Lessons Learned from Plant and Bacterial Toxins

K Sandvig1 and B van Deurs2 1Institute for Cancer Research, The Norwegian Radium Hospital, University of Oslo, Montebello, Oslo, Norway; and 2Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, University of Copenhagen, Denmark

A number of protein toxins of bacterial and plant origin have Some toxins enter from endosomes in response to low cytosolic targets, and knowledge about these toxins have endosomal pH, whereas others, including Shiga toxin and provided us with essential information about mechanisms that ricin, are transported all the way to the Golgi apparatus and can be used to gain access to the cytosol as well as detailed the ER before they are translocated to the cytosol. Plant knowledge about endocytosis and intracellular sorting. proteins such as gelonin that are without a binding moiety are Such toxins include those that have two moieties, one (the taken up only by fluid-phase endocytosis, and normally they B-moiety) that binds to cell surface receptors and another have a low toxicity. However, they can be used to test for (the A-moiety) with enzymatic activity that enters the cytosol, disruption of endosomal membranes leading to cytosolic as well as molecules that only have the enzymatically active access of internalized molecules. Similarly to toxins with moiety and therefore are inefficient in cell entry. The toxins a binding moiety they are highly toxic when reaching the discussed in the present article include bacterial toxins such cytosol, thereby providing the investigator with an efficient as Shiga toxin and diphtheria toxin, as well as plant toxins tool to study endosomal disruption and induced transport to such as ricin and ribosome-inactivating proteins without a the cytosol. In conclusion, the protein toxins are useful tools binding moiety, such as gelonin. Toxins with a binding moiety to study transport and cytosolic translocation, and they can can be used as vectors to translocate epitopes, intact be used as vectors for transport to the interior of the cell. proteins, and even nucleotides into the cytosol. The toxins Gene Therapy (2005) 12, 865–872. doi:10.1038/ fall into two main groups when it comes to cytosolic entry. sj.gt.3302525; Published online 7 April 2005

Keywords: toxin; Shiga toxin; diphtheria toxin; ricin; endocytosis

Introduction and as indicated in Figure 2, the toxins use two main pathways to gain access to the cytosol. They can be Bacteria produce a large variety of protein toxins with transferred from endosomes (early or late) in response intracellular targets, and also plants contain similar toxic to low pH, or they can enter the Golgi and then the ER proteins (for recent reviews about toxins containing both and be transferred to the cytosol from this destination. a binding moiety and an enzymatically active moiety, Toxins normally entering from acidic endosomes can as well as toxic proteins without a binding moiety, see even in some cases, as ﬁrst shown for diphtheria toxin,7,8 Sandvig and van Deurs,1 Hartley and Lord,2 Sandvig be induced to enter directly from the cell surface in et al,3,4 Turton et al5). The schematic structure and the response to external low pH. Not only do the toxins activity of some of these toxins are shown in Figure 1. reveal the different intracellular mechanisms that can be All of the plant toxins studied so far and several of the used to enter the cytosol, it turns out that they can in bacterial toxins (the Shiga toxins) block protein synthesis themselves be used as vectors to bring in epitopes to be by removing one adenine from the 28S ribosomal RNA presented by MHC class I,9 they can bring in intact of the 60S subunit, thereby blocking protein synthesis. proteins,10,11 and in some cases it turns out that they Diphtheria toxin and Pseudomonas exotoxin A inhibit might even be used to transfer nucleotides into the protein synthesis by inactivation of elongation factor 2. cytosol and the nucleus of cells.12–19 These aspects of the Bacterial toxins have a number of different target toxins are described in the paragraphs below. molecules, ranging from kinases to actin and Rho proteins.1,6 Common to all these toxins, regardless of the target molecule, is that they have to enter the cytosol to exert their effects. Therefore, studies of these toxins Endocytic mechanisms: ports of entry used have provided us with knowledge of how an external by protein toxins molecule might reach the cytosol. As described below, The variety of endocytic mechanisms in a cell has partly been revealed by studies of protein toxins that seem to Correspondence: Professor K Sandvig, Institute for Cancer Research, 1,20 The Norwegian Radium Hospital, University of Oslo, Montebello, exploit all of them in order to attack a given cell. The 0310 Oslo, Norway plant toxin ricin, which binds all over the cell surface Published online 7 April 2005 since it binds to both glycoproteins and glycolipids with Lessons learned from plant and bacterial toxins K Sandvig and B van Deurs 866

