Cholera toxin (CT) causes the onset of disease symptoms by binding its B unit to the ganglioside GM1 receptor on epithelial cells and trafficking retrograde from the cell surface and the trans-Golgi network (TGN) into the endoplasmic reticulum (ER). Once in ER, a portion of the A subunit unfolds and is retrotransported to the cytosol where it induces toxicity. In previous studies, Saslowsky et al. determined that in addition to retrotrafficking, binding to the GM1 also allows CT to cross epithelial cells from the apical to the basolateral surface by transcytosis. However, the mechanism of this form of membrane sorting into the absorptive basolaterally directed transyctotic pathway is poorly understood. Thus, in the highlighted study, Saslowsky et al. investigated the intracellular transcytotic itinerary of CT across polarized epithelial cells. Using a mutant CT (CT-GS) containing sulfation and glycosylation motifs, a TGN/ER trafficking assay was employed by selectively biotinylating basolateral or apical cell surfaces. Immunoprecipitation followed by a pull-down assay with avidin-coupled beads allowed the isolation of the fraction of CTB-GS that had been transcytosed. SDS-PAGE followed by immunoblot precipitation revealed quantities of 35S-radiolabeled toxin that transited through the TGN and ER. No 35S-radiolabeled toxin was detected in the transcytosed fraction, therefore indicating that the retrograde and transcytotic pathways do not intersect and instead represent distinct itineraries.
In addition, the authors investigated the role of structural dependence of the ceramide chain of GM1 species on transcytosis from the apical to basolateral membranes. GM1 lipids containing ceramide domains differing only in the structure of their acyl chains (C12:0, C16:0, or C16:1) and with Alexa Fluor 568 attached were synthesized and introduced into either polarized canine kidney MDCK or human intestinal T84 monolayers. Confocal microscopy of the live cells elucidated that only the species containing a single cis-unsaturated 16-carbon acyl chain in the ceramide domain were transported from the apical to the basolateral membranes. Immunoprecipitation of the monolayers revealed that GM1 species with a short 12-carbon ceramide chain were also rapidly transported across T84 monolayers, and this data collectively suggests that both the cis-unsaturated and short acyl chains are needed for efficient sorting into retrograde and transcytotic pathways.
Ergo, with intracellular lipid sorting being previously poorly understood, Saslowsky et al. successfully provided a possible mechanism for sorting which may be extended to other intracellular pathways. Furthermore, this information also provides great potential for research surrounding mechanisms of drug delivery across mucosal surfaces.
For further reading, please visit: Cholera