Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system.

CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and ß2-microglobulin (ß2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with ß2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 µg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

Structural basis of NKT cell inhibition using the T-cell receptor-blocking anti-CD1d antibody 1B1.

Natural killer T (NKT) cells are a subset of T lymphocytes that recognize glycolipid antigens presented by the CD1d molecule (CD1d). They rapidly respond to antigen challenge and can activate both innate and adaptive immune cells. To study the role of antigen presentation in NKT cell activation, previous studies have developed several anti-CD1d antibodies that block CD1d binding to T-cell receptors (TCRs). Antibodies that are specific to both CD1d and the presented antigen can only be used to study the function of only a limited number of antigens. In contrast, antibodies that bind CD1d and block TCR binding regardless of the presented antigen can be widely used to assess the role of TCR-mediated NKT cell activation in various disease models. Here, we report the crystal structure of the widely used anti-mouse CD1d antibody 1B1 bound to CD1d at a resolution of 2.45 Å and characterized its binding to CD1d-presented glycolipids. We observed that 1B1 uses a long hydrophobic H3 loop that is inserted deep into the binding groove of CD1d where it makes intimate nonpolar contacts with the lipid backbone of an incorporated spacer lipid. Using an NKT cell agonist that has a modified sphingosine moiety, we further demonstrate that 1B1 in its monovalent form cannot block TCR-mediated NKT cell activation, because 1B1 fails to bind with high affinity to mCD1d. Our results suggest potential limitations of using 1B1 to assess antigen recognition by NKT cells, especially when investigating antigens that do not follow the canonical two alkyl-chain rule.

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

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.