boCD1d-down: 5′-gactgtcgacatgcggtacctaccatggctgttgctgtgg gcattcctacaggtctggggacaatctccagccccgcaaacgcc-3′.

boCD1ab-up: 5′-gaggatccttagtgatggtgatggtgatgccagtagaggatgatgtcctgg-3′.

b2Mfus-down: 5′-atacaattgatccagcgtcctccaaagattc-3′.

b2Mfus-up: 5′-ttgcggccgcgatgatcctcctccgcttcctgatcctccgcttcctcctcctcccatgtctcgatcccacttaac-3′.

boCD1dfus-f: 5′-aagcggccgcaagccccgcaaacgcctttc-3′.

boCD1dfus-r: 5′-ataggatccgcgcggcaccagtccccagtacaggatgatgtcctg-3′.

mCD1d-D153N-f: 5′-tcaaagtgctcaacgctaatcaagggacaagtgca-3′.

boCD1d-N151D: 5′-caaggtgctcaatcaggaccaagggaccaagga-3′.

boCD1d-QN-AD: 5′-ggtcatcaaggtgctcaatgcggaccaagggaccaagg-3′.

The boCD1d ectodomain, in which the endogenous boCD1d leader sequence was replaced with that of boCD1b3 was amplified by PCR using the primers boCD1d-down and boCD1ab-up. The resulting PCR fragment was restricted with SalI and SphI and ligated into the boCD1b3 containing dual-promotor baculovirus transfer vector pBACp10pH by replacing the boCD1b3 fragment [10]. The resulting plasmid contained both boCD1d with a C-terminal hexa-histidine tag, as well as bovine β2-microglobulin (boβ2M). This construct was used for crystallization.

For functional studies boCD1d was also expressed as a single chain Fc-fusion protein, in which boβ2M (without leader sequence, amplified using the primers b2M-fus-down and –up) was connected to the N-terminus of the boCD1d heavy chain through a (G₄S)₃ amino acid linker contained within the primer b2Mfus-up, while the C-terminus of the heavy chain (amplified with primers boCD1dfus-f and-r) was further fused to the Fc-region of human IgG1 that had previously been cloned into the baculovirus transfer vector pAcGP67A (BD Biosciences). The recombinant soluble bovine CD1d-β2m heterodimeric molecule without Fc tag was produced to homogeneity using the baculovirus expression system as previously described for mouse CD1d [17] and stored at −80°C for lipid loading studies and crystallization. The single chain boCD1d-Fc fusion protein was purified from a 5 L culture of Spodoptera frugiperda (SF9) insect cells (EMD Biosciences) that had been infected with the corresponding recombinant baculovirus at an MOI ∼3 for 72h at 27.4°C. Briefly, SF9 cells were removed from the 5 L culture by gentle centrifugation (1,000×g, 10 min) and the supernatant concentrated to 300 mL using a tangential flow through filtration device (Pelllicon 2, Millipore) while exchanging the buffer to PBS. The boCD1d-Fc fusion protein containing supernatant was passed through a protein A column (GE Healthcare, Hi Trap™ rProtein A), washed with 3 column volumes of PBS and eluted with 20 mM glycine, 50 mM NaCl, pH 3.0. Fractions of 0.7 ml were collected into tubes prefilled with 0.3 ml 1 M Tris pH 8.5 for neutralization. Fractions containing boCD1d-Fc were analyzed by 4–20% SDS-PAGE, pooled and buffer exchanged against PBS, using centrifugal filtration devices (Amicon, 30 kDa molecular weight cut-off).

