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Protein toxins with intracellular targets
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
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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 <5 .3 . Treatment of the cells with any one of a large number of compounds that increase pH in endosomes, such as NH4, monensin, and others, protect the cells against intoxication .' In the presence of some of these drugs it is, however, possible to induce toxin entry by exposing the cells to medium with pH <5 .3 .' Under these conditions, diphtheria toxin that is present at the cell surface is translocated directly across the surface membrane . This experimental approach mimics at the cell surface a process that normally occurs in the endosomes . Toxin bound to the cell surface can be efficiently removed by pronase treatment of the cells . 78 To study which parts of the toxin that enter upon exposure to low pH, cells were incubated with 125 1-labelled toxin (mol . wt . 58 kDa), exposed to pH 4 .5,
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and then treated with pronase to remove toxin exposed to the exterior . Under these conditions two fragments were protected against the enzyme . These fragments had electrophoretic migration rates corresponding to 21 kDa and 25 kDa . The smaller polypeptide represents the A-fragment (mol . wt . 21 kDa), whereas the 25 kDafragment is derived from the B-fragment (mol . wt . 38 kDa) . Upon fractionation of the cells, the 21 kDa fragment was consistently found in a soluble fraction comprising the cytosol, while most of the 25 kDa polypeptide was associated with the membrane fraction .' $ This indicates that the A-fragment had been translocated to the cytosol . For translocation of the A-fragment to the cytosol to occur it is not sufficient that cell-bound toxin is exposed to low pH . If the cytosol is also acidified, the cells are not intoxicated and the A-fragment is not shielded, indicating that it is not translocated to the cytosol . 18 The 25 kDa fragment is, however, protected even under these conditions, indicating that at the low pH a portion of the B-fragment is inserted into the membrane whether or not the cytosol is acidified . In addition to an inward directed proton gradient, permeant anions play a role in the translocation, and inhibitors of anion transport inhibit both binding and translocation of the toxin . It is not yet clear in which way anion transport is linked to toxin entry . 4 Diphtheria toxin and toxin fragments containing the hydrophobic regions of the Bfragment are able to form ion-conducting channels in lipid bilayers and in cells ." When the toxin is inserted into Vero cells at low pH, there is a strong increase in the influx of "Na' and 86 Rb' and in the efflux of K' from the cells, but no increased permeability to CI - or SO' - . This indicates that the channels are selective for cations . Small monovalent cations (H', Na' and K') pass the toxin-induced channels more readily than larger cations (choline, glucosamine) and divalent cations pass very inefficiently . When the internal pH is subsequently reduced due to influx of H', the channels are closed . They also become closed when the cytosol is acidified in other ways . Furthermore, the channels were closed when the cells were transferred to neutral medium . Apparently, therefore, an inward directed proton gradient is required for the channels to remain open . The cation-selective channels may be required for toxin translocation . Thus Cd", which closes the channels, protected the cells against intoxication ." Vero cells contain 5-10X10 4 diphtheria toxin receptors, whereas most other cells possess a much lower receptor number . The surface receptors appear to be essential for efficient translocation of the A-fragment across the plasma membrane . Artificially bound toxin (e .g . biotinylated toxin bound to avidin-treated cells) could not be translocated across the surface membrane and did not induce formation of cationselective channels . The possibility therefore exists that the receptor participates directly in the translocation of the toxin to the cytosol . The nature of the receptor is not clear . Treatment of Vero cells with low concentrations of trypsin strongly reduced the ability of the cells to bind the toxin, suggesting that the receptor is a protein . However, treatment with phospholipase C was even more efficient in destroying the binding capability of the cel ls . 4 The step least well understood in the mechanism of action of the toxins is how the A-moiety penetrates the membrane to reach the cytosol . Normally this process does not occur through the cell surface membrane, but across membranes of intracellular organelles . In the case of diphtheria toxin, the transfer appears to occur in the endosomes as soon as their interior reaches the required pH value of 5 .3 . Other toxins may enter from compartments distal to the endosomes . It is possible that some of the toxins must be processed, e .g . by proteolytic cleavage or by glycosylation before the translocation can occur . This could be the reason why
Protein toxins with intracellular targets
