How do tetanus and botulinum toxins bind to neuronal membranes?

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for measuring the amounts of A G E D N A and protein linked to D N A by A G E . In addition, potential mechanisms for repair of the A G E - D N A need to be explored. T h e loss of biological function by the non-enzymatic reaction of glucose with proteins and nucleic acids offers a new way to explain the chemical basis of aging. This hypothesis is attractive because it could explain both the agerelated changes n o t e d with extracellular proteins as well as cells.

Note added in proof A recent study has revealed that aminoguanidine can inhibit, in vitro and in vivo, glucose derived protein crosslinks noted above 29.

Acknowledgement This work was supported by the National Institutes of Health Grant AM19655 and The Brookdale Foundation.

References 1 Maillard, L. C. (1912) C. R. Seances Soc. Biol. Paris 72, 599~01

2 Reynolds, T. M. (1963) Adv. Food Res. 12, 1-52 3 Reynolds, T. M. (1%5) Adv. Food Res. 14, 167-283 4 Cerami, A. and Koenig, R. J. (1978) Trends Biochem. Sci. 3, 73-75 5 Brownlee, M. and Cerami, A. (1981) Annu. Rev. Biochem. 50, 385-432 6 Mortensen, H. B. and Christophersen, C. (1983) Clin. Chim. Acta 134, 317-326 7 Pongor, S., Ulrich, P. C., Bencsath, F. A. and Cerami, A. (1984) Proc. Natl Acad. Sci. USA 81, 2684-2688 8 Chang, J. C. F., Ulrich, P. C., Bucala, R. and Cerami, A. (1985) J. Biol. Chem. 260, 7070--7074 9 Monnier, V. M., Kohn, R. R. and Cerami, A. (1984) Proc. Natl Acad. Sci. USA 81,583-587 10 Monnier, V. M., Vishwanath, V., Frank, K. E., Elmets, C. A. Danchot, P. and Kohn, R. R. (1986)N. Engl. J. Med. 314,403-408 ll Kohn, R. R., Cerami, A. and Monnier, V. M. (1984) Diabetes 33, 57-59 12 Harrison, D. E. and Archer, J. R. (1978) Exp. Gerontol. 13,754~2 13 Benedek, G. B. (1971) Appl. Ophthalmol. 10, 459-473 14 Nicholson, D. H., Harkness, D. R., Benson, W. E. and Peterson, C. M. (1976) Arch. Ophthalmol. 94, 927-930 15 Monnier, V. M., Stevens, V. J. and Cerami, A. (1979) J. Exp. Med. 150, 1098-1107 16 Manabe, S., Bucala, R. and Cerami, A. (1984)

How do tetanus and botulinum toxins bind to neuronal membranes? Cesare Montecucco The conflicting findings on tetanus a n d b o t u l i n u m toxins bi ndi ng to neuronal m e m branes are reviewed a n d discussed in terms o f a d o u b l e receptor f o r m e d b y both a G lb ganglioside a n d a protein compone nt .

Tetanus toxin (TeTx) and botulinum toxins (eight immunologically distinct subtypes, BoTx) are extremely potent neurotoxins produced by anaerobic bacteria of the Clostridium genus. In most animal species their LDs0 is in the range 1-5 ng kg - I , i.e. around 108 molecules are sufficient to kill a mouse. TeTx causes spastic paralysis by blocking presynaptic transmitter release mainly on the central nervous system (CNS). A t high doses, it also acts on the peripheral system t-3. TeTx gains access to the CNS by uptake at the nerve terminals, retrograde axonal transport within the m o t o n e u r o n and trans-synaptic migration into the inhibitory interneuron C. Montecucco is at the CNR Mitochondrial Physiology Centre and Laboratory of Molecular Biology and Pathology, Institute of General Pathology, Padova University, 35131 Padova, lta(v. ~) 1986, Elsevier Science Publishers B .V., Amsterdam

(Ref. 4 and references cited therein). BoTx acts at the peripheral level by inhibiting the release of acetylcholine at the neuromuscular junction thus causing a flaccid paralysis 5. It is thought that these toxins diffuse through the extracellular fluid from the site of release to the neuromuscular junction. A f t e r binding to nerve endings both toxins are internalized by an unknown, energy d e p e n d e n t process and appear in smooth vesicles where they are protected (at least partially) from degradation 4,6,7. While TeTx is taken up by all peripheral and central neurons 4, BoTx is specific for m o t o n e u r o n s 6. Available evid e n c e - i n d i c a t e s that the sequence of binding and internalization is a prerequisite for the intoxication process to occur.