(2R,3S,4S)-3,4-di-O-benzyl-1-O-(2,3-di-O-benzyl-4,6-O-benzylidene-α-D-galactopyranosyl)-2-palmitoylamino-1,3,4-octadecanetriol. To a solution of known azide (1, Figure 1c) (150 mg, 0.16 mmol) in 1.6 ml tetrahydrofuran (THF) at room temperature, a 1M solution of PMe₃ in THF (1.6 ml, 1.6 mmol) was added dropwise. After stirring for 2.5 h at room temperature, 2.9 ml of a 1M NaOH solution was added and the reaction mixture was allowed to stir for an additional 2h. The reaction was extracted with EtOAc and the combined organic layer was washed with a saturated aqueous NaCl solution (brine), dried over Na₂SO₄, filtered, and evaporated to afford the crude amine. A mixture of the crude amine, 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC, 60 mg, 0.31 mmol) and palmitic acid (80 mg, 0.31 mmol) in 2,4 ml dichloromethane (DCM) was stirred overnight at room temperature. The reaction mixture was diluted with DCM, washed with H₂O (2×5 ml) and brine (1×5 ml), dried over Na₂SO₄, filtered, and evaporated to dryness. Purification of the residue by flash chromatography (silica gel, hexane:EtOAc 7∶3) afforded 131 mg of compound 2 (Figure 1c) (72% yield) as a white solid. Exact mass (ESI-MS) for C₇₅H₁₀₈NO₉ [M+H]⁺ found, 1166.8021 calculated, 1166.8024.

(2R,3S,4S)-1-O-(α-D-galactopyranosyl)-2-palmitoylamino-1,3,4-octadecanetriol (C_16∶0-αGalCer). A solution of 2 (130 mg, 0.11 mmol) in 4 ml EtOH/CH₃Cl 3∶1 was hydrogenated for 1 h under atmospheric pressure in the presence of Pd black (20 mg). The solution was diluted with 5 ml of pyridine, filtered through celite and evaporated to dryness. The residue was purified by flash chromatography (silica gel, DCM:MeOH 9∶1) to give 46 mg of compound 3 (58% yield) as a pale yellow solid.

¹H NMR (300 MHz, pyridine-d₅) δ ppm 0.76–1.10 (m, 6 H, CH₃ terminal) 1.15–1.48 (m, 46 H, CH₂) 1.61–1.99 (m, 6 H, CH₂) 2.29 (br s, 1 H, OH) 2.46 (t, J = 7.4 Hz, 2 H, H-2′) 4.25 - 4.49 (m, 6 H, H-3″, H-4″, H-5″, H-3, H-4 and H-6″) 4.53 (t, J = 6.2 Hz, 1 H, H-3″) 4.57 (d, J = 3.1 Hz, 1 H, Ha-1) 4.63–4.73 (m, 2H, Hb-1 and H-2″) 5.24–5.34 (m, 1 H, H-2) 5.59 (d, J = 3.7 Hz, 1 H, H-1″) 8.48 (d, J = 8.5 Hz, 1 H, NH(CO)).

¹³C NMR (75 MHz, pyridine-d₅) δ ppm 11.52, 14.60, 14.69, 23.35, 23.62, 24.47, 26.80, 26.92, 29.57, 30.03, 30.16, 30.22, 30.27, 30.34, 30.40, 30.41, 30.44, 30.56, 30.78, 31.08, 32.54, 34.78, 37.21, 39.48, 51.83, 63.08, 68.58, 69.06, 70.71, 71.42, 72.02, 72.91, 73.45, 77.15, 101.94, 173.63.

Exact mass (ESI-MS) for C₄₀H₇₉NNaO₉ [M+Na]⁺ found, 740.5665 calculated, 740.5653.

Brain porcine sulfatide extract, C₁₂-di-sulfatide and total brain gangliosides were purchased from Avanti Polar Lipids Inc. and purified trisialoganglioside G_T1b from bovine brain was obtained from Sigma Aldrich (minimum purity 96%). Lipids were dissolved at 1 mg/ml in DMSO to assess in vitro loading of antigens by boCD1d. Aliquots of 5 µg of purified boCD1b3 at a concentration of 10 µM were loaded overnight at room temperature in the presence of 30 or 60 µM of each ligand (in DMSO, final concentration of DMSO 3–5%) with or without Tween 20 at a final concentration of 0.05%. Loading was performed in the presence of 100 mM TrisHCl pH 7. The binding of charged lipids was assessed by isoelectric focusing on a PhastGel IEF 3–9 using a PhastSystem (GE Healthcare) followed by staining with Coomassie dye.