Figure 1 Schematic overview of some bacterial and plant toxins. The enzymatically active subunit is denoted A. However, as indicated, in some cases this

subunit is cleaved into A1 and A2, where A1 is the one with enzymatic activity. The B moiety is the part of the toxin responsible for binding to cell surface receptors. In the ﬁgure, also the enzymatic activity and the intracellular targets are listed. The arrows indicate sites where cellular enzymes cleave and activate the toxins. For the properties and structure of more toxins, please see recent reviews, such as Sandvig and van Deurs.1

terminal galactose, provided some of the early evidence clathrin-dependent and -independent processes operate for clathrin-independent endocytosis.21,22 In fact, cla- in the same cell, and as described below in the paragraph thrin-dependent endocytosis was during many years about transcytosis, the clathrin-independent mechan- believed to account for all uptake from the cell surface. ism(s) can be subjected to regulation by a number of It is now known that several mechanisms exist, both signalling pathways. Furthermore, when it comes to

Gene Therapy Lessons learned from plant and bacterial toxins K Sandvig and B van Deurs 867 can induce pinching off of some caveolae, or whether some cholera toxin becomes internalized by a small fraction of caveolae which for unknown reasons are not stabilized at the plasma membrane. The internalization of caveolae may at least in some cases be dependent on tyrosine phosphorylation and actin reorganization33 or regulated by addition of cholesterol and glycolipids.34 Importantly, it has recently been published that cholera toxin taken up from caveolar structures ends up in normal acidified endosomes,35 simlarly as we have found for diphtheria toxin bound to a receptor with a GPI anchor (see below).36 That endocytic compartments are able to exchange contents makes it less critical in the present context whether a ligand is taken up from caveolae or clathrin-coated pits. Diphtheria toxin is ideal to investigate whether the receptor molecule it binds to is internalized into an acidic compartment. For instance, when diphtheria toxin was bound to its receptor modified to contain a GPI-anchor instead of the normal transmembrane/cytosolic domain, the toxin was, in Figure 2 Transport of protein toxins in cells. Toxins can be endocytosed by contrast to when bound to its normal receptor, inter- 36 clathrin-dependent (1) or clathrin-independent endocytosis (2), they can be nalized by clathrin-independent endocytosis. This GPI- internalized by macropinocytosis (3), recycled (4), transported to the Golgi anchored receptor was internalized into an acidified apparatus (5) and the ER (6) from where some of them are translocated to organelle, presumably an endosome, since the toxin was the cytosol (open arrow). Alternatively, after endocytosis, some of the translocated to the cytosol and intoxicated the cell. Thus, toxins are transported to the cytosol from acidified endosomes (early/late; although there are – as with other approaches – pitfalls open arrows). A variable, cell-type-dependent fraction of the toxin is transported from early to late endosomes (7) and toxins may then continue when it comes to studies of endocytosis using protein to lysosomes (8) for degradation. EE, early endosomes; SE sorting toxins, they have proven very important for our under- endosomes; LE, late endosomes; LY, lysosomes. standing of the endocytic mechanims. clathrin-independent processes, different molecules