Abrin and ricin — two toxic lectins

T I B S - January 1978

References I Crick, F.H.C. and Watson. J.D. (1956) Nature (London) 177,473-475 2 Caspar, D. L. D. and Klug, A. (19611 Cold Sprino Harbor Syrup. Quant. Biol. 27, 1-24 3 Winkler, F.K., Sehutt. C.E., Harrison, S.C. and Brieogne. G. (1977) Nature (London) 265. 509513 4 Weber, K., Rosenbusch. J. and Harrison. S.C. (1970) Virology41,763-765 5 Bawden,F.C. and Pirie, N. W. (1938)Brit, J. Exp. PathoL 19, 25 I 6 Bernal, J.D. and Frankuchen. 1. (1941) I. Gem Physiol. 25, 147-165 7 Leberman.R. and Finch,J.T. (1970) d. Mol. Biol. 50, 209-213 8 Richardson, J,S., Richardson, D.C., Thomas, K.A., Silverton, E.W. and Davies. D.R. (1976] J. Mol. Biol. 102,221-235 9 Huber, R. (1976) Trends Biochem.Sci. 1, 174

10 Schiffer, M., Girling, R.L., Ely, K.R. and Edmundson, A.B. (1973) Biochemistry 12. 4620463 I II Champness. J.N., Bloomer, A.C.. Bricogne, G., Butler, P.J.G. and Klug. A. [1976) Nature (London) 259,20 12 Butler, P.J.G., Bloomer. A.C., Brieogne, G., Champness,J.N., Graham, J., Guilley, H., Klug, A. and Zimmern. D. (I 976)in Structure-Fu,ction Relationships q[ Proteins (Markham, R. and Home, R,W,, eds) pp, 101-110, North-Holland, Amsterdam 13 Stubbs, G,, Warren, S. and Holmes, K.C. 11977) Nature (London) 267, 216-221 14 Caspar, D.L.D. (1976) in Structure-Function Relationships of Proteins (Markham, R. and Home, R.W., eds} pp, 85-99, North-Holland, Amsterdam

