Interaction of diphtheria toxin fragment A and of elongation factor 2 with Cibacron blue

Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10^–5 M and a 11 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH₂)P are able to displace EF2 from blue Sepharose.     

Studies of the diphtheria toxin receptor on chinese hamster cells

Concanavalin A, wheat germ agglutinin and the ovalbumin glycopeptide are all inhibitors of the cytotoxic effect of diphtheria toxin on Chinese hamster cells. Ovalbumin glycopeptide loses its inhibitory property after treatment with β-N-acetylglucosaminidase. This demonstrates the importance of the glycopeptide structure for the mechanism of inhibition. The glycopeptide may be a toxin cell-surface receptor analogue. Diphtheria toxin-resistant mutants were isolated in order to search for cells that might have an altered toxin receptor. One mutant was 10-to 15-fold more resistant to diphtheria toxin than wild-type cells when protein synthesis was measured as a function of toxin concentration. However, when protein synthesis was measured as a function of time at a high toxin concentration, the time before onset of inhibition was identical in the mutant and wild-type cells. We present evidence indicating that the resistance of this mutant can be accounted for by a decreased affinity of toxin for a cell-surface receptor.     

A comparison of three strategies for biopanning of phage-scFv library against diphtheria toxin

The biopanning process is a critical step in phage display for isolating peptides or proteins with specific binding properties. Conventional panning methods are sometimes not so effective and may result in nonspecific or low-yield positive results. In this study, three different strategies including soluble antibody-capturing, pH-stepwise elution, and conventional panning were used for enrichment of specific clones against diphtheria toxoid. The reactivity of the selected clones was evaluated using an indirect enzyme-linked immunosorbent assay. The positive clones were screened using Vero cell viability assay. The neutralizing clones were expressed in HB2151 strain of Escherichia coli and soluble single-chain fragment variable (scFv) fragments were purified by nickel-nitrilotriacetic acid affinity chromatography. Finally, the ability of scFv fragments for neutralizing diphtheria toxin (DT) were evaluated again using Vero cell viability assay. After four rounds of panning, the soluble antibody-capturing method yielded 15 positive phage-scFv clones against diphtheria toxoid. Conventional panning and pH-stepwise elution model resulted from nine and five positive phage-scFv clones, respectively. Among all positive clones, three clones were able to neutralize DT in Vero cell viability assay. Two of these clones belonged to a soluble antibody-capturing method and one of them came from conventional panning. Three neutralizing clones were used for soluble expression and purification of scFvs fragments. It was found that these soluble scFv fragments possessed neutralizing activity ranging from 0.15 to 0.6 µg against two-fold cytotoxic dose 99% of DT. In conclusion, the results of our study indicate that soluble antibody-capturing method is an efficient method for isolation of specific scFv fragments.     

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.     

Polymerisation of chemically cross-linked actin:thymosin beta(4) complex to filamentous actin: alteration in helical parameters and visualisation of thymosin beta(4) binding on F-actin.

The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.     

Soluble expression and characterization of a GFP-fused pea actin isoform（PEAc1）

A pea actin isoform PEAcl with green fluorescent protein (GFP) fusion to its C-terminus and His-tag to its Nterminus, was expressed in prokaryotic cells in soluble form, and highly purified with Ni-Chelating Sepharose^TM Fast Flow column. The purified fusion protein (PEAcl-GFP) efficiently inhibited DNase I activities before polymerization,and activated the myosin Mg-ATPase activities after polymerization. The PEAcl-GFP also polymerized into green fluorescent filamentous structures with a critical concentration of 0.75μM. These filamentous structures were labeled by TRITC-phalloidin, a specific agent for staining actin microfilaments, and identified as having 9 nm diameters by negative staining. These results indicated that PEAc 1 preserved the essential characteristics of actin even with His-tag and GFP fusion, suggesting a promising potential to use GFP fusion protein in obtainning soluble plant actin isoform to analyze its physical and biochemical properties in vitro. The PEAcl-GFP was also expressed in tobacco BY2 cells,which offers a new pathway for further studying its distribution and function in vivo.     

