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Primary structure at the site in beef and wheat elongation factor 2 of ADP-ribosylation by diphtheria toxin
... In the second step, Dph5 is involved in the trymethylation of the diphthamide intermediate [28] and, up to now, no protein involved in the last step of the diphthamide biosynthesis has been identified. Diphthamide can be found in all eukaryotic organisms and in archaebacteria except eubacteria, suggesting a relevant role in cell physiology [33,34]. The diphthamide residue is located at the tip of a domain loop in EF2 that mimics the anticodon loop of a tRNA. ...
... In addition of being the target of DT and ETA, it is assumed that the diphthamide residue present on EF-2 plays an important biological role since it is present in all eukaryotic organisms and in archaebacteria [33,34]. Nevertheless, CHO cells mutated for diphthamide formation are able to synthesize proteins and grow as well as wild-type cells [27,31]. ...
Diphtheria toxin (DT), Pseudomonas aeruginosa Exotoxin A (ETA) and cholix toxin from Vibrio cholerae share the same mechanism of toxicity; these enzymes ADP-rybosylate elongation factor-2 (EF-2) on a modified histidine residue called diphthamide, leading to a block in protein synthesis. Mutant Chinese hamster ovary cells that are defective in the formation of diphthamide have no distinct phenotype except their resistance to DT and ETA. These observations led us to predict that a strategy that prevents the formation of diphthamide to confer DT and ETA resistance is likely to be safe. It is well documented that Dph1 and Dph2 are involved in the first biochemical step of diphthamide formation and that these two proteins interact with each other. We hypothesized that we could block diphthamide formation with a dominant negative mutant of either Dph1 or Dph2. We report in this study the first cellular-targeted strategy that protects against DT and ETA toxicity. We have generated Dph2(C-), a dominant-negative mutant of Dph2, that could block very efficiently the formation of diphthamide. Cells expressing Dph2(C-) were 1000-fold more resistant to DT than parental cells, and a similar protection against Pseudomonas exotoxin A was also obtained. The targeting of a cellular component with this approach should have a reduced risk of generating resistance as it is commonly seen with antibiotic treatments.
... diphthamide residue. The amino acid sequences around diphthamide are well conserved in rat, bovine, yeast, wheat germ and hamster EF-2s [15]. Kohno et al. also obtained EF-2 cDNA clones from co-dominant DT-resistant hamster cells containing non-ADP-ribosylatable EF-2 by mutation in its structural gene [16]. ...
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.
... A single molecule of the toxin in the cytoplasm is sufficient to kill mammalian cells (2). This toxin-catalyzed activity is specific for EF-2 and occurs at a unique posttranslational histidine derivative, diphthamide (3,4), found in a conserved amino acid sequence (5) in the EF-2 of all eukaryotes and archaeobacteria examined thus far (6). Rare eukaryotic mutant cell lines defective in this posttranslational modification possess EF-2 that cannot serve as a substrate for diphtheria toxin, and they are thus resistant to its cytotoxic effects (7). ...
Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.
... function in protein synthesis is unknown. Diphthamide is synthesizedpost-translationally (6) and occurs within a highly conserved amino acid sequence (7) in the EF-2 of all eucaryotes and archaebacteria (8). Biosynthetic labeling studies in the yeast Saccharomyces cereuisiue (9) suggest that diphthamide is unique to EF-2 and is likely synthesized by adding the four-carbon backbone from methionine to the pre-existing histidine residue in EF-2 followed by trimethylation and amidation of the resulting amino and carboxylic groups of the added side chain. ...
The inactivation of elongation factor 2 (EF-2) by diphtheria toxin requires the presence of a post-translationally modified histidine residue in EF-2. This residue, diphthamide, has the structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. The present work was undertaken to study the pathway of diphthamide biosynthesis using diphtheria toxin-resistant yeast mutants (Chen. J.-Y., Bodley, J. W., and Livingston, D. M. (1985) Mol. Cell. Biol. 5, 3357-3360) which are defective in diphthamide formation. We demonstrate here that one of these mutants (dph5) contains a toxin-resistant form of EF-2 which can be converted in vitro to a toxin-sensitive form through the action of an enzyme present in other yeast strains. Both this toxin-resistant EF-2 and its modifying enzyme have been partially purified and evidence is presented that the modifying enzyme is a specific S-adenosylmethionine:EF-2 methyltransferase. In vitro complementation to diphtheria toxin sensitivity required S-adenosylmethionine, and when partially purified components were incubated with [methyl-3H]S-adenosylmethionine, label was incorporated specifically into EF-2. Hydrolysis of labeled EF-2 yielded diphthine (the unamidated form of diphthamide) and a single chromatographically separable labeling intermediate. We conclude that the S-adenosylmethionine:EF-2 methyltransferase adds at least the last two of the three methyl groups present in diphthine and that this modification is sufficient to create diphtheria toxin sensitivity. Evidence is also presented for the existence of an ATP-dependent amidating enzyme which catalyzes the final step in the biosynthesis of diphthamide in EF-2.
