Complete nucleotide sequence and characterization of the 5'-flanking region of mammalian elongation factor 2 gene

The hamster elongation factor 2 gene was isolated from genomic libraries of diphtheria toxin- and Pseudomonas aeruginosa exotoxin A-resistant cells containing non-ADP-ribosylatable elongation factor 2, and its structure was determined by a combination of restriction endonuclease mapping and DNA sequence analysis. The entire gene is about 6 kilobases long and has 13 exons. Almost all the introns are about 90-200 bases long, except the first and third, which are about 1 kilobase and 400 bases long, respectively. The first exon is processed just after the initiation codon for translation. The promoter of this gene was also characterized. As this gene contains the mutation conferring resistance to diphtheria toxin and P. aeruginosa exotoxin A, introduction of this gene into mammalian cells results in expression of toxin resistance. Using this characteristic, gene expression by deletion mutants of the 5'-flanking region were examined, and results showed that about 60 base pairs upstream of the TATA sequence were most efficient for expression of the elongation factor 2 gene.     

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

Targeted introduction of a diphtheria toxin-resistant point mutation into the chromosomal EF-2 locus by in vivo homologous recombination.

We have introduced a specific point mutation into the gene for chromosomal elongation factor 2 (EF-2) in Chinese hamster ovary cells (CHO-K1) by in vivo homologous recombination. To obtain a selectable construct for gene-targeting, we modified a diphtheria toxin-resistant mutant EF-2 gene (Gly717 to Arg) by deleting its promoter and first exon so that homologous recombinants could be distinguished from randomly integrated transformants, and also by inserting a second positive selection marker, the neomycin-resistance gene, into the 3'-flanking region to increase the selective accuracy. More than 80% of the clones surviving after selection for resistance to both the toxin and neomycin were the desired homologous recombinants in which the wild-type, toxin-sensitive EF-2 gene was replaced by the modified gene giving resistance to both the toxin and neomycin. This result shows that the specific point mutation was co-introduced with a second selective marker into an endogenous chromosomal gene and that the modified gene was expressed.     

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.     

Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.     

Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity.

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1-4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat- and mouse-derived cell lines were very insensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line-specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.     

The role of the diphthamide-containing loop within eukaryotic elongation factor 2 in ADP-ribosylation by Pseudomonas aeruginosa exotoxin A

eEF2 (eukaryotic elongation factor 2) contains a post-translationally modified histidine residue, known as diphthamide, which is the specific ADP-ribosylation target of diphtheria toxin, cholix toxin and Pseudomonas aeruginosa exotoxin A. Site-directed mutagenesis was conducted on residues within the diphthamide-containing loop (Leu693-Gly703) of eEF2 by replacement with alanine. The purified yeast eEF2 mutant proteins were then investigated to determine the role of this loop region in ADP-ribose acceptor activity of elongation factor 2 as catalysed by exotoxin A. A number of single alanine substitutions in the diphthamide-containing loop caused a significant reduction in the eEF2 ADP-ribose acceptor activities, including two strictly conserved residues, His694 and Asp696. Analysis by MS revealed that all of these mutant proteins lacked the 2'-modification on the His699 residue and that eEF2 is acetylated at Lys509. Furthermore, it was revealed that the imidazole ring of Diph699 (diphthamide at position 699) still functions as an ADP-ribose acceptor (albeit poorly), even without the diphthamide modification on the His699. Therefore, this diphthamide-containing loop plays an important role in the ADP-ribosylation of eEF2 catalysed by toxin and also for modification of His699 by the endogenous diphthamide modification machinery.     

A catalytic loop within Pseudomonas aeruginosa exotoxin A modulates its transferase activity.