Spectrin-4.1-actin complex of the human erythrocyte: Molecular basis of its ability to bind cytochalasins with high-affinity and to accelerate actin polymerization in vitro

The spectrin-4.1-actin complex isolated from the cytoskeleton of human erythrocyte [3] was found to be similar to muscle F-actin in several aspects: Both the complex and F-actin nucleate cytochalasin-sensitive actin polymerization; both bind dihydrocytochalasin B with similar binding constants; both can be depolymerized by DNase I with loss of cytochalasin binding activity. From these results, we conclude that the actin in the complex is in an oligomeric form. However, the presence of spectrin and band 4.1 in the complex not only stabilized the actin in the complex as evidenced by its resistance to depolymerization in low-ionic-strength conditions and to DNase I as compared with F-actin, but also altered the characteristics of the binding site(s) for cytochalasins believed to be located at the “barbed” (polymerizing) end of the oligomeric actin.     

Mutated form (G52E) of inactive diphtheria toxin CRM197: molecular simulations clearly display effect of the mutation to NAD binding

Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.     

Growth inhibition of cancer cells by co-transfection of diphtheria toxin A-chain gene plasmid with bovine leukemia virus-tax expression vector

We constructed a plasmid containing bovine leukemia virus (BLV)-tax gene driven by SR alpha promoter, designated as pME-BLVtax, to activate the promoter of the long terminal repeat (LTR) of BLV in various tumor cells. Activation of the promoter of BLV-LTR by pME-BLVtax was confirmed by luciferase assay. When the cells, such as COS-1, C8, and KU-1, were transfected with a plasmid pBLV-LUC1, which contained the luciferase gene under the control of BLV-LTR, and pME-BLVtax, luciferase was expressed in these cells, whereas no luciferase gene expression was observed when only pBLV-LUC1 was introduced into the cells. Activation of the BLV-LTR promoter was regulated by pME-BLVtax and 0.5 microg of pME-BLVtax was sufficient for the expression of the gene under the control of BLV-LTR. Furthermore, pME-BLVtax was used to direct the cell expression of the gene for diphtheria toxin A-chain under the control of BLV-LTR (pLTR-DT) to various tumor cell lines, KU-1, C8, COS-1, BL2M3, and HeLa cells. The transfection was carried out with cationic liposomes. In this experiment, co-transfection of pLTR-DT with pME-BLVtax exerted selective growth inhibitory effects on the tumor cell lines. Moreover, three co-introductions of pLTR-DT with pME-BLVtax into the cell lines resulted in significant inhibition of the cell growth. This result suggests that the delivery of the pLTR-DT and pME-BLVtax genes into tumor cells by the use of cationic liposomes may be potentially useful as a novel approach for the treatment of tumor cells.     

Refined structure of dimeric diphtheria toxin at 2.0 A resolution.

The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.     

Disassembly of actin filaments by botulinum C2 toxin and actin-filament-disrupting agents induces assembly of microtubules in human leukaemia cell lines

BACKGROUND INFORMATION: C(2) toxin produced by Clostridium botulinum types C and D ADP-ribosylates actin monomers and inactivates their polymerization activities. The disassembly of actin filaments by C(2) toxin induces a polarization of cultured human leukaemia cell lines. RESULTS: The polarization induced by C(2) toxin was temperature dependent and was prevented by nocodazole, a microtubule-disrupting agent, whereas it was promoted by paclitaxel, a microtubule-stabilizing agent. The fluorescence staining of polarized cells indicated an increase in microtubule assembly accompanying disassembly of actin filaments. Furthermore, several actin-filament-disrupting agents, other than C(2) toxin, also induced microtubule assembly and cell polarization, irrespective of their different mechanisms of action. The effects induced by some of the agents, which have lower binding affinities for actin, were reversible in response to the re-assembly of actin filaments. CONCLUSIONS: Thus the disassembly of actin filaments by C(2) toxin and actin-filament-disrupting agents induces assembly of microtubules followed by polarization of human leukaemia cell lines, indicating that the assembly/disassembly equilibrium of actin filaments influences the dynamics of microtubules, which control cell morphology and, in turn, diverse cellular processes.     

