Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation
Edited by Mitchell Kronenberg, La Jolla Institute for Allergy and Immunology, La Jolla, CA, and accepted by the Editorial Board October 28, 2013 (received for review May 29, 2013)
Significance
Understanding how to optimize lipid-loading onto CD1d molecules is important to better harness invariant natural killer T (iNKT) cells’ central role at the interface between innate and adaptive immunity. We report that the lipid transfer proteins saposins play an essential role in modulating human iNKT cell autoreactivity to antigen-presenting cells activated by inflammatory stimuli. Lipid-loading occurs in an endo-lysosomal compartment, where saposins work as “lipid editors,” capable of fine-tuning loading and unloading of CD1d molecules and increasing the off-rate of CD1d-bound lipids.
Abstract
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 off-rate 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.
Acknowledgments
We thank Prof. Alain R. Townsend (University of Oxford) for the gift of 293T cells secreting biotin-tagged human CD1d molecules. This work was supported by The Wellcome Trust Grants 084923/B/08/Z (to G.S.B.) and 084923Z/08/Z (to V.C.), Cancer Research UK Programme Grant C399/A2291 (to V.C.), Advanced Immunization Technologies Grant 280873 (to V.C.), and The Medical Research Council. G.S.B. acknowledges support in the form of a Personal Research Chair from Mr. James Bardrick.
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References
1
DC Barral, MB Brenner, CD1 antigen presentation: How it works. Nat Rev Immunol 7, 929–941 (2007).
2
DI Godfrey, HR MacDonald, M Kronenberg, MJ Smyth, L Van Kaer, NKT cells: What’s in a name? Nat Rev Immunol 4, 231–237 (2004).
3
A Bendelac, PB Savage, L Teyton, The biology of NKT cells. Annu Rev Immunol 25, 297–336 (2007).
4
V Cerundolo, JD Silk, SH Masri, M Salio, Harnessing invariant NKT cells in vaccination strategies. Nat Rev Immunol 9, 28–38 (2009).
5
SK Dougan, et al., Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells. J Exp Med 202, 529–539 (2005).
6
S Zeissig, et al., Hepatitis B virus-induced lipid alterations contribute to natural killer T cell-dependent protective immunity. Nat Med 18, 1060–1068 (2012).
7
F Winau, et al., Saposin C is required for lipid presentation by human CD1b. Nat Immunol 5, 169–174 (2004).
8
W Yuan, et al., Saposin B is the dominant saposin that facilitates lipid binding to human CD1d molecules. Proc Natl Acad Sci USA 104, 5551–5556 (2007).
9
D Zhou, et al., Editing of CD1d-bound lipid antigens by endosomal lipid transfer proteins. Science 303, 523–527 (2004).
10
SJ Kang, P Cresswell, Saposins facilitate CD1d-restricted presentation of an exogenous lipid antigen to T cells. Nat Immunol 5, 175–181 (2004).
11
L León, et al., Saposins utilize two strategies for lipid transfer and CD1 antigen presentation. Proc Natl Acad Sci USA 109, 4357–4364 (2012).
12
Y Kishimoto, M Hiraiwa, JS O’Brien, Saposins: Structure, function, distribution, and molecular genetics. J Lipid Res 33, 1255–1267 (1992).
13
M Hiraiwa, et al., Lysosomal proteolysis of prosaposin, the precursor of saposins (sphingolipid activator proteins): Its mechanism and inhibition by ganglioside. Arch Biochem Biophys 341, 17–24 (1997).
14
JS O’Brien, Y Kishimoto, Saposin proteins: Structure, function, and role in human lysosomal storage disorders. FASEB J 5, 301–308 (1991).
15
A Darmoise, P Maschmeyer, F Winau, The immunological functions of saposins. Adv Immunol 105, 25–62 (2010).
16
A Bendelac, M Bonneville, JF Kearney, Autoreactivity by design: Innate B and T lymphocytes. Nat Rev Immunol 1, 177–186 (2001).
17
M Brigl, L Bry, SC Kent, JE Gumperz, MB Brenner, Mechanism of CD1d-restricted natural killer T cell activation during microbial infection. Nat Immunol 4, 1230–1237 (2003).
18
C Paget, et al., Activation of invariant NKT cells by toll-like receptor 9-stimulated dendritic cells requires type I interferon and charged glycosphingolipids. Immunity 27, 597–609 (2007).
19
M Salio, et al., Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation. Proc Natl Acad Sci USA 104, 20490–20495 (2007).
20
M Brigl, et al., Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection. J Exp Med 208, 1163–1177 (2011).
21
KO Yu, et al., Modulation of CD1d-restricted NKT cell responses by using N-acyl variants of alpha-galactosylceramides. Proc Natl Acad Sci USA 102, 3383–3388 (2005).
22
JD Silk, et al., Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells. J Immunol 180, 6452–6456 (2008).
