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 Å 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.

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

“…This coil-helix transition upon ligand binding may to some degree explain the ''tightening'' of secondary structure we observe when SCP2 is loaded with stearoyl CoA. ''Soft'' ESI-MS has frequently been used to study biomolecular complexes in a nearly native state in the gas phase, for example protein/protein [35], enzyme/cofactor [36] and protein/lipid [25,37]. We have adapted this kind of assay to analyse SCP2/lipid.…”

The cellular and biochemical rules of lipid antigen presentation

“…On the contrary, soluble CD1d that is immediately secreted upon ER exit without trafficking to Ly mostly associates with phosphatidylinositol [23,24] and sphingomyelin [22], whereas CD1d that is retained within the ER is associated with PC [22]. Soluble CD1b secreted without lysosomal recycling is associated with phosphatidylinositol [23] and PC [25]. Native mass spectrometry analysis of the secreted CD1b-lipid complex has also revealed the concomitant presence of an additional ''spacer'' molecule whose nature remains unknown [25].…”

“…Endogenous lipids bind during CD1 assembly and probably prevent their collapse. CD1b binds PC together with a 42-44 carbon-long molecule that remains embedded into the lipid-binding groove [25]. Mouse CD1d associates with PC and a C 16 -long fatty acid [32,38].…”

“…The ER membrane is thinner than other cell membranes, is poor in lipids that facilitate its packing and thus allows fast translocation of PL even in the absence of dedicated flippases. This mechanism may explain why PC, which is one of the most abundant PL, associates with nascent CD1b and CD1d molecules [25,32]. Ceramide, which constitutes the apolar component of GSL, is also synthesized on the cytoplasmic leaflet of ER and then transported to the cytoplasmic leaflet of cis-Golgi membranes where glucosylceramide is synthesized.…”

“…Soluble CD1b secreted without lysosomal recycling is associated with phosphatidylinositol [23] and PC [25]. Native mass spectrometry analysis of the secreted CD1b-lipid complex has also revealed the concomitant presence of an additional ''spacer'' molecule whose nature remains unknown [25].Whether these endogenous lipids are involved in thymic selection of CD1-restricted T cells and can be recognized in some autoimmune diseases remains unknown. …”