Weber Alexander N R, Gangloff Monique, Moncrieffe Martin C, Hyvert Yann, Imler Jean-Luc, Gay Nicholas J
Department of Biochemistry, Cambridge University, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.
J Biol Chem. 2007 May 4;282(18):13522-31. doi: 10.1074/jbc.M700068200. Epub 2007 Feb 26.
The cytokine Spätzle is the ligand for Drosophila Toll, the prototype of an important family of membrane receptors that function in embryonic patterning and innate immunity. A dimeric precursor of Spätzle is processed by an endoprotease to produce a form (C-106) that cross-links Toll receptor ectodomains and establishes signaling. Here we show that before processing the pro-domain of Spätzle is required for correct biosynthesis and secretion. We mapped two loss-of-function mutations of Spätzle to a discrete site in the pro-domain and showed that the phenotype arises because of a defect in biosynthesis rather than signaling. We also report that the pro-domain and C-106 remain associated after cleavage and that this processed complex signals with the same characteristics as the C-terminal fragment. These results suggest that before activation the determinants on C-106 that bind specifically to Toll are sequestered by the pro-domain and that proteolytic processing causes conformational rearrangements that expose these determinants and enables binding to Toll. Furthermore, we show that the pro-domain is released when the Toll extracellular domain binds to the complex, a finding that has implications for the generation of a signaling-competent Toll dimer.
细胞因子斯帕兹勒（Spätzle）是果蝇Toll蛋白的配体，Toll蛋白是膜受体重要家族的原型，在胚胎模式形成和先天免疫中发挥作用。斯帕兹勒的二聚体前体由一种内切蛋白酶加工，产生一种形式（C-106），该形式可交联Toll受体胞外域并建立信号传导。在此我们表明，在加工之前，斯帕兹勒的前结构域对于正确的生物合成和分泌是必需的。我们将斯帕兹勒的两个功能丧失突变定位到前结构域中的一个离散位点，并表明该表型是由于生物合成缺陷而非信号传导缺陷引起的。我们还报告说，前结构域和C-106在切割后仍保持关联，并且这种加工后的复合物以与C末端片段相同的特征发出信号。这些结果表明，在激活之前，C-106上与Toll特异性结合的决定簇被前结构域隔离，蛋白水解加工导致构象重排，从而暴露这些决定簇并使其能够与Toll结合。此外，我们表明，当Toll胞外域与复合物结合时，前结构域会被释放，这一发现对产生具有信号传导能力的Toll二聚体具有重要意义。
