The project entitled 'Immune recognition of glycolipids' fits into a collaborative Program Project byaddressing biochemical and structural questions that complement biological studies led by Dr.Bendelac, andby using extensively chemical compounds developed in collaboration with Dr.Savage. Our 3 Specific Aimswill 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 CD1molecules in professional antigen presenting cells. In vivo studies in knock-out mice will evaluate thecontribution of each protein in the selection of NKT cells as well as in anti-bacterial responses. In vitrostudies will attempt to reconstitute the complex situation of the lysosome where CD1 resides with a mixtureof small LTPs and a wide spectrum of glycolipids. Biophysical measurements and structural studies will alsoattempt to understand the mechanisms of transfer of lipids between CD1 and LTPs using real time FRETand SPR. Extracellular Lipid Transport and Uptake. Extracellular lipids are kept in solution by associatingwith lipoproteins particles or lipid transport proteins. The delivery of immunogenic lipids, such as bacteriallipids, follows some of the same trafficking pathways. We will determine which pathway are important forknown 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 andbiochemistry. Lipid uptake and trafficking to the lysosome will be determined by cell biology and the usage ofknock-out mice for surface receptors. The consequences of in vivo modifications, such as oxidation thataccompanies inflammation, will also be studied with respect to transport. Finally, we will attempt some of theknowledge 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 thisapproach is highly unpredictable but we also know that it is unlikely that a sustained effort will not deliversome 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 TCRsto recognize ligands as different as CD1-iGb3 and CD1.-a-GalCer. To attain this goal new proteinengineering is required and will be exposed in details. Strong preliminary results will help us in the design ofthese experiments. Additional CD1-lipid structures studies will also be proposed to understand the influenceof acyl chain unsaturation and head group modifications. Finally, we will also carry the systematicmutagenesis of the Va14 chain to understand the thermodynamics of the reaction Va14/CD1.
Showing the most recent 10 out of 33 publications
