Table Of ContentCurrent Topics in
Microbiology
175 and Immunology
Editors
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Birmingham/Alabama· H. Koprowski, Philadelphia
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La Jolla/California
ADP-Ribosylating
Toxins
Edited by K. Aktories
With 23 Figures and 4 Tables
Springer-Verlag
Berlin Heidelberg New York
London Paris Tokyo
Hong Kong Barcelona
Budapest
Professor Dr. Dr. KLAUS AKTORIES
Institut fOr Pharmakologie
und Toxikologie
der Universitat des Saarlands
6650 Homburg/Saar, FRG
ISBN-13:978-3-642-76968-9 e-ISBN-13:978-3-642-76966-5
001: 10.1007/978-3-642-76966-5
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Preface
ADP-ribosylating toxins have been the focus of intensive
research for more than 30 years. Researchers from diverse
fields of science have taken an interest in these bacterial toxins;
they are studied, for example, by microbiologists, biochemists,
cell biologists, and pharmacologists. There are two principal
reasons for the broad and still growing interest in ADP
ribosylating toxins. First, insights into the structure and functions
of the toxins might be the key to prevention and treatment of
diseases caused by the toxin-producing infectious micro
organisms. Second, the ADP-ribosylating toxins provide potent
and often unique pharmacological tools for the study of the
physiological functions of their target proteins. The latter is
especially the case with cholera and pertussis toxins, which
both modify the IX-subunits of heterotrimeric G-proteins
involved in signal transduction pathways. These toxins have
proved invaluable in extending our basic understanding of
the regulation of hormone-controlled signal transduction.
This volume provides a review and an update of recent
studies on the basic properties of bacterial ADP-ribosylating
tbxins and/or exoenzymes. Our current knowledge of the cel
lular entry mechanisms of ADP-ribosylating toxins is reviewed
by MADSHUS and STENMARK. WILSON and COLLIER then deal with
recent insights into the enzyme mechanism and active site
structure of diphtheria toxin and Pseudomonas aeruginosa
exotoxin A, which modify elongation factor 2. Toxins which
ADP-ribosylate heterotrimeric G-proteins involved in trans
membrane signal transduction are the subject of the next two
chapters. SERVEN11, Moss and VAUGHAN describe the regulation
of adenylate cyclase by cholera toxin and the enhancement
of the cholera toxin-induced ADP-ribosylation by guanine
nucleotide-binding proteins, the so-called ADP-ribosylation
factors, and the review of pertussis toxin by GIERSCHIK lays
emphasis on toxin structure-function relationship, toxin-G
protein interactions and the functional consequences.of
G-protein ADP-ribosylation. A chapter by AKTORIES, WILLE, and
VI Preface
JUST discusses recently described actin-ADP-ribosylating
toxins such as Clostridium botulinum C2 toxin, C. perfringens
iota toxin, and other iota-like toxins. Together with the myco
toxins cytochalasin and phalloidin, these ADP-ribosylating
toxins appear to be important new tools with which to study the
role of actin in various physiological processes. AKTORIES,
MOHR, and KOCH deal with clostridial exoenzymes which modify
small GTP-binding proteins. C. botulinum C3 ADP-ribosyltrans
ferase, C. limosum exoenzyme, and their eukaryotic protein
substrates Rho and Rac are discussed in detail. Last but not
least, COBURN briefly reviews P. aeruginosa exoenzyme S. Much
less is known about this ADP-ribosyltransferase and its eukaryo
tic substrates. Recent studies indicate that, again, small
GTP-binding proteins (Ras proteins) are the preferred subs
trates of the enzyme.
This book offers an exciting and comprehensive account
of recent developments in the field of ADP-ribosylating toxins
and should inspire both further research in this area and the
use of these toxins as tools in biological science.
K. AKTORIES
List of Contents
I. H. MADSHUS and H. STENMARK: Entry of ADP-
Ribosylating Toxins into Cells ......... .
B. A. WILSON and R. J. COLLIER: Diphtheria Toxin
and Pseudomonas aeruginosa Exotoxin A:
Active-Site Structure and Enzymic Mechanism. . 27
I. M. SERVENTI, J. Moss, and M. VAUGHAN: Enhancement
of Cholera Toxin-Catalyzed ADP-Ribosylation
by Guanine Nucleotide-Binding Proteins . . . . . . .. 43
P. GIERSCHIK: ADP-Ribosylation of Signal-Transducing
Guanine Nucleotide-Binding Proteins
by Pertussis Toxin .......... . . . . . . . . . . . 69
K. AKTORIES, M. WILLE, and I. JUST:
Clostridial Actin-ADP-Ribosylating Toxins 97
K. AKTORIES, C. MOHR, and G. KOCH:
Clostridium botulinum C3 ADP-Ribosyltransferase 115
J. COBURN: Pseudomonas aeruginosa Exoenzyme S 133
Subject Index . . . . . . . . . . . . . . . . . . . . . . . 145
List of Contributors
(Their addresses can be found at the beginning of their res
pective chapters.)