... The ADP-ribose is attached to EF-2 via a modified histidine residue, termed diphthamide (24), which has been found only in this protein (8,22). This unique amino acid, 2-[3-carboxyamido-3(trimethylammonio)propyl]histidine (2,25), is synthesized posttranslationally (26) and occurs within a highly conserved amino acid sequence (6) in the EF-2 of all eucaryotes and archaebacteria (22). The biological role of this amino acid is unknown. ...
We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae
(Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the
toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed
survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants
comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged
storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the
resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant
phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective
in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition
by diphtheria toxin.
We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.
We examined the nature of the diphtheria toxin fragment A recognition site in the protein synthesis translocating factor present in cell-free preparations from the archaebacteria Thermoplasma acidophilum and Halobacterium halobium. In agreement with earlier work (M. Kessel and F. Klink, Nature (London) 287:250-251, 1980), we found that extracts from these organisms contain a protein factor which is a substrate for the ADP-ribosylation reaction catalyzed by diphtheria toxin fragment A. However, the rate of the reaction was approximately 1,000 times slower than that typically observed with eucaryotic elongation factor 2. We also demonstrated the presence of diphthine (the deamidated form of diphthamide, i.e., 2-[3-carboxyamide-3-(trimethylammonio)propyl]histidine) in acid hydrolysates of H. halobium protein in amounts comparable to those found in hydrolysates of similar preparations from eucaryotic cells (Saccharomyces cerevisiae and HeLa). Diphthine could not be detected in hydrolysates of protein from the eubacterium Escherichia coli. Whereas both archaebacterial and eucaryotic elongation factors contain diphthamide, they differ importantly in other respects.
Diphtheria in humans is an upper respiratory infection which can progress to localized necrosis, forming a pseudomembrane which may cause death by suffocation. In addition, muscle weakness and lethargy are observed, and death is often ascribed to heart failure.
The steps of protein synthesis in both prokaryotes and eukaryotes involve similar reactions. One exception is the initiation process which involves more factors and appears to be more complicated in eukaryotes than in prokaryotes. The steps of elongation and termination appear to be quite similar and this chapter will summarize some aspects of peptide chain elongation and termination in eukaryotes.
Corynebacterium diphtheriae, Clostridium tetani, and Bordetella pertussis produce potent toxins that are entirely or in part responsible for the severe diseases caused by these micro-organisms. The clinical manifestations of diphtheria and tetanus can be thoroughly reproduced by the systemic administration of the toxins. Both diseases can be prevented by administration of toxin-neutralizing antibodies or by immunization with partially purified and detoxified toxins (toxoids). Diphtheria and tetanus vaccines are commonly considered as composed only of the detoxified form of the toxins. However, it should be emphasized that diphtheria and tetanus vaccines usually contain approximately only 75% and 50% of detoxified toxins, respectively. The remaining impurities are antigens produced by the bacteria that may play a role in controlling colonization.
Mono-adenosine diphosphate (ADP) ribosylation is a post-translational modification of proteins that occurs in viruses, bacteria and eukaryotic cells (Ueda and Hayaishi 1985; Althaus and Richter 1987). During this reaction, the ADP—ribose moiety of nicotinamide adenine dinucleotide (NAD) is transferred onto an acceptor amino acid of the substrate molecule, which is usually forced to undergo a functional change (Fig. 1). When the reaction is mediated by a toxin, this event ultimately results in either malfunction or death of the target eukaryotic cells.
Studies on the amino acid sequences and three-dimensional structures of plant proteins have always lagged far behind those of proteins from other sources. Thus in 1969, only six complete sequences for plant proteins had been established compared to over 230 from other sources (see Dayhoff 1972). At that time the tertiary structure of only one plant protein had been established with a reasonable degree of certainty (Drenth et al. 1968). The major reason for this apparent lack of interest in plant proteins probably lies in the relative difficulty in obtaining sufficient quantities of material for study from plants when compared to other sources. This is due to both the lower intrinsic yields of the plant proteins and to specific difficulties in their preparation. Thus even a decade later plant enzymes have still been barely studied because the conservative nature of many of the biochemical pathways means that similar enzymes are more readily available elsewhere. Even when there are enzymes unique to plants, such as photosynthetic enzymes, these have not been examined.
