Wang Yue, Herrera Amy H, Li Ying, Belani Kiran K, Walcheck Bruce
Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN 55108, USA.
J Immunol. 2009 Feb 15;182(4):2449-57. doi: 10.4049/jimmunol.0802770.
L-selectin is constitutively expressed by neutrophils and plays a key role in directing these cells to sites of inflammation. Upon neutrophil activation, L-selectin is rapidly and efficiently down-regulated from the cell surface by ectodomain shedding. We have directly shown that A disintegrin and metalloprotease 17 (ADAM17) is a primary and nonredundant sheddase of L-selection by activated neutrophils in vivo. Following cell activation, intracellular signals lead to the induction of ADAM17's enzymatic activity; however, the target of this inducer mechanism remains unclear. Our study provides evidence of an activation mechanism that involves the extracellular region of the mature form of cell surface ADAM17 and not its intracellular region. We demonstrate that the catalytic activity of purified ADAM17 lacking a prodomain and its intracellular region is diminished under mild reducing conditions by DTT and enhanced by H(2)O(2) oxidation. Moreover, H(2)O(2) reversed ADAM17 inhibition by DTT. The treatment of neutrophils with H(2)O(2) also induced L-selectin shedding in an ADAM17-dependent manner. These findings suggest that thiol-disulfide conversion occurring in the extracellular region of ADAM17 may be involved in its activation. An analysis of ADAM17 revealed that within its disintegrin/cysteine-rich region are two highly conserved, vicinal cysteine sulfhydryl motifs (cysteine-X-X-cysteine), which are well-characterized targets for thiol-disulfide exchange in various other proteins. Using a cell-based ADAM17 reconstitution assay, we demonstrate that the cysteine-X-X-cysteine motifs are critical for L-selectin cleavage. Taken together, our findings suggest that reduction-oxidation modifications of cysteinyl sulfhydryl groups in mature ADAM17 may serve as a mechanism for regulating the shedding of L-selectin following neutrophil stimulation.
L-选择素由中性粒细胞组成性表达，在引导这些细胞至炎症部位中起关键作用。在中性粒细胞激活后，L-选择素通过胞外域脱落迅速且有效地从细胞表面下调。我们已直接表明，解整合素和金属蛋白酶17（ADAM17）是体内活化中性粒细胞对L-选择素进行切割的主要且非冗余的蛋白酶。细胞激活后，细胞内信号导致ADAM17酶活性的诱导；然而，这种诱导机制的靶点仍不清楚。我们的研究提供了一种激活机制的证据，该机制涉及细胞表面成熟形式的ADAM17的胞外区域而非其胞内区域。我们证明，在温和还原条件下，缺乏前结构域及其胞内区域的纯化ADAM17的催化活性会被二硫苏糖醇（DTT）降低，而被过氧化氢（H₂O₂）氧化增强。此外，H₂O₂可逆转DTT对ADAM17的抑制作用。用过氧化氢处理中性粒细胞也以ADAM17依赖的方式诱导L-选择素脱落。这些发现表明，ADAM17胞外区域发生的硫醇-二硫键转换可能参与其激活过程。对ADAM17的分析表明，在其解整合素/富含半胱氨酸区域内有两个高度保守的相邻半胱氨酸巯基基序（半胱氨酸- X - X -半胱氨酸），这是各种其他蛋白质中硫醇-二硫键交换的典型靶点。使用基于细胞的ADAM17重组试验，我们证明半胱氨酸- X - X -半胱氨酸基序对L-选择素的切割至关重要。综上所述，我们的发现表明，成熟ADAM17中半胱氨酰巯基基团的氧化还原修饰可能是调节中性粒细胞刺激后L-选择素脱落的一种机制。