Crystallization was performed in a 96-well format by a nanoliter dispensing liquid handling robot (Phoenix, Art Robbins Ltd.) using commercially available crystallization screens (PEG/Ion I & II, Wizard I & III, Hampton Research and Emerald Biosciences). Sitting drops of 100 nl precipitant were overlayed with 100 nl of protein solution (6.7 mg/ml in 10 mM Hepes, 30 mM NaCl, pH 7.5). A single diffraction quality crystal of boCD1d-C₁₆-αGalCer was obtained from the PEG/Ion screen (20% polyethylene glycol 4000, 0.2 M potassium sulfate) and used for subsequent data collection. Promising conditions for the boCD1d-di-SLF complex were optimized manually and diffraction quality crystals were grown by sitting drop vapor diffusion at 22°C, mixing 0.5 µl of protein at 6.7 mg/ml with 0.5 µl of precipitant (20% polyethylene glycol 3350, 0.2 M sodium formate).

Crystals were flash-cooled at 100K in crystallization solution containing 20% glycerol. Diffraction data of the bo-CD1d-di-SLF crystal were collected at beamline 5.0.3 of the Advanced Lightsource (ALS, Berkeley, CA) and data of the boCD1d-C₁₆-αGalCer crystal were collected at beamline 11.1 at the Stanford Synchrotron Radiation Lightsource (SSRL, Stanford, CA) and processed with the HKL2000 software to a resolution of 2.9 Å and 2.4 Å, respectively [18]. Molecular replacement was performed using Phaser [19], and the homology model of boCD1d generated from the boCD1db3 structure (PDB code 3L9R) using the Swiss Model server [20]. The asymmetric unit (ASU) of the boCD1d-di-SLF crystal contained two CD1d-β2m heterodimers, while the ASU of boCD1d-C₁₆-αGalCer contained one heterodimer. The initial phases derived from the molecular replacement solution were refined using maximum-likelihood restrained refinement in Refmac [21]. Refinement was intercalated with rounds of manual model building into 2F_o-F_c and F_o-F_c maps in Coot [22]. Tight NCS constraints between the two boCD1d-di-SLF molecules present in the ASU were imposed and maintained throughout refinement. TLS refinement was performed at later stages for the boCD1d-C₁₆-αGalCer structure in Refmac [21], [23]. Three TLS groups per boCD1d-β2M complex were defined, containing the α1, α2 domains and the C₁₆-αGalCer ligand (group 1), the α3 domain (group 2) and the β2m chain (group 3). A set of ∼ 1000 reflections (4%) were set aside for the calculation of R_free to monitor refinement progress. The quality of the final model was assessed with Molprobity [24]. Data collection and refinement statistics are presented in Table 1.

Structure factors and coordinates for the boCD1d-C₁₆-αGalCer and boCD1d-di-SLF structures have been deposited into the PDB database (http://www.rcsb.org/) with accession codes 4F7E and 4F7C, respectively.

The cell-free Ag presentation assay for stimulation of mouse iNKT cell hybridomas by recombinant mouse, human and bovine CD1d was carried out according to published protocols [25], [26] with the following modifications. Briefly, 1 µg soluble hCD1d, mCD1d, mCD1d Asp153Asn, boCD1d, boCD1d Asn151Asp and boCD1d Asn151Asp/Gln150Ala proteins were coated in 96-well flat-bottom plates at 37 °C for 1 h. Plates were blocked for 1 h with PBS containing 10% FBS and 100µl of glycolipids αGalCer (KRN7000) or C₁₆-αGalCer (dissolved in 0.05% Tween 20 and 0.9%NaCl) were added to each well at various concentrations and incubated for 20 h at 37 °C. After washing, 5x10⁴ hybridoma cells in 200 µl complete media were added to each well and incubated at 37 °C for 16 h in a CO2 incubator. IL-2 release in the supernatant (100 µl) was measured in a sandwich ELISA, as previously described [25], [26].