This chapter focuses on ADP-ribosylating toxins, which are a variety of bacterial proteins with totally unrelated structures that have in common only one feature: they contain an enzyme with ADP-ribosyltransferase activity. This growing family of enzymes shows that ADP-ribosylation is also an enzymatic reaction with an important role in the posttranslational modification of the eukaryotic cells. Based on their overall structure the toxins can be divided into A/B toxins, binary toxins, or A/only toxins. The A domain contains the enzymatic toxic activity, while the B domain is a non-toxic part that functions as a carrier or delivery system for the A domain. Two types of ADP-ribosylation reactions are known to occur in nature: poly- and mono-ADP-ribosylation. Since the target proteins of ADP-ribosylating toxins are all located in the cytosol or in the inner face of the cytoplasmic membrane, the toxins need to cross the cell membrane in order to reach their intracellular targets. The molecular, functional, and evolutionary aspects of ADP-ribosylating toxins have been discussed, along with different types of bacterial toxins.
Radical S-adenosylmethionine (SAM) enzymatic reactions are remarkably diverse, ranging from simple H-atom abstractions to generate product radicals, to H-atom abstractions that initiate a cascade of extraordinary chemical transformations. The members of the radical SAM superfamily exhibit only limited sequence homology. Radical SAM chemistry plays critical roles in numerous biosynthetic pathways including antibiotic production, posttranslational modifications, synthesis of protein cofactors, and catalyzing the synthesis of the nonprotein ligands that impart chemical reactivity to some of the most complex biological metal clusters known. The utilization of a universal protein fold with one of the most ubiquitous metal cofactors in biology, the [4Fe-4S] cluster, together with a simple organic molecule, SAM, is apparently a quite remarkable and adaptable method to carry out a wide variety of difficult transformations.
The unusual amino acid hypusine, so far considered to be specific for the eukaryotic translation initiation factor eIF-4D, has been isolated by Chromatographic methods not only from protein hydrolysates of various eukaryotic sources including flagellates and higher plants, but also from those of the aerobic archaebacteria Sulfolobus acidocaldarius, Halobacterium cutirubrum and Thermoplasma acidophilum. Hypusine was identified by co-chromatography with a standard, by oxidative cleavage with periodic acid and by in vivo labelling with [3H]lysine. No hypusine could be detected in eubacteria and in strictly anaerobic archaebacterial organisms. This distribution of hypusine is apparently of phylogenetic significance. A perceptible but not striking correlation of hypusine content with growth rate in Saccharomyces cerevisiae and Sulfolobus acidocaldarius may indicate that the function of the hypusine containing protein has been conserved throughout evolution.
The sequences of ADP-ribosylated tryptic peptides from three archaebacterial elongation factors — aEF-2 from Methanobacterium thermoautotrophicum, from Sulfolobus acidocaldarius and from Desulfurococcus mucosus — were established. The rates of ADP-ribosylation by diphtheria toxin of these factors and of two others — aEF-2 from Methanococcus vannielii and from Thermoplasma acidophilum — were determined. Enzymatically active EF's and purified ribosomes from the two sulphur metabolizing strains were prepared. The phenylalanine polymerization activity of factors and ribosomes from these two strains and from T. acidophilum and Mc. vannielii in various combinations was analyzed. The results of these experiments allow, in connection with previously published findings (Klink et al., 1983; Gehrmann et al., 1985), the following statements and conclusions:
IntroductionDescription of the OrganismPathogenesisEpidemiologyClinical ManifestationsLaboratory Diagnosis of DiphtheriaManagement, Prevention and Control of C. Diphtheriae InfectionsOther Potentially Toxigenic CorynebacteriaOther CorynebacteriaReferences
Toxins were the first bacterial virulence factors to be identified and were also the first link between bacteria and cell biology. Cellular microbiology was, in fact, naturally born a long time ago with the study of toxins, and only recently, thanks to the sophisticated new technologies, has it expanded to include the study of many other aspects of the interactions between bacteria and host cells. This chapter covers mostly the molecules that have been classically known as toxins; however, the last section also mentions some recently identified molecules that cause cell intoxication and have many but not all of the properties of classical toxins. Tables 22.1 and 22.2 show the known properties of all bacterial toxins described in this chapter, while Fig. 22.1 shows the subunit composition and the spatial organization of toxins whose structures have been solved either by X-ray crystallography or by quick-freeze deep-etch electron microscopy. © 2013 Springer-Verlag Berlin Heidelberg. All rights are reserved.
