Caveolar endocytosis and microdomain association of a glycosphingolipid analog is dependent on its sphingosine stereochemistry

We have previously shown that glycosphingolipid analogs are internalized primarily via caveolae in various cell types. This selective internalization was not dependent on particular carbohydrate headgroups or sphingosine chain length. Here, we examine the role of sphingosine structure in the endocytosis of BODIPYtrade mark-tagged lactosylceramide (LacCer) analogs via caveolae. We found that whereas the LacCer analog with the natural (D-erythro) sphingosine stereochemistry is internalized mainly via caveolae, the non-natural (L-threo) LacCer analog is taken up via clathrin-, RhoA-, and Cdc42-dependent mechanisms and largely excluded from uptake via caveolae. Unlike the D-erythro-LacCer analog, the L-threo analog did not cluster in membrane microdomains when added at higher concentrations (5-20 microm). In vitro studies using small unilamellar vesicles and giant unilamellar vesicles demonstrated that L-threo-LacCer did not undergo a concentration-dependent excimer shift in fluorescence emission such as that seen with BODIPYtrade mark-sphingolipids with natural stereochemistry. Molecular modeling studies suggest that in d-erythro-LacCer, the disaccharide moiety extends above and in the same plane as the sphingosine hydrocarbon chain, while in L-threo-LacCer the carbohydrate group is nearly perpendicular to the hydrocarbon chain. Together, these results suggest that the altered stereochemistry of the sphingosine group in L-threo-LacCer results in a perturbed structure, which is unable to pack closely with natural membrane lipids, leading to a reduced inclusion in plasma membrane microdomains and decreased uptake by caveolar endocytosis. These findings demonstrate the importance of the sphingolipid stereochemistry in the formation of membrane microdomains.