Natural Ligand of Mouse CD1d1: Cellular Glycosylphosphatidylinositol

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Science  06 Mar 1998:
Vol. 279, Issue 5356, pp. 1541-1544
DOI: 10.1126/science.279.5356.1541


Mouse CD1d1, a member of the CD1 family of evolutionarily conserved major histocompatibility antigen–like molecules, controls the differentiation and function of a T lymphocyte subset, NK1+natural T cells, proposed to regulate immune responses. The CD1d1 crystal structure revealed a large hydrophobic binding site occupied by a ligand of unknown chemical nature. Mass spectrometry and metabolic radiolabeling were used to identify cellular glycosylphosphatidylinositol as a major natural ligand of CD1d1. CD1d1 bound glycosylphosphatidylinositol through its phosphatidylinositol aspect with high affinity. Glycosylphosphatidylinositol or another glycolipid could be a candidate natural ligand for CD1d1-restricted T cells.

The CD1 region encodes a family of evolutionarily conserved proteins that closely resemble the classical antigen presenting major histocompatibility complex (MHC) molecules (1, 2). CD1d controls the function of NK1+ natural T (NKT) cells (3, 4), an unusual subset of T lymphocytes that express receptors for natural killer cells (NKRP-1C) and T cells [αβ or γδ T cell receptor (TCR)] (5). NKT cells are thought to play an immunoregulatory role in responses to foreign and self antigens (6, 7). Maturation of NKT cells depends on the interaction of the TCR with CD1d1 (8-10). It is unclear whether this interaction requires the display of specific ligands in the CD1d1 groove akin to those presented by MHC molecules. Because the TCR repertoire of NKT cells is highly restricted (11-13), they probably interact with ligand-free CD1d1 or with CD1d1 displaying a highly conserved ligand. The three-dimensional structure of CD1d1 revealed a large hydrophobic binding site occupied by ligands whose chemical nature could not be determined with the diffraction data (2).

Natural ligands of CD1d1 were isolated from purified CD1d1 molecules expressed by TAP (transporters associated with antigen processing)-deficient human T2 cells infected with a recombinant vaccinia virus expressing the CD1d1 gene (3, 14). Low molecular weight ligands associated with CD1d1 and HLA class I molecules (the latter serving as controls) were isolated and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) as described (15, 16). Several peaks were observed at 210 nm (detects peptide and double bonds) that were associated only with CD1d1-derived material (Fig. 1A). Very little, if any, absorbance was detected at 254 or 280 nm (detects aromatic amino acids) (17). The major peak present in CD1d1-derived material (Fig. 1A, arrow) corresponds to a minor peak in the fractions from HLA-associated ligands. Each of the six fractions constituting the major peak (about 82 and 85 min) were refractionated by RP-HPLC with a mixture of C18 and cation-exchange (1:1) matrix (18). This yielded single peaks that behaved in an indistinguishable manner (17). Similar refractionation of the HLA class I–associated material eluting between about 82 and 85 min did not yield any signal (17), probably because the chemical constitution of this material prevented its recovery.

Figure 1

CD1d1 binds glycosylated PI. (A) RP-HPLC profile at A 210 of CD1d1 (solid line) and control endogenous HLA class I (broken line)–associated ligands isolated as described (16). An aliquot of each fraction eluting between 58 and 95 min of CD1d1 and HLA-associated ligands was analyzed by MALDI-MS. A complex array of ion spectra was observed for both samples (17). (B) MALDI-MS analysis of each of the fractions constituting a refractionated peak (16, 18) of the CD1d1-associated ligand showing a representative positive ion mode mass spectrum of the ligand that contains ions at m/z 614, 1211, and 1227. (C) A positive ion mass spectrum upon adding saturated ammonium sulfate results in a new peak at m/z 887 and the loss of ions atm/z 1211 and 1227. (D) The negative ion mode spectrum of sample in (C) in the presence of saturated ammonium sulfate. The peak at m/z 885.3 is the (MH)ion of the species observed at m/z 886 in the positive ion mode. An additional peak is observed at m/z 705.6 that corresponds to the loss of a hexose, presumably inositol. Note that different fragments derived from the same molecule form stable positive (C) and negative (D) ions. Together the positive and negative ion spectra provide complementary structural information.

