Conformational changes in diphtheria toxoids

Bigio, Massimo; Rossi, Roberta; Nucci, Daniele; Antoni, G.; Rappuoli, Rino; Ratti, Giulio

doi:10.1016/0014-5793(87)81060-8

Cited by 24 publications

(16 citation statements)

References 31 publications

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“…Three distinct domains are present, the N-terminal catalytic domain (fragment A), an a-helical transmembrane domain and an all-p strand receptor-binding domain which together constitute fragment B. CRM197 contains a Gly-Glu substitution at position 52 in the catalytic domain (Giannini et al, 1984;Choe et al, 1992) which abolishes NAD binding and the ADP-ribosylation activity of the protein. It has been shown by a number of techniques that the conformation of CRM197 and diphtheria toxin differ significantly (Lory et al, 1980;Mekada and Uchida, 1985;Bigio et al, 1987). The mutation causes changes not only in the local conformation of the active site, but also in the more distant receptor-binding domain.…”

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confidence: 99%

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Comparison of the Diphtheria Mutant Toxin, Crm197, with a Haemophilus Influenzae Type‐b Polysaccharide‐Crm197 Conjugate by Optical Spectroscopy

Crane

Bolgiano

Jones

1997

European Journal of Biochemistry

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A Haemophilus influenzae type-b capsular polysaccharide-CRM197 protein conjugate vaccine was compared with unconjugated CRM197 and diphtheria toxin, its parent molecule. Using CD and fluorescence spectroscopy, it has been possible to observe differences in structure and stability to pH and temperature due to the G52-E mutation in CRM197 and the 'glycosylation' of CRM197 in the conjugate. begins unfolding at slightly lower temperatures (25-35 "C) than native material (> 35 "C). In the conjugate, tryptophan residues are more accessible to the non-ionic flourescence quencher acrylamide at 35 "C. The conformational change observed at pH4-6 for diphtheria toxin is also observed for CRM197, but in the conjugate begins at higher pH. This may result from the presence of charged oligosaccharide residues on the surface or the conjugation methods used. The consequences of these changes in conformation and solution behaviour of the carrier protein in terms of its ability to induce a protective, T-celldependent response to H. influenzae polysaccharide remain to be determined.

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confidence: 99%

“…The mutation causes changes not only in the local conformation of the active site, but also in the more distant receptor-binding domain. Immunological evidence suggests that CRM197 exists in a more 'open' conformation than the native toxin at neutral pH (Bigio et al, 1987).…”

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confidence: 99%

Comparison of the Diphtheria Mutant Toxin, Crm197, with a Haemophilus Influenzae Type‐b Polysaccharide‐Crm197 Conjugate by Optical Spectroscopy

Crane

Bolgiano

Jones

1997

European Journal of Biochemistry

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“…Not only do the two proteins have different circular dichroism spectra (22,23) and proteolytic susceptibilities (20), but fluorescence emission measurements (20), membrane binding and insertion experiments (20,24), and antibody binding studies (25) shtow that the hydrophobic domains of CRM197 are more exposed to water than those of DTx. Moreover, the studies suggest that the structure of CRM197 at neutral pH is more similar to the structure of DTx at low pH.…”

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confidence: 99%

Diphtheria toxin and its ADP-ribosyltransferase-defective homologue CRM197 possess deoxyribonuclease activity.

