| CD1 proteins are a family of MHC class I-like antigen presentation molecules that present both self and microbial lipid antigens to T cells. Given that most CD1 antigens are amphipathic molecules that intercalate into cellular membranes, it is not known how lipid antigens embedded in cellular membranes mobilize across an aqueous luminal environment to reach the lipid-binding grooves of CD1 molecules. The mobilization of monomeric lipid units from the membrane bilayer to an aqueous cellular environment is predicted to be energetically expensive; passive mobilization is therefore not likely to occur without the assistance of lipid-binding or lipid-transfer proteins. Saposins are a family of four small, non-enzymatic proteins that were initially described for their ability to facilitate glycosphingolipid degradation, and have been recently found to facilitate the CD1 lipid loading. The available data suggest that, while saposin B (SapB) and saposin C (SapC) share high-sequence homology, they use strikingly different mechanisms to facilitate glycosphingolipid degradation by hydrolytic enzymes. It has been proposed that SapB functions as a "solubilizer" where it extirpates target glycosphingolipids from a bilayer membrane and presents the terminal end of the glycosphingolipid carbohydrate headgroup to lysosomal hydrolases as a soluble SapB-glycosphingolipid complex. In contrast, SapC functions as a "liftase" where it enhances the spatial availability of hydrolytic glycosphingolipid substrates by embedding directly into the lipid bilayer thereby disrupting the tightly packed lipids that comprise the membrane. In this study, we delineate two mechanisms of saposin-facilitated CD1 lipid loading. Our findings indicate that SapB and SapC use two distinct mechanisms to mediate transfer of membrane-derived lipid antigens to CD1 molecules. These studies support the "solubilizer" and "liftase" models proposed for SapB and SapC in glycosphingolipid degradation, respectively, and further suggest that these distinct mechanisms are also used by saposins to facilitate lipid loading of CD1 molecules. Our findings describe how SapB and SapC, proteins characterized as important co-factors of glycosphingolipid digestion, interact with membrane bilayers and membrane-derived lipids to facilitate lipid loading. |
