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，热休克蛋白除了作为分子伴侣外，还具有危险信号的功能。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信号的天然免疫的分子机制。我们首先研究了内质网驻留的HSP70蛋白Bip与非抑制性丝氨酸分子热变性卵白蛋白的分子相互作用。BiP不与天然分子相互作用，但显著识别一些仅在母鸡卵清蛋白热变性时暴露的疏水性多肽。定量分析表明，这种相互作用的平衡常数K_d约为0.5μM。其次，研究了小鼠可诱导热休克蛋白72被天然免疫细胞识别的结构要求。当重组HSP72及其C-末端缺失蛋白被产生时，C-末端部分的α-螺旋区域被发现是HSP72寡聚体形成的原因。我们发现，Hop72可被巨噬细胞、树突状细胞、NK细胞等天然免疫细胞以及抗原提呈细胞的B细胞识别。最后，我们试图鉴定与HSP72结合的P388D1细胞上的受体分子。用蛋白酶K或胰酶处理P388D1细胞，可显著取消HSP72的结合，提示该蛋白分子是一种表面受体。抑制HSP72与P388D1细胞结合的分子是清道夫受体配体的成员，这一事实表明，P388D1细胞上HSP72的候选受体可能是迄今发现的清道夫受体或其他与清道夫受体具有相似结合性质的蛋白质分子。

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.