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Shiga toxin can be internalized by clathrin-dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin–receptor complex to clathrin-coated pits, but whether this process is concentration-dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp-2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A-subunit of cell surface-bound toxin. Surface-bound B-subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin-dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa-labeled Shiga toxin and Cy5-labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A-subunit of cell surface-bound toxin stimulates clathrin-dependent uptake of the toxin. Possible explanations for this phenomenon are discussed.
The Shiga toxins consist of one enzymatically active A-subunit noncovalently linked to a stable pentamer of binding subunits (StxB), which in most cases bind specifically to the glycosphingolipid Gb3 in the plasma membrane . To exert its toxic effect, the holotoxin must be endocytosed and retrogradely transported via the Golgi apparatus to the endoplasmic reticulum, where the A-subunit is translocated to the cytosol and inhibits protein synthesis [2,3]. The Shiga family of toxins is divided into two main groups, based on antigenic differences. Shiga toxin (Stx) produced by Shigella dysenteriae and Shiga toxin 1 (Stx1) secreted by certain strains of Eshcerichia coli (Shiga toxin producing E. coli; STEC) are virtually identical and differ only in one amino acid. The toxins in the Shiga toxin 2 family (Stx2, Stx2c, Stx2d and Stx2e) are also secreted by STEC and have a similar structure to the toxins in the first group, but differ both functionally and immunologically [1,4,5]. To understand the differences between the effects of the various Stx, it is important to clarify the uptake mechanisms for these toxins.
It has been shown that Stx is internalized via clathrin-dependent endocytosis in several cell types, although there is evidence that also clathrin-independent mechanisms are partly involved [2,6–8]. Importantly, several studies have been concerned with the uptake and intracellular transport of the Stx B-subunit [8–21] but so far, investigations of a possible role of the A-subunit for uptake and intracellular routing of the toxin have been few. By electron microscopy studies, intact Stx has been shown to preferentially localize to clathrin-coated pits in HeLa cells [22,23], and both acidification and potassium depletion of cytosol, which inhibit clathrin-dependent endocytosis, have been shown to protect the cells against Stx [22–24]. Furthermore, induced expression of antisense to clathrin heavy chain (CHC) in a baby hamster kidney (BHK) cell line [25–27], thereby blocking the clathrin-dependent endocytosis, also protects against Stx  and reduces the endocytic uptake of the toxin . By using this BHK cell line, we could quantify the initial Stx internalization, both when clathrin-dependent endocytosis was operating and when it was blocked by antisense expression. Toxin uptake was decreased by about 50% upon inhibition of clathrin-dependent endocytosis, but it should be noted that a block in clathrin-dependent endocytosis might lead to increased toxin uptake via other mechanisms.
Importantly, Stx seems to be able to induce its own entry from clathrin-coated pits. Electron microscopy studies revealed that bound Stx is evenly distributed on the cell surface at low temperature, while after shifting to 37 °C, the toxin is aggregated in clathrin-coated pits [22,23]. There is no obvious explanation as to how the Stx–Gb3 complex is recruited to clathrin-coated pits, given that the receptor is a glycosphingolipid and not a protein with specific sequences required for interaction with the sorting machinery. One possible explanation is that the Stx–Gb3 complex interacts with another protein that is internalized via clathrin-dependent endocytosis. In fact, it has been shown by crosslinking experiments that members of the Shiga family of toxins can interact with 27 and 40 kDa molecules at the cell surface of Vero and CaCo2 cells , suggesting that interaction with accessory proteins might facilitate clathrin-mediated uptake. However, other possible explanations do exist. The toxin could induce toxin-specific signaling leading to recruitment of clathrin. Furthermore, crosslinking of Gb3 and perhaps of lipid rafts might be important for this process. Interesting in this connection, is the finding that the epidermal growth factor receptor present in lipid rafts recruits clathrin to the membrane upon binding of epidermal growth factor .
We here demonstrate that the rate of Stx uptake is up-regulated by increasing toxin concentrations by a mechanism in which the A-subunit of the surface-bound toxin is required. Importantly, this increased endocytosis is clathrin-dependent, and it seems to be caused by an increased recruitment of the toxin–receptor complex to clathrin-coated pits.
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In the present article, we show that Stx is able to stimulate its own entry by a process dependent on the toxin concentration, the surface-binding of the complete AB5 toxin and clathrin-dependent endocytosis. This self-stimulatory effect of Stx uptake seems to be mediated by an increased recruitment of the Stx–Gb3 complex to clathrin-coated pits.