Lipid structures were prepared in ChemDraw (CambridgeSoft), all molecular representations were prepared using PyMol (Schroedinger). Protein cavities were prepared using the fpocket webserver (http://bioserv.rpbs.univ-paris-diderot.fr/fpocket/) [27] and visualized in PyMol. Lipid binding groove volumes were calculated using the CASTp server (http://sts-fw.bioengr.uic.edu/castp/) [28].

Bovine CD1d (boCD1d) was previously considered a pseudogene but is translated and expressed at the cell surface, when transfected into 293 T cells using the native CD1d leader peptide [8], [15]. To investigate whether boCD1d can bind glycolipids in vitro, we have recombinantly expressed the ectodomain of boCD1d in insect cells and assessed the glycolipid binding properties of boCD1d using native isoelectric focusing gel electrophoresis. Several charged ligands (Figure 1a) were incubated with boCD1d and successful loading was visualized by native IEF gel (Figure 1b). Loading of a negatively charged lipid resulted in a gel shift toward the cathode, caused by the charge difference of the CD1d-lipid complex. While boCD1d bound C_12∶0-di-sulfatide, total gangliosides and GT1b, it failed to bind bovine brain sulfatides (Figure 1B). Besides differences in the headgroups, which generally do not affect the ability of the antigen to bind to CD1, the glycolipids only differ in the length and saturation of the fatty acid, whereas the long chain base (sphingosine) is identical (Figure 1A). While gangliosides and GT1b contain predominantly C_18∶0 fatty acids, di-sulfatide is significantly shorter (C_12∶0) and the fatty acids of bovine brain sulfatides typically range from 22 to 24 carbons in length. This indicated that bovine brain sulfatides could potentially exceed the chain length for binding to boCD1d. For comparison, the lipid binding groove of mouse and human CD1d can accommodate fatty acids with up to 26 carbons in the A’ pocket [17], [29], [30]. Next, we tested whether increasing the molar ratio of C_12∶0-di-sulfatide and/or including 0.05% Tween 20 detergent during lipid loading could increase the loading efficiency (Figure 1B, right panel). Unexpectedly, increasing lipid excess did not increase loading efficiency, which already appeared optimal, while in contrast, the addition of Tween 20 prevented C_12∶0-di-sulfatide binding altogether. This observation could be specific for C_12∶0-di-sulfatide and indicates that this lipid is predominantly trapped in mixed lipid/detergent micelles, therefore being unavailable for loading to boCD1d. BoCD1d ran as a triplet on the IEF gel, with the majority of the protein migrating as the center band. Upon addition of Tween 20, however, the middle band remains unchanged, while the fainter top and bottom bands disappeared, potentially indicating extraction of single positively and negatively charged endogenous lipids that had been acquired during protein expression and folding in the ER of insect cells.

