Microbial Pathogenesis 1990 ; 8 : 163-168
Mini-review Protein toxins with intracellular targets Sjur Olsnes, Harald Stenmark, Jan oivind Moskaug, Stephen McGill, Inger Helene Madshus and Kirsten Sandvig Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway
Introduction In the normal functioning of cells proteins are translocated across a number of cellular membranes, such as into the endoplasmic reticulum, mitochondria, peroxisomes and chloroplasts ."' In all these cases the proteins are translocated away from the cytosol . However, certain proteins are also transported in the opposite direction, from the exterior to the cytosol . The only well-established examples are certain bacterial and plant toxins that exert their effect in the cytosol . 3-5 Considering the large and increasing list of toxins that act in the cytosol (Table 1), it remains an interesting possibility that physiologically important proteins may enter by the same mechanisms . A common property of protein toxins with intracellular action is that they contain two functionally different domains, in many cases consisting of two disulfide-linked polypeptides . In the case of the plant toxins abrin, ricin, modeccin, volkensin and
Table 1
Toxins with intracellular sites of action
Toxin Diphtheria toxin Pseudomonas aeruginosa exotoxin A Cholera toxin Escherichia co/i heat labile toxin Pertussis toxin Clostridium botulinum C toxin Clostridium perfringens E iota toxin Clostridium spiroforme toxin Clostridium difficile toxin Abrin Ricin Modeccin Volkensin Viscumin Shigella toxin Shiga-like toxins Bacillus anthracis invasive adenylate cyclase Bordetel/a pertussis invasive adenylate cyclase Botulinum neurotoxin ? Tetanus toxin
0882-4010/90/030163+06$03 .00/0
Intracellular target Elongation factor 2 Elongation factor 2 G-regulatory protein G-regulatory protein G-regulatory protein Actin Actin Actin Actin Ribosomes Ribosomes Ribosomes Ribosomes Ribosomes Ribosomes Ribosomes
© 1990 Academic Press Limited
S . Olsnes et al.
164
viscumin, an enzymatically active A-chain is disulfide-linked to a B-chain . The B-chain is a galactose-binding lectin that binds to carbohydrates at the cell surface and somehow facilitates the entry of the enzymatically active A-chain into the cytosol .' The bacterial diphtheria toxin is produced as a single polypeptide chain, but it is easily split ('nicked') by trypsin and trypsin-like proteases into two disulfide-linked fragments, A and B . The nicked toxin therefore closely resembles the structure of the plant toxins . 3 In Shigella toxin, cholera toxin and related proteins the binding part consists of a noncovalently bound complex of five identical polypeptides . However, the enzymatically active part, the A-chain, is also in these toxins a single polypeptide . This polypeptide is easily split at its C-terminal end into two disulfide-linked fragments . The smaller fragment (3-5 kDa) appears to link the A-chain to the B-subunits, while the larger fragment (24-27 kDa) is enzymatically active .'' The subunit structure is less clear in the case of exotoxin A of Pseudomonas aeruginosa . This toxin consists of three domains, one of which carries the enzymatic activity, whereas another one apparently binds to cell-surface receptors . 8 The toxin also contains a third domain to which a function has so far not been assigned . This domain may be involved in the translocation to the cytosol .
Intracellular targets Diphtheria toxin and Pseudomonas aeruginosa exotoxin A have the same intracellular target, elongation factor 2, which is an enzyme required for protein synthesis .' The toxins ADP-ribosylate this enzyme and thereby inactive it . In this way the toxins inhibit protein synthesis and, as a consequence, induce cell death . Elongation factor 2 contains a unique amino acid, diphthamide, which is formed by post-translational modification of a histidine residue . As indicated in Fig . 1 the covalent binding of ADPribose occurs to this unusual amino acid .' Cholera toxin, pertussis toxins, Escherichia coli toxin and certain Clostridium toxins also act by ADP-ribosylation of proteins, but, unlike diphtheria toxin, they do not modify diphthamide, but arginine, histidine and cysteine residues in various GTPbinding proteins, such as actin and the G-regulatory proteins of adenylate cyclases .""' A third group of toxins inactivate the 60 S ribosomal subunit . These toxins include Shigella toxin, Shiga-like toxins, ricin, abrin, modeccin, volkensin and viscumin which remove a single adenine residue from one particular adenosine residue 12 which is located in a highly conserved region near the 3'-end of 28 S ribosomal RNA (Fig . 2) . The phosphoribose backbone of the RNA is not cleaved in the process . As a consequence of the modification the ribosomes are unable to bind elongation factors and, therefore, protein synthesis stops .
