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Natural Lipid Ligands Associated with Human CD1d Targeted to Different Subcellular Compartments ¹

Weiming Yuan^*, Suk-Jo Kang^2,*, James E. Evans and Peter Cresswell^3,*

^* Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06520; and Mass Spectrometry Facility, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts, Worcester, MA 01655

CD1d is an MHC class I-like membrane glycoprotein that presents lipid Ags to NKT cells. Despite intensive biochemical, genetic, and structural studies, the endogenous lipids associated with CD1d remain poorly defined because of the biochemical challenges posed by their hydrophobic nature. In this study, we report the generation of a protease-cleavable CD1d variant with a similar trafficking pattern to wild-type CD1d that can be purified in the absence of detergent and allows the characterization of the naturally associated lipids. In addition, we used soluble variants of CD1d that are secreted or retained in the endoplasmic reticulum (ER) to survey their acquired lipids. By using multiple mass spectrometry methods, we found that CD1d retained in the ER is predominantly loaded with the most abundant phospholipid in the cell, phosphatidyl choline, while the protease cleavable version of CD1d contains bound sphingomyelin and lysophospholipids in addition to phosphatidyl choline. The secreted soluble version of CD1d, in contrast, lacks detectable phosphatidyl choline and the only detectable associated lipid is sphingomyelin. The data suggest that, in the absence of infection or stress, CD1d molecules survey the ER, the secretory pathway, and the endocytic pathway, and accumulate the most abundantly available lipids present in these compartments.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Howard Hughes Medical Institute and National Institutes of Health Grant R01 AI059167 (to P.C.). W.Y. was supported by a postdoctoral fellowship provided by the Cancer Research Institute.

² Current address: Howard Hughes Medical Institute, Departments of Medicine and Microbiology/Immunology, University of California San Francisco, San Francisco, CA 94143-0795

³ Address correspondence and reprint requests to Dr. Peter Cresswell, Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, 300 Cedar Street, PO Box 208011, New Haven, CT 06520-8011. E-mail address: peter.cresswell{at}yale.edu

⁴ Abbreviations used in this paper: β₂m, β₂-microglobulin; ER, endoplasmic reticulum; MTP, microsomal triglyceride lipid transfer protein; PI, phosphatidyl inositol; MS, mass spectrometry; pclCD1d, protease-cleavable CD1d; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; GILT, IFN-inducible lysosomal thioreductase.

⁵ The online version of this article contains supplemental material.