Cross-linking of glycosphingolipids at the plasma membrane: consequences for intracellular signaling and traffic

Abstract

Glycosphingolipids (GSLs) are predominantly found in the outer leaflet of the plasma membrane, where they play a role in important processes such as cell adhesion, migration and signaling. However, by which mechanisms GSLs regulate these processes remains elusive. In this study, we therefore took advantage of the fact that some GSLs also serve as receptors for certain protein toxins, which rely on receptor binding for internalization and intoxication. Here, we demonstrate that Shiga and cholera toxins, which both possess multivalent GSL-binding capacity, induce dissociation of the cytosolic cPLA2α-AnxA1 complex in HeLa and HMEC-1 cells. The dissociation is mediated through an increase in cytosolic calcium levels and activation of the tyrosine kinase Syk. Ricin, a protein toxin that does not cross-link surface molecules, has no effect on the same complex. Importantly, we find that antibody-mediated cross-linking of Gb3 and GM1, the GSL receptors for Shiga and cholera toxin, respectively, also induces dissociation. These data demonstrate that cross-linking of GSLs at the plasma membrane mediates the intracellular signaling events resulting in dissociation of the complex. After dissociation, cPLA2α and AnxA1 are translocated to intracellular membranes where they are known to function in regulating membrane transport processes. In conclusion, we have characterized a novel mechanism for cell surface-induced initiation of intracellular signaling and transport events.

Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin’s Enzymatic Subunit Interfere with Intracellular Toxin Transport

“…Our results are consistent with R70, PB10, SyH7, GD12, and IB2 having similar effects on ricin. As noted above, we are particularly interested in the possibility that neutralizing mAbs influence the valency of ricin at the cell membrane and, thus, alter receptor crosslinking and endocytosis 26 . Indeed, we have demonstrated that recombinant, bispecific camelid antibodies are particularly potent at neutralizing ricin in vitro and in vivo 27 .…”

The role of lipid species in membranes and cancer-related changes

“…The mechanisms responsible for this are not known but could be related to signaling induced upon crosslinking of glycosphingolipids. Both addition of Shiga toxin to cells as well as addition of antibodies against Gb3 can induce a flux of Ca 2+ , cytosolic tyrosine kinase activation, changes in life-time of clathrin-coated pits and activation of the phospholipase A2, with subsequent downstream effects in cells [137][138][139][140]. Similarly, cholera toxin has been shown to induce signaling [141], but since this toxin also can interact with proteins it is a more complex picture.…”

Exogenous lysophospholipids with large head groups perturb clathrin‐mediated endocytosis

“…Growth factor‐mediated signaling has been shown to induce ruffling and macropinocytosis, whereas galectins and other ligands that can cross‐link proteins and glycosphingolipids, such as Shiga toxin and cholera toxin, might induce bending of the membrane with the subsequent formation of an endocytic vesicle . Importantly, cross‐linking of glycolipids such as Gb3 and GM1 can induce signaling and affect the number and lifetime of clathrin‐coated pits (CCPs) …”