Bruce

Baldwin

Lessnick

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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The cytotoxic mechanism of diphtheria toxin (DTx) is associated with its ability to inhibit protein synthesis by ADP-ribosylation of elongation factor 2. Although DTx intoxication leads to internucleosomal DNA cleavage and cell lysis, these events do not occur when protein synthesis is inhibited by alternative treatments (e.g., cycloheximide). Here we show that endonucleolytic degradation of DNA is an intrinsic activity of DTx and also of the crossreactive mutant protein CRM197. Assays using DNA-impregnated gels as well as linear and supercoiled DNA in solution revealed not only that CRM197 has nuclease activity but also that its specific activity is actually significantiy greater than that' of the wild-type molecule. Since CRM197 contains a single amino acid substitution that renders it incapable of ADP-ribosylation, we propose that the active sites for ADP-ribosyltrasferase and nuclease activities are distinct.Diphtheria toxin (DTx) is a well-characterized toxic protein (1, 2). It has two functional domains, A and B, which can be resolved by limited proteolytic digestion and reduction of an interchain disulfide bond. Intoxication of cells by DTx involves endocytosis of receptor-bound toxin. Once the toxin is sequestered in endosomes, acid-triggered conformational changes promote the translocation of fragment A to the cytosol (3-5). Fragment A then proceeds to catalyze the transfer ofthe ADP-ribosyl group ofNAD to the diphthamide residue of translational elongation factor 2 (EF-2). Recently we have discovered that DTx also exhibits a cationdependent nuclease activity (6). In this report we explore the relationship between the toxin's ADP-ribosyltransferase and nuclease activities. CRM197, a mutant DTx with a Gly -* Glu52 substitution in its A domain (7), is unable to ADPribosylate EF-2 in vitro because of its extremely low affinity for NAD (8,9 Nuclease Activity Assay in DNA-Containing Gels. Samples of cleaved DTx monomer, cleaved CRM197, and "intact" CRM197 were solubilized in SDS reducing buffer (containing 0.01% 2-mercaptoethanol) and electrophoresed in a SDS/ 12.5% polyacrylamide gel (10) prepared with 32P-labeled DNA [0.2 ng/ml; 800,000 cpm/ng of sonicated salmon sperm DNA radiolabeled by the primer-extension method (11, 12), as described (6)]. The stacking gel was omitted. The gels were pre-run at 170 V for 90 min. For nondenaturing gels, SDS was omitted and samples were not boiled. After electrophoresis, the DTx lanes were separated from the CRM197 lanes and each gel piece was washed three times (40 min per wash) with 100 ml of 40 mM Tris/0.04% NaN3, pH 7.6, at 22TC. The gel pieces were then incubated with the same buffer containing 2 mM CaCl2 and 2 mM MgCl2 for 24 hr at 300C (for SDS gels) or for 12 hr at 370C (for nondenaturing gels), prior to autoradiography (-850C, Kodak XAR film, DuPont Cronex screen).Cleavage of Supercoiled and Linear Plasmid DNA. CRM197, DNase I, and -DTx were incubated in 10 Al of 10 mM Tris, pH 7.6/2.5 mM CaCl2/2.5 mM MgCl2 with 0.2 ,Ag of linear (EcoRI-cleaved) pBluescr...

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“…The exotoxin of toxigenic strains of C. diphtheriae is responsible for local and systemic toxicity seen during diphtheria (Mandell and Dolin, 1995). Immunity to diphtheria is mainly provided by IgG antibodyies made against the exotoxin, which is a powerful antigen (Bigio et al, 1987). Some epidemiological studies were carried out by this antigen (Holmes, 2000) and some studies were used this antigen for vaccination (Lobeck et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

Cloning and expression of Corynebacterium diphtheriae toxin gene

Mohammadi¹,

Mojgan²,

ehpour³

et al. 2011

Afr. J. Microbiol. Res.

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Diphtheria is an acute bacterial disease caused by oxygenic strains of Corynebacteria. Fatality rate of this disease, between 5 and 10%. In children under 5 years and adults, the fatality rate may be as much as 20%. Outbreaks, although very rare, still occur worldwide, even in developed nations. Diphtheria toxin (DT) is the major virulence factor for these organisms. The aim of this study was cloning and expression diphtheria toxin gene to produce recombinant protein and application in next investigations. The bacterial DNA was extracted and amplification of diphtheria toxin gene was carried out with specific primers. This gene was cloned in pTZ57R/T vector and sub cloned into pETDuet-1 expression vector then recombinant plasmid was transformed into BL21 of Escherichia coli strain and induced by IPTG. Diphtheria toxin gene was amplified successfully and cloned in pTZ57R. Recombinant plasmid was digested by restriction enzymes and released fragment (diphtheria toxin gene) sub cloned in pETDuet-1 expression vector and expressed protein was analysed in serological assay. In this study, the diphtheria toxin gene was cloned in pETDuet-1 expression vector and confirmed by sequencing and restriction analysis then recombinant plasmid was transformed in BL21 expression cell.

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Conformational changes in diphtheria toxoids

Cited by 24 publications

References 31 publications

Comparison of the Diphtheria Mutant Toxin, Crm197, with a Haemophilus Influenzae Type‐b Polysaccharide‐Crm197 Conjugate by Optical Spectroscopy

Comparison of the Diphtheria Mutant Toxin, Crm197, with a Haemophilus Influenzae Type‐b Polysaccharide‐Crm197 Conjugate by Optical Spectroscopy

Diphtheria toxin and its ADP-ribosyltransferase-defective homologue CRM197 possess deoxyribonuclease activity.

Cloning and expression of Corynebacterium diphtheriae toxin gene

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