BIOSYNTHESIS AND FUNCTION OF GLYCOSPHINGOLIPIDS AND OTHER GLYCOCONJUGATES

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Ganglioside GM1 is the cell surface receptor for cholera toxin (CT). The pentameric B subunit of CT binds to GM1 whereas the A subunit of CT is involved in activation of adenylylcyclase (AC). There is a distinct lag period between toxin binding and activation of adenylylcyclase. During this phase, the A subunit is reduced to the A1 peptide which ADP~ribosylates the stimulatory G protein (Gs) of the cyclase. We recently established that CT is oriented with its A subunit facing away from the cell surface when it binds, and that the holotoxin is internalized. In addition, we found that brefeldin A, which causes disassembly of the Golgi apparatus and disruption of intracellular membrane trafficking, is a potent blocker of CT action. Although brefeldin A does not prevent the internalization of CT, it does block its conversion to the A1 peptide. We are now in the process of delineating the pathway by which CT enters cells, the site where A1 is generated, and how the latter gains access to Gs. As a model, we are using human intestinal Caco~2 cells, which behave in culture as differentiated enterocytes, the natural target for CT. We are employing a combination of nondenaturing gel electrophoresis and subcellular fractionation. The goal is to identify the pathway of toxin disassembly and the site(s) where disassembly occurs. We have found that the stability of CT in solution or bound to Caco membranes was pH sensitive and began to dissociate to its A and pentameric B subunits at pH 5.5, which is the pH of endosomes. In addition, small amounts of A1 peptide were formed from membrane~bound CT in a pH~dependent manner. The presence of a membrane~associated reductase was supported by showing the activity was sensitive to N~ethylmaleimide which alkylates sulfhydral groups.