the sibling hybridoma 3DT18.11, which responds to (TG)-A--L plus I-Ad. Conversely, the monoclonal anti-I-Ad antibody MK-D6 inhibited stimulation of 3DT18.11 but not 3DT52.5. Although it is clear that 3DT52.5 recognizes a class I antigen coded for in the H-2D region, the precise molecular nature of the antigen is unknown. The structure of the antigen receptor on this hybridoma may prove to be of interest when it can be compared with receptors found on T cell hybridomas restricted by class II histocompatibility antigens.     

Regulation of insulin receptor kinase activity by insulin mimickers and an insulin antagonist

Three agents which mimic insulin action in intact cells (concanavalin A, wheat germ agglutinin, and polyclonal insulin receptor antibody), mimicked insulin's ability to stimulate the kinase activity of purified insulin receptors. In contrast, monoclonal insulin receptor antibody, an antagonist of insulin action, did not stimulate the phosphorylation of the insulin receptor either in intact IM-9 cells or in purified receptor preparations. This antibody, however, antagonized the ability of insulin to stimulate the phosphorylation of the receptor both in intact cells and in the purified receptor. These studies with insulin mimickers and an insulin antagonist are consistent with a role for the kinase activity of the receptor mediating the actions of insulin.     

The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197.

The amino-acid sequences of two diphtheria toxin-related, non-toxic proteins, CRM45 and CRM197 , were deduced from the complete sequence of their genes: tox 45 and tox 197. CRM45 lacks the last 149 C-terminal amino-acid residues, but is otherwise identical to diphtheria toxin: a single C----T transition introduces an "ochre" (TAA) termination signal in tox 45, after the codon for threonine-386. A single G----A transition was also found in tox 197, leading to the substitution of glycine-52, present in the wild-type toxin, with glutamic acid in CRM197 . This aminoacid change is responsible for the loss of the NAD:EF2 ADP-ribosyltransferase activity in CRM197 , due most probably to an alteration of the NAD+ binding site.     

The anti-tumor effect of cross-reacting material 197, an inhibitor of heparin-binding EGF-like growth factor, in human resistant ovarian cancer

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. Cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, has been proven to represent possible chemotherapeutic agent for ovarian cancer. However, the effect of CRM197 on the resistant ovarian carcinoma cells has not been sufficiently elucidated. Here, we found that HB-EGF was over-expressed in a paclitaxel-resistant human ovarian carcinoma cell line (A2780/Taxol) and a cisplatin-resistant cell line (A2780/CDDP), as well as the xenograft mouse tissue samples with these cells. To investigate the possible significance of the HB-EGF over-expression in A2780/Taxol and A2780/CDDP cells, we inhibited HB-EGF expression by CRM197 to investigate the effect of CRM197 treatment on these cells. We observed that CRM197 significantly induced anti-proliferative activity in a dose-dependent manner with the cell-cycle arrest at the G0/G1 phase and enhanced apoptosis in A2780/Taxol and A2780/CDDP cells. The sensitive ovarian carcinoma parental cell line (A2780), A2780/Taxol and A2780/CDDP cells formed tumors in nude mice, and enhanced tumorigenicity was observed in drug-resistant tumors. Furthermore, we observed that CRM197 significantly suppressed the growth of drug-resistant ovarian cancer xenografts in vivo (p<0.001). These results suggest that CRM197 as an HB-EGF-targeted agent has potent anti-tumor activity in paclitaxel- and cisplatin-resistant ovarian cancer which over-express HB-EGF.