Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids
CD1d is an atypical MHC class I molecule which binds endogenous and exogenous lipids and can activate natural killer T (NKT) cells through the presentation of lipid antigens. CD1d surveys different cellular compartments including the secretory and the endolysosomal pathway and broadly binds lipids t...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-07-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2022.897873/full |
| _version_ | 1811325794928033792 |
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| author | Maren Rudolph Maren Rudolph Yuting Wang Yuting Wang Yuting Wang Theresa Simolka Theresa Simolka Emilie Huc-Claustre Emilie Huc-Claustre Lingyun Dai Gijsbert Grotenbreg Gurdyal Singh Besra Anna Shevchenko Andrej Shevchenko Sebastian Zeissig Sebastian Zeissig |
| author_facet | Maren Rudolph Maren Rudolph Yuting Wang Yuting Wang Yuting Wang Theresa Simolka Theresa Simolka Emilie Huc-Claustre Emilie Huc-Claustre Lingyun Dai Gijsbert Grotenbreg Gurdyal Singh Besra Anna Shevchenko Andrej Shevchenko Sebastian Zeissig Sebastian Zeissig |
| author_sort | Maren Rudolph |
| collection | DOAJ |
| description | CD1d is an atypical MHC class I molecule which binds endogenous and exogenous lipids and can activate natural killer T (NKT) cells through the presentation of lipid antigens. CD1d surveys different cellular compartments including the secretory and the endolysosomal pathway and broadly binds lipids through its two hydrophobic pockets. Purification of the transmembrane protein CD1d for the analysis of bound lipids is technically challenging as the use of detergents releases CD1d-bound lipids. To address these challenges, we have developed a novel approach based on Sortase A-dependent enzymatic release of CD1d at the cell surface of live mammalian cells, which allows for single step release and affinity tagging of CD1d for shotgun lipidomics. Using this system, we demonstrate that CD1d carrying the Sortase A recognition motif shows unimpaired subcellular trafficking through the secretory and endolysosomal pathway and is able to load lipids in these compartments and present them to NKT cells. Comprehensive shotgun lipidomics demonstrated that the spectrum and abundance of CD1d-associated lipids is not representative of the total cellular lipidome but rather characterized by preferential binding to long chain sphingolipids and glycerophospholipids. As such, sphingomyelin species recently identified as critical negative regulators of NKT cell activation, represented the vast majority of endogenous CD1d-associated lipids. Moreover, we observed that inhibition of endolysosomal trafficking of CD1d surprisingly did not affect the spectrum of CD1d-bound lipids, suggesting that the majority of endogenous CD1d-associated lipids load onto CD1d in the secretory rather than the endolysosomal pathway. In conclusion, we present a novel system for the analysis of CD1d-bound lipids in mammalian cells and provide new insight into the spectrum of CD1d-associated lipids, with important functional implications for NKT cell activation. |
| first_indexed | 2024-04-13T14:39:12Z |
| format | Article |
| id | doaj.art-ce90314b62184a15b77ee1f947741d7e |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-04-13T14:39:12Z |
| publishDate | 2022-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-ce90314b62184a15b77ee1f947741d7e2022-12-22T02:42:57ZengFrontiers Media S.A.Frontiers in Immunology1664-32242022-07-011310.3389/fimmu.2022.897873897873Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated LipidsMaren Rudolph0Maren Rudolph1Yuting Wang2Yuting Wang3Yuting Wang4Theresa Simolka5Theresa Simolka6Emilie Huc-Claustre7Emilie Huc-Claustre8Lingyun Dai9Gijsbert Grotenbreg10Gurdyal Singh Besra11Anna Shevchenko12Andrej Shevchenko13Sebastian Zeissig14Sebastian Zeissig15Department of Medicine I, University Medical Center Dresden, Technische Universität (TU) Dresden, Dresden, GermanyCenter for Regenerative Therapies Dresden (CRTD), Technische Universität (TU) Dresden, Dresden, GermanyDepartment of Medicine I, University Medical Center Dresden, Technische Universität (TU) Dresden, Dresden, GermanyCenter for Regenerative Therapies Dresden (CRTD), Technische Universität (TU) Dresden, Dresden, GermanyMax