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J. Clin. Invest. 74, 1803-1810 17 Beswick, H. T. and Harding, J. J. (1984) Biochem. J. 223,221-227 18 Harding, J. J. and Ri'xon, K. C. (1985) Lancet i, 762 19 Monnier, V. M. and Cerami, A. (198l) Science 211, 491493 20 Brownlee, M., Pongor, S. and Cerami, A. (1983) J. Exp. Med. 158, 1739-1744 21 Brownlee, M., Vlassara, H. and Cerami, A. (1985) Diabetes 34, 938-941 22 Steffes, M. W. and Mauer, S. M. (1984) in International Review of Experimental Pathology

23 24 25 26 27 28 29

(Richter, G. W. and Epstein, M. A., eds), pp. 147-177, Academic Press Brownlee, M., Vlassara, H. and Cerami, A. Diabetes (in press) Vlassara, H., Brownlee, M. and Cerami, A. (1984)Z Exp. Med. 160,197-207 Vlassara, H., Brownlee, M. and Cerami, A. (1985) Proc. Natl Acad. Sci. USA 82, 5588-5592 Bucala, R., Model, P. and Cerami, A. (1984) Proc. Natl Acad. Sci. USA 81,105-109 Bojanovic, J. J., Jevtovic, A. D., Pantic, V. S., Dugandzic, S. M. and Javonovic, D. S. (1970) Gerontologia 16,304-312 Bucala, R., Model, P., Russel, M. and Cerami, A. (1985) Proc. Natl Acad. Sci. USA 82, 8439~,442 Brownlee, M., Vlassara, H., Kooney, A., Urich, P. and Cerami, A. (1986) Science 2321 1629-1632

transmitter release is unknown. TeTx is effective on several types of synapse while BoTx acts on cholinergic nerve endings (but see Ref. 8); it is likely, however, that the mechanisms of action of TeTx and BoTx differ quantitatively rather than qualitatively (see Ref. 9 and references cited therein). Their potency and some similarity to other bacterial toxins suggest that they possess an enzyme activity which acts on a cytoplasm-facing m e m b r a n e structure involved in the exocytosis s. The hunt for a cellular model system amenable to study this problem at the biochemical level is open; it has already been shown that BoTx type D inhibits catecholamine release in bovine chromaffin cells 8. H o w a water-soluble protein can cross the hydrophobic m e m b r a n e barrier to reach the cytoplasm constitutes a m a j o r unsolved aspect of the action of all bacterial toxins with intraceUular targets. Experiments with model systems support the idea that acidification of the vesicle lumen induces a conformational rearrangement of TeTx and BoTx with exposure of hydrophobic surfaces that allow t h e m to insert into the lipid bilayer10-13.

Toxin structure The active form of these toxins is made of two polypeptide chains (Fig. la): H (100 kDa) and L (50 kDa) joined by a