Mutagenesis techniques were used to replace two loop regions within the catalytic domain of Pseudomonas aeruginosa exotoxin A (ETA) with functionally silent polyglycine loops. The loop mutant proteins, designated polyglycine Loops N and C, were both less active than the wild-type enzyme. However, the polyglycine Loop C mutant protein, replaced with the Gly(483)-Gly(490) loop, showed a much greater loss of enzymatic activity than the polyglycine Loop N protein. The former mutant enzyme exhibited an 18,000-fold decrease in catalytic turnover number (k(cat)), with only a marginal effect on the K(m) value for NAD(+) and the eukaryotic elongation factor-2 binding constant. Furthermore, alanine-scanning mutagenesis of this active-site loop region revealed the specific pattern of a critical region for enzymatic activity. Binding and kinetic data suggest that this loop modulates the transferase activity between ETA and eukaryotic elongation factor-2 and may be responsible for stabilization of the transition state for the reaction. Sequence alignment and molecular modeling also identified a similar loop within diphtheria toxin, a functionally and structurally related class A-B toxin. Based on these results and the similarities between ETA and diphtheria toxin, we propose that this catalytic subregion represents the first report of a diphthamide-specific ribosyltransferase structural motif. We expect these findings to further the development of pharmaceuticals designed to prevent ETA toxicity by disrupting the stabilization of the transition state during the ADP-ribose transfer event.     

The histidine residue of codon 715 is essential for function of elongation factor 2

Several mutant cDNAs of elongation factor 2 (EF-2) were constructed by site-directed mutagenesis and their products expressed in mouse cells were investigated. Amino acid substitution for the histidine residue of codon 715, which is modified post-translationally to diphthamide, resulted in non-functional EF-2 and this substitution did not render EF-2 resistant to Pseudomonas aeruginosa exotoxin A, which inactivates EF-2 transferring ADP-ribose to the diphthamide residue. These non-functional EF-2s with replacements of the histidine-715 residue showed various extents of inhibition of protein synthesis by competing with functional EF-2 in vivo. These results suggest that histidine-715 is essential for the translocase activity of EF-2 and that the region around diphthamide functions in recognition of, and/or binding to ribosomes. Substitution of proline for the alanine-713 residue and substitution of glutamine for the glycine-717 residue converted EF-2 to partially toxin-resistant forms. Two-dimensional gel analysis with fragment A of diphtheria toxin of these toxin-resistant EF-2s revealed that their ADP-ribosylations by toxin were much less than that of wild-type EF-2.     

Crystal structure of ADP-ribosylated ribosomal translocase from Saccharomyces cerevisiae

The crystal structure of ADP-ribosylated yeast elongation factor 2 in the presence of sordarin and GDP has been determined at 2.6 A resolution. The diphthamide at the tip of domain IV, which is the target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A, contains a covalently attached ADP-ribose that functions as a very potent inhibitor of the factor. We have obtained an electron density map of ADP-ribosylated translation factor 2 revealing both the ADP-ribosylation and the diphthamide. This is the first structure showing the conformation of an ADP-ribosylated residue and confirms the inversion of configuration at the glycosidic linkage. Binding experiments show that the ADP-ribosylation has limited effect on nucleotide binding affinity, on ribosome binding, and on association with exotoxin A. These results provide insight to the inhibitory mechanism and suggest that inhibition may be caused by erroneous interaction of the translation factor with the codon-anticodon area in the P-site of the ribosome.     

Pseudomonas aeruginosa exotoxin A: alterations of biological and biochemical properties resulting from mutation of glutamic acid 553 to aspartic acid

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) was identified earlier as a putative active-site residue by photoaffinity labeling with NAD. Here ETA-E553D, a cloned form of the toxin in which Glu-553 has been replaced by aspartic acid, was purified from Escherichia coli extracts and characterized. Cytotoxicity of the mutant toxin for mouse L-M cells was less than 1/400,000 that of the wild type. The mutation caused a 3200-fold reduction in NAD:elongation factor 2 ADP-ribosyltransferase activity, as estimated by assays with an active fragment derived from the toxin by digestion with thermolysin. NAD glycohydrolase activity was reduced somewhat less, by a factor of 50, and photoaffinity labeling with NAD by a factor of 2. We detected less than 2-fold change in the values of KM for NAD or elongation factor 2 and no change in KD for NAD, as determined by quenching of protein fluorescence. The drastic reduction of ADP-ribosyltransferase activity therefore results primarily from an effect of the mutation on kcat, implying that Glu-553 plays an important and possibly direct role in catalyzing this reaction. The effects of the E553D mutation are similar to those of the E148D mutation in diphtheria toxin, supporting the notion that these two Glu residues perform the same function in their respective toxins.     

