Lipid Rafts Act as Specialized Domains for Tetanus Toxin Binding and Internalization into Neurons

Abstract

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons. INTRODUCTIONTetanus (TeNT) and botulinum neurotoxins (BoNTs) block neurotransmitter release and are responsible for tetanus and botulism, respectively. These toxins share a common structure comprised of a heavy (H, 100 kDa) and a light (L, 50 kDa) chain linked by a disulfide bond. The H chain mediates binding and internalization in neurons, whereas the L chain is a metallo-protease that selectively cleaves synaptic proteins (Niemann et al., 1994;Schiavo et al., 2000). TeNT and BoNTs bind to the neuromuscular junction, but their intracellular actions take place at different levels of the nervous system. TeNT undergoes retrograde transport to the cell body of spinal cord motor neurons (MNs), is transcytosed, and cleaves the synaptic vesicle protein vesicle-associated membrane protein (VAMP)/synaptobrevin in inhibitory synapses. Instead, BoNTs act at a peripheral level by blocking acetylcholine release at the motor nerve terminal. This differential sorting has been interpreted as a consequence of binding to different surface receptors (Habermann and Dreyer, 1986;Herreros et al., 1999).TeNT and BoNTs bind to polysialogangliosides of the G1b series (Halpern and Neale, 1995). However, the fact that their binding is sensitive to proteases (Lazarovici and Yavin, 1986;Pierce et al., 1986;Yavin and Nathan, 1986) suggests the existence of specific protein receptors. Thus, a model in which TeNT and BoNTs interact with a complex constituted by both lipid and protein receptors has been proposed (Montecucco, 1986). Despite several efforts, these protein receptors have not been conclusively identified. Several BoNT serotypes interact with synaptotagmins (Nishiki et al., 1994;Li and Singh, 1998), but the role of these proteins as physiological BoNT receptors remains controversial (Evans et al., 1986;Bakry et al., 1997). We have previously followe...

Association of Helicobacter pylori Vacuolating Toxin (VacA) with Lipid Rafts

“…7C). These results are consistent with the existence of heterogeneity among DRMs (58), such that certain types of DRMs may be differentially affected by cholesterol depletion.…”

“…Two toxins with intracellular enzymatic activity, cholera toxin and tetanus toxin, bind to GM1 and a specific GPI-anchored protein, respectively, which are enriched in lipid raft microdo- mains (43-45, 58, 67). It has been proposed that lipid rafts play an important role in the internalization and intracellular trafficking of these toxins (44,58,67). The conclusion that these bacterial protein toxins require lipid rafts for intoxication of cells has been based mainly on experiments that utilize cyclodextrin or other cholesterol-interacting drugs to alter lipid rafts.…”

“…This interpretation is consistent with the concept that raft-dependent mechanisms are important for intracellular sorting events (68 -71). We speculate that there is heterogeneity among DRMs (58) and that functional properties of certain types of DRMs relevant for intracellular trafficking processes may be especially susceptible to disruption by drugs such as methyl-␤-cyclodextrin and nystatin. In future studies, it will be important to investigate further the role of lipid rafts and cellular cholesterol content on the intracellular trafficking of various bacterial protein toxins, viruses, and bacterial pathogens.…”

The Dystonia-associated Protein TorsinA Modulates Synaptic Vesicle Recycling

“…PC12 cells were grown on poly-L-lysine-coated coverslips and differentiated with 100 ng/ml nerve grow factor in Dulbecco's modified Eagle's medium for 72 h (24).…”

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

“…The identification of synaptotagmins (Syts) I and II as protein receptors for BoNT/B and BoNT/G, and SV2 for BoNT/A supported this concept (15)(16)(17)(18)(19). Recent results (20)(21)(22) suggested that, unlike BoNTs, TeNT uses glycosyl-phosphatidylinositol (GPI)-anchored glycoproteins of neuronal rafts as receptors.…”