315

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above-mentioned findings. Glb gangliosides are considered to act as a first receptor for the toxins (Tx) (step 1). The binding is reversible with a binding constant, K G, that varies with toxin, ganglioside type and temperature. Ganglioside binding is supposed to account for the low affinity site. The membrane-bound ganglioside-Tx complex then moves laterally to reach and bind the toxin specific protein receptor (Rxx) (step 2), a transmembrane protein that is involved in Tx internalization. Few Rxx molecules are supposed to be present on a synapse and the receptor is expressed only, or at least preferentially, in nerve cells to account for the high potency and specificity of these toxins. The toxin binds to Rxx with a binding constant, KR, that may vary with Tx binding to gangliosides if the latter induce a conformational change in the toxin that alters its affinity for Rxx. Only when the ternary complex gangliosideTx-Rxx is formed is the toxin binding in its high affinity binding conformation and the binding of Tx to the cell productive. At this stage the association of the toxin with the nerve becomes very fight because the membrane binding constant in now KCR = K 6 . K R. Moreover, given a certain value of K R, the toxin occupancy of the receptor is much higher when the toxin is pre-bound to gangliosides with respect to its water-soluble form because the two partners of the binding reaction are now restricted to the two dimensional plane of the plasma membrane, rather than in the threedimensional water phase. The ganglioside binding step is actually equivalent to concentrating the toxin and its protein receptor in a much smaller volume. For this reason K R may even be low and yet be very relevant to the process of binding if the toxin is first complexed with membrane gangliosides. A low K R would not be surprising considering that toxin receptors are unlikely to be the expression of a suicide tendency of cells; it is more likely that they serve unknown functions and are simply exploited by toxins to intoxicate cells. An additional kinetic effect is also to be considered. Since Glb gangliosides are dispersed on the neuronal surface 26, are present in a very large excess with respect to the toxin and are expected to diffuse laterally as fast as the other membrane lipids (in the order of 10-s cm2/s), they would form an effective system to catch the toxin and deliver it to its protein receptor. Practically any 'hit' of the toxin molecule on the membrane results in a binding, which is completed when

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single disulfide bridge. Papain cleaves the heavy chain of TeTx into two fragments: a COOH-terminal part (fragment C), which retains the toxin ability to bind gangliosides, and the remaining part (fragment B), comprising the light chain and the NH2-terminal part of the heavy chain, which causes flaccid, rather than spastic, paralysis when injected at very high doses t. Membrane binding Both toxins bind to the nervous tissue and, after the discovery that gangliosides (sialic acid-containing glycolipids) are involved 14, many efforts (reviewed in Refs 1-3 and 5) have been dedicated to demonstrating the role of gangliosides as TeTx and BoTx receptors, similar to previous studies on cholera toxin 15. Summarizing briefly: (i) both toxins show a preference for gangliosides of the Gtb series (Fig. lb); (ii) the interaction is strong with half-saturation for TeTx in the range of ganglioside concentrations of 10-8--10 -9 M; (iii) one ganglioside binds to one molecule of TeTx; (iv) the ganglioside binding site is located in fragment C. These data, together with the nearly exclusive location of Gtb gangliosides on neuronal cells 16, favour Glb gangliosides as the receptors of TeTx and BoTx much like GM1 is the receptor of cholera toxin. However, several other findings and considerations suggest that the Glb gangliosides are not solely responsible for TeTx and BoTx binding to neuronal membranes. (1) There is only a partial correlation between toxin binding and Glb content of neuronal cells and synaptosomes

(Refs 17, 18 and references cited therein). (2) The binding is trypsin sensitivel7,18. (3) There appear to be two classes of binding sites with different affinities2,9,18-20. (4) TeTx and BoTx do not show for any ganglioside the high specificity shown by cholera toxin for the Gra 1 ganglioside. (5) The ganglioside-binding part of TeTx, fragment C, is less competitive than TeTx for the membrane binding of iodinated TeTx21,22; moreover, membrane-bound iodinated TeTx cannot be completely displaced by unlabelled TeTx 22. (6) GTt and a mixture of brain gangliosides cause a 40% inhibition of the retrograde axonal transport of TeTx while GM1 completely blocks that of cholera toxin 23. (7) Gangliosides are present in enormous excess with respect to TeTx (at clinical doses): in the mouse CNS it has been estimated that the Gotb:TeTx molar ratio in vivo is over 109 (Leeden and Meilanby, quoted in Ref. 3) and in vitro, using synaptic membranes and comparable TeTx concentrations, the molar ratio of Go1 b to bound toxin is the order of 105 or higher 2. (8) Birds with a high content of Glb gangliosides24,25 are highly resistant to tetanus2,3. Taken together these data indicate that in addition to gangliosides another component(s) is involved in the binding of TeTx and BoTx to neuronal membranes. Double receptors? Figure 2 shows a model for TeTx and BoTx binding to the nerve plasma membrane that explains several aspects of their action and accommodates the

