Determination of Cellular Lipids Bound to Human CD1d Molecules

Abstract

CD1 molecules are glycoproteins that present lipid antigens at the cell surface for immunological recognition by specialized populations of T lymphocytes. Prior experimental data suggest a wide variety of lipid species can bind to CD1 molecules, but little is known about the characteristics of cellular ligands that are selected for presentation. Here we have molecularly characterized lipids bound to the human CD1d isoform. Ligands were eluted from secreted CD1d molecules and separated by normal phase HPLC, then characterized by mass spectroscopy. A total of 177 lipid species were molecularly identified, comprising glycerophospholipids and sphingolipids. The glycerophospholipids included common diacylglycerol species, reduced forms known as plasmalogens, lyso-phospholipids (monoacyl species), and cardiolipins (tetraacyl species). The sphingolipids included sphingomyelins and glycosylated forms, such as the ganglioside GM3. These results demonstrate that human CD1d molecules bind a surprising diversity of lipid structures within the secretory pathway, including compounds that have been reported to play roles in cancer, autoimmune diseases, lipid signaling, and cell death.

Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphingolipids presented by CD1d

“…A variety of lipids have been eluted from human CD1d molecules including phospholipids, sphingomyelin, and GSL, suggesting that CD1 molecules can accommodate diverse lipids (22,30,31). The majority of studies to date that examined the repertoire of lipids bound by CD1d have used transfected B cell lines and identified PI, PC, lysophospholipids, GM3, and sphingomyelin (20,22,30,31).…”

Discovery of deoxyceramides and diacylglycerols as CD1b scaffold lipids among diverse groove-blocking lipids of the human CD1 system

“…Actual measurements of mouse CD1d complexes formed in cells first suggested that cellular ligands were dominated by one class of phosphatidylinositol-containing molecule (2), and CD1b was reported to bind PC in a selective way (27). However, subsequent study of mouse and human CD1d detected several types of phospho-and sphingolipid ligands (12)(13)(14), suggesting greater diversity of lipid ligands for CD1d. Using cellular CD1 proteins lacking endosomal targeting motifs and captured under two different conditions that avoid the acid-mediated unloading effects, our comparative lipidomics approach provides evidence for substantial diversity of CD1 ligands for each of the four human CD1 proteins, detecting hundreds of molecular features whose retention times vary greatly.…”

“…The folding of nascent CD1 heavy chains is thought to involve cotranslational binding of self lipids in the ER (11). This expectation is supported by experiments that detect self-diacylglycerols, monoacylglycerols, and sphingolipids eluted from CD1 proteins that have ER retention signals or lack endosomal recycling motifs (2,12,13). Some of these endogenous lipids activate T cells, so are true autoantigens (14).…”

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

“…By vesicular and nonvesicular transport through the cell, β-GlcCer and other self-lipids might then access the endolysosomal compartment, where exposure to acidic hydrolases and lipid transfer proteins, such as GM2 activator (GM2a) and saposins, could orchestrate their loading onto recycling CD1d molecules. Cellular lipids bound to human CD1d molecules isolated from different cellular compartments have been determined by high-resolution mass spectrometry (39)(40)(41). Neo-synthesized CD1d molecules have been shown to acquire phosphatidylcholine in the ER, which is then exchanged for sphingomyelin when CD1d traffics through the Golgi (41).…”

“…Indeed, in preliminary experiments we have observed that prosaposin enhances loading of β-GlcCer on plate-bound CD1d molecules at pH 7. Whether β-GlcCer could be identified as a major species associated with secreted or recycling CD1d molecules remains an open question, as the previous analysis was performed on resting Epstein-Barr virus-B cells (39,41) or in myeloid cells after digestion with ceramide glycanase, which does not cleave monohexoses from the ceramide backbone (40).…”