H
Adenine
H 1,N CH 2
Adenine
Nicotinamide
I I . Ribose-P-P-Ribose
CH2 H I -N'(CH3)3 CONH2 Diphthamide
H~ 'I Ribose -P-P-Ribose-N Y N
(DTA)
NAD
C, H 2 . CH2 HC - N'(CH3 ) 3 CONH2
Nicotinamide
ADP-ribosyl-diphthamide
Fig . 1 . Enzymatic activity of diphtheria toxin fragment A . The A-fragment cleaves NAD and links ADPribose covalently to a unique amino acid, diphthamide, which is present only in elongation factor 2 . Diphthamide is formed by post-translational modification of a histidine residue .
Protein toxins with intracellular targets
1 65
R
Cleavage by ricin A
G-C U-A C-G U C-G A-U A-U
0
OH
- 0- P=0 0
Fig . 2 . Enzymatic activity of ricin A-chain . Ricin A-chain is a specific N-glycosidase that cleaves adenine from a single adenosine residue located in a highly conserved region near the 3' end of 28 S RNA . The RNA backbone is not cleaved .
Invasive adenylate cyclases from Bacillus anthracis and Bordetella pertussis represent a fourth group of toxins acting in the cytosol ." , "
Uptake and translocation The toxins are taken up by endocytosis and subsequently transferred to endosomes, a process which appears to be required for toxic effect . Some toxins are subsequently transferred to lysosomes and to the Golgi region ." Diphtheria toxin and Pseudomonas aeruginosa exotoxin A are taken up by endocytosis from coated pits ." Although ricin is in part taken up from coated pits, this toxin appears to a large extent also to be taken up by a process that does not involve this kind of endocytosis ." Toxin taken up by this alternative route was highly efficient in intoxicating the cells . The endocytosed ricin rapidly appears in vesicular and tubular elements of the endosomal system as revealed by experiments with ricin-gold and ricin-horseradish peroxidase conjugates . Some of the internalized ricin is delivered to elements of the Golgi complex ." This may be necessary for entry of the toxin to the cytosol . Thus, a number of conditions that either prevent transport to the Golgi region or that inactivate the toxin once there, protect cells against ricin . The mechanism of toxin penetration is best understood in the case of diphtheria toxin . The toxin is endocytosed from coated pits and translocation is induced as soon as the pH in the endosome reaches values
Mini-review Protein toxins with intracellular targets Sjur Olsnes, Harald Stenmark, Jan oivind Moskaug, Stephen McGill, Inger Helene Madshus and Kirsten Sandvig Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway
Introduction In the normal functioning of cells proteins are translocated across a number of cellular membranes, such as into the endoplasmic reticulum, mitochondria, peroxisomes and chloroplasts ."' In all these cases the proteins are translocated away from the cytosol . However, certain proteins are also transported in the opposite direction, from the exterior to the cytosol . The only well-established examples are certain bacterial and plant toxins that exert their effect in the cytosol . 3-5 Considering the large and increasing list of toxins that act in the cytosol (Table 1), it remains an interesting possibility that physiologically important proteins may enter by the same mechanisms . A common property of protein toxins with intracellular action is that they contain two functionally different domains, in many cases consisting of two disulfide-linked polypeptides . In the case of the plant toxins abrin, ricin, modeccin, volkensin and
Table 1
Toxins with intracellular sites of action
Toxin Diphtheria toxin Pseudomonas aeruginosa exotoxin A Cholera toxin Escherichia co/i heat labile toxin Pertussis toxin Clostridium botulinum C toxin Clostridium perfringens E iota toxin Clostridium spiroforme toxin Clostridium difficile toxin Abrin Ricin Modeccin Volkensin Viscumin Shigella toxin Shiga-like toxins Bacillus anthracis invasive adenylate cyclase Bordetel/a pertussis invasive adenylate cyclase Botulinum neurotoxin ? Tetanus toxin
0882-4010/90/030163+06$03 .00/0
Intracellular target Elongation factor 2 Elongation factor 2 G-regulatory protein G-regulatory protein G-regulatory protein Actin Actin Actin Actin Ribosomes Ribosomes Ribosomes Ribosomes Ribosomes Ribosomes Ribosomes
© 1990 Academic Press Limited
S . Olsnes et al.
