Endogenous phosphatidylcholine and a long spacer ligand stabilize the lipid-binding groove of CD1b

Abstract

CD1 proteins present lipid antigens to T cells. The antigens are acquired in the endosomal compartments. This raises the question of how the large hydrophobic CD1 pockets are preserved between the moment of biosynthesis in the endoplasmic reticulum and arrival to the endosomes. To address this issue, the natural ligands associated with a soluble form of human CD1b have been investigated. Using isoelectric focusing, native mass spectrometry and resolving the crystal structure at 1.8 A resolution, we found that human CD1b is simultaneously associated with endogenous phosphatidylcholine (PC) and a 41-44 carbon atoms-long spacer molecule. The two lipids appear to work in concert to stabilize the CD1b groove, their combined size slightly exceeding the maximal groove capacity. We propose that the spacer serves to prevent binding of ligands with long lipid tails, whereas short-chain lipids might still displace the PC, which is exposed at the groove entrance. The data presented herein explain how the CD1b groove is preserved, and provide a rationale for the in vivo antigen-binding properties of CD1b.

Figure 1

IEF analysis of shCD1b lipid binding. (A) SDS–PAGE gel (12%) of purified shCD1b expressed in mouse cells (first lane) or in insect cells (third lane). Note that shCD1b heavy chains are glycosylated. The middle lane contains the molecular weight marker: from top to bottom 108, 90, 50.7, 35.5, 28.6 and 21.2 kDa. (B) IEF gel of mouse-expressed shCD1b (top) and insect-expressed shCD1b (bottom) after incubation with 50 μM of the indicated lipids. (C) IEF gel after incubation of mouse-expressed shCD1b with no detergent (first lane), or with Triton X-100, CTAB or C₁₂DAO. The four lanes to the right are from a similar experiment using mouse-expressed shCD1b that had been preloaded with PI and purified by size-exclusion chromatography.

Figure 2

MS detection of natural ligands associated to shCD1b. (A) Positive-ion mode native ESI-MS from mouse-expressed endoF₃-deglycosylated shCD1b. Ion peaks arising from shCD1b, shCD1b:UL (unknown ligand), shCD1b:PC (phosphatidylcholine) and shCD1b:UL:PC species are labeled A, B, C and D, respectively. The labels are completed with a number indicative of their multi-charged state. (B) Neutral mass spectrum obtained by deconvolution, averaging over all charged states, of the spectrum of panel A. This spectrum was recorded using harsh desolvation energy (black line). A second deconvoluted spectrum was obtained by repeating the experiment at lower energy (gray line). The lower desolvation of the protein induces a broadening of the signals and a 100–150 Da displacement towards higher values. (C) The D14 precursor ion of panel A (indicated with an asterisk) was selected and subjected to tandem MS. Three other major species appear in the high-mass range, originating from the loss of either PC (−PC) or UL (−UL), or both (−UL, −PC). Signals corresponding to the UL and PC ligands were also detected in the low-mass range of this positive-ion mode ESI-mass spectrum. This mass region is enlarged in the inset. The y-axis scale in the mass range 540–700 m/z is zoomed five-fold in this inset.

Figure 3

The C′ portal is closed in natively folded hCD1b. Representation of the protein surface at the entry portal to the C′ channel, as observed in the crystal structure of hCD1b in complex with PI (A), or in the structure of natively folded hCD1b (B). This portal was proposed to allow egress of the lipid (shown in yellow) from the interior of the protein. The Cα and side-chain atoms of residues lining the portal are drawn as balls and sticks and colored in blue. The separation between the Cα atoms of Arg159 and Ala129 is indicated.

Figure 4

Natural ligands in the binding groove of shCD1b. (A) Electron density for the PC and the spacer molecule in the shCD1b crystal structure. Both lipids are drawn as sticks. The PC is colored by atom type (carbon green, oxygen red, phosphorous violet and nitrogen blue), the spacer molecule in yellow. The electron density is from the final 2F_o−F_c map contoured at 1.0σ and represented as a blue mesh. Letter labels indicate the groove channels occupied by the close portions of electron density. The inset shows the same map for the polar head region of PC, contoured at 0.6σ. (B) Disposition of ligands in the CD1b lipid groove. The crystal structures of shCD1b presented in this work (cyan cartoon representation) and of hCD1b in complex with PI (in blue) were superimposed. For clarity, the helices of the α2 domain are represented as loops. PC and UL ligands are represented as balls and sticks and colored as in panel A. The PI and the two detergents from the CD1b:PI structure are represented as sticks. The polar head of PI is colored by atom type, the fatty acid chains in violet and the two detergent molecules in orange. The distance between the Cα atom of Ile99 and the topmost atom of the glycerol moiety of PI or PC is indicated for both structures. The inset shows a top view of the region where the acyl chains of PC and PI stop, at the connection between the A′ channel and the T′ tunnel.