Underlying mechanisms of regulation of glycosphingolipid synthesis in cells

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Glycosphingolipids (GSLs) are sugar-lipid conjugates which play a key role in cell membrane processes and their deficiency or accumulation can result in many diseases. However, a central regulatory step in the synthesis of GSLs is essentially unknown. GSLs are made in a vesicular structure in cells called the Golgi. However, the one sugar GSL glucosyl ceramide(GlcCer), from which all more complex GSLs are derived, is made on the outside membrane of the Golgi. An enzyme called a 'flippase' must translocate glucosyl ceramide from the outside to the inside membrane of the Golgi. This regulates GSL synthesis but no GlcCer flippase has yet been identified. This is a major missing step in understanding GSL synthesis. We found the multiple drug resistance pump, MDR1, mediates ~50% of Golgi GlcCer flipping in spleen, kidney and liver. We will define the other GlcCer flippase(s) which translocate GlcCer into the Golgi in these, and the other, mouse organs. We will use molecular inhibitory technology for candidate flippases we identified by sequence alignment, and crosslinking to a photoaffinity derivative of GlcCer we will make, to identify these missing GlcCer flippases. Synthesis of acidic GSLs (containing sialic acid but also derived from GlcCer) is unaffected by MDR1, suggesting acidic GSLs can be made from a separate GlcCer source in the Golgi (translocated by a different flippase?). We showed cholesterol-GSL binding changes membrane GSL sugar from exposed/protruding, to flat/obscured. This change could inhibit enzymes making certain GSLs from GlcCer. As cholesterol content increases from early to late Golgi, the position of GlcCer in the Golgi stacks could define the type of GSL made. Different flippases may translocate GlcCer into different parts of the Golgi to provide different GlcCer pools (with different conformations).We showed loss of cell cholesterol induces synthesis of a new GSL, consistent with cholesterol control of GlcCer availability as a GSL precursor. Effect of increasing/decreasing cell cholesterol on GSL synthesis and action of the GlcCer flippases we identify will be determined. These studies will reveal a completely new regulation of cellular GSLs and define the major missing step in GSL metabolism.