Thus, the major peak eluting between about 82 and 85 min represents a CD1d1 ligand. To identify this material, we subjected it to matrix-assisted laser-desorption/ionization mass spectrometric (MALDI-MS) analysis (19). The positive ion mass spectra of each of the six refractionated fractions contained peaks atm/z 598, 614, 636, 1211, and 1227 (Fig. 1B), conclusively showing their identity to one another. MALDI-MS analysis after digestion of these samples with either carboxypeptidase or aminopeptidase indicated that the ligands associated with CD1d1 were not peptidic (17).

The CD1d1-associated natural ligand was incubated with ammonium sulfate (19) to enhance the ion signal in the mass spectrometer (20). This resulted in the loss of signals at m/z1211 and 1227 and the gain of signal at m/z 887 (Fig. 1C). The peak at m/z 887 was consistent with the mass of a protonated molecular ion (MH+) of a phospholipid (21). The negative ion mass spectrum (19) of the sample treated with ammonium sulfate revealed a (MH) ion of the same species at m/z 885.3 and a fragment ion atm/z 705.6 (Fig. 1D) resulting from the characteristic, and nearly diagnostic, loss of an inositol head group (22).

From these three mass spectra, one solution to the identity of the peak at m/z 1211 is that it is the MH+ ion for a phosphatidylinositol-diglycoside containing stearic and arachidonic acids, glucosamine, and mannose, with a calculated mass of 1211.4 daltons. Because it contains a negatively charged phosphate group, it is unstable and hence of relatively low intensity in the positive ion mass spectrum (Fig. 1B). Under high laser power it decomposes, cleaving the C2-C3 bond of glycerol to form a stable fragment ion at m/z 614 that lacks a phosphate. Them/z 614 ion was the most abundant ion in the mass spectrum and was used to determine the identities of the acyl groups as stearic acid and arachidonic acid. The peak at m/z 1227, 16 mass units greater than the peak at m/z 1211 (Fig. 1B), was a related structure that appears to result from the decomposition of larger glycosylphosphatidylinositol (GPI) structures. Treatment of the sample material with ammonium sulfate (Fig. 1, C and D) may have resulted in hydrolysis of the diglycoside linked to inositol, leading to the observation of the MH+ and (MH) ions of phosphatidylinositol (PI) in these two mass spectra, respectively. Thus, the material recovered from CD1d1 is probably GPI.

The biological significance of GPI association with CD1d1 was potentially undermined by the conditions of its isolation; namely, the use of detergent to solubilize CD1d1, vaccinia virus to express CD1d1, or the monoclonal antibodies (mAbs) used for HLA depletion and CD1d1 recovery. Therefore we repeated this analysis with CD1d1 molecules purified in a completely different manner. Soluble CD1d1 (sCD1d1), tagged with His6 at its carboxyl terminus, secreted by engineered mouse cells was purified by Ni-affinity chromatography (23). Several peaks at 210 nm were associated only with the ligands isolated from sCD1d1 [HPLC conditions were different from those used in the experiment in Fig. 1 (16, 18,23), which accounts for the different profile] but not from soluble H-2Db (Db-sol) (Fig.2A). MALDI-MS analysis of these peaks revealed ions with m/z identical to that observed with the ligand eluted from CD1d1 expressed by T2 cells (Fig. 2B) (17) as well as additional ions arising from larger GPI structures (Fig. 2B). Similar larger ion species, m/z> 1227, observed in Fig. 2B were also observed in the ion spectra described in Fig. 1 but were of lower intensity (17), probably owing to their loss or decomposition during multiple RP-HPLC. Additionally, the smaller ion species, m/z < 886, were also observed in the ion spectrum shown in Fig. 2B but were of very low intensity (17). Thus, the recovery of material from CD1d1, tentatively identified as GPI by MALDI-MS, occurs under different conditions of CD1d1 expression and purification and is a bona fide ligand for CD1d1 in cells. The identified natural CD1d1-associated ligand resembled the mammalian GPI moiety of the glycolipid-anchored proteins (24).

