Kim Tae Kwon, Tirloni Lucas, Radulovic Zeljko, Lewis Lauren, Bakshi Mariam, Hill Creston, da Silva Vaz Itabajara, Logullo Carlos, Termignoni Carlos, Mulenga Albert
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA; Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Int J Parasitol. 2015 Aug;45(9-10):613-27. doi: 10.1016/j.ijpara.2015.03.009. Epub 2015 May 5.
Tick saliva serine protease inhibitors (serpins) facilitate tick blood meal feeding through inhibition of protease mediators of host defense pathways. We previously identified a highly conserved Amblyomma americanum serpin 19 that is characterised by its reactive center loop being 100% conserved in ixodid ticks. In this study, biochemical characterisation reveals that the ubiquitously transcribed A. americanum serpin 19 is an anti-coagulant protein, inhibiting the activity of five of the eight serine protease blood clotting factors. Pichia pastoris-expressed recombinant (r) A. americanum serpin 19 inhibits the enzyme activity of trypsin, plasmin and blood clotting factors (f) Xa and XIa, with stoichiometry of inhibition estimated at 5.1, 9.4, 23.8 and 28, respectively. Similar to typical inhibitory serpins, recombinant A. americanum serpin 19 forms irreversible complexes with trypsin, fXa and fXIa. At a higher molar excess of recombinant A. americanum serpin 19, fXIIa is inhibited by 82.5%, and thrombin (fIIa), fIXa, chymotrypsin and tryptase are inhibited moderately by 14-29%. In anti-hemostatic functional assays, recombinant A. americanum serpin 19 inhibits thrombin but not ADP and cathepsin G activated platelet aggregation, delays clotting in recalcification and thrombin time assays by up to 250s, and up to 40s in the activated partial thromboplastin time assay. Given A. americanum serpin 19 high cross-tick species conservation, and specific reactivity of recombinant A. americanum serpin 19 with antibodies to A. americanum tick saliva proteins, we conclude that recombinant A. americanum serpin 19 is a potential candidate for development of a universal tick vaccine.
蜱唾液丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂）通过抑制宿主防御途径的蛋白酶介质来促进蜱的吸血过程。我们之前鉴定出一种高度保守的美洲钝眼蜱丝氨酸蛋白酶抑制剂19，其特征在于其反应中心环在硬蜱中100%保守。在本研究中，生化特性表明普遍转录的美洲钝眼蜱丝氨酸蛋白酶抑制剂19是一种抗凝血蛋白，可抑制八种丝氨酸蛋白酶凝血因子中的五种的活性。毕赤酵母表达的重组美洲钝眼蜱丝氨酸蛋白酶抑制剂19抑制胰蛋白酶、纤溶酶以及凝血因子Xa和XIa的酶活性，抑制化学计量估计分别为5.1、9.4、23.8和28。与典型的抑制性丝氨酸蛋白酶抑制剂类似，重组美洲钝眼蜱丝氨酸蛋白酶抑制剂19与胰蛋白酶、fXa和fXIa形成不可逆复合物。在重组美洲钝眼蜱丝氨酸蛋白酶抑制剂19摩尔过量较高时，fXIIa被抑制82.5%，凝血酶（fIIa）、fIXa、胰凝乳蛋白酶和类胰蛋白酶被中度抑制14 - 29%。在抗止血功能测定中，重组美洲钝眼蜱丝氨酸蛋白酶抑制剂19抑制凝血酶，但不抑制ADP和组织蛋白酶G激活的血小板聚集，在重新钙化和凝血酶时间测定中使凝血延迟长达250秒，在活化部分凝血活酶时间测定中延迟长达40秒。鉴于美洲钝眼蜱丝氨酸蛋白酶抑制剂19在蜱种间具有高度保守性，以及重组美洲钝眼蜱丝氨酸蛋白酶抑制剂19与美洲钝眼蜱唾液蛋白抗体的特异性反应，我们得出结论，重组美洲钝眼蜱丝氨酸蛋白酶抑制剂19是开发通用蜱疫苗的潜在候选物。