can Direct transport from endosomes be involved (perhaps in a cell-type specific manner?). For to the cytosol instance, it has been described that uptake of GPI- anchored proteins can be dependent on cdc42 in CHO To which extent can transport of proteins and nucleo- cells,23 whereas this does not seem to be the case in HeLa tides to the cytosol occur from endosomes? It is now cells.24 Furthermore, the clathrin-independent endo- 25 years since it was discovered that diphtheria toxin cytosis can be dependent on lipid rafts (domains rich enters from endosomes in response to the relatively low in cholesterol and sphingolipids),25 or it can be raft- pH found in this organelle, and that direct entry from independent.26,27 Clathrin-independent endocytosis can the cell surface can be induced by exposing cells with also operate with25 and without dynamin26,27 as an surface-bound toxin to low pH, thereby mimicking the essential molecule. Thus, clathrin-independent uptake conditions in the endosomes (see Table 1).7,8 of the IL2 receptor is dependent on lipid rafts,25 whereas Thus, it became clear that low pH is sufficient to drive ricin and cholera toxin can be internalized under the translocation of the receptor-bound toxin across the conditions where clathrin-dependent uptake is inhibited membrane. The low endosomal pH triggers a conforma- and cholesterol has been depleted from the rest of the tional change in the toxin molecule,37 leading to exposure membrane.26,28 In the case of anthrax toxin, the uptake is of hydrophobic areas that are inserted into the mem- dependent on lipid rafts for the initiation of processing brane, and then the enzymatically active part of the toxin (protective antigen (PA) heptamerization, cleavage of PA is translocated. During membrane insertion and translo- and subsequent binding of lethal factor (LF)), and then cation of the toxin there is a concomitant formation of the toxin (the complex of PA and LF) is taken in by a cation-selective pores,38 but the role of the pores is not clathrin-dependent process.29 known. It is still not clarified whether the A-fragment One danger in using protein toxins to study a given actually passes through the pore. It is important to note process is that the toxin might induce signalling that that although a number of studies have been performed could affect the process. For instance, both Shiga toxin with diphtheria toxin and artificial lipid membranes,39 and cholera toxin, which bind to the glycosphingolipids these systems do not necessarily reflect what goes on Gb3 and GM1, respectively, are known to affect signal- during toxin transport in an intact cell. For instance, in ling pathways.3,30,31 In this context, it should be noted cells the receptor seems to be essential for transloca- that both these toxins lead to crosslinking of their tion,36,38 and changes of the cytosolic tail of the receptor receptors since they are multivalent. A subtype of lipid have been shown to protect against the toxin.40 rafts are caveolae, 50–70 nm invaginated portions of the During the last two decades, a number of other protein plasma membrane associated with the protein caveolin. toxins have been found to use a similar mechanism (for Caveolae are normally quite stable,32 but they can be recent reviews, see Sandvig and van Deurs,1 Turton involved in uptake of cholera toxin. However, so far it is et al4), and in several cases the same test system as not known to which extent cholera toxin in some cells applied to diphtheria toxin has been used: Can the