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attempts to induce translocation from the cell surface of toxins other than diphtheria toxin have so far been unsuccessful . The possibility must also be considered that the translocation is facilitated by membrane proteins only present in the wall of certain intracellular organelles . In the case of diphtheria toxin there are indications that interaction with a membrane protein is necessary for translocation . Thus, treatment with inhibitors of anion transport prevents the insertion of the toxin into the membrane and, as a consequence, precludes delivery of the A-fragment to the cytosol . During the translocation, charged and polar residues in the A-fragment must be transported through the hydrophobic lipid bilayer . Possibly, the inserted part of the B-fragment shields these residues during the transport . It is also possible that a membrane protein, such as an anion antiporter shields the polar and charged residues during the translocation . The B-fragment could, together with the hydrophilic channel assumed to be present in the antiporter, accommodate these residues while the A-fragment passes across the membrane . The hydrophobic parts of the A-fragment would then face the lipids . The inward directed proton gradient could provide energy for the transport . The energy could be transferred to the system by unfolding of the A-fragment at low pH and refolding at neutral pH . The energy gain obtained when the A-fragment refolds in the cytosol could pull the protein across the membrane . Therapeutic potential In recent years much effort has been made to improve the specificity of cytostatic drugs used in cancer treatment and in immunosuppression . Often antibodies against surface antigens have been used as a delivery system to target toxic components to the cells in question . True tumor-specific antigens are rare, but the re-expression of developmental antigens on many tumor cells may provide the required selectivity . The amounts of antibodies that reach and bind to the target cells are often small and the antibodies must therefore be armed with very active compounds . Cytocidal toxins of the group here described may fulfil this requirement . A list with examples of immunotoxins is given in Table 2 . In humans immunotoxins have proved valuable in purging of bone marrow for Tlymphocytes in vitro before transplantation to avoid graft-versus-host reaction (see reviews in Frankel 20 ) . Moreover, clinical trials with patients treated systemically with immunotoxins have been initiated, and encouraging results have been reported in a study on acute graft-versus-host reactions as well as in one on malignant melanomas . Immunotoxins were first considered as anticancer drugs, but more recent research Table 2 proteins
Examples of immunotoxins and related
Conjugate Ricin A/anti-T101 Ricin A/anti-melanoma Ricin A/anti-CD1 9 Ricin A/anti-Id-antibody to acetylcholine receptor Ricin A/IgE Ricin A/CD4 Pseudomonas toxin-CD4 Diphtheria toxin-IL 2 Diphtheria toxin-MSH
Binding site CD5 220 kDa-antigen CD1 9 Antibodies on B-cells Fc-receptors gp 120 gp 120 IL 2-receptor MSH-receptor
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indicates that their potential applications reach much further . The use of antiT-cell immunotoxins in immunosuppression has already been mentioned, and conjugates with anti-idiotype antibodies to the acetylcholine receptor may eradicate B-cells producing antibodies to the acetylcholine receptor in myasthenia gravis . Allergic conditions might be treated with IgE-toxin conjugates that bind to and destroy mast cells and basophil leukocytes . It should also be noted that the protein synthesis machinery of eukaryotic parasites is sensitive to the toxins, and the possibility of making immunotoxins against such organisms is currently being explored . 20
J .O .M . is a fellow of the Norwegian Research Council for Science and Humanities . The work was supported by the Norwegian Cancer Society and by Harbitz Legat .
References 1 . Wickner WT, Lodish HF . Multiple mechanisms of protein insertion into and across cells . Science 1985 ; 230: 400-7 . 2 . Schatz G . Signal guiding proteins to their correct locations in mitochondria . Eur J Biochem 1987 ; 165 : 1-6 . 3 . Pappenheimer AM Jr . Diphtheria toxin . Ann Rev Biochem 1977 ; 46 : 69-94 . 4 . Olsnes S, Sandvig K . Entry of polypeptide toxins into animal cells . In Pastan I, Willingham MC, eds . Endocytosis . New York : Plenum, 1985 ; 195-234 . 5 . Middlebrook JL, Dorland RB . Bacterial toxins : cellular mechanisms of action . Microbiol Rev 1984; 48 : 199-221 . 6 . Olsnes S, PihI A . Toxic lectins and related proteins . In Cohen P, van Heyningen S, eds . Molecular action of toxins and viruses . Amsterdam : Elsevier/North Holland, 1982 ; 51-105 . 7 . Olsnes S, Reisbig R, Eiklid K . Subunit structure of Shigella toxin . J Biol Chem 1981 ; 256 : 8732-8 . 8 . Alured VS, Collier RJ, Carroll SF, McKay DB . Structure of exotoxin A of Pseudomonas aeruginosa at 3 .0 A resolution . Proc Natl Acad Sci USA 1986 ; 83 : 1320-4 . 9 . Van Ness BG, Hovard JB, Bodley JW . ADP-ribosylation of elongation factor 2 by diphtheria toxin . NMR spectra and proposed structure of ribosyl-diphthamide and its hydrolysis products . J Biol Chem 1980 ; 255 :10710-6 . 10 . Gill DM . Seven toxic peptides that cross cell membranes . In Jeljaszewicz J, Wadstrom T, eds . Bacterial toxins and cell membranes . New York : Academic Press, 1978 ; 291-332 . 