399 Peripheral Cellular Mechanisms of Induction of Visceral Hypersensitivity By Chronic Stress

Abstract

Cholera toxin (CT) secreted by the bacterium Vibrio cholerae causes the massive secretory diarrhea seen in Asiatic cholera. CT binds to five GM1 gangliosides on the plasma membrane (PM), which carries CT retrograde from PM via the Golgi to the endoplasmic reticulum (ER) where the toxin crosses into the cytosol to induce disease. GM1 consists of an extracellular oligosaccharide to which cholera toxin binds, and a hydrophobic ceramide domain containing sphingosine and an amide-linked fatty acyl chain that can be structurally diverse. How membrane lipids, such as GM1, act as sorting motifs in the retrograde pathway remains unknown. One hypothesis is that lipid sorting is stochastic and dependent on lipid structures that allow for or restrict movement across membrane curvature during budding and tubule formation; another invokes sorting by association with lipid rafts. Our AIM was to test these ideas and whether ceramide structure explains how GM1 sorts retrograde in the secretory pathway. METHODS: GM1 isoforms were synthesized that contained Alexa fluorophores attached to the oligosaccharide head group but differed in the ceramide domain by their fatty acyl chain. GM1 isoforms were tested whose fatty acid chains were: long chain (C16, C18), short chain (C12), or long chain and “kinked” via cisdouble bond (C16:1). Lipids were incorporated into A431 human epithelial cells stably expressing GFP-fusion proteins that define early, recycling, or late endosomes, and the trans-Golgi Network (TGN). Live cells were imaged by confocal microscopy under steady-state and real-time conditions. RESULTS and DISCUSSION: GM1 with either short-chain (C12) or “kinked” (C16:1) fatty acids localized predominantly to the PM and recycling endosomes, with a smaller fraction (8%) localizing to the TGN. GM1 containing long saturated fatty acids (C16 or C18) localized predominantly to late endosomes. Thus, the length and the saturation of the fatty acyl chain of GM1 affects the steady-state distribution in live cells; consistent with sorting by membrane curvature. Only the short chain and kinked GM1 isoforms co-localized with the TGN, a compartment required for CT transport from PM to ER and subsequently for toxicity. Clustering of short-chain or “kinked” GM1 with cholera toxin diverted the ganglioside from recycling to late endosomes; but the fraction localizing to the Golgi remained stable. This result cannot be stochastic. Thus, some factor rescues the trafficking of unsaturated ceramides when cross-linked to the Golgi, suggesting a mechanism of sorting by lipid rafts.