AKTORIES, K. 97, MOHR, C. . . . . . . . 115
115 Moss, J. ... 43
COBURN, J. . . 133 SERVENTI, I. M. .... 43
COLLIER, R. J. 27 STENMARK, H. 1
GIERSCHIK, P. 69 VAUGHAN, M ..... 43
KOCH, G. ....... 115 WILLE, M. 97
MADSHUS, I. H. . . . . 1 WILSON, B. A. .. 27
This book is dedicated
to my mentor in toxinology,
Prof. Dr. Ernst Habermann
Entry of ADP-Ribosylating Toxins into Cells*
I. H. MADSHUS and H. STENMARK
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Structure of ADP-Ribosylating Toxins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1 Cholera Toxin and Escherichia coli Heat-Labile Toxins. . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2 Pseudomonas aeruginosa Exotoxin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3 Diphtheria Toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.4 Pertussis Toxin ............................................................. 4
2.5 Botu!inum C2 Toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3 Binding of ADP-Ribosylating Toxins to Cells ................................... 5
3.1 Cholera Toxin and E. coli Heat-Labile Toxins .................................. 5
3.1.1 Cellular Receptors .......................................................... 5
3.1.2 Receptor-Binding Domains. .. . . . .. . .. . . . . . . .. . . ... . .. . ... . . . . . . . . .. . 6
3.2 Pseudomonas Toxin ........................................................ 7
3.2.1 Cellular Receptors .......................................................... 7
3.2.2 Receptor-Binding Regions ................................................... 7
3.3 Diphtheria Toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.3.1 Cellular Receptors .................................................... 8
3.3.2 Receptor-Binding Regions ................................................... 9
3.4 Pertussis Toxin ............................................................. 10
3.5 Botulinum C2 Toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4 Endocytosis of Cholera Toxin, Pseudomonas Toxin, and Diphtheria Toxin ......... 11
4.1 Cholera Toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . 11
4.2 Pseudomonas Toxin. . . . . . . . . .. . . .. . .. . . ... . . . .. . . ... . .. . . . . .... . ... . . . . . .. . 12
4.3 Diphtheria Toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5 Membrane Translocation of ADP-Ribosylating Toxins ........................... 13
5.1 Cholera Toxin. . . . .. .. . . . . . . . . . .. . .. . . . . . . . . . . . . . . . .. . ... . . ... . . . .. . . . . . .. . . 13
5.2 Pseudomonas Toxin ........................................................ 14
5.3 Diphtheria Toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
5.3.1 Conformational Alterations at Low pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
5.3.2 Translocation Directly Across the Plasma Membrane. . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5.3.3 Diphtheria Toxin·lnduced Ion Channels ....................................... 18
5.4 Pertussis Toxi n ....................................................... . . . . . . 18
5.5 Botulinum C2 Toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6 Conclusions .............................................................. . 19
References .................................................................... . 20
Department of Biochemistry, Institute for Cancer Research at the Norwegian Radium Hospital,
Montebello, N-031O Oslo 3, Norway
* The authors were supported by the Norwegian Cancer Society.
Current TopIcs In Microbiology and Immunology, Vol. 175
© Springer-Verlag Berlin· Heidelberg 1992
2 I. H. Madshus and H. Stenmark
1 Introduction
A number of bacterial toxins exert their action by ADP-ribosylating proteins
essential for normal cellular function. In all cases known the substrate of these
enzymes is either located free in the cytosol or associated with the cytoplasmic
side of the plasma membrane. A key to understanding intoxication is therefore
to understand how the toxins enter cells and get access to their cytoplasmic
targets.
Most of the toxins can be divided functionally into two moieties, A and B.
The A moiety carries the ADP-ribosyltransferase activity, whereas the B moiety
mediates cellular uptake. The entry of ADP-ribosylating toxins into cells starts
with their binding to specific cell surface receptors. At least in the cases of
pseudomonas toxin and diphtheria toxin, the next step in the entry process
involves endocytic uptake. Finally, a membrane translocation event is necessary
to transpqrt the enzymatically active moiety to the cytoplasmic side of the
membrane.
In this review we will first describe the structure of the different ADP
ribosylating toxins and then sequentially discuss the binding, endocytosis
(where relevant), and the membrane translocation step. We have chosen to
deal with the different toxins individually. This review does not intend to cover
the entry of all ADP-ribosylating toxins known, as the entry mechanisms of
several toxins are to date unclear.
2 Structure of ADP-Ribosylating Toxins
2.1 Cholera Toxin and Escherichia coli Heat-Labile Toxins
Cholera toxin, an exotoxin produced by Vibrio cholerae, produces a massive
noninflammatory secretory diarrhea when released in the small bowel, due to
stimulation of adenylate cyclase (FINKELSTEIN 1973). The toxin is composed of
five B subunits (12 kDa) that mediate the binding to cell surface receptors
and one A subunit (27 kDa) which is responsible for the enzymatic effect of
the toxin (Fig. 1; for review, see EIDELS et al. 1983). From evidence of a
stoichiometry of AB5 it was proposed that the A subunit lies on the central axis
of a ring of B subunits (GILL 1977; LAI 1980; DWYER and BLOOMFIELD 1982). The
A subunit is. divided by proteolytic cleavage and chemical reduction into two
fragments, A 1 (22 kDa), which has catalytic activity, and A2 (5 kDa), presumed
to mediate the interaction be.tween A 1 and the B subunits. Both cleavage and
reduction seem to be required for catalytic activity (MEKALANOS et al. 1979).
Cholera toxin has been crystallized (SIGLER et al. 1977; MAULIK et al. 1988);
however, the complete atomic structure has so far not been reported.