A-chain and the B-chain. The two chains are connected by a single SS-bond (Fig. 1). When we treated the two toxins with 2mercaptoethanol, the SS-bond was easily reduced and the two chains could be separated by ion-exchange chromatography 1,4-1.The separated A- and B-chains proved to contain less than 1To of the toxicity of the intact toxins and also a mixture of the chains was not very toxic to animals or to cells in culture. If 2-mercaptoethanol was removed by dialysis, the interchain SSbond was reformed, as revealed by polyacrylamide gel electrophoresis and, significantly, there was an almost complete restoration of the toxicity. Furthermore, in experiments where mixtures of abrin AI III chain and ricin B-chain, or vice versa, were II dialyzed to remove 2-mercaptoethanol, highly toxic hybrid molecules were formed in good yield. It is therefore clear that the requirement for toxicity to animals and cultured cells is an A-chain bound by a disulfide bond to a B-chain. It is of little importance which of the A-chains is bound to which of the B-chains. Some physical properties of the intact lectins and the separated chains are given in Table 1, together with their toxicities. Abrin attd ricin are two peculiar proteins, each having two peptide chains with different The effeetor moiety is an enzyme fimctions. One chain, the "effectonter', is an enzpne capable of inactit,atin 9 eucaryotic ribosomes. The other chain, the ~haptomer', is a lectin which binds the toxin to cells and faciliOur early experiments showed that tates the entry of the effectomer into the cytoplasm. The toxins are useful in studies of abrin and ricin are potent inhibitors of celhtlar uptake of proteins and may be vahtable in the treatment of cancer. protein synthesis in cell-free systems, due to an irreversible inactivation of the riboThe lectins are a poorly defined group of The properties of abrin and ricin were somes I-6,7]. Quantitative considerations proteins which are able to bind to and ag- extensively studied in the late 19th century suggested that one toxin molecule inactiglutinate cells. The best characterized lec- by many workers, including Paul Ehrlich, vated many ribosomes, indicating a catatins are present in plant material and par- who discovered some of the fundamental lytic activity of the toxins. This was even ticularly in plant seeds, but lectins have principles in immunology by working with clearer after 2-mercaptoethanol treatment also been found in animal tissue such as rat abrin, ricin and antisera against them (for of the toxins. In contrast to the situation liver and rabbit muscle I-I-3]. The mam- review, see ref. 4). in living animals and in cells in culture malian lectins are present in low amounts Lethal doses ofabrin and ricin are about where such treatment resulted in an almost in the plasma membrane and possibly play 1 ,ug toxin/kg body weight in the mouse, complete loss of toxicity, the ability of the a role in cellular recognition and inter- rat and dog, whereas the rabbit is about reduced toxins to inhibit cell-free protein action. Plant lectins like concanavalin A 10 times more sensitive. There is always a synthesis was strongly increased. Experiand phytohemagglutinin from Phaseohts lag period of several hours before the ments with the isolated chains established vul#aris are often found in high amounts animals become sick. This lag period inthat only the free A-chain is able to inhibit (up to a few per cent ofthe total dry weight) creases with decreasing doses of toxin. protein synthesis in cell-free systems [8,9]. in the endosperm of the seeds, but their Experiments with cells in culture have Clearly this was the effector-moeity of the physiological role in the plant is not shown that the earliest demonstrable effect toxin and it was termed 'effectomer'. known. Most lectins so far isolated are not of the toxins is inhibition of protein synFurther experiments by Sperti et al. [10] very toxic to animals, except when very thesis [5], an effect seen even at concen- and in our laboratory [11] have shown high doses (milligrams per kg body weight) trations considerably below 1 ng/ml. In that the target for the toxins is the 60S are given. accordance with findings made with other ribosomal subunit, which is modified in a Abrin and ricin, the first two lectins de- inhibitors of protein synthesis, the intoxi- still unknown way. This modification scribed, are present in the seeds from Abrus cated cells may survive for many hours, as somehow interferes with the GTPase site precatorius and Ricinus communis. In con- measured by dye exclusion tests after on the ribosomes. Thus, the ability of trast to most other lectins, they are highly protein synthesis has 'completely ceased. toxin-treated ribosomes to support elontoxic. In fact, they are amongst the most As will be outlined below, the toxins have gation factors, EF-I and EF-2, dependent toxic substances known. Their concentra- many features in common with diphtheria hydrolysis of G T P was clearly reduced [4]. tion in the seeds, in comparison to their toxin, which also inhibits protein synthesis. Apparently, the binding site for EF-2 on toxicity, is high. Thus, about 1 mg of pure the ribosomes is identical with or partly toxin can be isolated from 1 g of seed. Structure-function relationships overlaps with the target for the toxin AAbrin and ricin are both glycoproteins chain. Thus, prebound EF-2 strongly proThe authors are at the Norsk Hydro's Institute for Cancer Research,Montebello, Oslo 3, Norway. and consist of two polypeptide chains, the tects the ribosomes from being inactivated