23
TI Prigozy, et al., Glycolipid antigen processing for presentation by CD1d molecules. Science 291, 664–667 (2001).
24
V Arrunategui-Correa, HS Kim, The role of CD1d in the immune response against Listeria infection. Cell Immunol 227, 109–120 (2004).
25
PJ Brennan, et al., Invariant natural killer T cells recognize lipid self antigen induced by microbial danger signals. Nat Immunol 12, 1202–1211 (2011).
26
M Cernadas, et al., Lysosomal localization of murine CD1d mediated by AP-3 is necessary for NK T cell development. J Immunol 171, 4149–4155 (2003).
27
YH Chiu, et al., Distinct subsets of CD1d-restricted T cells recognize self-antigens loaded in different cellular compartments. J Exp Med 189, 103–110 (1999).
28
YH Chiu, et al., Multiple defects in antigen presentation and T cell development by mice expressing cytoplasmic tail-truncated CD1d. Nat Immunol 3, 55–60 (2002).
29
D Elewaut, et al., The adaptor protein AP-3 is required for CD1d-mediated antigen presentation of glycosphingolipids and development of Valpha14i NKT cells. J Exp Med 198, 1133–1146 (2003).
30
X Chen, et al., Distinct endosomal trafficking requirements for presentation of autoantigens and exogenous lipids by human CD1d molecules. J Immunol 178, 6181–6190 (2007).
31
VE Ahn, KF Faull, JP Whitelegge, AL Fluharty, GG Privé, Crystal structure of saposin B reveals a dimeric shell for lipid binding. Proc Natl Acad Sci USA 100, 38–43 (2003).
32
JS Im, et al., Kinetics and cellular site of glycolipid loading control the outcome of natural killer T cell activation. Immunity 30, 888–898 (2009).
33
G Denkberg, et al., Phage display-derived recombinant antibodies with TCR-like specificity against alpha-galactosylceramide and its analogues in complex with human CD1d molecules. Eur J Immunol 38, 829–840 (2008).
34
C McCarthy, et al., The length of lipids bound to human CD1d molecules modulates the affinity of NKT cell TCR and the threshold of NKT cell activation. J Exp Med 204, 1131–1144 (2007).
35
N Dalchau, et al., A peptide filtering relation quantifies MHC class I peptide optimization. PLOS Comput Biol 7, e1002144 (2011).
36
K Hanada, et al., Molecular machinery for non-vesicular trafficking of ceramide. Nature 426, 803–809 (2003).
37
AH Futerman, H Riezman, The ins and outs of sphingolipid synthesis. Trends Cell Biol 15, 312–318 (2005).
38
G D’Angelo, et al., Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide. Nature 449, 62–67 (2007).
39
D Cox, et al., Determination of cellular lipids bound to human CD1d molecules. PLoS ONE 4, e5325 (2009).
40
K Muindi, et al., Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphingolipids presented by CD1d. Proc Natl Acad Sci USA 107, 3052–3057, and correction (2010) 107(13):6118. (2010).
41
W Yuan, SJ Kang, JE Evans, P Cresswell, Natural lipid ligands associated with human CD1d targeted to different subcellular compartments. J Immunol 182, 4784–4791 (2009).
42
M Hiraiwa, et al., Isolation, characterization, and proteolysis of human prosaposin, the precursor of saposins (sphingolipid activator proteins). Arch Biochem Biophys 304, 110–116 (1993).
43
M Hiraiwa, S Soeda, Y Kishimoto, JS O’Brien, Binding and transport of gangliosides by prosaposin. Proc Natl Acad Sci USA 89, 11254–11258 (1992).
44
SJ Kang, P Cresswell, Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules. EMBO J 21, 1650–1660 (2002).
45
J Jayawardena-Wolf, K Benlagha, YH Chiu, R Mehr, A Bendelac, CD1d endosomal trafficking is independently regulated by an intrinsic CD1d-encoded tyrosine motif and by the invariant chain. Immunity 15, 897–908 (2001).
46
JE Gumperz, et al., Murine CD1d-restricted T cell recognition of cellular lipids. Immunity 12, 211–221 (2000).
47
F Ciaffoni, et al., Saposin B binds and transfers phospholipids. J Lipid Res 47, 1045–1053 (2006).
48
F Facciotti, et al., Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus. Nat Immunol 13, 474–480 (2012).
49
C Paduraru, et al., Role for lysosomal phospholipase A2 in iNKT cell-mediated CD1d recognition. Proc Natl Acad Sci USA 110, 5097–5102 (2013).
50
T Kolter, K Sandhoff, Principles of lysosomal membrane digestion: Stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids. Annu Rev Cell Dev Biol 21, 81–103 (2005).
51
N Remmel, S Locatelli-Hoops, B Breiden, G Schwarzmann, K Sandhoff, Saposin B mobilizes lipids from cholesterol-poor and bis(monoacylglycero)phosphate-rich membranes at acidic pH. Unglycosylated patient variant saposin B lacks lipid-extraction capacity. FEBS J 274, 3405–3420 (2007).
