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.

Proteins in the aqueous phase of MTBE extraction were reduced with dithiothreitol, alkylated with iodacetamid and digested with trypsin (modified, sequencing grade, Promega). Resulting peptide mixtures were spiked with labelled peptide standards (New England Peptides) with final concentration 10fmol/μl and analyzed by LC-MS/MS. The analysis was performed on a nano-UPLC Ultimate 3000 interfaced on-line to a Q Exactive HF Hybrid Quadrupole Orbitrap mass spectrometer (both Thermo Fischer Scientific). The UPLC system was equipped with Acclam PepMaptm 100 75 µm x 2cm trapping column and 75µm x 50cm separating column packed with 3 µm diameter C18 particles (Thermo Fischer Scientific). Peptides were separated using 80min linear gradient (solvent A – 0.1% formic acid in water, solvent B – 0.1% formic acid in acetonitrile). Spectra were acquired in parallel reaction monitoring (PRM) mode, a full mass spectrum at 240000 resolution (AGC target 3e6, 150 ms maximum injection time, m/z 350–1700) was followed by PRM scans at 120000 resolution (AGC target 1e5, 200 ms maximum injection time) triggered by a scheduled inclusion list. To set up PRM parameters, the information about target peptides (m/z, charge state, retention time) was obtained from prior experiments with recombinant proteins. Stability of labelled peptide standards was tested in a separate LC-MS/MS experiment. Data processing was performed by SkyLine software (1) , quantitative values were calculated based on sum of extracted peak areas of precursor and two isotopes.

L cells were harvested and washed twice using 10 ml of tris(hydroxymethyl)aminomethane (Tris) buffer (50 mM Tris-HCl, 150 mM NaCl, 1.8 mM CaCl2, pH 7.5 at 37 °C). The L cells were incubated with Sortase (Δ59SortA) and 3G-Twin-Strep-tag (GGGWSHPQFEKGGGSGGGSGGSAWSHPQFEK, purity ≥ 95 %) (peptides&elephants) for 3 h at 37 °C while shaking at 600 rpm. After incubation, the cells were centrifuged (300 × g, 5 min, 4 °C). The collected supernatant was centrifuged at 4,500 × g for 20 min at 4 °C, and again centrifuged at 118,244 × g for 75 min at 4 °C. SortA activity in the cell supernatant was inhibited by adding [2-(Trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET) (Toronto Research Chemicals) (final conc. 5 mM). Samples were dialyzed against Tris buffer (100 mM Tris-HCl pH 8 at RT, 150 mM NaCl, 1 mM EDTA) and concentrated. Purification of tagged proteins was done using Strep-Tactin® Superflow high capacity® 50 % suspension (Iba) as resin material. The purification was performed according to manufacturer's instructions.