Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

Alattia, Jean‐René; Shaw, James E.; Yip, Christopher M.; Privé, Gilbert G.

doi:10.1073/pnas.0704998104

Cited by 73 publications

(60 citation statements)

References 45 publications

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“…These findings support a mechanism in which SapC inserts directly into the lipid bilayer, thereby disrupting the tightly packed lipids that comprise the target membrane. These SapC-liposome interactions are reminiscent of SapC-membrane interactions observed in the context of glycosphingolipid (GSL) degradation whereby SapC inserts into membrane bilayers to provide lysosomal hydrolases greater accessibility to GSL substrates embedded in model membranes (19)(20)(21)(22). Because membraneassociated SapC facilitates GSL degradation by positioning hydrolytic enzymes, such as β-glucosidase, next to disorganized membrane environments where GSL substrates are accessible (23,24), we tested whether SapC also interacts with CD1 molecules on the surface of model membranes.…”

Section: Resultsmentioning

confidence: 99%

“…Because our liposome sedimentation and crosslinking studies suggest that CD1c-Fc is recruited to the liposome surface by membrane-associated SapC molecules, we hypothesized that CD1c-Fc molecules could access liposome-embedded lipid antigens by co-opting a similar membrane-associated mechanism used by lysosomal hydrolases to mediate GSL degradation (19,22,23,26). In support of this hypothesis, our results demonstrate that only liposome-associated SapC molecules had the capacity to load CD1c-Fc and evoke T-cell activation when the antigen is provided in a membrane-bound form (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Saposins utilize two strategies for lipid transfer and CD1 antigen presentation

León

Tatituri

Grenha

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Saposins utilize two strategies for lipid transfer and CD1 antigen presentation

León

Tatituri

Grenha

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…2, 6, and 7), the results suggest either allosteric activation of GBA1 or increased access of GBA1 to its substrate glucosylceramide. One possibility of allosteric regulation would involve saposin C (9, 31, 32), a known cofactor for GBA1 that enhances interaction of GBA1 with substrate (32), such that PKC activation may facilitate the dynamic association of saposin C and GBA1. PKC␦ is likely to act upstream of GBA1; however, the specific mechanism remains to be identified.…”

Section: Discussionmentioning

confidence: 99%

Involvement of Acid β-Glucosidase 1 in the Salvage Pathway of Ceramide Formation

Kitatani

Sheldon

Rajagopalan

et al. 2009

Journal of Biological Chemistry

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In the present study, we examined whether acid ␤-glucosidase 1 (GBA1), which hydrolyzes glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated formation of ceramide from recycled sphingosine. Glucosylceramide levels declined after treatment of MCF-7 cells with a potent PKC activator, phorbol 12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs significantly attenuated acid glucocerebrosidase activity and decreased PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced degradation of glucosylceramide and generation of sphingosine, the source for ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased ceramide levels. These observations indicate that GBA1 activation can generate the source (sphingosine) for PMA-induced formation of ceramide through the salvage pathway. Next, the role of PKC␦, a direct effector of PMA, in the formation of ceramide was determined. By attenuating expression of PKC␦, cells failed to trigger PMA-induced alterations in levels of ceramide, sphingomyelin, and glucosylceramide. Thus, PKC␦ activation is suggested to stimulate the degradation of both sphingomyelin and glucosylceramide leading to the salvage pathway of ceramide formation. Collectively, GBA1 is identified as a novel source of regulated formation of ceramide, and PKC␦ is an upstream regulator of this pathway.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Salio

Ghadbane

Dushek³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Lipid transfer proteins, such as molecules of the saposin family, facilitate extraction of lipids from biological membranes for their loading onto CD1d molecules. Although it has been shown that prosaposin-deficient mice fail to positively select invariant natural killer T (iNKT) cells, it remains unclear whether saposins can facilitate loading of endogenous iNKT cell agonists in the periphery during inflammatory responses. In addition, it is unclear whether saposins, in addition to loading, also promote dissociation of lipids bound to CD1d molecules. To address these questions, we used a combination of cellular assays and demonstrated that saposins influence CD1d-restricted presentation to human iNKT cells not only of exogenous lipids but also of endogenous ligands, such as the self-glycosphingolipid β-glucopyranosylceramide, up-regulated by antigen-presenting cells following bacterial infection. Furthermore, we demonstrated that in human myeloid cells CD1d-loading of endogenous lipids after bacterial infection, but not at steady state, requires trafficking of CD1d molecules through an endo-lysosomal compartment. Finally, using BIAcore assays we demonstrated that lipid-loaded saposin B increases the offrate of lipids bound to CD1d molecules, providing important insights into the mechanisms by which it acts as a "lipid editor," capable of fine-tuning loading and unloading of CD1d molecules. These results have important implications in understanding how to optimize lipid-loading onto antigen-presenting cells, to better harness iNKT cells central role at the interface between innate and adaptive immunity. autoreactivity | inflammation | innate immunity | lipid binding proteins

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Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

Cited by 73 publications

References 45 publications

Saposins utilize two strategies for lipid transfer and CD1 antigen presentation

Saposins utilize two strategies for lipid transfer and CD1 antigen presentation

Involvement of Acid β-Glucosidase 1 in the Salvage Pathway of Ceramide Formation

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

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