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

Douglas, Cameron M.; Collier, R. J.

doi:10.1128/jb.169.11.4967-4971.1987

Cited by 116 publications

(80 citation statements)

References 18 publications

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“…Furthermore, a histidine, a glutamate, and a tryptophan residue are conserved at nearly identical distances from each other in CT, diphtheria toxin, ETA, and the Si subunit of pertussis toxin (Si), and LTh-I contains an aspartate residue instead of a glutamate residue at the corresponding position (47). In diphtheria toxin and ETA, these glutamate residues (Glu-148 and Glu-553, respectively) are required for ADP-ribosylating activity and are believed to be part of the active site (10,45). Substitution of Asp for Glu at these positions in diphtheria toxin and ETA by site-directed mutagenesis lowered ADP-ribosylating activity (38).…”

Section: Discussionmentioning

confidence: 99%

Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli

et al. 1989

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Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-Ilb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II-producing strains of E. coli also suggested that additional variants of LT-II exist.More than a decade ago, the heat-labile enterotoxins of Escherichia coli and Vibrio cholerae were recognized to be a family of related protein toxins with similarities in structure, mode of action, and immunochemistry (12,13 Recently, new heat-labile enterotoxins were discovered (17,19,21), and the V. cholerae-E. coli enterotoxin family was divided into two distinct antigenic groups (36). Cholera toxin (CT) (12) and the type I E. coli heat-labile enterotoxins (LT-I), including the antigenic variants LTh-I and LTp-I (22), belong to serotype I, and antiserum to any one of them will neutralize the other type I toxins. In contrast, type II E. coli enterotoxins (LT-II) are not neutralized by antisera against type I toxins, but they are neutralized by antiserum to the prototype 21,36). Two antigenic variants of type II enterotoxin, designated LT-IIa and LT-IIb, were characterized (19, 21). Both consist of A and B subunits which are similar in size to the subunits of CT and LT-I. The ADP-ribosyltransferase activity of these LT-II toxins is similar to that of CT and LT-I (8; P. P. Chang, S.-C. Tsai, R. Adamik, B. C. Kunz, J. Moss, E. M. Twiddy, and R. K. Holmes, submitted for publication), but the gangliosidebinding specificities of LT-IIa and LT-IIb are different from those of CT and LT-I and from each other (14,19,21 (20). The plasmids pBR322, p...

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Section: Discussionmentioning

confidence: 99%

Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli

et al. 1989

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“…We also constructed a clone 2014-PE38 E553D encoding a negative control protein containing the E553D mutation in the PE38 domain that inactivates its ADP ribosylation activity. 93 The corresponding expression plasmids are designated pDC6 and pDC8, respectively. As a control we employed BL22-PE38, directed against CD22 antigen, 94 generously provided by Ira Pastan (NCI, NIH).…”

Section: Discussionmentioning

confidence: 99%

Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein

Chatterjee

Chandran

Berger

2012

mAbs

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“…They are BL22 (9), LMB-2 (55), M1 (56), LMB-9 (57), SS1P (10), and T6 (58) and their inactive mutants (59,60). We also made both domains of PE38 (domain II and III) separately (61).…”

Section: The Immunotoxins and Their Recombinant Target Proteinsmentioning

confidence: 99%

“…e E553 is the NAD binding site and R276 is one of the basic amino acids that constitute the furin cleavage site (59,60). Both sites are essential for cytotoxicity (11).…”

Section: Production Of Mabsmentioning

confidence: 99%

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Characterization of the B Cell Epitopes Associated with a Truncated Form of Pseudomonas Exotoxin (PE38) Used to Make Immunotoxins for the Treatment of Cancer Patients

Onda

Nagata

FitzGerald

et al. 2006

The Journal of Immunology

129

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Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

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

Cited by 116 publications

References 18 publications

Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli

Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli

Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein

Characterization of the B Cell Epitopes Associated with a Truncated Form of Pseudomonas Exotoxin (PE38) Used to Make Immunotoxins for the Treatment of Cancer Patients

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