Comparison of high-resolution structures of the diphtheria toxin repressor in complex with cobalt and zinc at the cation-anion binding site.

The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is a divalent-metal activated repressor of chromosomal genes responsible for siderophore-mediated iron-uptake and of a gene on several corynebacteriophages that encodes diphtheria toxin. Even though DtxR is the best characterized iron-dependent repressor to date, numerous key properties of the protein still remain to be explained. One is the role of the cation-anion pair discovered in its first metal-binding site. A second is the reason why zinc exhibits its activating effect only at a concentration 100-fold higher than other divalent cations. In the presently reported 1.85 A resolution Co-DtxR structure at 100K, the sulfate anion in the cation-anion-binding site interacts with three side chains that are all conserved in the entire DtxR family, which points to a possible physiological role of the anion. A comparison of the 1.85 A Cobalt-DtxR structure at 100K and the 2.4 A Zinc-DtxR structure at room temperature revealed no significant differences. Hence, the difference in efficiency of Co2+ and Zn2+ to activate DtxR remains a mystery and might be hidden in the properties of the intriguing second metal-binding site. Our studies do, however, provide a high resolution view of the cationanion-binding site that has most likely evolved to interact not only with a cation but also with the anion in a very precise manner.     

白喉毒素突变体CRM197在大肠杆菌中的高效可溶表达、纯化及免疫原性分析

CRM197(Cross-reacting material 197)是白喉毒素的无毒突变体,在生物制药领域具有非常广泛的应用前景。为了实现CRM197在大肠杆菌中的无标签、可溶表达,以替代现有昂贵、复杂的白喉杆菌分泌发酵工艺,文中对目的蛋白的序列进行优化,转化大肠杆菌经IPTG诱导,目的蛋白获得了可溶性高表达,目的蛋白约占碎菌上清总蛋白的40%。超声破菌后、经阴离子交换、肝素亲和以及脱盐三步柱上层析获得纯度高于95%的CRM197样品。细胞毒性实验证明,CRM197的IC_(50)值是白喉毒素IC_(50)值的2.1×10~7倍,是白喉类毒素IC_(50)值的9.6倍,表明文中表达的CRM197是安全无毒的。随后,比较了高、低剂量的CRM197在小鼠体内的免疫原性,发现2μg组三免后的抗体滴度与20μg组的相当,可达1︰409 600。文中建立了CRM197的无标签、高效、可溶表达的大肠杆菌表达系和快速纯化体系,获得了具有较好免疫原性和安全性的重组蛋白,为该蛋白的进一步应用奠定了基础。     

Eukaryotic elongation factor-2 (eEF2): its regulation and peptide chain elongation

Regulation at the level of translation in eukaryotes is feasible because of the longer lifetime of eukaryotic mRNAs in the cell. The elongation stage of mRNA translation requires a substantial amount of energy and also eukaryotic elongation factors (eEFs). The important component of eEFs, i.e. eEF2 promotes the GTP-dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome. Mostly the eEF2 is regulated by phosphorylation and dephosphorylation by a specific kinase known as eEF2 kinase, which itself is up-regulated by various mechanisms in the eukaryotic cell. The activity of this kinase is dependent on calcium ions and calmodulin. Recently it has been shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and mTOR signalling pathway. There are also various stimuli that control the peptide chain elongation in eukaryotic cell; some stimuli inhibit and some activate eEF2. These reports provide the mechanisms by which cells likely serve to slow down protein synthesis and conserve energy under nutrient deprived conditions via regulation of eEF2. The regulation via eEF2 has also been seen in mammary tissue of lactating cows, suggesting that eEF2 may be a limiting factor in milk protein synthesis. Regulation at this level provides the molecular understanding about the control of protein translocation reactions in eukaryotes, which is critical for numerous biological phenomenons. Further the elongation factors could be potential targets for regulation of protein synthesis like milk protein synthesis and hence probably its foreseeable application to synthetic biology.     