To address the question of why boCD1d does not bind bovine brain sulfatides to a measurable degree, we crystallized boCD1d in complex with synthetic C_12∶0-di-sulfatide (di-SLF) and determined the crystal structure to a resolution of 2.9 Å (Table 1 and Figure 2A). The obtained structural information encouraged us to synthesize a shorter form of αGalCer with a C_16∶0 instead of the typical C_26∶0 fatty acid (Figure 1C), which we also crystallized bound to boCD1d. The crystal structure of the boCD1d-C_16∶0-αGalCer complex was determined to a resolution of 2.4 Å (Table 1 and Figure 2B). The asymmetric unit of the boCD1d-di-SLF crystal contained two separate boCD1d-boβ2M-di-SLF complexes that are very similar, especially in the presentation of the bound glycolipid. Therefore, we will present structural information for only one complex. The overall structure of boCD1d is very similar to that of other CD1 or MHC I molecules, in which the boCD1d heavy chain is organized in 3 domains, α1 and α2 that form the central binding groove and α3, which non-covalently associates with boβ2M (Figure 2) [1]. The glycolipids are bound in the hydrophobic binding groove that can further be divided into the two main pockets, A and F. While the A pocket binds the acyl chain of each glycolipid, the F pocket binds the sphingosine, leaving the structurally distinct carbohydrate headgroups exposed above the boCD1d binding groove for immune recognition by cognate T cells (Figure 2). Unambiguous electron density is observed for the galactosyl-di-sulfate headgroup and the C_12∶0 fatty acid of di-sulfatide, while in case of C_16∶0 -αGalCer, electron density is well defined for the entire lipid backbone but less well ordered for the galactose headgroup (Figure 2C, D and Figure S1). This is in contrast to the well ordered electron density of the αGalCer headgroup, when presented by either mouse or human CD1d, indicating that the interaction between the galactose headgroup and boCD1d is not conserved with that of human or mouse CD1d [29], [30]. Details about the headgroup presentation by boCD1d will be discussed later.

The boCD1d α1-α2 superdomain (residues 1–180) shares 60% sequence identity (BLAST search) with human CD1d and 55% with mouse CD1d. Superposition of the entire boCD1d heavy chain from the di-sulfatide complex with that of huCD1d and mCD1d yielded rmsd values of 0.92 Å (PDB 1ZT4) and 1.13 Å (PDB 1Z5L), respectively, indicating a very similar topology of the boCD1d molecules. Interestingly, superposition of both boCD1d crystal structures resulted an rmsd of 0.62 Å, suggesting a degree of flexibility of the binding groove when it accommodates different glycolipids.

Cross-species conserved N-linked oligosaccharides are observed at residues Asn20 and 42, while the third conserved site on the α2-helix of CD1d (N163 in human, N165 in mouse) is lost in boCD1d (H163) (Figure 2).

Our in vitro lipid loading studies indicated that lipids with fatty acid chains lengths of 24 carbons or more, such as C_24∶1-sulfatide or αGalCer, are unable to bind to boCD1d. As the acyl chain is bound within the A’ pocket of CD1d, we compared the A’ pockets of boCD1d containing C12∶0-di-sulfatide or C₁₆-αGalCer with that of human CD1d containing full-length C_26∶0-αGalCer (Figure 3A–D). We made two observations that have not been observed before for any CD1d molecule. Firstly, the shape and size of the boCD1d A’ pocket differs when either C_12∶0-di-sulfatide or C₁₆-αGalCer are bound indicating flexibility within the A’ pocket (Figure 3B, C). This is a result of a different conformation of the conserved residue Trp40, which can either increase the size of the small side pocket, likely induced through contacts with the terminal carbons of the C_12∶0 acyl chain of di-sulfatide, or restrict the size when the longer C_16∶0 fatty acid is further inserted into the Á pocket. Secondly, the conserved disulfide bond in human and mouse CD1d that anchors the α2-helix to the β-sheet platform (Cys166-Cys102) is absent in boCD1d. Here, Trp166 adopts a rare rotamer, stabilized by interaction with the side chain of Thr100, pointing inside the A’ pocket and as such blocking the remainder of the A’ pocket (Figure 3D). This results in a shorter, more restricted A’ pocket of boCD1d, which can maximally bind glycolipid antigens with an acyl chain not exceeding 18 carbons in length. The restricted A’ pocket also results in a smaller boCD1d lipid binding groove, which is 1680 ų in volume, compared to mouse (1970 ų) and human (1960 ų) CD1d.