Abrin and ricin- two toxic lectins Sjur Olsnes and Alexander Pihl

T I B S - January 1978

8

cells and animals. It needs a carrier which brings the A-chain 'war-head' to the cells Properties of toxin-resistant cell Hnes In recent years a number of cell variants and possibly facilitates its entry through , ~ have been isolated which tolerate higher the cell membrane. "~l~lomlt" ricin concentrations than the parent lines. Ehrlich had already assumed that abrin and ricin must be bound to cells before The variants are obtained by incubating they can intoxicate them. In recent years, cells (with or without mutagens) in the E a I ~ I . lalcllvlil experiments with labelled toxins have presence of increasing toxin concentra¢lll -Iml iDlim directly shown that abrin and ricin indeed tions and then surviving clones are H SH / Inti¢l cells bind to cell-snrface receptors (for review, selected. From the known mechanism of ~,~,..!-<,.°z~ ~ ...... ',oz:" ...... action of ricin there may be various reasee ref. 4). Experiments in our laboratory showed that one HeLa cell contains 3 x 10 7 sons for the resistance. One possible reabinding sites for either toxm, with a K , of son would be a change in the ribosomes, 107 M - 1 to 10a M - 1 depending on the rendering them resistant to the toxin Atemperature [16,17]. When we tested the chain. So far, no such variant has been isolated A- and B-chains for binding to described. Other possible reasons are cells, it was evident that only the isolated changes in the surface receptors resulting Fig. I. Relationship between structure and fimction t~f B-chain is able to bind and we termed it in a redttced number of binding sites for the 'haptomer'. The B-chain also binds the toxins. Finally, the resistance may be the toxins. to free galactose or lactose [18], although due to a decreased rate of internalization by the toxins and, furthermore, once the with a much lower affinity (Ka of the order of surface-bound toxin molecules. ribosomes are inactivated after toxin treat104 M - t ) than to intact cells. In spite of Gottlieb et al. [19] isolated a variant of ment, their ability to bind EF-2 is strongly this, lactose is an efficient inhibitor of the Chinese hamster ovary cells which tolereduced [12-13]. binding to cells, indicating that the cell- rated 80 times higher ricin concentrations The inactivation of pure ribosomes by surface binding sites have features in com- than the parent cell line and contained the isolated A-chains was studied in a mon with lactose. Apparently, the binding only 1.3",; as many binding sites. Obviquantitative way by Olsnes et al. [14]. sites contain terminal non-reducing galac- ously, in this case the resistance can be The data showed that one A-chain mole- tose residues. accounted for by the reduction in the numcule is able to inactivate about 1,500 ribober of binding sites. Also some of the variSeveral lines of evidence indicate that somes per minute. The Qto was found to only toxin molecules bound to the cell ants isolated by Meager et al. [20] have a be 1.8 and the Km about l - 2 x l0 -~ M. surface can intoxicate cells. Thus, addition strongly reduced number of binding sites. The inactivation could be stopped at any of lactose to the cell culture medium There appear to be at least two common time by adding specific anti-A-chain anti- strongly reduces the amount of toxin reasons for the reduction in the number bodies. This clearly shows that the A- bound to the cells and concomitantly the of ricin-binding sites. Thus, ricin-resistant chains arc enzymes acting directly on the toxic effect is reduced to the same extent variants isolated in 3 different laboratories 60S subunits without any intermediates. [17]. A similar effect is obtained after pre- proved to be deficient in N-acetylglycosThe most easily demonstrable effect of treatment of the cells with isolated (and aminyl transferase [20-22]. Since N-acetabrin and ricin A-chains in cell-free sys- non-toxic) ricin B-chain to block the major ylglucosamine is often penultimate to tertems is inhibition of peptide chain elonga- part of the binding sites. Furthermore, minal galactose residttes, such cells have a tion [6,7]. Our recent data indicate that treatment of cells with neuraminidase, reduced ability to complete binding sites inactivation of one or a few ribosomes per which removes terminal sialic acid residues for abrin and ricin. The same variants polysome is sufficient to stop the elonga- often results in the exposure of penultimate have an increased number of binding sites tion [15]. galactose residues which may act as bind- for concanavalin A, apparently due to the Recently, we have also shown that pro- ing sites for abrin and ricin. In most cases presence of incomplete oligosaccharide tein synthesis in systems made dependent tested (but not all, see below) the sensitivity chains ending in mannose. Other variants on initiation of new peptide chains is of the cells to the toxins increases to the contain more membrane-bound sialic acid strongly inhibited by the toxin A-cl~ains. same extent as the increase in the number than the parent strain (ref. 23 and S. Olsnes, The association of the 40S initiation com- of binding sites. submitted for publication). In these variplex with the 60S subunit to form the 80S Although the B-chains have only a single ants terminal galactose residues are apparinitiation complex is the only initiation binding site for lactose per molecule, in ently covered by sialic acid which prevents step inhibited by the toxins. Interestingly, concentrations of 10 pg/ml they do agglu- toxin binding. this process, which is dependent upon tinate the cells, probably due to the presIn other cases the resistant cell lines bind hydrolysis of GTP, appears to be even ence of a small amount of B-chain dimers approximately as much toxin as the sensimore sensitive to the toxin A-chains than [18]. Thus the B-chain fulfills the require- tive parent lines [20,24]. Since we have the process of chain elongation (Skorve, ments for being a lectin. shown that at least in some such variants Abraham, Olsnes and Pihl, submitted for publication). TABLE I Propertiesof abrin, ricin and their constituent peptidechains. Lectin properties of the B-chains AenlH