TIBS 11- August 1986

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5 ®lifo. Fig. 2. (a) The two sequential steps of the interaction of tetanus and botulinum toxins with nerve membranes are depicted here: step 1, binding to GIb gangliosides (G), characterized by a binding constant Ka; step 2, binding to the toxin protein receptor R rx after lateral movement of the ganglioside.-toxin complex in the plane of the membrane. The binding of the toxin to Rrx is characterized by a binding constant K R. The formation of the ternary complex ganglioside-toxin-Rrx involves an association constant." KaR = K a. K R. (b) Binding of.fragment B (step 3) to the protein receptor, Rrx. For simplicity, a constant, K R, is associated to this step even though its value may differ ~ o m the K R of step 3, which is equal to the ratio KcR:K G. (c) Binding of ~agment C of tetanus toxin to gangliosides. The association constant, indicated here as Ka, may differ from that of the intact toxin shown in step 1 (see texO.

RTx is encountered. This kind of sink effect has been proposed for the chemoreception of insects by Adam and Delbruck27 and has been later evaluated for different membrane systems (Ref. 28 and references quoted therein, Ref. 29). The specific characteristics of TeTx and BoTx binding to neuronal membranes appear to satisfy all the conditions posed for an effective rate enhancement by reduction of dimensionality28,29. A further possibility is that the binding to gangliosides induces a conformational rearrangement of the toxin structure so as to increase its affinity for RTx. It has been found that ganglioside binding alters the mode of interaction of TeTx with the lipid bilayer at low pH 13. That both proteins and lipids can act as receptors for hormones, toxins and interferons has already been considered on the basis of binding experiments to thyroid membranes3°. A 'double receptor model' for tetanus and botulinum toxins binding to the nerve cell easily accommodates their high neuronal selectivity because Glb gangliosides are more concentrated in the nervous system than in other t i s s u e s 17 and because the receptor, RTx is supposed to be expressed only or preferentially by nerve cells. Moreover, it pro-

vides an explanation for the ability of the miniscule amount of toxin needed to give clinical symptoms to bind to its specific target. The existence of both a ganglioside and a protein receptor is consistent with points (1), (2) and (3) and also explains the findings of point (5) because fragment C only competes for the ganglioside binding step and because iodinated TeTx bound to both the ganglioside and RTx would not be easily displaceable by unlabelled TeTx. The large excess of gangliosides (point 7) is no longer a difficulty here because they are only part of the binding structure of TeTx and the birds' resistance to TeTx intoxication may result from a reduced number or affinity of their protein receptors. Because the number of gangliosides is very large compared to the number of toxin molecules, the toxin would be expected to bind first via gangliosides and only later bind to the protein receptor. However, if large amounts of toxin are incubated with ganglioside-poor membranes a direct binding to RTx might occur. This binding would not be detected in immunoblotting assays such as those described in point (7) if K R is low or if RTx is irreversibly denatured by SDS.

Direct binding to Rxx would be the only option available for fragment B (step 3), which lacks the ganglioside binding region. The fragment B-RTx complex is still able to be taken up by the cell and the large amount of fragment B needed to cause a biological effect is explained here by the lower value of K R compared with KGR and by the absence of the membrane concentration and of the 'catch and deliver' effect. The value of K R for fragment B may be different from that of the intact toxin if conformational changes are caused in the toxin structure by binding to ganglioside or by its papain cleavage. This model also provides an explanation for the competition in membrane binding between TeTx fragment C and BoTx 31 and for the different targets of the two toxins in mammals by supposing that they share the ganglioside adhesion step, but not their protein receptors (RTeTx different from RBoTx), which can also be expressed at different levels in different cells. The model would also accommodate the possibility that different BoTx subtypes show differential binding properties because of differential binding to gangliosides and to RBoTx; moreover, there is the possibility that the various subtypes of BoTx do not share the same protein receptor. Both TeTx and BoTx can be endocytosed and transported retroaxonally (reviewed in Ref. 5) and yet only TeTx can enter the inhibitory nerve. This is explained here by assuming that RTeTx, but n o t RBoTx, is present at the level of the presynapse of the inhibitory neuron with the result that TeTx is taken up while BoTx is not.

