Pseudomonas exotoxin A: toxoid preparation by photoaffinity inactivation.

Marburg, Stephen; Tolman, Richard L.; Callahan, L T

doi:10.1073/pnas.80.10.2870

Cited by 12 publications

(11 citation statements)

References 15 publications

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“…Subsequently we observed ultraviolet-induced linking between fragment A and NAD (13) (26,27), and photochemical labeling occurred only with the activated form.…”

Section: Discussionmentioning

confidence: 96%

NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD.

Carroll¹,

Collier²

1984

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

145

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We showed earlier that exposing mixtures of NAD and diphtheria toxin fragment A to ultraviolet radiation (253.7 nm) induced the formation of covalently linked proteinligand photoproducts. Here we report that when [carbonyl-14C]NAD was employed in such procedures, the efficiency of labeling of the protein approached 1 mol/mol, and at least 94% of the incorporated label was associated with a single residue, glutamic acid at position 148. Fragment A photolabeled in this manner was enzymically inactive. The efficiency of photolabeling was much lower (<0.2 mol/mol) when NAD radiolabeled in either the adenine moiety or the adenylate phosphate was used, and the label was attached to different site(s) within fragment A. Efficient photochemical transfer of label from [carbonyl-'4C]NAD occurred under identical conditions with the nucleotide-free form of whole diphtheria toxin, CRM-45, or activated exotoxin A from Pseudomonas aeruginosa, but not with nucleotide-bound diphtheria toxin, CRM-197, native exotoxin A, or any of several NAD-linked dehydrogenases. On the basis of these and other results we suggest that part or all of the nicotinamide moiety of NAD is efficiently transferred to glutamate-148 of fragment A under the influence of ultraviolet irradiation and that this residue is located within the nicotinamide subsite. This location implies that glutamate-148 is at or near the catalytic center of the toxin. Our data provide direct evidence for the location of the NAD site in an ADP-ribosylating toxin and demonstrate highly efficient and specific photo-Several bacterial exotoxins act by catalyzing transfer of the ADP-ribose moiety of NAD into covalent linkage with specific target proteins of mammalian cells. For both diphtheria toxin (DT) and exotoxin A from Pseudomonas aeruginosa the target protein is elongation factor 2 (EF-2) (1). ADP-ribosylation inactivates the factor, thereby inhibiting protein synthesis and ultimately causing cell death. For cholera toxin and related enterotoxins the target is a subunit of the adenylate cyclase system (2). ADP-ribosylation enhances the activity of the cyclase, and the resulting increase in cAMP concentrations produces any of a variety of physiological changes, depending upon the type of cell affected. Recently, pertussis toxin has also been shown to ADP-ribosylate a component of adenylate cyclase (3).DT (Mr, 58,342) is released from Corynebacterium diphtheriae as a single, 535-residue polypeptide of known primary structure (4, 5). The intact toxin is inactive in catalyzing the ADP-ribosylation of EF-2 and must be subjected to mild proteolysis and reduction for this activity to be expressed. Such treatment cleaves the toxin into an enzymically active A fragment (Mr,21,167), and a B fragment (Mr, 37,195) involved in receptor recognition and membrane translocation.In earlier studies we characterized the enzymic and ligandbinding properties of fragment A (6). The . Both the efficiency of labeling (approaching 1 mol/mol) and the specificity of attachment are significan...

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“…Subsequently we observed ultraviolet-induced linking between fragment A and NAD (13) (26,27), and photochemical labeling occurred only with the activated form.…”

Section: Discussionmentioning

confidence: 96%

NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD.

Carroll¹,

Collier²

1984

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

145

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“…Photoaffinity inactivation has been employed directly to create derivatives of ETA with reduced activity (1,13). Using azido-substituted analogs of NAD, Marburg et al (13) generated a photo-inactivated protein whose toxicity for L cells was reduced 200-fold.…”

Section: Discussionmentioning

confidence: 99%

“…Using azido-substituted analogs of NAD, Marburg et al (13) generated a photo-inactivated protein whose toxicity for L cells was reduced 200-fold. Potential toxoids which retained immunoreactivity were formed with either 8-azidoadenosine or 8-azidoadenine was used in the photolysis; a derivative of NAD did not inactivate the enzyme, presumably because of the inaccessibility of the binding site in the unactivated toxin.…”

Section: Discussionmentioning

confidence: 99%

Exotoxin A of Pseudomonas aeruginosa: substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity

1987

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Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) has been identified by photoaffinity labeling as a residue within the NAD binding site (S.F. Carroll and R.J. Collier, J. Biol. Chem. 262:8707-8711, 1987). To explore the function of Glu-553 we used oligonucleotide-directed mutagenesis to replace this residue with Asp in cloned ETA and expressed the mutant gene in Escherichia coli K-12. ADP-ribosylation activity of Asp-553 ETA in cell extracts was about 1,800-fold lower and toxicity for mouse L-M929 fibroblasts was at least 10,000-fold lower than that of the wild-type toxin. Extracts containing Asp-553 ETA inhibited the cytotoxicity of authentic ETA on L-M929 fibroblasts, suggesting that the mutant toxin competes for ETA receptors. The results indicate that Glu-553 is crucial for ADP-ribosylation activity and, consequently, cytotoxicity of ETA. Substitution or deletion of this residue may be a route to new ETA vaccines.

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“…Exotoxin A tqxoid. Exotoxin A toxoid was generated by photoaffinity labeling as described by Marburg et al (17). Briefly, 150 jig of exotoxin A in 1 ml of phosphate-buffered saline was mixed with 1 ml of 2.6 mM 8-azido adenosine.…”

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confidence: 99%

Variables which affect suppression of the immune response induced by Pseudomonas aeruginosa exotoxin A

Holt¹,

Misfeldt²

1986

Infect Immun

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Pseudomonas exotoxin A has been shown previously to induce suppression of the murine immune response. In the present study, various parameters were examined which may have an effect on immunosuppression. The addition of 10-4 ng of exotoxin A induced suppression of the immune response to trinitrophenylated Ficoll from days 3 to 10, while 10 ng of toxin exerted no suppressive effect over the same examination periods. When the toxin was administered 1 or 2 days before antigen stimulation, suppression of the response was observed with both 10 and 10-4 ng. Priming splenocytes with toxin either in vivo or in vitro for 1 or 2 days suppressed the response of fresh cultured splenocytes to antigenic stimulation. Heated toxin, photoaffinity-labeled toxin, or preincubation of the toxin with rabbit anti-exotoxin A antiserum eliminated the toxin-induced suppression. These results suggest that Pseudomonas exotoxin A can generate multiple biological effects.Exotoxin A is a protein toxin produced by the gramnegative bacterium Pseudomonas aeruginosa. The toxin has been shown to be directly cytotoxic for a number of mammalian cells primarily through the ADP ribosylation of protein elongation factor 2, thereby inhibiting cellular polypeptide synthesis (18,20,21). We have been studying the effects of exotoxin A on the murine immune response and have found that the toxin can induce suppression in euthymic nul+ mice to thymus-dependent and thymusindependent antigens both in vitro and in vivo (11). The suppression was observed over a toxin range of 1 to 10-6 ng, while 10 and 100 ng had no apparent effect (11). A number of parameters can influence the overall action of immunomodifiers. Tubercle bacilli have been observed to elicit an immunoenhancing effect when administered with the antigen (7) but to induce suppression of the response when given before the antigen (31). A similar effect has also been observed for cholera toxin (13, 16) and Bordetella pertussis vaccine (6). We now report that timing of exotoxin A administration relative to antigenic stimulation can have a dramatic effect on the ability of the toxin to modify the immune response. Given concomitantly with the antigen, 10 ng of exotoxin A is not immunosuppressive. However, this same dose of toxin induced suppression when administered 1 or 2 days before the antigen stimulation. Modification of the toxin by heating, photoaffinity labeling, or preincubation with rabbit anti-exotoxin A antibodies eliminated the capacity of exotoxin A to induce suppression. These studies support the concept that Pseuidomonas exotoxin A is a potent biological response modifier. MATERIALS AND METHODS Mice. NFR/N (H-2q) nul+ mice were derived from brother-and-sister breeding pairs maintained in the animal care facilities at the University of Missouri-Columbia School of Medicine from breeding stock originally obtained from Carl * Corresponding author. t Present address: Veterinary Toxicology and Entomology Research Laboratory, U.S. Department of Agriculture, College Station, TX 77841.Hansen, Nationa...

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Pseudomonas exotoxin A: toxoid preparation by photoaffinity inactivation.

Cited by 12 publications

References 15 publications

NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD.

NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD.

Exotoxin A of Pseudomonas aeruginosa: substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity

Variables which affect suppression of the immune response induced by Pseudomonas aeruginosa exotoxin A

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