164
viscumin, an enzymatically active A-chain is disulfide-linked to a B-chain . The B-chain is a galactose-binding lectin that binds to carbohydrates at the cell surface and somehow facilitates the entry of the enzymatically active A-chain into the cytosol .' The bacterial diphtheria toxin is produced as a single polypeptide chain, but it is easily split ('nicked') by trypsin and trypsin-like proteases into two disulfide-linked fragments, A and B . The nicked toxin therefore closely resembles the structure of the plant toxins . 3 In Shigella toxin, cholera toxin and related proteins the binding part consists of a noncovalently bound complex of five identical polypeptides . However, the enzymatically active part, the A-chain, is also in these toxins a single polypeptide . This polypeptide is easily split at its C-terminal end into two disulfide-linked fragments . The smaller fragment (3-5 kDa) appears to link the A-chain to the B-subunits, while the larger fragment (24-27 kDa) is enzymatically active .'' The subunit structure is less clear in the case of exotoxin A of Pseudomonas aeruginosa . This toxin consists of three domains, one of which carries the enzymatic activity, whereas another one apparently binds to cell-surface receptors . 8 The toxin also contains a third domain to which a function has so far not been assigned . This domain may be involved in the translocation to the cytosol .
Intracellular targets Diphtheria toxin and Pseudomonas aeruginosa exotoxin A have the same intracellular target, elongation factor 2, which is an enzyme required for protein synthesis .' The toxins ADP-ribosylate this enzyme and thereby inactive it . In this way the toxins inhibit protein synthesis and, as a consequence, induce cell death . Elongation factor 2 contains a unique amino acid, diphthamide, which is formed by post-translational modification of a histidine residue . As indicated in Fig . 1 the covalent binding of ADPribose occurs to this unusual amino acid .' Cholera toxin, pertussis toxins, Escherichia coli toxin and certain Clostridium toxins also act by ADP-ribosylation of proteins, but, unlike diphtheria toxin, they do not modify diphthamide, but arginine, histidine and cysteine residues in various GTPbinding proteins, such as actin and the G-regulatory proteins of adenylate cyclases .""' A third group of toxins inactivate the 60 S ribosomal subunit . These toxins include Shigella toxin, Shiga-like toxins, ricin, abrin, modeccin, volkensin and viscumin which remove a single adenine residue from one particular adenosine residue 12 which is located in a highly conserved region near the 3'-end of 28 S ribosomal RNA (Fig . 2) . The phosphoribose backbone of the RNA is not cleaved in the process . As a consequence of the modification the ribosomes are unable to bind elongation factors and, therefore, protein synthesis stops .
H
Adenine
H 1,N CH 2
Adenine
Nicotinamide
I I . Ribose-P-P-Ribose
CH2 H I -N'(CH3)3 CONH2 Diphthamide
H~ 'I Ribose -P-P-Ribose-N Y N
(DTA)
NAD
C, H 2 . CH2 HC - N'(CH3 ) 3 CONH2
Nicotinamide
ADP-ribosyl-diphthamide
Fig . 1 . Enzymatic activity of diphtheria toxin fragment A . The A-fragment cleaves NAD and links ADPribose covalently to a unique amino acid, diphthamide, which is present only in elongation factor 2 . Diphthamide is formed by post-translational modification of a histidine residue .
Protein toxins with intracellular targets
1 65
R
Cleavage by ricin A
G-C U-A C-G U C-G A-U A-U
0
OH
- 0- P=0 0
Fig . 2 . Enzymatic activity of ricin A-chain . Ricin A-chain is a specific N-glycosidase that cleaves adenine from a single adenosine residue located in a highly conserved region near the 3' end of 28 S RNA . The RNA backbone is not cleaved .
Invasive adenylate cyclases from Bacillus anthracis and Bordetella pertussis represent a fourth group of toxins acting in the cytosol ." , "
Uptake and translocation The toxins are taken up by endocytosis and subsequently transferred to endosomes, a process which appears to be required for toxic effect . Some toxins are subsequently transferred to lysosomes and to the Golgi region ." Diphtheria toxin and Pseudomonas aeruginosa exotoxin A are taken up by endocytosis from coated pits ." Although ricin is in part taken up from coated pits, this toxin appears to a large extent also to be taken up by a process that does not involve this kind of endocytosis ." Toxin taken up by this alternative route was highly efficient in intoxicating the cells . The endocytosed ricin rapidly appears in vesicular and tubular elements of the endosomal system as revealed by experiments with ricin-gold and ricin-horseradish peroxidase conjugates . Some of the internalized ricin is delivered to elements of the Golgi complex ." This may be necessary for entry of the toxin to the cytosol . Thus, a number of conditions that either prevent transport to the Golgi region or that inactivate the toxin once there, protect cells against ricin . The mechanism of toxin penetration is best understood in the case of diphtheria toxin . The toxin is endocytosed from coated pits and translocation is induced as soon as the pH in the endosome reaches values