Acknowledgements I would like to thank R. Bisson, B. Bizzini, P. Bouquet, J. Meldolesi, T. Pozzan, M. Roa and M. Tomasi for useful criticisms and comments.

References 1 Bizzini, B. (1979) Microbiol. Rev. 43, 224-240 2 Mellanby, J. and Green, J. (1981) Neuroseience 6,281-300 3 Wellhoner, H. H. (1982) Rev. Physiol. Biochem. Pharmacol. 93, 1~o8 4 Schwab, M. E., Suda, K. and Thoenen, H. (1979) J. Cell. Biol. 82,798-810 5 Simpson, L. L. (1981) Pharmacol. Rev. 33, 155-188 6 Dolly, J. O., Black, J., Williams, R. S. and Melling, J. (1984) Nature 307, 457-460 7 Critchley, D. R., Nelson, P. G., Habig, W. H. and Fishman, P. H. (1985) J, Cell Biol. 100, 1499-1507 8 Knight, D. E., Tonge, D. A. and Baker, P. F. (1985) Nature 317,719-721 9 Mellanby, J. (1984) Neuroscience 11, 29-34

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10 Boquet, P. and Duflot, E. (1982) Proc. Natl Acad. Sci. USA 79, 7614-7618 11 Roa, M. and Boquet, P. (1985) J. Biol. Chem. 260, 6827--6835 12 Hoch, D. H., Romero-Mira, M., Ehrlich, B. E., Finkelstein, A., Das-Gupta, B. R. and Simpson, L. L. (1985) Proc. Natl Acad. Sci. USA 82, 1692-1696 13 Montecucco, C., Schiavo, G., Brunner, J., Duflot, E., Boquet, P. and Roa, M. (1986) Biochemistry 25,919-924 14 Van Heyningen, W. E. and Miller, P. A. (1961) J. Gen. Microbiol. 24,107-119 15 Fishman, P. H. (1982) J. Membr. Biol. 69, 85-97 16 Wiegandt, H. (1985) in Glycolipids (Wiegandt, H. ed.), pp. 199-260, Elsevier Science Publishers 17 Yavin, E. and Nathan, A. (1986) Eur. Z

Biochem. 154,403-407 18 Evans, D. M., Williams, R. S., Shone, C. C., Hambleton, P., Melling, J. and Dolly, J. O. (1986) Eur. J. Biochem. 154, 409-~16 19 Agui, T., Syuto, B., Oguma, K., Iida, H. and Kubo, S. (1983)J. Biochem. 94, 521-527 20 Agui, T., Syuto, B., Oguma, K., Iida, H. and Kubo, S. (1985)J. Biochem. 97,213-218 21 Morris, N. P., Consiglio, E., Kohn, L. D., Habig, W. H., Hardegree, M. C. and Helting, T. B. (1980) J. Biol. Chem. 255, 6071-6076 22 Goldberg, R. L., Costa, T., Habig, W. H., Kohn, L. D. and Hardegree, M. C. (1981) Mol. Pharmacol. 20, 565-570 23 Stoeckel, K., Schwab, M. and Thoenen (1977) Brain Res. 132,273-285 24 Avrova, N. F. (1981) J. Neurochem. 18, 667~574 25 Sonnino, S., Ghidoni, R., Chigorno, V.,

