Saposins utilize two strategies for lipid transfer and CD1 antigen presentation

Abstract

Transferring lipid antigens from membranes into CD1 antigenpresenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein-lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin-lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens. . Although the molecular mechanisms governing MHC class I and II peptide loading are well appreciated, the mechanisms of CD1 lipid loading remain poorly understood. In the case of MHC class I molecules, proteasome-derived peptides are loaded onto MHC class I molecules by proteins of the peptide loading complex whose components include calnexin, calreticulin, ERp57, tapasin, and TAP (2). MHC class II molecules are loaded in endosomal compartments, where HLA-DM facilitates the exchange of the class II associated invariant peptide for high-affinity peptides derived from lysosomal processing of exogenous proteins (3, 4). The capacity of the MHC class I and II processing and antigen loading mechanisms are major factors determining which peptides are antigenic. In contrast to MHC-presented peptides, CD1-restricted lipid antigens are amphipathic molecules that are typically embedded in cellular membranes or micelles (5) and thus may be largely inaccessible to luminal proteins. Given the nonpolar component of lipids, mobilization of membrane-derived lipids across an aqueous endocytic environment likely requires extraction from membranes and, in most cases, transport across aqueous biological buffers into the lipid-binding grooves of CD1 molecules. These related processes are predicted to have unfavorable dissolution energetics and is unlikely to occur efficiently without the assistance of lipid-transfer proteins or other molecular mediators (6, 7).Recent studies have shown that saposins, a family of small, nonenzymatic lipid-binding proteins, facilitate CD1 lipid loading in endosomal compartments (6,(8)(9)(10). To a relative extent, all saposins enhance CD1 lipid loading in cellular and in vitro assays, but they appear to have a differential capacity to load particular lipids into individual CD1 isoforms. In one study, saposin B (SapB) was shown to facilitate α-galactosylceramide (αGalCer) loading onto human CD1d molecules more efficiently than other saposin isoforms (9). In cellular studies, saposin C (SapC), but not other saposins, restored CD1b presentation of long-chain mycobacterial antigens in saposin-deficient antigen presenting cells (8). Furthermore, direct visualization of lipid-l...

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

“…Together, these results highlight the important role of saposins as lipid-editors for CD1d antigen presentation, in addition to GM2a (9) and Niemann Pick type C2 protein (NPC2) (58). In contrast to DM molecules, which edit the repertoire of peptides bound to MHC class II molecules (59), saposins do not have an enzymatic mechanism of action and we and others have not been able to demonstrate physical association with human CD1d molecules (8), unlike what has been shown for murine CD1d and saposin A (9) or human CD1b and CD1c and saposin C (7,11). Despite extensive in vivo and in vitro studies, the ultimate mechanism of hydrolase activation by saposins is unclear and that of lipid transfer onto CD1d molecules remains unknown.…”

“…Despite high homology, the four saposins have different lipid specificities, as demonstrated by the phenotype of patients with selective deficiency of individual molecules (53). We limited our study to the role of saposin B, because evidence from our own works and from the literature (8,11,32) suggested its dominant role in loading soluble lipids onto CD1d molecules. We determined the binding affinity of saposin B to a radiolabeled version of ThrCer at pH5 and pH7 (1.8 μM and 4.5 μM, respectively), and observed that it reached a plateau when saposin B was present in molar excess.…”

“…Despite extensive in vivo and in vitro studies, the ultimate mechanism of hydrolase activation by saposins is unclear and that of lipid transfer onto CD1d molecules remains unknown. The two main proposed modes of action of saposin (11,60,61) are the "detergent-like or solubilizer" (saposin B and GM2a), in which saposins extract lipids from bilayers and present them to hydrolases as a soluble protein-lipid complex, and the "liftase model" (saposin C), whereby saposins remodel the bilayer surface facilitating binding of the hydrolases to the relative substrates. We speculate that saposin B molecules, dimeric at neutral and low pH and with a large hydrophobic cavity (31), may readily solubilize lipids and offer them to recycling CD1d molecules throughout the endocytic compartment, actively promoting lipid exchange in the groove of CD1d molecules by increasing the off-rate of already bound ligands.…”

“…In the endoplasmic reticulum (ER), the microsomal triglyceride transfer protein has been shown to assist loading of nascent CD1d molecules with self-phospholipids (5,6). In the lysosomal compartment, lipid transfer proteins belonging to the saposin family have been shown to play an important role in loading lipids onto CD1d and CD1b molecules (7)(8)(9)(10)(11). Saposins are a group of four, small, heat-stable glycoproteins required for hydrolysis of sphingolipids by lysosomal hydrolases and membrane remodeling (12).…”

Deciphering the Role of CD1e Protein in Mycobacterial Phosphatidyl-myo-inositol Mannosides (PIM) Processing for Presentation by CD1b to T Lymphocytes

“…In contrast, Sap-C was completely ineffective in this in vitro digestion assay. Recent studies comparing the mode of action of Sap-B and Sap-C showed that the two investigated saposins use different strategies for structurally diverse lipid antigen presentation (24). Although Sap-B forms soluble saposin-lipid complexes and can directly load CD1 proteins, Sap-C inserts itself directly into the membrane bilayer, thereby disrupting the tightly packed lipid bilayer and thus facilitating antigen loading onto the presenting CD1 protein.…”