The elongation factor 2 (aEF-2) from the extreme thermo-acidophilic archaebacterium Sulfolobus solfataricus, is the only cytosolic target protein which is ADP-ribosylated by diphtheria toxin in presence of NAD. Once ADP-ribosylated, aEF-2 is no longer able to sustain poly(Phe) synthesis in vitro. aEF-2 displays a great thermoresistance: at the growth temperature of the archaebacterium, 87 degrees C, its half-life is 3 h. The amino acid sequence of the N-terminal region of aEF-2 has been determined up to residue 22. In the first 15 positions such a sequence is identical to that of EF-2 from Sulfolobus acidocaldarius and very similar to that of EF-2 from other archaebacteria or eukaryotes. The same is true for the primary structure of the peptide containing the ADP-ribosylation site. The fact that the primary structure of EF-2 at the ADP-ribosylation site is highly conserved ensures either the correct recognition of the histidine residue by the enzymes involved in its modification to diphthamide, or the proper interaction with the diphtheria toxin.
The gene coding for ADP-ribosylatable elongation factor 2 (EF-2) from the extreme thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been cloned and its sequence is reported. Amino acid sequence comparisons showed that EF-2 from S. acidocaldarius is more closely related to eukaryotic EF-2 than to eubacterial EF-G. Consensus sequences are derived from comparison of a region around the unique amino acid diphthamide, which is the target for ADP-ribosylation by diphtheria toxin in archaebacteria and eukaryotes. The conserved positions are likely to constitute a recognition site for the toxin and the histidine-modifying enzymes.
A single transcript of approximately the size of the EF-2 gene was observed in Northern blot experiments. Transcription initiation and termination signals were identified in the immediate vicinity of the respective translation start and stop codons of the gene. These results indicate that, in contrast to all prokaryotic EF-2 genes studied previously, the gene of S. acidocaldarius is not located within the streptomycin operon but is transcribed separately.
A Caenorhabditis elegans lambda ZAP cDNA library was screened using a fragment amplified from highly conserved regions of the mammalian and Drosophila elongation factor 2 (EF-2). Two types of cDNA clones were obtained, corresponding to two mRNA species with 3'-untranslated regions of 60 and 115 nucleotides, both encoding identical polypeptides. Sequence analysis of these clones and comparisons with hamster and Drosophila EF-2 sequences suggests that they encode C. elegans EF-2. Clone pCef6A, encoding the entire C. elegans EF-2 mRNA sequence including 45 nucleotides of 5'-untranslated region, contains a 2,556-bp open reading frame which predicts a polypeptide of 852 amino acid residues (Mr 94,564). The deduced amino acid sequence is greater than 80% identical to that of mammalian and Drosophila EF-2. Conserved sequence segments shared among a variety of GTP-binding proteins are found in the amino-terminal region. The carboxy-terminal half contains segments unique to EF-2 and its prokaryotic homolog, EF-G, as well as the histidyl residue which is ADP-ribosylated by diphtheria toxin. The C. elegans protein contains a 12-amino-acid insertion between positions 90 and 100, and a 13-amino-acid deletion between positions 237 and 260, relative to hamster EF-2. Partial sequencing of a genomic clone encoding the entire C. elegans EF-2 gene (named eft-2) has so far revealed two introns of 48 and 44 bp following codons Gln-191 and Gln-250, respectively. Southern and Northern blot analyses indicate that eft-2 is a single-copy gene and encodes a 3-kb mRNA species which is present throughout nematode development.
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.
Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing [32P]orthophosphate and 10% (vol/vol) fetal bovine serum. A 32P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The 32P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The 32P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as [32P]AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the 32P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and [32P]NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When 32P-labeled cultures were incubated in medium containing unlabeled phosphate, the 32P label was lost from the EF-2 within 30 min.
A cDNA clone, pHGR81, encoding 358 amino-acid residues of the C-terminal region of human elongation factor 2 (EF-2), was isolated from a human ovarian granulosa cell cDNA library. The deduced amino-acid sequence of pHGR81, when compared with the known identical amino-acid sequences of hamster as well as rat EF-2 revealed a substitution of a glutamine by an alanine residue in the partially determined human sequence. The 15 amino-acid-residue sequence comprising the histidine-715, supposed to be of importance for the biological function of EF-2, is preserved in human EF-2. The coding region of the cDNA insert of pHGR81 displays a homology of 87% to hamster and of 88% to rat EF-2 cDNA. In Northern-transfer analysis, pHGR81 specifically hybridizes with an mRNA species of 3.1 kb.
