Fernandez-Botran R, Vitetta E S
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
J Exp Med. 1991 Sep 1;174(3):673-81. doi: 10.1084/jem.174.3.673.
The present studies were undertaken to determine whether the interleukin 4 binding proteins (IL-4BPs) previously identified in the biological fluids of mice are soluble forms of IL-4Rs. We also studied the binding properties of IL-4BPs in order to gain insight into their physiological role in vivo. Affinity-purified IL-4BPs and recombinant soluble IL-4Rs generated similar one-dimensional (Cleveland) peptide maps after digestion with either Staphylococcus aureus V8 protease or trypsin, indicating structural similarities. Furthermore, a rat mAb directed against the murine IL-4Rs immunoprecipitated the IL-4BPs and completely inhibited binding of 125I-IL-4 to a purified preparation of IL-4BPs. Taken together these data indicate that the IL-4BPs are soluble IL-4Rs. At 4 degrees C the IL-4BPs competitively inhibited the binding of IL-4 to membrane IL-4Rs but their ability to prevent binding of IL-4 to cells at 37 degrees C, at the same concentrations, was significantly reduced. Kinetic binding studies of soluble IL-4BPs vs. membrane IL-4Rs disclosed important differences in their rates of dissociation from IL-4. Whereas dissociation at 4 degrees C was slow for both, dissociation of IL-4 from IL-BPs at 37 degrees C was considerably faster (t 1/2 of 2 min) than dissociation of IL-4 from membrane IL-4Rs (t 1/2 of approximately 69 min). Temperature-dependent changes in dissociation kinetics were reversible, and could not be accounted for by either inactivation of the IL-4BPs at 37 degrees C or receptor internalization. Additional experiments also demonstrated that when IL-4BPs bind to IL-4 at 37 degrees C, the IL-4/IL-4BPs complex can rapidly dissociate, allowing IL-4 to bind to membrane IL-4Rs. In addition, binding of IL-4 by the IL-4BPs protects IL-4 from proteolytic degradation. Taken together, these results suggest that the IL-4BPs are naturally occurring forms of soluble IL-4Rs and that some of their properties (fast dissociation kinetics and protection of IL-4 from proteolysis) are consistent with a potential role as carrier proteins for IL-4 in the circulation.
开展本研究以确定先前在小鼠生物体液中鉴定出的白细胞介素4结合蛋白（IL-4BPs）是否为IL-4受体的可溶性形式。我们还研究了IL-4BPs的结合特性，以便深入了解其在体内的生理作用。用金黄色葡萄球菌V8蛋白酶或胰蛋白酶消化后，亲和纯化的IL-4BPs和重组可溶性IL-4受体产生了相似的一维（考马斯）肽图，表明结构相似。此外，一种针对小鼠IL-4受体的大鼠单克隆抗体免疫沉淀了IL-4BPs，并完全抑制了125I-IL-4与纯化的IL-4BPs制剂的结合。综合这些数据表明，IL-4BPs是可溶性IL-4受体。在4℃时，IL-4BPs竞争性抑制IL-4与膜IL-4受体的结合，但在相同浓度下，它们在37℃时阻止IL-4与细胞结合的能力显著降低。可溶性IL-4BPs与膜IL-4受体的动力学结合研究揭示了它们与IL-4解离速率的重要差异。虽然在4℃时两者的解离都很慢，但在37℃时，IL-4从IL-BPs的解离（半衰期为2分钟）比从膜IL-4受体的解离（半衰期约为69分钟）快得多。解离动力学的温度依赖性变化是可逆的，并且不能用37℃时IL-4BPs的失活或受体内化来解释。额外的实验还表明，当IL-4BPs在37℃与IL-4结合时，IL-4/IL-4BPs复合物可以迅速解离，使IL-4能够与膜IL-4受体结合。此外，IL-4BPs对IL-4的结合保护IL-4免受蛋白水解降解。综合来看，这些结果表明，IL-4BPs是可溶性IL-4受体的天然存在形式，并且它们的一些特性（快速解离动力学和保护IL-4免受蛋白水解）与作为循环中IL-4载体蛋白的潜在作用一致。