Gene Therapy Lessons learned from plant and bacterial toxins K Sandvig and B van Deurs 868 Table 1 Basic mechanisms – important aspects of protein delivery into cells discovered by using protein toxins

1979 Some of the ﬁrst studies demonstrating recycling of endocytosed material was made by using ricin.54 1980 Direct membrane penetration of a protein in response to low pH: studies with diphtheria toxin.7,8 1982 Endocytosis is required for transport to the cytosol also of toxins not requiring low pH.48 1985–1987 Evidence for clathrin-independent endocytosis.21,22 1986 The valency of an internalized toxin is essential for its intracellular destiny.51 1992 First demonstration of retrograde transport all the way from the cell surface to the Golgi apparatus, the ER and the nuclear envelope.57 These studies were performed by using Shiga toxin. 1999 A GPI-anchored diphtheria toxin receptor is endocytosed by clathrin- and caveolin-independent endocytosis into an acidic compartment.36

surface-bound toxin enter the cytosol directly across the it does not mediate release of gelonin and intoxication plasma membrane when exposed to low pH? The group of cells that are resistent to diphtheria toxin due to a of toxins that enters in response to low endosomal pH mutation of elongation factor 2 (our own unpublished includes in addition to diphtheria toxin, anthrax toxin, data). If the endosomes had been disrupted, released Clostridial neurotoxins, cytotoxic necrotizing factor 1 gelonin would have attacked the ribosomes. Also, the (CNF1), Clostridium botulinum C2 toxin, Clostridium lysosomotropic compound chloroquine turns out to difficile toxin B, and Pasteurella multodica toxin (PMT). increase transfer of DNA to the cytosol.45 The mechanism Although the toxins do not disrupt the endosomal behind this is not clear. The increase could be due to an membrane leading to a general release of vesicle content inhibition of transfer to the lysosome since endosome to (see below), they can still be used to transport other lysosome transport is inhibited by increased pH,47 or it molecules into the cell. For instance, ricin A-chain can be could be due to swelling of endosomes and destabiliza- brought into the cytosol when bound to diphtheria toxin tion of the membrane, thereby inducing some leakiness. B-chain via a disulfide bond,41 and similarly as found Interestingly, the ionophore monensin that can also with diphtheria toxin, the cells are protected against this increase endosomal pH is able to sensitize cells to the molecule by compounds that abolish low pH in endo- protein toxin ricin.48 Again, the mechanism behind this somes. Furthermore, acidic fibroblast growth factor can, sensitization is not known. It could be due to increased bound to diphtheria toxin, be brought into the cytosol,10 retrograde transport to the Golgi and the ER, and and also C. botulinum exoenzyme C3 can enter the cytosol importantly, there is some sentization also at monensin of cells by coupling it (on a genetic level) to diphtheria concentrations that are so low that they are unlikely to toxin B.11 However, the mechanism of entry is not affect endosomal pH. Whether these conditions can identical to that of diphtheria toxin since it cannot be affect DNA transfer have, to our knowledge, not been pulsed in from the cell surface. Also, cell-specific tested. differences can give rise to surprises when it comes to the abililty of a given compound to enter the cytosol. Cell differences may for instance explain some of the Transport to the lysosomes apparent disagreements on the ability or inability of cell permeable peptides with or without additional cargo A certain fraction of internalized toxins are transported to enter cells.42 Both toxins and peptides may function to the lysosomes where they are then degraded. as vectors to bring in other molecules although the However, the protein toxin ricin is very resistant to mechanisms involved may differ from what one origin- proteolytic degradation, making the protein a useful tool ally thought. also to study the transport of a membrane-marker from Toxins can also be used to test for disruption of the cell surface to lysosomes. By using ricin, we earlier found evidence for the involvement of actin in this endosomes caused by other agents. The toxin gelonin, 49 which belongs to a group of toxins with an A-chain only process. Actually disruption of actin inhibited ricin (also called RIPs for ribosome-inactivating proteins),2,43 degradation more than was the case after disruption of microtubules. Furthermore, ricin was used to quantify can inactivate ribosomes after entry into the cytosol. sorting to the various compartments such as the Golgi Without a binding moiety this toxin is taken up only apparatus versus lysosomes,50 and it was discovered that by fluid-phase endocytosis and is free to diffuse into the the valency of this ligand was important for its destiny.51 cytosol after disruption of the endosomal membrane. For Multivalent complexes of ricin and HRP as well as ricin instance, gelonin is released to the cytosol after treatment coupled to gold particles were sent directly to lysosomes, of cells with phorphyrins and subsequent light treatment whereas monovalent ricin had the ability to be trans- that leads to disruption of endosomes and release of ported also to the Golgi apparatus. Similarly, evidence content.44 A number of different agents have been used for the importance of valency for sorting has been to increase release of DNA into the cytosol.45 Such agents obtained from other systems.52,53 Thus, how one pro- include virus, cationic polymers, and lysosomotropic duces a compound (the valency of the resulting agents such as chloroquine.45,46 In the case of adenovirus conjugate) can define destiny of the resulting product. and peptides derived from Heamophilus influenza hemagglutinin from influenza virus, low pH-induced effects on the virus or the virus peptides are involved in Recycling of internalized material the disruption of endosomes and increased DNA transfer to the cytosol. This is in contrast to diphtheria A fraction of endocytosed molecules are, instead of being toxin that does not seem to disrupt endosomes, since delivered to the lysosomes, transported to the Golgi