Gene trap mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2

OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we isolated a novel mutant resistant to diphtheria toxin and Pseudomonas exotoxin A from the gene trap insertional mutants library of Chinese hamster ovary cells. In this mutant, the Ovca1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin-resistant phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2, the target of bacterial ADP-ribosylating toxins. A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes.     

Characterization of the endogenous ADP-ribosylation of wild-type and mutant elongation factor 2 in eukaryotic cells.

Anti-[ADP-ribosylated elongation factor 2 (EF-2)] antiserum has been used to immunoprecipitate the modified form of EF-2 from polyoma-virus-transformed baby hamster kidney (pyBHK) cells [Fendrick, J. L. & Iglewski, W. J. (1989) Proc. Natl Acad. Sci. USA 86, 554-557]. This antiserum also immunoprecipitates a 32P-labelled protein of similar size to EF-2 from a variety of primary and continuous cell lines derived from many species of animals. One of these cell lines, chinese hamster ovary CHO-K1 cells was further characterized. The time course of labelling of ADP-ribosylated EF-2 with [32P]orthophosphate was similar in pyBHK cells and in CHO-K1 cells. The kinetics of labelling were more rapid for cells cultured in 2% serum than 10% serum, with incorporation of 32P reaching a maximum at 6 h and 10 h, respectively. EF-2 mutants of pyBHK and CHO-K1 cells resistant to diphtheria-toxin-catalyzed ADP-ribosylation of EF-2 remain sensitive to cellular ADP-ribosylation of EF-2. The 32P-labelled moiety of ADP-ribosylated EF-2 was digested by snake venom phosphodiesterase and the product was identified as AMP. The same 32P-labelled tryptic peptide was modified by toxin in wild-type EF-2 and by the cellular transferase in mutant EF-2. When purified EF-2 from pyBHK cells was incubated with [carbonyl-14C]nicotinamide and diphtheria toxin fragment A, under conditions for reversal of the ADP-ribosylation reaction, [14C]NAD was generated. The results suggest that cellular ADP-ribosylated EF-2 exists in a variety of cell types, and the ribosylated product is identical to that produced by toxin ADP-ribosylation of EF-2, except in diphthamide mutant cells. Studies with the mutant cell lines indicate that the toxin and the cellular transferase, however, recognize different determinants at the ADP-ribose acceptor site in EF-2. The cellular transferase does not require the diphthamide modification of the histidine ring in the amino acid sequence of EF-2 for the transfer of ADP-ribose to the ring. Therefore, we would expect the cellular transferase active site to be similar to, but not identical to, the critical amino acids demonstrated in the active site of diphtheria toxin and Pseudomonas exotoxin A.     

Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.

Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.     

Retroviral insertional mutagenesis identifies a small protein required for synthesis of diphthamide, the target of bacterial ADP-ribosylating toxins

Retroviral insertional mutagenesis was used to produce a mutant Chinese hamster ovary cell line that is completely resistant to several different bacterial ADP-ribosylating toxins. The gene responsible for toxin resistance, termed diphtheria toxin (DT) and Pseudomonas exotoxin A (ETA) sensitivity required gene 1 (DESR1), encodes two small protein isoforms of 82 and 57 residues. DESR1 is evolutionally conserved and ubiquitously expressed. Only the longer isoform is functional because the mutant cell line can be complemented by transfection with the long but not the short isoform. We demonstrate that DESR1 is required for the first step in the posttranslational modification of elongation factor-2 at His(715) that yields diphthamide, the target site for ADP ribosylation by DT and ETA. KTI11, the analog of DESR1 in yeast, which was originally identified as a gene regulating the sensitivity of yeast to zymocin, is also required for diphthamide biosynthesis, implicating DESR1/KTI11 in multiple biological processes.     

