Natural lipid ligands associated with human CD1d targeted to different subcellular compartments

Abstract

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.

Figure 1

The CD1d constructs used for identification of endogenous lipid ligands. (A). Schematic illustration of ER-retained (ERCD1d), secretory (secCD1d) and cleavable CD1d (pclCD1d) constructs. SS, signal sequence, TM, transmembrane domain. CT, cytoplasmic tail. The tetrameric ER-retention sequence KDEL (light gray) was fused C-terminal to the ectomain to generate ERCD1d. A lysosomal protease-processing site from GILT (dark gray) was introduced between the ectodomain and transmembrane domain to generate pclCD1d (B–C). Immunofluorescence analysis of ERCD1d (B) and pclCD1d (C) transiently expressed in HeLa cells. The cells were co-stained with the CD1d51 anti-CD1d mAb and and a rabbit antiserum against the ER marker calnexin. (D). Percoll density gradient separation of membranes from .221.pclCD1d cells. Fractions were separated by SDS-PAGE and western blotted for CD1d (top panel), ERp57 (ER), and anti-HLA-DM (late endosomes/lysosomes), (lower panel).

Figure 2

Purification of CD1d from stable .221 transfectants. Purified proteins were separated by SDS-PAGE and stained with Coomassie Blue.

Figure 3

Mass spectrometry of purified lipids from cleavable CD1d (pclCD1d) by (A) MALDI-TOF and (B) linked scan MS/MS. In B), the spectrum shows the result of m/z 184 parent ion linked scans for phosphocholine containing species. The major PC and sphingomyelin (SM) species detected are indicated, together with their molecular species composition. (C.) Lysophospholipids are associated with pclCD1d. Lipids isolated from pclCD1d were subjected to linked scan MS/MS and scanned for neutral loss of m/z 141 (phosphoethanolamine, top), neutral loss of m/z 185 (phosphoserine, middle) and parents m/z 184 (phosphocholine, bottom). Lyso-PC and lyso-PE were detected in these analyses. The peaks of m/z 522.4 (18:2a) and m/z 524.3 (18:1a), highlighted in bold, were used to compare lyso-PC content with .221cells and FBS (see Figure S2A and B).

Figure 4

Mass spectrometry of purified lipids from ER CD1d by (A) MALDI-TOF and (B) linked scan MS/MS. In B), the spectrum shows the result of m/z 184 precursor ions for phosphocholine containing species. The major PC species detected are indicated, together with their molecular species composition.

Figure 5

Mass spectrometry of purified lipids from sec CD1d by (A) MALDI-TOF and (B) linkes scan MS/MS. In b), the spectrum shows the result of m/z 184 precursorions for phosphocholine containing species. The major sphingomyelin (SM) molecular species detected are indicated.