Figure 2

GPI is the natural ligand of CD1d1. (A) RP-HPLC profile at A 210 of the sCD1d1 (solid line) and control Db-sol (broken line)–associated ligands eluted on a different gradient than that used in Fig. 1A (16, 18, 23). (B) A representative positive ion mass spectrum of the sCD1d1-associated natural ligand. All sCD1d1-associated peaks analyzed showed similar ion spectra. In addition to the ions at m/z 887 and 1227, several new ion peaks were observed at m/z 1791.2, 1862.8, and 2022.5; the ion at m/z 2043 could be the MNa+ salt of the dominant ion at m/z 2022.5.

To independently confirm that CD1d1 binds GPI, cells expressing sCD1d1 and control cells expressing Db-sol were metabolically labeled with components of GPI: [3H]arachidonic acid, [3H]mannose, or [3H]ethanolamine (25). Each of the radiolabels was recovered with purified CD1d1 but not with Db-sol (Fig. 3A). Because [3H]mannose can also be incorporated into the carbohydrate modification of sCD1d1 heavy chain, the ligand associated with [3H]mannose-labeled sCD1d1 and Db-sol were separated from the heavy and light chains by Microcon-10 filtration (25). [3H]Mannose label was recovered only from sCD1d1 and not from Db-sol, which collected into the filtrate (Fig. 3A, insets), showing that mannose is incorporated into GPI.

Figure 3

(A) The core components of GPI are associated with CD1d1. Cells expressing sCD1d1 and Db-sol were metabolically labeled with the indicated 3H-labeled compound. CD1d1 (left) and Db-sol (right) were Ni-affinity– and immunoaffinity purified, respectively, and the amount of radioactivity incorporated was monitored by scintillation counting (25). The background was derived from Db-sol supernatant (left) and sCD1d1 supernatant (right) that nonspecifically bound to Ni-Sepharose and B22-249–coupled protein A–Sepharose, respectively. sCD1d1 elutes into fractions 2 and 3 and Db-sol elutes into fractions 5, 6, and 7. [3H]Mannose-labeled ligand was separated from sCD1d1 and Db-sol by Microcon-10 filtration. The label was recovered from the filtrate of only CD1d1-associated material (insets). Thus, the label was incorporated into GPI. (B) CD1d1 specifically binds GPI through its PI aspect but does not bind H-2Db, a classical antigen presenting class I molecule, or immunoglobulins (17). (C) Scatchard analysis of [3H]PI binding to sCD1d1 (26) revealed a dissociation constant of ∼0.4 μM.

Based on the hydrophobic nature of the CD1d1 ligand binding site (2), GPI would be predicted to bind CD1d1 through its PI group. This was tested in an in vitro binding assay (26), which revealed that [3H]PI specifically bound sCD1d1 but not H-2Db (Fig. 3B). Binding of [3H]PI to sCD1d1 occurred in the micromolar range (17) with a dissociation constant of ∼0.4 μM calculated by Scatchard analysis (Fig. 3C).

Thus, GPI is a major detectable ligand of CD1d1, representing >90% of the low molecular weight material recovered (27). Peptides were previously identified as CD1d1 ligands (28). These peptides are highly hydrophobic, contain numerous aromatic residues (28), and would therefore be detected by absorption at 254 or 280 nm. Our recovery of extremely small amounts of CD1d1 ligands that absorb at 254 and 280 nm suggests that similar cellular peptides are not major natural ligands for CD1d1. The ubiquitous occurrence of GPI in different cell types is consistent with the pattern of NKT cell recognition of CD1d1 molecules. Alternatively, GPI may provide a chaperone function by occupying the groove of nascent CD1d1 molecules, preserving the folded conformation until an alternative self or a foreign glycolipid is loaded in a distal secretory compartment. Because GPI is present in the lumen of the endoplasmic reticulum (ER), where it is added to the carboxyl terminus of glycolipid-anchored proteins (29), GPI binding to CD1d1 molecules could occur there. This is consistent with the finding that sCD1d1 also naturally associates with GPI. CD1d1 molecules, like other CD1 family members, possess a motif in their cytosolic tails that direct them to endosomes (30), presumably for the loading of exogenous antigens. In this case, ER-loaded GPI would have to be removed to allow for association with the exogenous ligand. A specific mechanism may be required for this process, akin to the removal of invariant chain–derived CLIP peptide (MHC class II–associated invariant chain–derived peptide) from newly assembled MHC class II molecules by H2-M or HLA-DM glycoproteins (31).

  • * To whom correspondence should be addressed. E-mail: sjoyce{at}


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