Design of Natural Killer T Cell Activators: Structure and Function of a Microbial Glycosphingolipid Bound to Mouse CD1d

• Published in: Proceedings of the National Academy of Sciences - PNAS 2006-03, Vol.103 (11), p.3972-3977
• Main Authors: Wu, Douglass, Zajonc, Dirk M., Fujio, Masakazu, Sullivan, Barbara A., Kinjo, Yuki, Kronenberg, Mitchell, Wilson, Ian A., Wong, Chi-Huey
• Format: Article
• Language: English
• Subjects: Animals; Antigens; Antigens, CD1 - chemistry; Antigens, CD1 - metabolism; Antigens, CD1d; BASIC BIOLOGICAL SCIENCES; Binding Sites; Biochemistry; BIOLOGICAL FUNCTIONS; Biological Sciences; Fatty acids; Glycolipids; Glycosphingolipids; Glycosphingolipids - chemistry; Glycosphingolipids - metabolism; Hydrogen Bonding; Hydrogen bonds; Immunology; In Vitro Techniques; Killer Cells, Natural - immunology; Killer Cells, Natural - metabolism; Ligands; LIPIDS; Lymphocyte Activation; Macromolecular Substances; MICE; Models, Molecular; Molecular Structure; MORPHOLOGY; MORTALITY; Natural killer T cells; Other,OTHER; Physical Sciences; Protein Structure, Quaternary; Rodents; Sphingomonas; Sugars; T cell antigen receptors; T-Lymphocytes - immunology; T-Lymphocytes - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0600285103

Natural killer T (NKT) cells provide an innate-type immune response upon T cell receptor interaction with CDld-presented antigens. We demonstrate through equilibrium tetramer binding and antigen presentation assays with Vα14i-positive NKT cell hybridomas that the Sphingomonas glycolipid ct-galacturonosyl ceramide (GalA-GSL) is a NKT cell agonist that is significantly weaker than α-galac-tosylceramide (α-GalCer), the most potent known NKT agonist. For GalA-GSL, a shorter fatty acyl chain, an absence of the 4-OH on the sphingosine tail and a 6'-COOH group on the galactose moiety account for its observed antigenic potency. We further determined the crystal structure of mCDld in complex with GalA-GSL at 1.8-Å resolution. The overall binding mode of GalA-GSL to mCDld is similar to that of the short-chain α-GalCer ligand PBS-25, but its sphinganine chain is more deeply inserted into the F' pocket due to alternate hydrogen-bonding interactions between the sphinganine 3-OH with Asp-80. Subsequently, a slight lateral shift (>1 Å) of the galacturonosyl head group is noted at the CD1 surface compared with the galactose of α-GalCer. Because the relatively short$C_{14}$fatty acid of GalA-GSL does not fully occupy the A' pocket, a spacer lipid is found that stabilizes this pocket. The lipid spacer was identified by GC/MS as a mixture of saturated and monounsaturated palmitic acid ($C_{16}$). Comparison of available crystal structures of a-anomeric glycosphingolipids now sheds light on the structural basis of their differential antigenic potency and has led to the design and synthesis of NKT cell agonists with enhanced cell-based stimulatory activities compared with α-GalCer.