11 . Actories K, Wegner A. ADP-ribosylation of actin by clostridial toxins . J Cell Biol 1989; 109 : 1385-7 . 12 . Endo Y, Tsurugi T. RNA N-glycosidase activity of ricin A-chain . Mechanism of action of the toxic lectin ricin on eucaryotic ribosomes . J Biol Chem 1987; 262 : 8128-30 . 13 . Leppla SH . Anthrax toxin edema factor : a bacterial adenylate cyclase that increases cAMP concentrations in eucaryotic cells . Proc Natl Acad Sci USA 1982 ; 79 : 3162-6 . 14 . Hanski H, Farfel Z . Bordetella pertussis invasive adenylate cyclase. J Biol Chem 1985 ; 260 : 5526-32 . 15 . van Deurs B, Tonnessen TI, Petersen OW, Sandvig K, 0lsnes S . Routing of internalized ricin and ricinconjugates to the Golgi complex . J Cell Biol 1986 ; 102 : 37-47 . 16 . Morris RE, Saelinger CB . Reduced temperature alters Pseudomonas exotoxin A entry into the mouse LM cells . Infect Immun 1986 ; 52 : 445-53 . 17 . Sandvig K, 0lsnes S, Petersen OW, van Deurs B . Acidification of the cytosol inhibits endocytosis from coated pits . J Cell Biol 1987 ; 105 : 679-89 . 18 . Moskaug JO, Sandvig K, Olsnes S . Low pH-induced release of diphtheria toxin A-fragment to the cytosol . Biochemical evidence for transfer to the cytosol . J Biol Chem 1988; 263 : 2518-25 . 19 . Sandvig K, Olsnes S . Diphtheria toxin-induced channels in Vero cells selective for monovalent cations . J Biol Chem 1988 ; 263 :12352-9. 20 . Frankel A . E . (ed) . Immunotoxins . Boston : Kluwer, 1988 .
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
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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
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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 <5 .3 . Treatment of the cells with any one of a large number of compounds that increase pH in endosomes, such as NH4, monensin, and others, protect the cells against intoxication .' In the presence of some of these drugs it is, however, possible to induce toxin entry by exposing the cells to medium with pH <5 .3 .' Under these conditions, diphtheria toxin that is present at the cell surface is translocated directly across the surface membrane . This experimental approach mimics at the cell surface a process that normally occurs in the endosomes . Toxin bound to the cell surface can be efficiently removed by pronase treatment of the cells . 78 To study which parts of the toxin that enter upon exposure to low pH, cells were incubated with 125 1-labelled toxin (mol . wt . 58 kDa), exposed to pH 4 .5,
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atL
and then treated with pronase to remove toxin exposed to the exterior . Under these conditions two fragments were protected against the enzyme . These fragments had electrophoretic migration rates corresponding to 21 kDa and 25 kDa . The smaller polypeptide represents the A-fragment (mol . wt . 21 kDa), whereas the 25 kDafragment is derived from the B-fragment (mol . wt . 38 kDa) . Upon fractionation of the cells, the 21 kDa fragment was consistently found in a soluble fraction comprising the cytosol, while most of the 25 kDa polypeptide was associated with the membrane fraction .' $ This indicates that the A-fragment had been translocated to the cytosol . For translocation of the A-fragment to the cytosol to occur it is not sufficient that cell-bound toxin is exposed to low pH . If the cytosol is also acidified, the cells are not intoxicated and the A-fragment is not shielded, indicating that it is not translocated to the cytosol . 18 The 25 kDa fragment is, however, protected even under these conditions, indicating that at the low pH a portion of the B-fragment is inserted into the membrane whether or not the cytosol is acidified . In addition to an inward directed proton gradient, permeant anions play a role in the translocation, and inhibitors of anion transport inhibit both binding and translocation of the toxin . It is not yet clear in which way anion transport is linked to toxin entry . 4 Diphtheria toxin and toxin fragments containing the hydrophobic regions of the Bfragment are able to form ion-conducting channels in lipid bilayers and in cells ." When the toxin is inserted into Vero cells at low pH, there is a strong increase in the influx of "Na' and 86 Rb' and in the efflux of K' from the cells, but no increased permeability to CI - or SO' - . This indicates that the channels are selective for cations . Small monovalent cations (H', Na' and K') pass the toxin-induced channels more readily than larger cations (choline, glucosamine) and divalent cations pass very inefficiently . When the internal pH is subsequently reduced due to influx of H', the channels are closed . They also become closed when the cytosol is acidified in other ways . Furthermore, the channels were closed when the cells were transferred to neutral medium . Apparently, therefore, an inward directed proton gradient is required for the channels to remain open . The cation-selective channels may be required for toxin translocation . Thus Cd", which closes the channels, protected the cells against intoxication ." Vero cells contain 5-10X10 4 diphtheria toxin receptors, whereas most other cells possess a much lower receptor number . The surface receptors appear to be essential for efficient translocation of the A-fragment across the plasma membrane . Artificially bound toxin (e .g . biotinylated toxin bound to avidin-treated cells) could not be translocated across the surface membrane and did not induce formation of cationselective channels . The possibility therefore exists that the receptor participates directly in the translocation of the toxin to the cytosol . The nature of the receptor is not clear . Treatment of Vero cells with low concentrations of trypsin strongly reduced the ability of the cells to bind the toxin, suggesting that the receptor is a protein . However, treatment with phospholipase C was even more efficient in destroying the binding capability of the cel ls . 