Planck Institute of Molecular Cell Biology and Genetics, Dresden, GermanyDepartment of Medicine I, University Medical Center Dresden, Technische Universität (TU) Dresden, Dresden, GermanyCenter for Regenerative Therapies Dresden (CRTD), Technische Universität (TU) Dresden, Dresden, GermanyDepartment of Medicine I, University Medical Center Dresden, Technische Universität (TU) Dresden, Dresden, GermanyCenter for Regenerative Therapies Dresden (CRTD), Technische Universität (TU) Dresden, Dresden, GermanyDepartment of Geriatrics, First Affiliated Hospital of Southern University of Science and Technology (Shenzhen People’s Hospital), Shenzhen, ChinaMaze Therapeutics, South San Francisco, CA, United StatesSchool of Biosciences, University of Birmingham, Birmingham, United KingdomMax Planck Institute of Molecular Cell Biology and Genetics, Dresden, GermanyMax Planck Institute of Molecular Cell Biology and Genetics, Dresden, GermanyDepartment of Medicine I, University Medical Center Dresden, Technische Universität (TU) Dresden, Dresden, GermanyCenter for Regenerative Therapies Dresden (CRTD), Technische Universität (TU) Dresden, Dresden, GermanyCD1d is an atypical MHC class I molecule which binds endogenous and exogenous lipids and can activate natural killer T (NKT) cells through the presentation of lipid antigens. CD1d surveys different cellular compartments including the secretory and the endolysosomal pathway and broadly binds lipids through its two hydrophobic pockets. Purification of the transmembrane protein CD1d for the analysis of bound lipids is technically challenging as the use of detergents releases CD1d-bound lipids. To address these challenges, we have developed a novel approach based on Sortase A-dependent enzymatic release of CD1d at the cell surface of live mammalian cells, which allows for single step release and affinity tagging of CD1d for shotgun lipidomics. Using this system, we demonstrate that CD1d carrying the Sortase A recognition motif shows unimpaired subcellular trafficking through the secretory and endolysosomal pathway and is able to load lipids in these compartments and present them to NKT cells. Comprehensive shotgun lipidomics demonstrated that the spectrum and abundance of CD1d-associated lipids is not representative of the total cellular lipidome but rather characterized by preferential binding to long chain sphingolipids and glycerophospholipids. As such, sphingomyelin species recently identified as critical negative regulators of NKT cell activation, represented the vast majority of endogenous CD1d-associated lipids. Moreover, we observed that inhibition of endolysosomal trafficking of CD1d surprisingly did not affect the spectrum of CD1d-bound lipids, suggesting that the majority of endogenous CD1d-associated lipids load onto CD1d in the secretory rather than the endolysosomal pathway. In conclusion, we present a novel system for the analysis of CD1d-bound lipids in mammalian cells and provide new insight into the spectrum of CD1d-associated lipids, with important functional implications for NKT cell activation.https://www.frontiersin.org/articles/10.3389/fimmu.2022.897873/fullCD1dNKT cellSortase Ashotgun lipidomicssphingomyelin |
| spellingShingle | Maren Rudolph Maren Rudolph Yuting Wang Yuting Wang Yuting Wang Theresa Simolka Theresa Simolka Emilie Huc-Claustre Emilie Huc-Claustre Lingyun Dai Gijsbert Grotenbreg Gurdyal Singh Besra Anna Shevchenko Andrej Shevchenko Sebastian Zeissig Sebastian Zeissig Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids Frontiers in Immunology CD1d NKT cell Sortase A shotgun lipidomics sphingomyelin |
| title | Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids |
| title_full | Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids |
| title_fullStr | Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids |
| title_full_unstemmed | Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids |
| title_short | Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids |
| title_sort | sortase a cleavable cd1d identifies sphingomyelins as major class of cd1d associated lipids |
| topic | CD1d NKT cell Sortase A shotgun lipidomics sphingomyelin |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2022.897873/full |
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