The putative role of Rhipicephalus microplus salivary serpins in the tick-host relationship.
The putative role of Rhipicephalus microplus salivary serpins in the tick-host relationship.
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DOI：
10.1016/j.ibmb.2016.01.004
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发表时间：
2016-04
影响因子：
3.8
通讯作者：
da Silva Vaz I Jr
中科院分区：
文献类型：
--
作者：
Tirloni L;Kim TK;Coutinho ML;Ali A;Seixas A;Termignoni C;Mulenga A;da Silva Vaz I Jr
炎症和止血是宿主对蜱虫进食的第一道防线的一部分。这些系统部分是丝氨酸蛋白酶介导的，并由其内源性抑制剂严格控制，在丝氨酸蛋白酶抑制剂超家族（丝氨酸蛋白酶抑制剂）。从这个角度来看，蜱虫被认为在进食过程中使用蛇蛋白来逃避宿主的防御。牛蜱微小扇头蜱编码至少24种丝氨酸蛋白酶抑制剂，其中RmS-3、RmS-6和RmS-17先前在该蜱的唾液中鉴定。在这项研究中，我们筛选了这三种唾液丝氨酸蛋白酶抑制剂对整个哺乳动物防御途径的16种蛋白酶的抑制功能。我们的数据证实毕赤酵母表达的rRmS-3、rRmS-6和rRmS-17可能是促炎和促凝血蛋白酶的抑制剂。我们发现，rRmS-3抑制胰凝乳蛋白酶和组织蛋白酶G的化学计量抑制（SI）指数为1.8和2.0，胰腺弹性蛋白酶的SI高于10。同样，rRmS-6抑制胰蛋白酶，SI为2.6，胰凝乳蛋白酶、因子Xa、因子XIa和纤溶酶，SI高于10，而rRmS-17抑制胰蛋白酶、组织蛋白酶G、胰凝乳蛋白酶、纤溶酶和因子XIa，SI分别为1.6、2.6、2.7、3.4和9.0。此外，我们观察到rRmS-3与胰凝乳蛋白酶、rRmS-6/rRmS-17与胰蛋白酶以及rRmS-3/rRmS-17与组织蛋白酶G之间形成不可逆复合物，这与抑制性丝氨酸蛋白酶抑制剂的典型机制一致。在凝血试验中，rRmS-17在凝血时间试验中延迟血浆凝血60 s，而rRmS-3和rRmS-6没有任何影响。与抑制剂功能分析数据一致，2.0 µM rRmS-3和rRmS-17以剂量响应方式分别抑制组织蛋白酶G激活的血小板聚集高达96%和95%。重要的是，多克隆抗体阻断了三种丝氨酸蛋白酶抑制剂的抑制功能。同样值得注意的是，对美洲钝眼蜱、肩突硬蜱和R.血红蜱唾液蛋白与三种R. microplus唾液丝氨酸蛋白酶抑制剂，表明这些蛋白质作为通用抗蜱疫苗的候选者的潜力。
Inflammation and haemostasis are part of the host's first line of defense to tick feeding. These systems are in part serine protease mediated and are tightly controlled by their endogenous inhibitors, in the serpin superfamily (serine protease inhibitors). From this perspective ticks are thought to use serpins to evade host defenses during feeding. The cattle tick Rhipicephalus microplus encodes at least 24 serpins, of which RmS-3, RmS-6, and RmS-17 were previously identified in saliva of this tick. In this study, we screened inhibitor functions of these three saliva serpins against a panel of 16 proteases across the mammalian defense pathway. Our data confirm that Pichia pastorisexpressed rRmS-3, rRmS-6, and rRmS-17 are likely inhibitors of pro-inflammatory and pro-coagulant proteases. We show that rRmS-3 inhibited chymotrypsin and cathepsin G with stoichiometry of inhibition (SI) indices of 1.8 and 2.0, and pancreatic elastase with SI higher than 10. Likewise, rRmS-6 inhibited trypsin with SI of 2.6, chymotrypsin, factor Xa, factor XIa, and plasmin with SI higher than 10, while rRmS-17 inhibited trypsin, cathepsin G, chymotrypsin, plasmin, and factor XIa with SI of 1.6, 2.6, 2.7, 3.4, and 9.0, respectively. Additionally, we observed the formation of irreversible complexes between rRmS-3 and chymotrypsin, rRmS-6/rRmS-17 and trypsin, and rRmS-3/rRmS-17 and cathepsin G, which is consistent with typical mechanism of inhibitory serpins. In blood clotting assays, rRmS-17 delayed plasma clotting by 60 s in recalcification time assay, while rRmS-3 and rRmS-6 did not have any effect. Consistent with inhibitor function profiling data, 2.0 µM rRmS-3 and rRmS-17 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner by up to 96% and 95% respectively. Of significant interest, polyclonal antibodies blocked inhibitory functions of the three serpins. Also notable, antibodies to Amblyomma americanum, Ixodes scapularis, and R. sanguineus tick saliva proteins cross-reacted with the three R. microplus saliva serpins, suggesting the potential of these proteins as candidates for universal anti-tick vaccines.