4 The step least well understood in the mechanism of action of the toxins is how the A-moiety penetrates the membrane to reach the cytosol . Normally this process does not occur through the cell surface membrane, but across membranes of intracellular organelles . In the case of diphtheria toxin, the transfer appears to occur in the endosomes as soon as their interior reaches the required pH value of 5 .3 . Other toxins may enter from compartments distal to the endosomes . It is possible that some of the toxins must be processed, e .g . by proteolytic cleavage or by glycosylation before the translocation can occur . This could be the reason why
Protein toxins with intracellular targets
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attempts to induce translocation from the cell surface of toxins other than diphtheria toxin have so far been unsuccessful . The possibility must also be considered that the translocation is facilitated by membrane proteins only present in the wall of certain intracellular organelles . In the case of diphtheria toxin there are indications that interaction with a membrane protein is necessary for translocation . Thus, treatment with inhibitors of anion transport prevents the insertion of the toxin into the membrane and, as a consequence, precludes delivery of the A-fragment to the cytosol . During the translocation, charged and polar residues in the A-fragment must be transported through the hydrophobic lipid bilayer . Possibly, the inserted part of the B-fragment shields these residues during the transport . It is also possible that a membrane protein, such as an anion antiporter shields the polar and charged residues during the translocation . The B-fragment could, together with the hydrophilic channel assumed to be present in the antiporter, accommodate these residues while the A-fragment passes across the membrane . The hydrophobic parts of the A-fragment would then face the lipids . The inward directed proton gradient could provide energy for the transport . The energy could be transferred to the system by unfolding of the A-fragment at low pH and refolding at neutral pH . The energy gain obtained when the A-fragment refolds in the cytosol could pull the protein across the membrane . Therapeutic potential In recent years much effort has been made to improve the specificity of cytostatic drugs used in cancer treatment and in immunosuppression . Often antibodies against surface antigens have been used as a delivery system to target toxic components to the cells in question . True tumor-specific antigens are rare, but the re-expression of developmental antigens on many tumor cells may provide the required selectivity . The amounts of antibodies that reach and bind to the target cells are often small and the antibodies must therefore be armed with very active compounds . Cytocidal toxins of the group here described may fulfil this requirement . A list with examples of immunotoxins is given in Table 2 . In humans immunotoxins have proved valuable in purging of bone marrow for Tlymphocytes in vitro before transplantation to avoid graft-versus-host reaction (see reviews in Frankel 20 ) . Moreover, clinical trials with patients treated systemically with immunotoxins have been initiated, and encouraging results have been reported in a study on acute graft-versus-host reactions as well as in one on malignant melanomas . Immunotoxins were first considered as anticancer drugs, but more recent research Table 2 proteins
Examples of immunotoxins and related
Conjugate Ricin A/anti-T101 Ricin A/anti-melanoma Ricin A/anti-CD1 9 Ricin A/anti-Id-antibody to acetylcholine receptor Ricin A/IgE Ricin A/CD4 Pseudomonas toxin-CD4 Diphtheria toxin-IL 2 Diphtheria toxin-MSH
Binding site CD5 220 kDa-antigen CD1 9 Antibodies on B-cells Fc-receptors gp 120 gp 120 IL 2-receptor MSH-receptor
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indicates that their potential applications reach much further . The use of antiT-cell immunotoxins in immunosuppression has already been mentioned, and conjugates with anti-idiotype antibodies to the acetylcholine receptor may eradicate B-cells producing antibodies to the acetylcholine receptor in myasthenia gravis . Allergic conditions might be treated with IgE-toxin conjugates that bind to and destroy mast cells and basophil leukocytes . It should also be noted that the protein synthesis machinery of eukaryotic parasites is sensitive to the toxins, and the possibility of making immunotoxins against such organisms is currently being explored . 20
J .O .M . is a fellow of the Norwegian Research Council for Science and Humanities . The work was supported by the Norwegian Cancer Society and by Harbitz Legat .
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