Immune Recognition of Glycolipids

题为“糖脂的免疫识别”的项目属于一个合作计划项目， 解决生物化学和结构问题，补充Bendelac博士领导的生物学研究， 通过广泛使用与萨维奇博士合作开发的化合物。我们的三个具体目标 将是：脂质转移蛋白（LTP）。我们将研究saposins A到D的作用， Niemann-Pick C-2和GM 2激活蛋白在CD 1呈递的脂质库形成中的作用 专业抗原呈递细胞中的分子。在基因敲除小鼠中的体内研究将评估 每种蛋白质在NKT细胞的选择以及在抗细菌应答中的贡献。体外 研究将试图用混合物重建溶酶体的复杂情况， 小的LTP和广泛的糖脂。生物物理测量和结构研究也将 尝试使用真实的时间FRET来理解脂质在CD 1和LTP之间转移的机制 的SPR。细胞外脂质转运和摄取。细胞外脂质通过缔合而保持在溶液中 脂蛋白颗粒或脂质转运蛋白。免疫原性脂质，如细菌脂质的递送是有效的。 脂质遵循一些相同的运输途径。我们将确定哪些途径对 已知的NKT细胞激动剂如α-半乳糖基神经酰胺（α-GalCer）和细菌葡糖醛酸基神经酰胺。 转运蛋白的鉴定将通过使用生物素化的化合物来驱动， 生物化学脂质摄取和向溶酶体的运输将由细胞生物学和脂质代谢物的使用来确定。 表面受体基因敲除小鼠。体内修饰的后果，如氧化， 伴随炎症，也将研究关于运输。最后，我们将尝试一些 通过这一特定目标获得的知识设计用于体内递送脂质抗原的新策略。 Va 14 T细胞受体识别CD 1的结构研究。我们从经验中知道， 这种方法是高度不可预测的，但我们也知道，持续的努力不太可能不产生效果 一些结果。这种方法使我们能够确定重要的结构，如CD 1结合到一个- 但是需要更多的结构。该目的的主要目标是了解Va 14 TCR的能力， 识别CD 1-iGb 3和CD 1等不同的配体。-α-GalCer。为了实现这一目标， 工程是必需的，并将在细节中暴露。强有力的初步结果将有助于我们设计 这些实验。还将提出额外的CD 1-脂质结构研究，以了解其影响 酰基链不饱和和头基修饰。最后，我们还将进行系统的 诱变的Va 14链，以了解反应Va 14/CD 1的热力学。

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.