Stx can be internalized by clathrin-dependent endocytosis, despite the lipid nature of its receptor Gb3 [2,4,7,22,35]. To study this process we decided to investigate whether Stx uptake was dependent on the toxin concentration added to the cells, and indeed this seemed to be the case. When increasing concentrations of unlabeled Stx were added to different cell lines (HEp-2, BHK, Vero, HeLa and A431 cells), uptake of labeled Stx was increased approximately twofold in all the cell lines except A431 cells. This self-stimulated Stx uptake was found to be mediated by clathrin-dependent endocytosis, as suggested from experiments using BHK cells with inducible expression of antisense to CHC. Based on this result, one might speculate whether the difference in stimulation of Stx uptake between cell lines is due to differential involvement of clathrin-dependent endocytosis of Stx in the cells. Interestingly, there were large differences in the actual amount of Stx internalized in each cell line after 10 min at low toxin concentrations, ranging from 13% in A431 cells to 33% in BHK cells (internalized toxin as percent of total cell-associated toxin; data not shown). In general, clathrin-dependent endocytosis is much faster than clathrin-independent mechanisms studied so far, due to the ≈ 1 min half-life of clathrin-coated pits at the cell surface . Thus, a high rate of toxin uptake might reflect that a larger fraction of toxin endocytosis is clathrin-dependent, even at low toxin concentrations.
Upon stimulation of Stx endocytosis with increasing concentrations of toxin, clathrin-dependent and -independent endocytosis were not increased in general. There was no increase in the uptake of transferrin or ricin, rather the Stx uptake seemed to be increased specifically. This could be explained by an increased localization of Stx–Gb3 complexes in clathrin-coated pits, and was investigated by immunofluorescence using Alexa-labeled Stx (in the absence and presence of unlabeled toxin) and Cy5-labeled transferrin as a marker of clathrin-coated pits. Although Alexa-Stx gave a heterogeneous labeling of the cells, as reported previously by others , the results clearly showed an increased colocalization of Stx and transferrin in cells incubated with unlabeled Stx1 compared to control cells incubated with Alexa-Stx only. The increased colocalization of Stx and transferrin did not result from an increased amount of transferrin receptor on the plasma membrane, because neither the binding nor the endocytosis of transferrin was increased upon incubation with high concentrations of Stx1 (data not shown). Thus, it seems likely that Stx–Gb3 localization in transferrin-containing clathrin-coated pits is increased upon stimulation with high concentrations of Stx1. These new results can explain our previously published electron microscopy data regarding Stx redistribution to clathrin-coated pits upon shifting the temperature from 0 °C to 37 °C . In those experiments, Stx at a concentration as high as 133 nm was added to HeLa cells, and in agreement with the data in the present article, a high degree of Stx redistribution to clathrin-coated pits was observed by electron microscopy upon shifting the temperature.
It seems that only cell surface-bound toxin molecules are responsible for the self-stimulation of Stx uptake, as was shown by comparing the stimulation of toxin uptake in the presence or absence of free toxin molecules in the medium. This could be due to the much higher local toxin concentration at the cell surface compared to that in the medium. Calculations show that there is ≈ 10 000 times higher concentration of labeled Stx at the cell periphery than in the rest of the medium. (For the assumptions involved, see Experimental procedures.)
Importantly, binding of the complete AB5 Stx structure seems to be specifically required for the ability of high toxin concentrations to induce an increased rate of toxin endocytosis. Control experiments using CT showed that the observed stimulation of Stx endocytosis was not due to an unspecific aggregation of glycosphingolipids/lipid rafts in the plasma membrane, nor was a mere aggregation of Gb3 by Stx B-subunits sufficient to stimulate Stx internalization. Thus, the presence of the A-subunit of the Shiga holotoxin seems to be crucial for the self-stimulated uptake to occur. In order to explain this, one might envision that the A-subunits are directly involved in important interactions either to other A-subunits, which could possibly cluster the toxins, or to other plasma membrane proteins, which might facilitate toxin internalization. Alternatively, the A-subunit might influence the toxin internalization indirectly by affecting the exposure of its associated B-subunits. This could change the surface location of the toxin or facilitate interactions with other membrane proteins that might induce toxin internalization. Also, as mentioned above, both toxin subunits (A and/or B) might induce signaling that could mediate toxin internalization, and this toxin-induced signaling might differ, depending on whether the intact toxin or the B-subunit bind to Gb3. However, these signaling pathways are largely unknown.
Interestingly, increasing concentrations of Stx2, which also binds to Gb3 and has an A-subunit with 55% amino acid similarity to Stx1, gave only a slight stimulation of Stx uptake in HEp-2 cells, a further demonstration of the specificity of this process. Important in this connection is that although Stx1 and Stx2 are structurally very similar, the A-subunit of Stx2 has a different orientation with respect to the B-subunits than in Stx1 . This might influence the ability of Stx2 to stimulate Stx1 uptake. The finding that Stx2 behaves differently from Stx1 is in agreement with their different effects on cells and in disease (for review see [4,5]).
The data shown in this article clearly reveal that the Stx A-subunit bound to the cells via the B-pentamer is responsible for the stimulation of endocytosis. However, the requirement for the A-subunit could be mediated via an effect of the B-moiety. This illustrates the importance of detailed studies of the role of the different subunits/toxin domains for toxin uptake and intracellular transport. In order to understand the action of Stx and the Shiga-like toxins on the cellular level as well as in disease, each step along the retrograde pathway from the cell surface to the cytosol should be characterized.