52
S Locatelli-Hoops, et al., Saposin A mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: Patient variant Saposin A lacks lipid extraction capacity. J Biol Chem 281, 32451–32460 (2006).
53
H Schulze, T Kolter, K Sandhoff, Principles of lysosomal membrane degradation: Cellular topology and biochemistry of lysosomal lipid degradation. Biochim Biophys Acta 1793, 674–683 (2009).
54
CB Fluharty, J Johnson, J Whitelegge, KF Faull, AL Fluharty, Comparative lipid binding study on the cerebroside sulfate activator (saposin B). J Neurosci Res 63, 82–89 (2001).
55
NA Haig, et al., Identification of self-lipids presented by CD1c and CD1d proteins. J Biol Chem 286, 37692–37701 (2011).
56
LM Fox, et al., Recognition of lyso-phospholipids by human natural killer T lymphocytes. PLoS Biol 7, e1000228 (2009).
57
F Facciotti, et al., Fine tuning by human CD1e of lipid-specific immune responses. Proc Natl Acad Sci USA 108, 14228–14233 (2011).
58
N Schrantz, et al., The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells. J Exp Med 204, 841–852 (2007).
59
LK Denzin, P Cresswell, HLA-DM induces CLIP dissociation from MHC class II alpha beta dimers and facilitates peptide loading. Cell 82, 155–165 (1995).
60
JR Alattia, JE Shaw, CM Yip, GG Privé, Molecular imaging of membrane interfaces reveals mode of beta-glucosidase activation by saposin C. Proc Natl Acad Sci USA 104, 17394–17399 (2007).
61
JR Alattia, JE Shaw, CM Yip, GG Privé, Direct visualization of saposin remodelling of lipid bilayers. J Mol Biol 362, 943–953 (2006).
62
L Bai, et al., Lysosomal recycling terminates CD1d-mediated presentation of short and polyunsaturated variants of the NKT cell lipid antigen alphaGalCer. Proc Natl Acad Sci USA 106, 10254–10259 (2009).
63
A Traunecker, F Oliveri, K Karjalainen, Myeloma based expression system for production of large mammalian proteins. Trends Biotechnol 9, 109–113 (1991).
64
PJ Jervis, et al., Synthesis and biological activity of alpha-glucosyl C24:0 and C20:2 ceramides. Bioorg Med Chem Lett 20, 3475–3478 (2010).
65
N Veerapen, et al., Synthesis and biological activity of alpha-galactosyl ceramide KRN7000 and galactosyl (alpha1—>2) galactosyl ceramide. Bioorg Med Chem Lett 19, 4288–4291 (2009).
66
J Wojno, et al., Amide analogues of CD1d agonists modulate iNKT-cell-mediated cytokine production. ACS Chem Biol 7, 847–855 (2012).
67
YR Garcia Diaz, J Wojno, LR Cox, GS Besra, Synthesis of threitol ceramide and [14C]threitol ceramide, non-glycosidic analogues of the potent CD1d antigen a-galactosyl ceramide. Tetrahedron Asymmetry 20, 747–753 (2009).
68
X Qi, T Leonova, GA Grabowski, Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid beta-glucosidase. J Biol Chem 269, 16746–16753 (1994).
69
Z Chu, DP Witte, X Qi, Saposin C-LBPA interaction in late-endosomes/lysosomes. Exp Cell Res 303, 300–307 (2005).
70
AR Aricescu, W Lu, EY Jones, A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr 62, 1243–1250 (2006).
71
LM Schimanski, et al., In vitro binding of HFE to the cation-independent mannose-6 phosphate receptor. Blood Cells Mol Dis 43, 180–193 (2009).
72
SD Gadola, N Dulphy, M Salio, V Cerundolo, Valpha24-JalphaQ-independent, CD1d-restricted recognition of alpha-galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes. J Immunol 168, 5514–5520 (2002).
73
OV Naidenko, et al., Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules. J Exp Med 190, 1069–1080 (1999).
74
AN Odyniec, et al., Regulation of CD1 antigen-presenting complex stability. J Biol Chem 285, 11937–11947 (2010).
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Published online: November 18, 2013
Published in issue: December 3, 2013
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Acknowledgments
We thank Prof. Alain R. Townsend (University of Oxford) for the gift of 293T cells secreting biotin-tagged human CD1d molecules. This work was supported by The Wellcome Trust Grants 084923/B/08/Z (to G.S.B.) and 084923Z/08/Z (to V.C.), Cancer Research UK Programme Grant C399/A2291 (to V.C.), Advanced Immunization Technologies Grant 280873 (to V.C.), and The Medical Research Council. G.S.B. acknowledges support in the form of a Personal Research Chair from Mr. James Bardrick.
Notes
This article is a PNAS Direct Submission. M.K. is a guest editor invited by the Editorial Board.
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The authors declare no conflict of interest.
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