The effects of halothane on the DNase I activity in an isolated enzyme preparation and in the DNase I-G actin complex

The effects of halothane on the DNase I activity in an isolated enzyme preparation and in a DNase I-globular (G) actin complex was investigated. DNase I, DNase I-G actin complexes and G actin were exposed to various (0.2–4.0 vol./%) halothane concentrations for 3 h. Thereafter, DNase I was mixed with a DNA solution and the extinction of the acid soluble supernatant of the DNase I assay was determined as a measure of DNase I activity. After 10 min of halothane exposure the DNase I activity is inhibited in direct proportion to halothane concentrations between 0.6 and 4.0 vol/%. After 10 min halothane activates inactive DNase I by inhibiting G actin, an inhibitor of DNase I. G actin, exposed to halothane, does not inhibit the activity of DNase I. The results suggest a mechanism by which halothane may contribute to chromosomal defects and disturbances of DNA metabolism in cells.     

Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions

Eukaryotic elongation factor 2 can undergo ADP-ribosylation in the absence of diphtheria toxin under the action of an endogenous transferase. The investigation which aimed to gain insight into the nature of endogenous ADP-ribosylation revealed that this reaction may be, in some cases, due to covalent binding of free ADP-ribose to elongation factor 2. Binding of free ADP-ribose, and NAD- and endogenous transferase-dependent ADP-ribosylation were suggested to be distinct reactions by different findings. Free ADP-ribose could bind to elongation factor 2 previously subjected to ADP-ribosylation by diphtheria toxin or endogenous transferase. The binding of free ADP-ribose was inhibited by neutral NH2OH, L-lysine and picrylsulfonate, whereas endogenous ADP-ribosyltransferase was inhibited by NAD glycohydrolase inhibitors and L-arginine. The ADP-ribosyl-elongation factor 2 adduct which formed upon binding of free ADP-ribose was resistant to neutral NH2OH, but decomposed almost completely upon treatment with NaOH. The product of endogenous transferase-dependent ADP- ribosylation was partially resistant to NH2OH and NaOH treatment. Moreover, this reaction was reversed in the presence of diphtheria toxin and nicotinamide. Both types of endogenous ADP-ribosylation gave rise to inhibition of polyphenylalanine synthesis. This study thus provides evidence for the presence of two different types of endogenous ADP-ribosylation of eukaryotic elongation factor 2. The respective sites involved in these reactions are distinct from one another as well as from diphthamide, the site of attack by diphtheria toxin.     

Refined structure of monomeric diphtheria toxin at 2.3 A resolution.

The structure of toxic monomeric diphtheria toxin (DT) was determined at 2.3 A resolution by molecular replacement based on the domain structures in dimeric DT and refined to an R factor of 20.7%. The model consists of 2 monomers in the asymmetric unit (1,046 amino acid residues), including 2 bound adenylyl 3'-5' uridine 3' monophosphate molecules and 396 water molecules. The structures of the 3 domains are virtually identical in monomeric and dimeric DT; however, monomeric DT is compact and globular as compared to the "open" monomer within dimeric DT (Bennett MJ, Choe S, Eisenberg D, 1994b, Protein Sci 3:0000-0000). Detailed differences between monomeric and dimeric DT are described, particularly (1) changes in main-chain conformations of 8 residues acting as a hinge to "open" or "close" the receptor-binding (R) domain, and (2) a possible receptor-docking site, a beta-hairpin loop protruding from the R domain containing residues that bind the cell-surface DT receptor. Based on the monomeric and dimeric DT crystal structures we have determined and the solution studies of others, we present a 5-step structure-based mechanism of intoxication: (1) proteolysis of a disulfide-linked surface loop (residues 186-201) between the catalytic (C) and transmembrane (T) domains; (2) binding of a beta-hairpin loop protruding from the R domain to the DT receptor, leading to receptor-mediated endocytosis; (3) low pH-triggered open monomer formation and exposure of apolar surfaces in the T domain, which insert into the endosomal membrane; (4) translocation of the C domain into the cytosol; and (5) catalysis by the C domain of ADP-ribosylation of elongation factor 2.