Toxin-resistant polypeptide chain elongation factor 2 cDNA has been cloned from a mutant hamster cell line with only non-ADP-ribosylatable elongation factor 2. The mutation conferring resistance to diphtheria toxin and Pseudomonas aeruginosa exotoxin A is a G-to-A transition in the first nucleotide of codon 717. Codon 715 encodes a histidine residue that is modified post-translationally to diphthamide, which is the target amino acid for ADP-ribosylation by both toxins. Transfection of mouse L cells with a recombinant elongation factor 2 cDNA differing from the wild-type only by this G-to-A transition confers resistance to P. aeruginosa exotoxin A. The degrees of toxin-resistant protein synthesis of stable transfectants are dependent on the ratio of non-ADP-ribosylated elongation factor 2 to wild-type elongation factor 2, not the amount of non-ADP-ribosylated elongation factor 2. The mutation creates a new Mbo II restriction site in the elongation factor 2 gene. Several independently isolated diphtheria toxin-resistant Chinese hamster ovary cell lines show the same alteration in the Mbo II restriction pattern.
Diphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) [(1980) J. Biol. Chem. 255, 10710-10720]. The reaction requires NAD as ADP-ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1-N6-ethenoadenine dinucleotide (epsilon NAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the epsilon ADP-ribose, to the EF-2. As a consequence of the transfer of the epsilon ADP-ribosyl moiety to the EF-2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The epsilon ADP-ribosylated EF-2, like ADP-ribosylated EF-2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF-2 molecule for conformational or binding studies is discussed.
Complementary DNA clones, pHEW1 and pRE2, coding for hamster and rat polypeptide chain elongation factor 2 (EF-2), respectively, were isolated and sequenced. It was shown that the cDNA insert in pHEW1 contains a 2574-base-pair open reading frame coding for an 857-amino acid polypeptide with Mr 95,192, excluding the initiation methionine. Comparative studies of sequence homology among EF-2 and several GTP-binding proteins show that five regions in the amino-terminal position of EF-2, corresponding to about 160 amino acids, show homology with GTP-binding proteins, including protein synthesis elongation and initiation factors, mammalian ras proteins, and transducin. The carboxyl-terminal half of EF-2 contains several regions that have 34-75% homology with bacterial elongation factor G. These results suggest that the amino-terminal region of EF-2 participates in the GTP-binding and GTPase activity whereas the carboxyl-terminal region interacts with ribosomes. Finally, the sequence provides direct evidence that diphthamide (2-[3-carboxy-amido-3-(trimethylammonio)propyl]histidine), the site of ADP-ribosylation by diphtheria toxin, is produced by post-translational modification of a histidine residue in the primary translational product.
The antibiotic sensitivity of the archaebacterial factors catalyzing the binding of aminoacyl-tRNA to ribosomes (elongation factor Tu [EF-Tu] for eubacteria and elongation factor 1 [EF1] for eucaryotes) and the translocation of peptidyl-tRNA (elongation factor G [EF-G] for eubacteria and elongation factor 2 [EF2] for eucaryotes) was investigated by using two EF-Tu and EF1 [EF-Tu(EF1)]-targeted drugs, kirromycin and pulvomycin, and the EF-G and EF2 [EF-G(EF2)]-targeted drug fusidic acid. The interaction of the inhibitors with the target factors was monitored by using polyphenylalanine-synthesizing cell-free systems. A survey of methanogenic, halophilic, and sulfur-dependent archaebacteria showed that elongation factors of organisms belonging to the methanogenic-halophilic and sulfur-dependent branches of the "third kingdom" exhibit different antibiotic sensitivity spectra. Namely, the methanobacterial-halobacterial EF-Tu(EF1)-equivalent protein was found to be sensitive to pulvomycin but insensitive to kirromycin, whereas the methanobacterial-halobacterial EF-G(EF2)-equivalent protein was found to be sensitive to fusidic acid. By contrast, sulfur-dependent thermophiles were unaffected by all three antibiotics, with two exceptions; Thermococcus celer, whose EF-Tu(EF1)-equivalent factor was blocked by pulvomycin, and Thermoproteus tenax, whose EF-G(EF2)-equivalent factor was sensitive to fusidic acid. On the whole, the results revealed a remarkable intralineage heterogeneity of elongation factors not encountered within each of the two reference (eubacterial and eucaryotic) kingdoms.
The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5'-deoxy-5'-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.