[

A°thaln

atCIS

Inlltl c l l l l

i

The A-chain is like a 'war-head" of very high potential impact. Thus, only a few A-chain molecules ~n the cytoplasm might be sufficient to inactivate the major part of the ribosomes in a cell within one day and thus kill the cell [14]. However, the isolated A-chain as such is not toxic to

Abrin

Molecular weight pI Toxicity to mice (LDso!~j)

Ricin

Intact

A - c h a i n B-chain

Intact

A - c h a i n B-chain

65000 6.1

30000 4.6

35000 7.2

65~0 7.1

32000 7.2

34000 4.8

77

<0.1

<0.1

15

<0.1

<0.1

TIBS- January 1978 the ribosomes are fully sensitive to the enzymatic activity of the A-chains (S. Olsnes, submitted for publication), it is likely that in these instances the resistance is somehow due to a reduced efficiency in the uptake of bound toxin. Evidence that such a deficiency may exist was obtained in studies of two resistant variants with reduced number of binding sites. Thus, it was found that the number of bound toxin molecules required to give 50?,0 reduction in protein synthesis under standardized conditions was strongly increased, indicating that in these variants the bound toxins or their A-chains are less easily transferred from the cell-surface binding sites to their site of action, the ribosomes (K. Sandvig, S. Olsnes and A. Pihl, submitted for publication). Studies of surface-membrane proteins labelled by the lactoperoxidase/~ 251 method, which labels proteins containing tyrosine residues exposed at the cell surface, or by the galactose oxidase/[3H]borohydride method, which labels surface-exposed galactoproteins, have shown that the pattern of exposed proteins in the resistant cell lines differs from that in the parent cells. However, so far these changes have not been proved to be causally connected with

the resistance. Hopefully, studies of a greater number of resistant variants may reveal consistent membrane differences between sensitive and resistant cell lines. Selectivity for cancer cells Anti-cancer properties ofabrin and ricin have been observed by many authors, and Lin, Tung and Hsu in Taiwan have used the toxins in the treatment of human cancers, in many cases with good results [25,26]. In our laboratory cancers removed from patients referred to The Norwegian Radium Hospital were transplanted into nude (thymus-less) mice which were then treated with abrin and ricin. In some cases very good inhibition of tumor growth was observed [27]. This effect was comparable with, and in some cases even better than. that obtained with cytostatics in current clinical use. The reason for this selective effect on cancer cells is not known. Experiments with synchronized HeLa cells have shown that the cells are equally sensitive in all phases of the cell cycle, and there is no evidence that rapidly dividing cells are more sensitive than resting cells (our unpublished data). Interestingly, Nicolson et al. [28] found that malignant transformation of 3T3 cells

with SV-40 strongly increased the sensitivity of the cells to ricin without increasing the number of binding sites. Possibly the malignant cells are more efficient in internalizing the toxins. In contrast to most cytostatics, abrin and ricin do not depress the level of white blood cells and have only a moderate effect on the erythropoesis and thrombopoesis [26,27]. Therefore they may prove valuable in cases where bone marrow depression excludes the use of other cytostatics. The toxins, which in contrast to most cytostatics do not directly interfere with DNA synthesis and cell division, may also prove valuable in combination with other cytostatics. How do toxins penetrate into cells ? Since the 60S ribosomal subunits are the target for abrin and ricin, it is clear that the toxins or their A-chains in some way must enter the cytoplasm. A similar situation exists in the case of diphtheria toxin, which also has an intracellular target, the elongation factor EF-2 [29]. Diphtheria toxin consists of two functionally different domains which are easily split by mild proteolytic treatment. The resulting Afragment is an enzyme capable of inactivating EF-2 and thus inhibiting protein synthesis, whereas the B-fragment binds the toxins to cell-surface receptors. Obviously, the A-fragment of diphtheria toxin must somehow penetrate through the plasma membrane. It should be noted that the uptake into pinocytotic vesicles is not a sufficient requirement since in that case the toxins are still separated from the cytoplasm by a unit membrane. Nicolson et al. [28] have shown that ricin is indeed taken up by pinocytosis and Gonatas et al. [301 have shown that it may end up in the ,Golgi,: endoplasmic t reticulum, lysosome system. It is not known, however, if this uptake is involved in the expression of toxicity. We have recently observed that some HeLa cell variants which tolerate 50 times more cell-bound toxin than the parent HeLa cells internalize by pinocytosis the same amount of toxin as the parent HeLa cells. Since only a few toxin molecules need enter the cytoplasm to intoxicate the cells, the functionally significant uptake may be orders of magnitude lower than the bulk uptake by pinocytosis. Interesting results recently obtained with diphtheria toxin [31] could explain the uptake of toxic proteins in general. Studies of the binding of the non-ionic detergent Triton X-100 have shown that a certain region of diphtheria toxin Bfragment binds the detergent in high amounts similarly to many membrane proteins. The A-fragment does not bind the

