The project entitled """"""""Immune recognition of glycolipids"""""""" fits into a collaborative Program Project by addressing biochemical and structural questions that complement biological studies led by Dr.Bendelac, and by using extensively chemical compounds developed in collaboration with Dr.Savage. Our 3 Specific Aims will be: Lipid Transfer Proteins (LTPs). We will examine the contribution of saposins A through D, Niemann-Pick type C-2 and GM2 activator protein in the shaping of the repertoire of lipids presented by CD1 molecules in professional antigen presenting cells. In vivo studies in knock-out mice will evaluate the contribution of each protein in the selection of NKT cells as well as in anti-bacterial responses. In vitro studies will attempt to reconstitute the complex situation of the lysosome where CD1 resides with a mixture of small LTPs and a wide spectrum of glycolipids. Biophysical measurements and structural studies will also attempt to understand the mechanisms of transfer of lipids between CD1 and LTPs using real time FRET and SPR. Extracellular Lipid Transport and Uptake. Extracellular lipids are kept in solution by associating with lipoproteins particles or lipid transport proteins. The delivery of immunogenic lipids, such as bacterial lipids, follows some of the same trafficking pathways. We will determine which pathway are important for known agonists of NKT cells such as a-galactosyl ceramide (a-GalCer) and bacterial glucuronyl ceramides. The identification of transport proteins will be driven by the usage of biotinylated compounds and biochemistry. Lipid uptake and trafficking to the lysosome will be determined by cell biology and the usage of knock-out mice for surface receptors. The consequences of in vivo modifications, such as oxidation that accompanies inflammation, will also be studied with respect to transport. Finally, we will attempt some of the knowledge gained by this specific aim to devise new strategies for delivering lipid antigens in vivo. Structural Studies of CD1 recognition by Va14 T cell receptors. We know from experience that this approach is highly unpredictable but we also know that it is unlikely that a sustained effort will not deliver some results. This approach has allowed us to determine important structures such as CD1 bound to a- GalCer but more structures are required. The main goal of this aim is to understand the ability of Va14 TCRs to recognize ligands as different as CD1-iGb3 and CD1.-a-GalCer. To attain this goal new protein engineering is required and will be exposed in details. Strong preliminary results will help us in the design of these experiments. Additional CD1-lipid structures studies will also be proposed to understand the influence of acyl chain unsaturation and head group modifications. Finally, we will also carry the systematic mutagenesis of the Va14 chain to understand the thermodynamics of the reaction Va14/CD1.
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