# Shiga Toxin--A Model for Glycolipid-Dependent and Lectin-Driven Endocytosis
## 3 Formation of Membrane Invaginations and Tubular Endocytic Pits
This binding site geometry is preserved for the receptor-binding parts of cholera toxin and simian virus 40 (SV40) (Figure 3c), for which it was shown previously that they also have curvature-active properties, endowing them with the capacity to drive tubular membrane invaginations through interaction with their GSL receptor molecules [25], as observed for Shiga toxin [9]. Strikingly, these GSL-binding pathogenic lectins do not have any sequence similarity, which suggests that this binding site geometry might be the result of convergent evolution towards a common function: membrane mechanical work in relation to inward-oriented curvature generation for the construction of endocytic pits. Of note, cholera toxin and SV40 have indeed both been described to be efficiently internalized into cells in which the clathrin pathway is inhibited [18; 26]. Further pathogens and pathogenic factors exist that also interact with GSLs in one way or another to get into cells (reviewed for gangliosides in Reference [27]), suggesting that this mechanism is used more widely. An example of a cellular lectin that drives the GSL-dependent biogenesis of tubular endocytic pits for the uptake of endogenous cargo proteins via clathrin-independent carriers has indeed been described: galectin-3 (Gal3). Gal3 is a member of a family of 15 galectins, 12 of which are expressed in human [28]. This galactose-binding lectin has many physiological (cell migration, immune modulation, inflammation, signaling, etc.) and pathological (cancer, fibrosis, diabetes, etc.) functions, raising the question of how a single protein can manage all of this. The cell biology of Gal3 still remains very little explored. Interestingly, it has recently been shown that membrane-associated Gal3 drives the GSL-dependent formation of narrow tubular invaginations on model membranes [29], similar to Shiga toxin [9], cholera toxin, and SV40 [25]. On cells, Gal3 drives the GSL-dependent biogenesis of clathrin-independent carriers (CLICs), via which Gal3-interacting proteins such as the cell adhesion and migration factors CD44 and \(\beta\)1 integrin are internalized [29]. Based on the results published in Reference [29], the GlycoLipid-Lectin (GL-Lect) hypothesis was put forward [22]. According to this hypothesis, monomeric Gal3 in solution binds to glycosylated cargo proteins such as CD44 and \(\beta\)1 integrin on target cells (Figure 3d). This leads to the oligomerization of Gal3 and its capacity to interact with GSLs in a way such as to induce membrane bending and thereby, the biogenesis of endocytic pits, from which CLICs are then generated. According to this model, Gal3 functions like an endocytic adaptor that links glycosylated cargo proteins and membrane curvature-generating GSLs into the same compositional nanoenvironments from which tubular endocytic pits emerge. In contrast, the pathogenic lectins from Shiga toxin, cholera toxin, and SV40 are themselves cargoes, which are produced as stable pentamers (Figure 3c) such that they can immediately aim for the membrane mechanical part of the program, bypassing the cargo recognition step. Several aspects of the GL-Lect model are worth further discussion. One concerns the question of why monomeric Gal3 fails to bind GSLs directly. Most likely, the affinity of the sugar binding pocket of Gal3 for carbohydrates on GSLs is weak, such that efficient binding becomes possible only upon the formation of multiple-bond interactions (avidity) of each Gal3 oligomer with several GSLs. Another key question concerns the membrane-bound structure of Gal3. The C-terminal carbohydrate recognition domain of the protein has been crystallized in solution, while the N-terminal domain turned out to be unstructured [30]. It is still unknown when and how the N-terminal domain folds, and what type of oligomeric configuration Gal3 adopts on membranes. It has been proposed that the protein may form pentamers [31], but also, that even larger oligomers may arise [32]. One may finally point out that GSLs are basic fabric of raft-type membrane domains [33].