Sphingoglycolipid as a candidate receptor for stress signal to innate immunity

本研究的重点是应激信号对先天免疫系统功能的影响。近年来，热休克蛋白被认为是一种危险信号和分子伴侣。本研究主要探讨热震荡蛋白（Hsp） 70家族的结构与免疫功能之间的关系，并阐明先天免疫中识别Hsp70信号的分子机制。我们首先研究了BiP（一种ER-resident Hsp70）和热变性卵清蛋白（一种非抑制性蛇形蛋白分子）之间的分子相互作用。BiP不与天然分子相互作用，但能识别一些仅在母鸡卵白蛋白热变性条件下暴露的疏水肽。定量分析表明，该相互作用的平衡常数K_d约为0.5μM。其次，研究小鼠诱导的Hsp72对先天免疫细胞识别的结构要求。制备重组Hsp72及其c端缺失蛋白时，发现c端α-螺旋区域负责Hsp72低聚物的形成。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细胞株上结合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，可显著抑制Hsp72的结合，提示该蛋白分子可能是表面受体。抑制Hsp72与P388D1细胞结合的分子是清道夫受体（scavenger receptor， SR）配体的成员，这表明P388D1细胞上Hsp72的候选受体可能是目前已鉴定的SR或其他与SR具有相似结合特性的蛋白质分子。

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.