10

T I B S - January 1978

detergent.Possibly, the B-fragment enters the lipid moeity of the plasma m e m b r a n e and s o m e h o w pulls the hydrophilic A-fragment through the m e m b r a n e [31-1. Recent work has revealed that several other toxins also consist of two functionally different moieties, and it is possible that entry of an enzymatically active protein into the cytoplasm m a y be c o m m o n to toxic proteins. Possibly the toxins enter the cytoplasm by preformed routes originally developed for the uptake of s o m e physiologically active substances like peptide hormones or growth factors that may act on intracellular targets.

Acknowledgement This work was supported by the Norwegian Cancer Society.

References 1 Sharon, N. and Lis. H. (1972) Science 177,949 2 Ashwell, G. and Morell, A.G. (19771 Trends Biochem. Sci. 2, 76 3 De Waard, A.. Hickman, S. and Kornfeld, S. (1976) J. Biol. Chem. 251,7581 4" Olsnes, S. and Pihl, A. (1976) in The Specificity of Animal, Bacterial and Plant Toxins (Receptors

and Recognition, Series B vol. I) (Cuatrecasas. P., ed.) Chapman and Hall. London. p. 129 5 Lin,J.-Y.,Kao, W.-Y.,Tserng.K.-Y., Chen, C.-C. and Tung, T.-C.(1970) Cancer Res. 30. 2431 6 Olsnes,S. and Pihl, A. (1972) FEBS Lett. 20, 327 70lsnes, S. and Pihl, A. (1972) Nature (London) 238.459 8 Olsnes, S. and Pihl, A. (19731 Ear. J. Biochem. 35, 179 90lsnes, S. and Pihl, A. (19731 Biochemistry 12, 3121 10 Sperti, S.. Montanaro. L., Maniolo, A. and Stirpe, F. (1973) Biochem. J. 136.813

II Benson. S., Olsnes, S., Pihl, A., Skorve, J. and Abraham, K. A. (1975) Eur. J. BiochenL 59. 573 12 Montanaro, L.. Sperti, S., Mattiolo, A.. Testoni. G. and Stirpe, F. (1975) Biochem. J. 146, 127 13 Carrasco, L.. Fernandez-Puentes, C. and Vazquez, D..(1975) Eur. J. Biochem. 54, 499 14 Olsnes, S., Fernandez-Puentes, C.. Carrasco, L. and Vazquez,D. (1975) Eur. J. Biochem. 60, 281 15 Fodstado O. and Olsnes, S. (1977) Eur. J. Biochem. 74, 209 16 Sandvig, K.,'Olsnes,S. and Pihl, A. (1976) J. Biol. Chem. 251,3977 17 Olsnes. S., Sandvig, K.. Refsnes, K. and Pihl, A. (1976) J. Biol. Chem. 251, 3985 18 Olsnes, S., Saltvedt, E. and Pihl. A. (1974) d. Biol. Chem. 249, 803 19 Gottlieb, C.. Skinner, S.A.M. and Kornfeld, S. (1974) Proc. Nat. Acad. Sci. U.S.A. 71. 1078 20 Meager, A., Ungkitchanukit, A., Nairn, R. and Hughes, R.C. (1975) Nature (London) 257, 137 21 Gottlieb, C., Baenziger,J. and Kornfeld, S. (1975) J. Biol. Chem. 250, 3303 22 Stanley, P., Caillibot, V. and Siminovitch, L. (1975)Ce116, 121 23 Gottlieb. C. and Kornfeld, S. (1976) d. BioL Chem. 251. 776 I 24 Hyman, R.. Lacorbiere, M., Stavarek, S. and Nicolson. G. ( 1974).LNat. Cancer Inst. 52,963 25 Tung, T.-C., Hsu, C.-T. and Lin, J.-Y. (1971) J. Formosan Med. Ass. 70, 569 26 Hsu, C.-T., Lin, J.-Y. and Tung, T.-C. (1974) J. Formosan Med. Ass. 73,526 27 Fodstad, O., Olsnes. S. and Pihl, A. (1977) Cancer Res. in press 28 Nicolson, G. L., Lacorbiere, M. and Hunter. T. R. (1975) Cancer Res. 35, 144 29 Pappenheimer, A.M. Jr. and Gill. D.M. (1973) Science 182,353 30 Gonatas, N.K., Kim, S.U., Stieber. A. and Avrameas.S. (1977)J. Cell Biol. 73, 1 31 Boquet, P., Silverman, M.S., Pappenheimer, A.M. Jr. and Vernon, W.B. (1976) Proc, Natl. Acad. Sci. U.S.A. 73.4449