Structure and expression of elongation factor 2 gene during development of Dictyostelium discoideum

A cDNA library constructed from poly(A)+ RNA isolated from Dictyostelium discoideum cells at 12 h of development was screened with the hamster elongation factor 2 (EF-2) cDNA. Several different cDNA clones which hybridized were isolated after a second screening. A cDNA clone representing the 5'-end of the mRNA was obtained by primer extension. By comparing the amino acid sequence deduced from the nucleotide sequences of these clones with that of hamster EF-2, we found enough homology between them to conclude that the isolated clones were complementary to the mRNA of D. discoideum EF-2. The N terminus which is the GTP-binding domain and the C-terminal half where it interacts with a ribosome showed a high degree of homology. The amino acid sequence of the carboxyl half includes that it contain a site of ADP-ribosylation by diphtheria toxin. From the Northern blotting analysis, the size of the mRNA was estimated to be 2.6 kilobases. The expression of the mRNA was high in vegetative cells, became maximal at the aggregation stage, and decreased thereafter through development. Upon differentiation of prespore and prestalk cells, the mRNA was highly enriched in the former over the latter. ADP-ribosylation assay of EF-2 protein by diphtheria toxin showed nearly the same developmental changes for the protein as the mRNA. However, prestalk cells were found to contain the same amount of the protein as prespore cells. The Southern blot analyses indicated that the gene encoding EF-2 is unique.     

Identification of Nsp100 as elongation factor 2 (EF-2)

The nerve growth factor-sensitive phosphoprotein from PC12 cells, previously designated Nsp100, has been shown to be elongation factor 2 (EF-2). The criteria used for this identification include: (i) similarity of N-terminal sequence; (ii) phosphorylation by the same kinase; (iii) ADP-ribosylation mediated by diphtheria toxin; (iv) comparable function in cell-free protein synthesis. According to these criteria, Nsp100 and EF-2 are identical and the kinase that phosphorylates Nsp100 in PC12 cells is calcium/calmodulin kinase III.     

Circular dichroism of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and various derivatives

We have recorded the near- and far-ultraviolet circular dichroism spectra of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and derivatives of these toxins. The far-ultraviolet spectra of various forms of diphtheria toxin were virtually identical, implying that no major changes in secondary structure accompany proteolytic nicking or dimerization of toxin, or binding of the endogenous dinucleotide, adenylyl-(3'-5')-uridine 3'-monophosphate (AdoPUrdP). Alpha-helix content was estimated to be 29%, as compared with 8% for fragment A. Near-ultraviolet spectra were identical between nicked and intact diphtheria toxin. A broad negative transition with a minimum at 304 nm was assigned to the intrachain disulfide bridge within the B moiety. Dimeric diphtheria toxin showed perturbations of aromatic residues. Binding of AdoPUrdP to monomeric diphtheria toxin or of adenylyl-(3',5')-uridine (AdoPUrd) to fragment A perturbed one or more tryptophans. The latter results correlate with evidence for involvement of a tryptophan in NAD binding. Native exotoxin A was estimated to have 16% alpha-helix, and the activated form of exotoxin A, 11%. An enzymically active, 31 kDa proteolytic fragment of exotoxin A showed similar alpha-helix content (7%) to that of diphtheria toxin fragment A.     

Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain.

DNA including the coding sequence for the A chain of the mutant diphtheria toxin tox 176 was cloned. The cloned mature A-chain coding sequence showed a G-to-A transition at nucleotide 383 as the only difference from the wild-type sequence. This resulted in replacement of the glycine at position 128 by aspartic acid in the predicted amino acid sequence. A eucaryotic cell expression plasmid, pTH1-176, was constructed in which the tox 176 A-chain coding sequence was attached to a truncated metallothionein promoter. The toxicity of this construct, compared with that of the corresponding wild-type diphtheria toxin A-chain plasmid, pTH1, was assessed after transfection into the human 293 cell line by an indirect transient expression assay (I. H. Maxwell, F. Maxwell, and L. M. Glode, Cancer Res. 46:4660-4664, 1986). For the same effect, 15- to 30-fold more pTH1-176 than pTH1 was required, a result consistent with previous in vitro estimates of the diminished activity of the tox 176 A chain. Controlled expression of the cloned tox 176 A-chain coding sequence may provide a means of eliminating specific cell populations in an organism, for which purpose the wild-type diphtheria toxin A chain might prove too toxic.