Gene Therapy Lessons learned from plant and bacterial toxins K Sandvig and B van Deurs 869 apparatus, transported across the cell layer (transcytosis enter the same endosome and from there be sorted to the in polarized epithelial or endothelial cells), or recycled to Golgi apparatus.35,62 the cell surface.1 Actually, some of the earliest evidence Why is it necessary for a toxin to move all the way for the existence of recycling was obtained by studying from the cell surface to the ER for translocation to the the protein toxin ricin (Table 1).54 Since then, our cytosol to occur? There are several points to consider: knowledge about recycling of endocytosed molecules to some of the toxins are activated by cellular proteases that the cell surface is dramatically increased,55 and the they encounter on the way. For instance, Shiga toxin can molecules involved in the process are partly identified. be cleaved and activated by the enzyme furin,1 and in Also, recycling seems to involve more than one process. cells without this enzyme, alternative processing occur- It may occur directly from early sorting endosomes, it ring later along the pathway can activate the toxin can be mediated by the perinuclear-recycling compart- thereby making it toxic to more cell types than would ment, and there seems to be export to the surroundings otherwise have been the case. Also, the ER membrane is, even from later endocytic compartments. Recycling can due to its low cholesterol content, probably more be inhibited by certain drugs that increase endosomal permeable than the plasma membrane or the endosomal pH, for example, by monensin and chloroquine, that is membrane, a feature that might be important for transfer by conditions that might facilitate transport of some gene of molecules with carbohydrate attached to the protein conjugates to the cytosol.45 In polarized cells, recycling backbone. One could speculate that even transport of from the two poles are different (see below). Also in nucleotides into the cytosol might benefit from this polarized cells, the recycling pathways have been property. Interesting in this context is the finding that investigated not only by studies of fluid transport but cholesterol inhibits an early step in protein translocation also by quantifying the trafficking of membrane-bound from the cytosol to the ER.63 Most importantly there are, markers such as the plant toxin ricin.1 in the ER, chaperones and translocators responsible for ER to cytosol transport.64–67 There is evidence that protein disulfide isomerase (PDI) is involved in release of the enzymatically active part of cholera toxin,64 Retrograde toxin transport from endosomes Pseudomonas exotoxin A68 and ricin,69,70 and interaction to the Golgi, the ER, and the cytosol of Sec 61 with several of the protein toxins have been published.5,64,71 Recently, also other translocators, derlin In contrast to the group of toxins entering the cytosol 1 and 2, have been described,72,73 and it remains to be from endosomes, there is a second group of the protein studied to which extent these molecules can mediate toxins that travels all the way from the cell surface to transfer of toxins or conjugates made from toxins to the endosomes, through the Golgi appparatus and to the ER cytosol. At least these molecules seem to be involved in before they are translocated to the cytosol, presumably translocation of proteins from the ER to the cytosol, a through the Sec61p channel which is important for feature that makes them interesting as candidates for cotranslational translocation of peptides into the ER and transport of toxin/conjugates into cells. for transport of misfolded proteins out of the ER.1,5,56,57 The efficiency of routing between endosomes and the Golgi apparatus, that is, the fraction of endocytosed toxin Transcytosis in polarized epithelial cells that enters the Golgi is toxin- and cell-type dependent, and can even vary depending on the growth conditions. Both receptor-bound ligands and compounds in solution However, often not more than 5–10% of the endocytosed can be transported across epithelial cell layers by toxin is transported in the direction of the Golgi transcytosis, that is, the process by which vesicles pinch apparatus.1 Recent studies from a number of laboratories off from either the apical or basolateral pole of the have revealed that both endosome to Golgi transport and polarized cells and mediate transfer to the opposite side, retrograde transport from the Golgi to the ER comprise presumably via the endocytic compartment. Also for several pathways. Toxins such as ricin, cholera toxin and studies of transcytosis, protein toxins have proven Shiga toxin do not seem to travel through late endo- useful, and again, a number of results have been somes, but instead, they are transported from early obtained with the plant toxin ricin that can serve as a endosomes to the Golgi.1,5 However, even the require- membrane marker and which can be expected to provide ments for this transport step is different for ricin and information also about uptake and transcytosis of Shiga toxin.56 For instance, for efficient transport of Shiga compounds in solution. It is important to note that toxin, there is a requirement for clathrin,58,59 whereas this trancytosis of membrane is not necessarily subjected is not the case for ricin.5 However, it can be difficult to to the same type of regulation as certain receptors. study intracellular pathways by blocking certain routes. For instance, transcytosis of pIgA receptors from the By expressing dominant negative mutants of the proteins basolateral to the apical side is inhibited by the drug involved, alternative transport mechanisms can be brefeldin A which inhibits GTP loading of ARF1, and induced in the cells. One may find transport routes which might thereby inhibit recruitment of coats to that are normally nonexistent. This is the case when membranes.74 However, when transcytosis of ricin is a temperature-sensitive mutant of e-COP is expressed, studied in the same cell, there is no reduction.75 There- leading to disruption of the Golgi apparatus. Ricin is fore, it is important to study membrane and fluid-phase then still able to intoxicate the cells, presumably due to transcytosis and not only transepithelial transport of induction of an endosome to ER transport that does not certain ligands. operate under normal conditions.60,61 Importantly, it has Uptake of ligands and fluid can occur both from been demonstrated that toxins and other receptor-bound clathrin-coated pits and by clathrin-independent endo- ligands taken up by different endocytic mechanisms can cytosis in polarized epithelial cells (for instance MDCK

Gene Therapy Lessons learned from plant and bacterial toxins K Sandvig and B van Deurs 870 are required to obtain a better understanding of this process and the extent to which transcellular transport of vectors can be improved.

