Laboratory of Biochemistry, Instituto Butantan, São Paulo, 05503900, Brazil.
Programa Interunidades (USP, Instituto Butantan, IPT) de pós-graduação em Biotecnologia, Universidade de São Paulo, São Paulo, 05508000, Brazil.
Sci Rep. 2023 Feb 10;13(1):2389. doi: 10.1038/s41598-023-29576-y.
Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a K value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.
蜘蛛是昆虫的重要捕食者，其毒液在猎物捕获中起着至关重要的作用。蜘蛛毒液具有作为药物化合物和杀虫剂的多种潜在应用。然而，对蜘蛛消化系统（DS）的转录组学和蛋白质组学分析表明，DS 也是新的肽酶抑制剂分子的丰富来源。蜘蛛 Nephilingis cruentata 的粗 DS 提取物的生化、转录组学和蛋白质组学数据显示，DS 中存在具有肽酶抑制剂潜力的分子。因此，本工作的目的是分离和表征具有胰蛋白酶抑制活性的分子。在酸性条件下将禁食成年雌性蜘蛛的 DS 匀浆，并进行热处理。之后，将样品进行离子交换批处理和高效反相色谱。通过质谱确认具有胰蛋白酶抑制活性的馏分，鉴定出六种具有抑制潜力的分子。抑制剂 NcTI（Nephilingis cruentata 胰蛋白酶抑制剂）的动力学特性进行了表征，显示 K 值为 30.25 nM ± 8.13。使用 Alpha-Fold2 进行的分子建模的三级结构分析表明，抑制剂 NcTI 在结构上属于 MIT1 样 atracotoxin 家族。这是首次将丝氨酸肽酶抑制功能归因于这种结构家族，并确定了抑制剂反应性位点残基。序列分析表明，这些分子可能存在于其他蜘蛛的 DS 中，并且可能与蜘蛛摄入的猎物胰蛋白酶（丝氨酸肽酶）的失活有关。