The amino acid diphthamide is a complex post-translational derivative of histidine that exists in eukaryotic and Archaebacterial elongation factor 2 (EF-2). Diphtheria toxin and Pseudomonas exotoxin A catalyze the transfer of an ADP-ribose residue from NAD to diphthamide, causing the inactivation of EF-2. We have used cytosolic extracts of mutant CHO-K1 cells to study the biosynthesis of diphthamide in vitro. We have identified chromatographically a precursor form of diphthamide that exists in one complementation group of mutant cells and have documented the addition of 3 methyl residues from S-adenosylmethionine to this precursor. We have identified the presence of methyltransferase capable of carrying out this reaction in vitro in cells of 15 diverse eukaryotic species.
The elongation cycle in the course of protein biosynthesis is terminated by the translocation, which consists of a ribosome shift in the 5‵-3‵ direction along the mRNA, together with the transfer of the peptidyl-tRNA from the A to the P site and the release of the deacylated tRNA. The translocation is accompanied by the hydrolysis of a GTP molecule and is catalyzed, in the eukaryotic cells by a monomeric protein, the elongation factor 2 (EF-2), the molecular weight of which ranges between 90,000 and 110,000 in the sources so far examined (Bermek, 1978).
Protein synthesis elongation factor 2 (EF-2) from all archaebacteria so far analysed, is susceptible to inactivation by diphtheria
toxin, a property which it shares with EF-2 from the eukaryotic 80S translation system. To resolve the structural basis of
diphtheria toxin susceptibility, the structural gene for the EF-2 from an archaebacterium, Methanococcus vannielii, was cloned and its nucleotide sequence determined. It was found that (i) this gene is closely linked to that coding for
elongation factor 1α (EF-1α), (ii) the size of the gene product, as derived from the nucleotide sequence, lies between those
for EF-2 from eukaryotes and eubacteria, (iii) it displays a higher sequence similarity to eukaryotic EF-2 than to eubacterial
homologues, and (iv) the histidine residue which is modified to diphthamide and then ADP-ribosylated by diphtheria toxin is
present in a sequence context similar to that of eukaryotic EF-2 but it is not conserved in eubacterial EF-G. The EF-2 gene
from Methanococcus is expressed in transformed Saccharomyces cerevisiae but is not ADP-ribosylated by diphtheria toxin. This indicates that the Saccharomyces enzyme system is unable to post-translationally convert the respective histidine residue from the Methanococcus EF-2 into diphthamide.
While preparing human placenta elongation factor 2 (EF-2), whose purification and some molecular properties are reported, we noticed the presence of numerous protein fractions which did not have EF-2 activity, but were ADP-ribosylated by diphtheria toxin in the presence of NAD+. All these proteins, like EF-2, were selectively retained by a heparin-Sepharose column, which we used as an affinity-chromatography step. This was also observed when EF-2 was prepared, by this purification step, from other sources, i.e. ox liver and two species of yeasts. In order to assess whether these proteins were a degradation product of EF-2, independent proteins or a mixture of both, they were analysed by subjecting them, after [14C]ADP-ribosylation, to exhaustive trypsinolysis. Only one radioactive peptide was found, thus suggesting that those proteins originate from EF-2 by some proteolytic process. Our findings indicate that this proteolysis does not occur after cell disruption, but is more or less active in the intact cell, depending on the system considered.
Elongation factor 2 (EF-2) from eukaryotes and archaebacteria can be ADP-ribosylated by diphtheria toxin (DT) [(1977) Annu. Rev. Biochem. 46, 69-94; (1980) Nature 287, 250-251]. The primary structure of the ADP-ribose accepting region in EFs from the archaebacteria Thermoplasma acidophilum Halobacterium cutirubrum and Methanococcus vannielli was determined in order to elucidate the degree of conservation compared with 4 previously established eukaryotic sequences [(1971) FEBS Lett. 103, 253-255]. Within a 9-residue sequence including the site of ADP-ribosylation 5 positions were found to be occupied by the same amino acid in all the archaebacterial and eukaryotic factors studied. There were more differences among the 3 archaebacterial sequences than among the 4 eukaryotic ones.
The translocation of the enzymatic moiety of diphtheria toxin, fragment A, across the membranes of pure lipid vesicles was demonstrated. A new assay, which employed vesicles made to contain radiolabeled NAD and elongation factor-2, was used to measure the appearance of the enzymatic activity of the A fragment in the vesicles. When the vesicles were exposed to a low-pH medium in the presence of diphtheria toxin, small molecules, such as NAD, escaped into the extravesicular medium, whereas large molecules mostly remained inside the vesicles. The vesicle-entrapped elongation factor-2 became ADP-ribosylated, indicating the entry of fragment A into the vesicle. The translocation of the A fragment depended upon the pH of the medium, being negligible at pH greater than 7.0 and maximal at pH 4.5. The entire toxin molecule was needed for function; neither the A fragment nor the B fragment alone was able to translocate itself across and react with the sequestered substrates. After exposure of the toxin to low pH, the entry of the A fragment was rapid, being virtually complete within 2-3 min at pH 5.5, and within 1 min at pH 4.7. Translocation occurred in the absence of any protein in the vesicle membrane. These results are consistent with the notion that the diphtheria toxin molecule enters the cytoplasm of a cell by escaping from an acidic compartment such as an endocytic vesicle.
