Budagian Vadim, Bulanova Elena, Orinska Zane, Ludwig Andreas, Rose-John Stefan, Saftig Paul, Borden Ernest C, Bulfone-Paus Silvia
Center for Cancer Drug Discovery and Development, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
J Biol Chem. 2004 Sep 24;279(39):40368-75. doi: 10.1074/jbc.M404125200. Epub 2004 Jun 23.
This study shows that the high affinity alpha-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Ralpha (sIL-15Ralpha) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Ralpha is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-alpha-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Ralpha, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Ralpha. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Ralpha shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Ralpha, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Ralpha generation in mice. The existence of natural sIL-15Ralpha offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention.
本研究表明，白细胞介素（IL）-15受体的高亲和力α链不仅以膜锚定形式存在，还以可溶性形式存在。通过酶联免疫吸附测定、免疫沉淀和蛋白质印迹法可在小鼠血清和细胞条件培养基中检测到可溶性IL-15Rα（sIL-15Rα）。由于存在单个N-糖基化位点，该蛋白的分子量约为30 kDa，经N-糖苷酶处理后降至26 kDa。跨膜IL-15Rα通过涉及肿瘤坏死因子-α转换酶（TACE）的蛋白水解切割而组成性地转化为其可溶性形式，并且该过程通过佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）刺激而进一步增强。能够阻断TACE和密切相关的解整合素样金属蛋白酶10（ADAM10）的羟肟酸GW280264X有效抑制了IL-15Rα的自发和PMA诱导的切割，而优先阻断ADAM10的GI254023X则无效。在COS-7细胞中过表达TACE而非ADAM10增强了IL-15Rα的组成性和PMA诱导的切割。此外，缺乏TACE表达但不缺乏ADAM10表达的小鼠成纤维细胞在自发和诱导的IL-15Rα脱落方面显著减少，而在这些细胞中重建TACE恢复了sIL-15Rα的释放，从而表明TACE介导的蛋白水解可能是小鼠中sIL-15Rα产生的主要机制。天然sIL-15Rα的存在为IL-15的复杂生物学提供了新的见解，并设想了治疗干预的新水平。
