Abstract
Natural killer T (NKT) cells express a semi-invariant Vα14 T cell receptor (TCR) and recognize structurally diverse antigens presented by the antigen-presenting molecule CD1d that range from phosphoglycerolipids to α- and β-anomeric glycosphingolipids, as well as microbial α-glycosyl diacylglycerolipids. Recently developed antibodies that are specific for the complex of the prototypical invariant NKT (iNKT) cell antigen αGalCer (KRN7000) bound to mouse CD1d have become valuable tools in elucidating the mechanism of antigen loading and presentation. Here, we report the 3.1 Å resolution crystal structure of the Fab of one of these antibodies, L363, bound to mCD1d complexed with the αGalCer analog C20:2, revealing that L363 is an iNKT TCR-like antibody that binds CD1d-presented αGalCer in a manner similar to the TCR. The structure reveals that L363 depends on both the L and H chains for binding to the glycolipid-mCD1d complex, although only the L chain is involved in contacts with the glycolipid antigen. The H chain of L363 features residue Trp-104, which mimics the TCR CDR3α residue Leu-99, which is crucial for CD1d binding. We characterized the antigen-specificity of L363 toward several different glycolipids, demonstrating that whereas the TCR can induce structural changes in both antigen and CD1d to recognize disparate lipid antigens, the antibody L363 can only induce the F' roof formation in CD1d but fails to reorient the glycolipid headgroup necessary for binding. In summary, L363 is a powerful tool to study mechanism of iNKT cell activation for structural analogs of KRN7000, and our study can aid in the design of antibodies with altered antigen specificity.
Highlights
Antibodies that recognize glycolipids presented by the antigen-presenting molecule CD1d are useful tools in studying natural killer T cell biology
Using surface plasmon resonance (SPR), we show that L363 is able to bind to the glycolipids KRN7000, C20:2-␣GalCer, NU-␣GalCer, OCH, ␣-C-GalCer, as well as the bacterial antigen GalA-GSL from Sphingomonas spp., whereas in contrast to the T cell receptor (TCR), other microbial glycolipids that are based on a diacylglycerol backbone rather than a ceramide backbone are not recognized (e.g. B. burgdorferi BbGL-2c and S. pneumoniae GlcDAG-s2) (Fig. 1)
The L363 Fab binds to mCD1d in a parallel orientation and centered above the FЈ pocket, similar to the TCR of invariant NKT (iNKT) cells [8, 13, 25, 37]
Summary
Antibodies that recognize glycolipids presented by the antigen-presenting molecule CD1d are useful tools in studying natural killer T cell biology. Developed antibodies that are specific for the complex of the prototypical invariant NKT (iNKT) cell antigen ␣GalCer (KRN7000) bound to mouse CD1d have become valuable tools in elucidating the mechanism of antigen loading and presentation. INKT cells express a semi-invariant ␣ T cell receptor (TCR) that recognizes glycolipid antigens (Ag) presented by the nonclassical MHC class I-like molecule CD1d [2]. The crystal structures of CD1d with bound ␣GalCer, as well as in complex with the V␣14V8.2 TCR and V␣14V7 TCR of mouse iNKT cells, have been determined and illustrated the general binding of glycolipids to mCD1d, as well as a conserved docking mode of the TCR onto the mCD1d molecule [13, 14]. To elucidate the structural basis for the recognition of ␣GalCer by L363, as well as the antigen specificity of L363, we determined the binding kinetics of the Fab portion to different glycolipids, as well as determined the crystal structure of L363 Fab-mCD1d-C20:2-␣GalCer complex to a resolution of 3.1Å
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