Fragment A of diphtheria toxin and Pseudomonas toxin A intoxicate cells by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) resulting in an inhibition of protein synthesis [1-3]. A cellular enzyme from polyoma virus transformed baby hamster kidney (pyBHK) cells ADP-ribosylates EF-2 in an identical manner [4]. Here we describe a similar cellular enzyme from beef liver which transfers [adenosine-14C]ADP-ribose from NAD to EF-2. The 14C-label can be removed from the EF-2 by snake venom phosphodiesterase as a soluble product which comigrates with AMP on TLC plates, indicating the 14C-label is present on EF-2 as monomeric units of ADP-ribose. Furthermore, the forward transferase reaction catalyzed by the beef liver ADP-ribosyltransferase is reversible by excess diphtheria toxin fragment A, with the formation of 14C-labeled NAD, indicating that both transferases ADP-ribosylate the same site on the diphthamide residue of EF-2. Thus, beef liver and pyBHK mono(ADP-ribosyl)transferases both modify the diphthamide residue of EF-2, in a manner identical to diphtheria toxin fragment A and Pseudomonas toxin A. These results suggest the cellular enzyme is probably ubiquitous among eukaryotic cells.
This chapter discusses the inhibition of protein synthesis and activation of adenylate cyclase. Diphtheria toxin and Pseudomonas exotoxin A inhibit protein synthesis in susceptible cells and in cell-free systems by catalyzing the ADP-ribosylation of a single amino acid in elongation factor II (EF-II), which then becomes inactive in protein synthesis. These toxins apparently do not ADP-ribosylate other proteins, presumably because they lack the post-translationally modified histidine that serves as the ADP-ribose acceptor in EF-II. Choleragen has been a valuable tool in the work that has resulted in the isolation and characterization of the guanyl nucleotide-binding component (G/F) of the cyclase. In addition, demonstration of the enzymic activity of choleragen leads directly to the question whether animal cells employ ADP-ribosylation in a controlled way to regulate adenylate cyclase activity.
Two different preparations isolated from beef cerebrum have been used to compare the polyadenosine diphosphate ribose (polyADPR) polymerase activities in neuronal and glial nuclei: (1) nuclear suspensions (with or without DNase I treatment), and (2) 1 M NaCl nuclear extracts (soluble enzyme). The DNAse I treatment of nuclei and the solubilization of polyADPR polymerase by 1 M NaCl enhances the polyADPR polymerase activity. The polyADPR polymerase activity is similar in neuronal and glial nuclear suspensions, while the neuronal soluble enzyme activity is significantly higher than that of the glial soluble enzyme. Evidence is presented that the difference in soluble enzyme activities is not due to the effects of DNA or degrading enzymes. Some activating factor(s) seem to be present in neuronal soluble extracts, while both inhibiting and activating factor(s) seem to be present in glial soluble extracts.
Tryptic digestion of [¹⁴C]ADP-ribosylated elongation factor 2 (EF-2), purified from rat liver, yielded a single radioactive peptide. The pure peptide (62% yield) consists of 15 amino acid residues whose sequence is Phe-Asp-Val-His-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg; the ADP ribose is linked to the unknown residue, X. X is a weakly basic residue which does not correspond to any amino acid commonly occurring in proteins but which is present in unmodified EF-2, possibly to the extent of only 1 residue per mole of EF-2.
Polyphenylalanine synthesis in a wheat germ system requires GTP, polyuridylic acid, and two soluble aminoacyl transfer factors. Procedures are described for the complete resolution and partial purification of each of the aminoacyl transfer factors. One of the factors catalyzes a GTP and polyuridylic acid-dependent binding of phenylalanyl transfer RNA to ribosomes while the second factor facilitates the formation of peptidyl puromycin from nonenzymatically bound phenylalanyl-tRNA. Both of the factors catalyze a ribosome-dependent hydrolysis of GTP.
