Saposin B is the dominant saposin that facilitates lipid binding to human CD1d molecules

Abstract

CD1d molecules bind lipid antigens in the endocytic pathway, and access to the pathway is important for the development of CD1d-restricted natural killer T (NKT) cells. Saposins, derived from a common precursor, prosaposin, are small, heat-stable lysosomal glycoproteins required for lysosomal degradation of sphingolipids. Expression of prosaposin is required for efficient lipid binding and recognition of human CD1d molecules by NKT cells. Despite high sequence homology among the four saposins, they have different specificities for lipid substrates and different mechanisms of action. To determine the saposins involved in promoting lipid binding to CD1d, we expressed prosaposin deletion mutants lacking individual saposins in prosaposin-negative, CD1d-positive cells. No individual saposin proved to be absolutely essential, but the absence of saposin B resulted in the lowest recognition of α-galactosylceramide by NKT cells. When recombinant exogenous saposins were added to the prosaposin-negative cells, saposin B was the most efficient in restoring CD1d recognition. Saposin B was also the most efficient in mediating α-galactosylceramide binding to recombinant plate-bound CD1d and facilitating NKT cell activation. Saposin B could also mediate lipid binding to soluble CD1d molecules in a T cell-independent assay. The optimal pH for saposin B-mediated lipid binding to CD1d, pH 6, is higher than that of lysosomes, suggesting that saposin B may facilitate lipid binding to CD1d molecules throughout the endocytic pathway.

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

“…Indeed, using a combination of reagents that selectively recognize CD1d bound to ThrCer or GalGalCer, we have been able to demonstrate specific lipid exchange occurring in the SPR assay. In agreement with results shown by Zhou et al (9) and Yuan et al (8), saposin B alone (not precomplexed with an iNKT cell agonist) could not remove lipids already bound to CD1d molecules, unlike what has been shown for CD1e (57). However, we have shown that lipid-loaded saposin B specifically increased the offrate of lipids bound to CD1d molecules.…”

“…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.…”

“…cDNAs encoding full-length prosaposin (10) or mutants deleted of each individual saposin (8) were cloned in the retroviral expression vector pBMN and retroviruses produced according to published protocols (www.stanford. edu/group/nolan/protocols/pro_helper_dep.html).…”

“…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).…”