Plasmids as vehicles for gene cloning: impact on basic and applied research Donald R. Helinski The basic procedures utilising p l a s m i d elements as vehicles f o r cloning D N A f r o m an), source in the bacterium Escherichia coli are described. Consideration also is given to the impact o f this research on the biological sciences and the technical problems that must be overcome before the benefits o f this technology are f u l l y realized.

The development of the technology" for establishing genes of virtually any organism in the bacterium Escherichia coli has provided exciting new opportunities for basic biological research and the promise of exceptional benefits to society in the areas o f medicine and agriculture. This relThe author is at the Department of Biology, University of California. San Diego, La Jolla. CA 92093, U.S.A.

atively new field of research, labeled recombinant D N A research, gene cloning or genetic engineering, also has stirred m u c h controversy as to its potential danger to m a n and his environment. It has been argued that species have retained their identity over a multitude of generations as a result of certain natural barriers and to violate these barriers, given

our present state of knowledge (or ignorance), is to recklessly tamper with the evolutionary process and risk the 'creation' of dangerous new forms of life. It has been counter-argued that the exchange of genes between unrelated organisms is a continuous process in the natural environment, albeit at a low level in frequency, and the truly novel aspect of this technology is not the creation o f new life forms, but it is the opportunity to establish unrelated genes in a cell by a direct and predictable process. While the 'safety' of this research has been a point of much controversy, there is much less disagreement as to its potential benefits. One immediate benefit is the availability for the first time o f large quantities of specific D N A segments of the c h r o m o s o m e s of higher organisms. Biochemical analysis o f these D N A regions, cloned in E. coli with the aid of both plasmid and phage vectors, is providing fundamental information on the structure o f c h r o m o s o m a l D N A of eukaryotic D N A and powerful new insights to the problem of the m e c h a n i s m of control of gene expression in higher organisms. The groundwork also is being laid for the implantation of genes into E . c o l i and other ceils that specify the production of medically and agriculturally important substances. To date, the bulk of research in this area has employed the E. coli strain, K 12, as the recipient cell for the recombinant D N A . The usual requirement for nucleotide sequence homology in order to stably incorporate in a bacterial cell D N A from another source is circumvented with the use of two types of self-replicating elements, the lambdoid phages and plasmids. These elements are capable of existing independently of the c h r o m o s o m e of the cell and are used essentially as molecular vehicles or carriers for the stable maintenance of heterologous D N A in the cell. This treatment of gene cloning will deal exclusively with the use of plasmids as molecular vehicles for the establishment of foreign D N A in bacteria. Plasmids exist in bacterial cells as covalently-closed and circular duplex D N A molecules. Gene cloning in bacteria employing a plasmid element consists basically of isolating the plasmid molecule from the bacterial cell, cleaving the molecule with a site-specific enzyme, coupling the foreign D N A with the cleaved plasmid molecule and by the process of transformation re-establishing the plasmid molecule with its attached foreign D N A in the bacterial cell.

Plasmids Plasmids are e x t r a c h r o m o s o m a l genetic elements that are found in a wide variety