An analytical procedure which affords the precise amino acid composition of a protein or a peptide from a single hydrolysate is described. This method utilizes 4 N methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole, rather then 6N HCl as a catalyst for hydrolysis. The hydrolysis is carried out in vacuo (20 mu) at 115 degrees for 22 to 72 hours. Half-cystine is determined as S-sulfocysteine by treating the hydrolysate with dithiothreitol followed by an excess of tetrathionate. The values of all amino acids, including tryptophan and half-cystine, were close to the expected theoretical values for the proteins examined. The method has the advantage that the neutralized hydrolysate can be applied directly to an ion exchange column. Further, the method is capable of distinguishing between free sulfhydryl groups as S-carbosymethylcysteine and disulfides as S-sulfocysteine. A limitation of the procedure is that tryptophan remains sensitive to the presence of carbohydrate in the sample.
The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.
We have developed a method for the purification in micromole amounts of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2 (EF-2). EF-2 was partially purified (15 to 20% purity) by ammonium sulfate precipitation and DEAE-Sephadex chromatography. After [3H]ADP-ribosylation by [3H]nad+ and diphtheria toxin, EF-2 was digested with trypsin and a homogeneous [3H]ADP-ribosyl peptide was isolated by chromatography on DEAE-Sephadex and dihydroxyboryl-substituted cellulose. Based on the amount of ADP-ribose acceptor activity in the crude extract, the overall yield of the peptide was 35%. The yeast peptide contains an unusual amino acid (X) which is the site of ADP ribosylation and is apparently identical to the amino acid reported from rat liver EF-2 by Robinson et al. (Robinson, E. A., Hendriksen, O., and Maxwell, E.S. (1974) J. Biol. Chem. 249, 5088-5093). The sequence of the 15-residue yeast peptide was determined to be: Val-Asn-Ile-Leu-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg. The 11 COOH-terminal residues of this peptide and of the homologous 15-residue peptide reported by Maxwell and co-workers from rat liver EF-2 are identical.
In an effort to extend automated Edman degradation to nanomole quantities of protein, the method of sequenator analysis described by Edman and Begg (Edman, P., and Begg, G. (1967), Eur. J. Biochem. 1, 80) was modified to permit long degradations in the absence of carrier proteins. By using an aqueous 0.1 M Quadrol program with limited, combined benezene-ethyl acetate solvent extractions, as well as a change in the delivery system for heptafluorobutyric acid, it was possible to recover and identify the first 30 amino acid residues from a sequenator run on 7 nmol of myoglobin. For 3 nmol of myoglobin, 20 steps could be identified. PTH-amino acids were identified by gas-liquid chromatography and thin-layer chromatography on polyamide sheets. Without using a carrier protein the cup to prevent mechanical losses (Niall, H. D., Jacobs, J. W., Van Rietshoten, J., and Tregear, G. W. (1974), FEBS Lett. 41, 62), the repetitive yield using this program was 93-96%. The same program has been applied successfully to peptides of 14 or more residues with or without modification by Braunitzer's reagent and to a number of larger peptides and proteins including a 216 residue segment of rabbit antibody heavy chain in which a sequence of 35 steps was accomplished on 25 nmol.
Tryptic digestion of [14C]ADP-ribosylated elongation factor 2 (EF-2), purified from rat liver, yielded a single radioactive peptide. The pure peptide
(62% yield) consists of 15 amino acid residues whose sequence is Phe-Asp-Val-His-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg;
the ADP ribose is linked to the unknown residue, X. X is a weakly basic residue which does not correspond to any amino acid
commonly occurring in proteins but which is present in unmodified EF-2, possibly to the extent of only 1 residue per mole
of EF-2.
Polyphenylalanine synthesis in a wheat germ system requires GTP, polyuridylic acid, and two soluble aminoacyl transfer factors.
Procedures are described for the complete resolution and partial purification of each of the aminoacyl transfer factors. One
of the factors catalyzes a GTP and polyuridylic acid-dependent binding of phenylalanyl transfer RNA to ribosomes while the
second factor facilitates the formation of peptidyl puromycin from nonenzymatically bound phenylalanyl-tRNA. Both of the factors
catalyze a ribosome-dependent hydrolysis of GTP.
(1892) Hoppe-Seylzr's
- D Lundeli
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Lundeli, D. 4. and Howard, J. 3. (1978) J. Rio!.. Chem.
253,3422-34X_
[lo] Kruger;M_ (1892) Hoppe-Seylzr's
Z. Fhysiol. Chem. 16,
160-172.
- B G Van Ness
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Van Ness, B. G., Howard, J. B. and Bodley, J. IV.
(1978) J. Biol. Chem. 253, S687--8690.