26

27

28

29 30

31

Masserini, M. and Tettamanti, G. (1983) Anal Biochem. 128,104-114 Mirsky, R., Wendon, L. M. B., Black, P., Stolkin, C. and Bray, D. (1978) Brain Res. 148, 251-259 Adam, G. and Delbruck, M. (1968) in Structural Chemistry and Molecular Biology (Rich~ A. and Davidson, N, eds), pp. 198-215, Freeman Berg, O. G. and yon Hippel, P. H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 131-160 McCIoskey, M. A. and Poo, M-m. (1986) J. Cell Biol. 102, 88-96 Kohn, L. D. (1978) in Receptors and Recognition, Series A (Cuatrecasas, P. and Greaves, M. F. eds), pp. 133-212, Chapman & Hall Simpson, L. L. (1984) J. Pharmacol. Exp. Ther. 229,182-187

Talking Point Informational DNA: a useful concept? J.M. Barry Living organisms are built up according to structions' for cleavage are not here inherited instructions encoded in their D NA. encoded in embryonic DNA. The funcThis sentence is from a recent book on tion of embryonic D N A often begins the molecular biology of animal develop- with R N A transcription in the multinucment, and similar sentences in which lear embryo; and an early step in the forD N A is assigned the special function of mation of tissues of differing composiencoding 'instructions' or 'information' tion appears to be the synthesis of differcan be found in almost every modern ent R N A molecules by nuclei in different biology textbook. I shall consider here parts of the embryo. But the 'instrucwhether such statements contribute to tions' for this differential R N A synthesis our understanding of living organisms. come not from D N A but from unknown In the sentence quoted, the words 'in- molecules that differ in kind or concenstructions encoded in' are presumably tration in different regions of the embryo. It is true that the structure of intended to increase our understanding of the development of an adult organism these molecules may have been deterfrom the fertilized egg by arousing an mined in the previous generation by the image of a machine whose particular structure of DNA. But it is not primarily function is determined by inserting 'in- their structure but their arrangement structions' encoded in a linear series of within the embryo which results in cormarks on a tape or disc. But this analogy rectly regulated development. How this is imprecise: it overemphasizes the pre- arrangement is determined is unknown, eminence of D N A within the cell and but it can only remotely depend on D N A wrongly implies an unspecific function structure. D N A , we are repeatedly reminded, for other cell components. Let us consider whether D N A contains 'information' in the technical molecules do in fact show this pre-emi- sense used by information theorists. nence in a developing embryo. Develop- Since each subunit of D N A can exist in ment of a fertilized egg into an adult \ o n e of four forms, the structure of the begins with cleavage; but cleavage in molecule is analogous to the structure of some organisms can occur during sup- a word, or to the sequence of dots and pression of R N A synthesis and even in dashes in a morse code message. In fact, the absence of the nucleus 1. Hence 'in- messages could be sent in bottles containing D N A molecules which were synthesized by the sender and sequenced by J.M. Barry is at the Department of Plant Sciences, the recipient and deciphered according. University of Oxford, Agricultural Science Building, to an agreed code. Moreover, the transParks Road, Oxford OX1 3PF, UK.

cription of D N A into R N A and the translation of R N A into protein is analogous to the translation of a message from one code to another. So it is true that some aspects of cell metabolism have amusing similarities to the transmission of coded messages, and a few terms such as 'genetic code' and 'translation' possibly aid our understanding of the chemical reactions involved. But claims, sometimes made, that application of terms from information theory to the function of D N A represents a breakthrough in our understanding of living cells are grossly exaggerated. Moreover, the implication that D N A is an almost unique repository of 'information' within the cell appears to arise either from ignorance of cell function or from a misunderstanding of information theory according to which 'information' can be contained not only in words and other linear combinations of alternative symbols, but in any non-random structure. Hence, in the living cell 'information' can be, and is, also contained in other cell molecules and structures derived from them. D N A molecules are unique within the cell in that they determine the structure of all newly synthesized nucleic acid and protein molecules and, as a consequence, ultimately control the synthesis or entry into the cell of all other molecules. D N A can in part function in a foreign environment. For example, cells that will grow and divide have been formed by fusing non-dividing rat myoblast 'minicells', that have cell membranes and nuclei but little rat cytoplasm, with enucleated mouse fibroblasts 2. But the D N A molecules of a particular species appear able to function fully only in cells from the same or a

~) 1986,ElsevierSciencePublishersB.V.. Amsterdam 0376-5067/86/$02.00