The di-sulfo-galactosyl headgroup of di-SLF is presented in the for β-anomeric glycolipids typical upright orientation, between the α1- and α2 helices and stabilized through an intricate network of H-bonds that involve the core residues of CD1d (Arg79, Asp80, Asn151 and Thr154), as well as the polar moieties of the di-SLF ligand (Figure 4A). The 3′-SO₄ group contacts Asn151, while the 6′-SO₄ group is bound through a water-mediated hydrogen (H) bond to Arg79. The ceramide lipid backbone is oriented inside the binding groove by an H-bond between Thr154 and the N-amide nitrogen, as well as by Asp80 of CD1d binding the 3″-OH group of sphingosine. A total of 6 H-bonds are formed between boCD1d and di-SLF (four with the headgroup and two with the lipid), which gives rise to ordered electron density for the headgroup (Figure 2C). Overall, the orientation of di-SLF in the boCD1d binding groove, and the presentation at the opening of the groove is conserved with the presentation of sulfatide by mCD1d (Figure 4C).

As human and mouse iNKT cells are cross-reactive in regards to αGalCer, we investigated whether the presentation of C_16∶0-αGalCer by boCD1d is altered compared to that of either human or mouse CD1d. Indeed, the binding and presentation of C_16∶0-αGalCer is not conserved with that of either mouse or human CD1d (Figure 4B, D). BoCD1d presents C_16∶0-αGalCer in an untypically tilted orientation. The only polar interactions between C_16∶0-αGalCer and boCD1d is through α1-helix residue Arg79 that interacts with the 2′-OH of galactose, as well as the contact formed between Asp80 and the 3″-OH of the phytosphingosine chain. Loss of the intimate contacts between the α-anomeric galactose and the helix is likely caused by the exchange of Asp151 to Asn151. This otherwise conserved aspartate residue on the α2-helix (Asp 151 in hCD1d and Asp153 in mCD1d) is critical for the presentation of αGalCer and related antigens, by CD1d, as it binds the 2″- and 3″-OH of the galactose in human and mCD1d and substitutions to either tyrosine or alanine abrogate iNKT cell activation [31] (and data not shown).

We next assessed, whether mutating Asp153 of mouse CD1d to Asn (as found in boCD1d) alone is sufficient for the loss of mouse iNKT cell activation and conversely, if re-constitution of Asp151 residue in boCD1d (to mimic mouse and human CD1d) can restore mouse iNKT cell activation in a cell-free antigen-presentation assay (Figure 5). We also prepared the boCD1d double mutant Asn151Asp/Gln150Ala, which further introduces a mouse CD1d residue in an area that is in close contact with the CDR3β loop of the mouse iNKT cell hybridoma 2C12 [32]. While both human and mouse CD1d can activate the two mouse iNKT cell hybridomas 2C12 as well as 1.2 by presenting either full-length αGalCer or C₁₆-αGalCer, the mCD1d Asp153Asn mutant and all the bovine CD1d proteins (wt and mutants) are unable to activate both mouse iNKT cell hybridomas (Figure 5, and data not shown). Both hybridomas respond equally well to αGalCer when presented by either human or mCD1d, while 1.2 is activated better when C₁₆-αGalCer is presented by human CD1d. Our data further indicates that alteration of Asp153 of mouse CD1d is not tolerated, not even when replaced with similar amino acids, such as asparagine, thereby explaining the inability of boCD1d to activate murine NKT cell. However, replacing Asn151 of boCD1d with aspartate does not reconstitute cross-species reactivity, indicating that other amino acids that differ between mouse and bovine CD1d likely are equally important for either optimal glycolipid presentation or iNKT TCR binding to CD1d. Surprisingly, however, superposition of bovine CD1d onto the structure of the mouse CD1d-αGalCer –Vα14Vβ8.2 TCR complex (PDB ID 3HE6) [33], as well as the human ortholog complex (PDB ID 3HUJ) [34], did not reveal any other obvious amino acids that are conserved between mouse and human CD1d but differ with boCD1d, and which would prevent binding of the mouse iNKT TCR onto boCD1d, raising the question as to what residues actually limit cross-species reactivity.