Thrombin Cleavage of Osteopontin Disrupts a Pro-chemotactic Sequence for Dendritic Cells, Which Is Compensated by the Release of Its Pro-chemotactic C-terminal Fragment
Thrombin Cleavage of Osteopontin Disrupts a Pro-chemotactic Sequence for Dendritic Cells, Which Is Compensated by the Release of Its Pro-chemotactic C-terminal Fragment
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DOI：
10.1074/jbc.m114.572172
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发表时间：
2014-09-26
影响因子：
4.8
通讯作者：
Leung, Lawrence L. K.
中科院分区：
文献类型：
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作者：
Shao, Zhifei;Morser, John;Leung, Lawrence L. K.
凝血酶切割通过暴露整联蛋白结合位点并释放趋化性C末端片段来改变骨桥蛋白（OPN）的功能。在这里，我们研究了在树突状细胞（DC）迁移的背景下OPN的凝血酶切割，以确定其功能域。全长OPN（OPN-FL）、凝血酶切割的N端片段（OPN-R）、凝血酶和羧肽酶B2双切割的N端片段（OPN-L）和C端片段（OPN-CTF）不具有内在趋化活性，但都增强了CCL 21诱导的DC迁移。OPN-FL具有最高的效力，而OPNRAA-FL具有显著较低的活性，表明RGD的重要性。我们鉴定了OPN-FL上跨越凝血酶切割位点的保守（RSKSKKFRR 176）-R-168序列，并且其对CCL 21诱导的DC迁移表现出强的促趋化作用。OPN-FLR 168 A具有降低的活性，并且双突变体OPNRAA-FLR 168 A具有甚至更低的活性，表明这些功能结构域占OPN-FL的大部分促趋化活性。OPN-CTF也具有实质性的促趋化活性，其在凝血酶切割和其从完整蛋白释放时完全表达，因为OPN-CTF比在完整蛋白质内含有OPN-CTF序列的OPNRAA-FLR 168 A活性显著更高。OPN-R和OPN-L具有相似的效力，表明OPN-R中新暴露的C-末端SVVYGLR序列不参与趋化作用。OPN-FL和OPN-CTF不直接结合CD 44标准形式或CD 44 v6。总之，凝血酶裂解OPN破坏了完整OPN中的趋化原序列，其趋化原活性的丧失通过释放OPN-CTF来补偿，OPN-CTF呈现新的构象并具有增强趋化因子诱导的DC迁移的实质性活性。
Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. Here, we examined thrombin cleavage of OPN in the context of dendritic cell (DC) migration to define its functional domains. Full-length OPN (OPN-FL), thrombin-cleaved N-terminal fragment (OPN-R), thrombin-and carboxypeptidase B2-double-cleaved N-terminal fragment (OPN-L), and C-terminal fragment (OPN-CTF) did not have intrinsic chemotactic activity, but all potentiated CCL21-induced DC migration. OPN-FL possessed the highest potency, whereas OPNRAA-FL had substantially less activity, indicating the importance of RGD. We identified a conserved (RSKSKKFRR176)-R-168 sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. OPN-FLR168A had reduced activity, and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of the pro-chemotactic activity of OPN-FL. OPN-CTF also possessed substantial pro-chemotactic activity, which was fully expressed upon thrombin cleavage and its release from the intact protein, because OPN-CTF was substantially more active than OPNRAA-FLR168A containing the OPN-CTF sequence within the intact protein. OPN-R and OPN-L possessed similar potency, indicating that the newly exposed C-terminal SVVYGLR sequence in OPN-R was not involved in the pro-chemotactic effect. OPN-FL and OPN-CTF did not directly bind to the CD44 standard form or CD44v6. In conclusion, thrombin cleavage of OPN disrupts a pro-chemotactic sequence in intact OPN, and its loss of pro-chemotactic activity is compensated by the release of OPN-CTF, which assumes a new conformation and possesses substantial activity in enhancing chemokine-induced migration of DCs.