Protein toxins used as vectors for import of DNA Not only can the toxins here described be used to bring other proteins10,11 or epitopes for presentation by MHC class I9 into cells, but protein toxins or toxin domains can also be used to facilitate entry of DNA and obtain transcription/translocation of the gene that enters. For this purpose, both toxins that enter from endosomes and those that are normally targeted to the ER can be used, and in some instances only toxin domains are included in the conjugates. Details concerning construction of toxin-nucleotide vectors have recently been described by Figure 3 An overview of factors involved in toxin endocytosis and 85 12,13 transcytosis in polarized MDCK cells. For a detailed description, please see Uherek and Wels. Thus, diphtheria toxin and 16 the text. anthrax toxin, which normally enters from endosomes, as well as Pseudomonas exotoxin A,14,19 Shiga toxin,15 and cholera toxin,16–18 which all normally enter from the ER, cells). Importantly, and for reasons not yet understood, have been found to mediate gene transfer. Interestingly, caveolae are present only at the basolateral side of these some of the characteristics of toxin entry have been cells.76 Even when Caco-2 cells, which originally do not conserved. Entry mediated by both diphtheria toxin contain caveolin 1 and caveolae, are transfected with and Pseudomonas exotoxin A can at least in some caveolin, caveolae are formed only at the basolateral side. instances be inhibited by agents that counteract acid- Thus, the uptake by clathrin-independent endocytosis ification of internal organelles. It is however not obvious at the apical side occurs by a process that is also that intracellular routing of a modified toxin leads to independent of caveolae. Interestingly, this process turns release of attached material from the organelle that is out to be highly regulated, and also transcytosis is as a normally used by the toxin for entry into the cytosol. The consequence changed. As indicated in Figure 3, endo- possibility exists that the material might enter the cytosol cytosis at the apical side is upregulated by activation to some extent from another organelle, for instance from of heterotrimeric G proteins77 and activation of protein endosomes. However, as long as only a few molecules kinase C,78 by protein kinase A,77 and by addition of are required in the cytosol or the nucleus to obtain a brefeldin A.75 Also, it is regulated by the cyclooxygenase biological effect, such a transport or leakage into the pathway79 and by calmodulin.80 Upon addition of N- cytosol could contribute to get the desired effect. ethylmaleimide (NEM) to polarized MDCK cells, ruffling and formation of macropinosomes are induced, and this process seems to be dependent on phospholipase D.81 Conclusions Interestingly, overexpression of the Rab5-effector Raban- kyrin-5 was recently shown to stimulate apical and not Protein toxins have proven useful to study intracellular basolateral endocytosis.82 It is, however, possible that pathways in general, and such knowledge is essential different types of apical clathrin-independent processes for drug design. Investigation of the toxins and their might be responsible for the responses observed. Thus, interactions with cells are required not only to develop the growth conditions of a cell layer can clearly influence strategies to prevent their toxic effect and to cure disease uptake and transcytosis of a ligand taken up by clathrin- caused by infectious bacteria that secrete toxins, but the independent endocytosis and the transfer of solutes protein toxins can also be exploited to cure other diseases across epithelial cells. Whether this can be exploited to by using them as vectors to bring both proteins and influence transport in a physiological situation is not nucleotides into cells. clear. We still need to clarify which proteins are involved in the actual endocytic mechanism found in such cells. So far, clathrin-independent apical endocytosis of ricin and Acknowledgements the fluid-phase marker horseradish peroxidase (HRP) is Work performed by the authors of the present article is the only endocytic process that seems to occur without being supported by the Norwegian and Danish Cancer GTP-hydrolysis.83 Interestingly, by using permeabilized, Societies, the Norwegian Research Council for Science polarized MDCK cells, it was found that this process is and the Humanities, The Danish Medical Research dependent on the small GTP-binding protein RhoA. Council, The Novo Nordisk Foundation, the Jahre Clearly, different ligands are transcytosed and might Foundation, and Jeanette and Søren Bothners Legacy. be used to bring nucleotides across cell layers. However, the efficiencies might differ in ways we still cannot predict. For instance, transcytosis of Shiga-like toxin I References and II, which both bind to the neutral glycosphingolipid Gb3, are subjected to different regulation.84 Thus, future 1 Sandvig K, van Deurs B. Membrane traffic exploited by protein studies of transcytosis both of protein toxins and fluid toxins. Ann Rev Cell Dev Biol 2002; 18: 1–14.

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