Sphingoglycolipid as a candidate receptor for stress signal to innate immunity

这项研究的重点是压力信号对先天免疫系统功能的影响。最近，人们知道热休克蛋白既可以作为危险信号，也可以作为分子伴侣。在这里，我们主要研究了热休克蛋白（Hsp）70家族的结构与免疫功能之间的关系，并阐明了识别Hsp70信号的先天免疫的分子机制。我们首先研究了内质网驻留的Hsp70 BiP和热变性卵清蛋白（一种非抑制性丝氨酸蛋白酶抑制剂分子）之间的分子相互作用。 BiP 不与天然分子相互作用，但显着识别一些仅在母鸡卵清蛋白热变性时暴露的疏水性肽。定量分析表明，这种相互作用的平衡常数 K_d 估计约为 0.5μM。检查了先天免疫细胞识别的小鼠诱导型 Hsp72 的次要结构要求。当产生重组Hsp72及其C端缺失蛋白时，发现C端部分的α螺旋区域负责形成Hsp72寡聚体。我们发现Hop72可以被巨噬细胞、树突状细胞、NK细胞以及抗原呈递细胞的B细胞等先天免疫细胞识别。最后，我们尝试表征 P388D1 细胞系上结合 Hsp72 的受体分子。用蛋白酶 K 或胰蛋白酶对 P388D1 细胞进行蛋白酶处理，显着消除了 Hsp72 的结合，表明蛋白质分子作为表面受体。抑制 Hsp72 与 P388D1 细胞结合的分子是清道夫受体 (SR) 配体成员的事实表明，P388D1 细胞上 Hsp72 的候选受体可能是迄今为止鉴定的 SR 之一或与 SR 具有相似结合特性的其他蛋白质分子。

This study has the focus on the effect of stress signals on the function of innate immune system. Recently, heat shock proteins are known to function as a danger signal as well as molecular chaperone. Here, we mainly examined the relationship between the structure and the immune functions of heat shook protein (Hsp) 70 family, and also elucidated molecular mechanisms in innate immunity for recognizing a signal of Hsp70.We, first, studied the molecular interaction between BiP, an ER-resident Hsp70, and a heat-denatured ovalbumin, a non-inhibitory serpin molecule. BiP did not interact with the native molecule, but significantly recognized some hydrophobic peptides which were exposed only upon heat-denaturation of hen ovalbumin. Quantitative analysis showed that the equilibrium constant K_d for this interaction was estimated to be approximately 0.5μM. secondary, structural requirement of murine inducible Hsp72 for recognition by innate immune cells was examined. When the recombinant Hsp72 and its C-terminal deletion proteins were produced, the region of α-helices at the C-terminal portion was found to be responsible for formation of oligomers of Hsp72. We found that Hop72 was recognized by the innate immune cells such as macrophages, dendritic cells, and NK cells as well as B cells of an antigen-presenting cell. Finally, we tried to characterize the receptor molecule on P388D1 cell line that con bind Hsp72. Protease treatment of P388D1 cells with proteinase K or trypsin significantly abolished the binding of Hsp72, suggesting the proteinaceous molecule as a surface receptor. The fact that the molecules that inhibit the binding of Hsp72 to P388D1 cells are the members of ligands for scavenger receptors (SR) suggests that a candidate receptor for Hsp72 on P388D1 cells may be one of the SR identified so far or other proteinaceous molecule with similar binding properties to that of SR.