Shedding of the lymphocyte L-selectin adhesion molecule is inhibited by a hydroxamic acid-based protease inhibitor - Identification with an L-selectin-alkaline phosphatase reporter
Shedding of the lymphocyte L-selectin adhesion molecule is inhibited by a hydroxamic acid-based protease inhibitor - Identification with an L-selectin-alkaline phosphatase reporter
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DOI：
10.1074/jbc.271.12.7019
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发表时间：
1996-03-22
影响因子：
4.8
通讯作者：
Kishimoto, TK
中科院分区：
文献类型：
--
作者：
Feehan, C;Darlak, K;Kishimoto, TK
l -选择素粘附分子的表达可以通过调节近端膜处的蛋白水解而迅速下调。l -选择素分泌酶仍然不明确，分泌酶活性对广泛的常见蛋白酶抑制剂具有抗性。我们开发了l -选择素碱性磷酸酶报告基因，包括人胎盘碱性磷酸酶的外域融合到l -选择素的近端膜切割、跨膜和细胞质结构域。为了帮助筛选l -选择素分泌酶抑制剂，一种羟肟酸基金属蛋白酶抑制剂KD-IX-73-4以剂量依赖的方式抑制l -选择素碱性磷酸酶报告基因的释放，羟肟酸基肽也被发现抑制野生型l -选择素从肉芽肿酸激活的外周血淋巴细胞和植物血凝素刺激的淋巴细胞表面下调。l -选择素蛋白水解裂解片段分析证实KD-IX-73-4抑制l -选择素蛋白水解。当淋巴细胞l -选择素与甲酰基蛋氨酸-亮氨酸-苯丙氨酸刺激的中性粒细胞共培养时，l -选择素没有下调，这表明假定的分泌酶与膜结合的l -选择素是顺式作用的。这些结果表明，l -选择素分泌酶活性可能涉及细胞表面锌依赖的金属蛋白酶，尽管l -选择素的脱落不受EDTA的影响，并且可能与最近描述的参与膜结合tnf - α加工的活性有关。
Expression of the L-selectin adhesion molecule can be rapidly down-modulated by regulated proteolysis at a membrane-proximal site, The L-selectin secretase has remained undefined, and the secretase activity is resistant to a broad panel of common protease inhibitors, We have developed an L-selectin-alkaline phosphatase reporter, consisting of the ectodomain of human placental alkaline phosphatase fused to the membrane-proximal cleavage, transmembrane, and cytoplasmic domains of L-selectin, to aid in the screening for L-selectin secretase inhibitors, A hydroxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited release of the L-selectin-alkaline phosphatase reporter in a dose-dependent manner, The hydroxamic acid-based peptide was also found to inhibit wild type L-selectin down-regulation from the surfaces of phorbol myristate acetate-activated peripheral blood lymphocytes and phytohemagglutinin-stimulated lymphoblasts. Analysis of the proteolytic cleavage fragments of L-selectin confirmed that KD-IX-73-4 inhibited L-selectin proteolysis. Lymphocyte L-selectin was not down-regulated when co-cultured with formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting that the putative secretase acts in cis with the membrane-bound L-selectin. These results suggest that the L-selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, although L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane-bound TNF-alpha.