Institute of Biomedicine, Hubei Key Laboratory of Embryonic Stem Cell Research, and Hubei Key Laboratory of Wudang Local Chinese Medicine Research, College of Basic Medicine, Hubei University of Medicine, Shiyan 442000, China.
Hubei Key Laboratory of Wudang Local Chinese Medicine Research, Hubei University of Medicine, Shiyan 442000, China.
Toxins (Basel). 2024 Oct 21;16(10):450. doi: 10.3390/toxins16100450.
Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system.
最近的研究表明，凝血系统通过在凝块中捕获细菌并产生抗菌肽来在哺乳动物先天防御中发挥作用。因此，细菌抵御宿主凝血系统的能力对于其生存至关重要，这表明细菌外毒素可能是新型抗凝剂的来源。在本研究中，我们分析了 和一个新的细菌外毒素蛋白的基因组序列，鉴定出一个具有五个 Kunitz 结构域的新型细菌外毒素蛋白 F6W77，分别命名为 KABP1-5。每个 Kunitz 型结构域都具有经典的六半胱氨酸框架，由三个保守的二硫键连接，这与动物 Kunitz 结构域肽明显相似，但与植物 Kunitz 结构域肽不同。抗凝功能评价表明，对于内源性凝血途径，KABP1 和 KABP5 具有明显的抑制活性，KABP4 具有较弱的抑制活性，而 KABP2 和 KABP3 即使在 20μg/ml 的高浓度下也没有作用。五个 Kunitz 结构域肽，KABP1-5，对外源性凝血途径没有抑制活性。酶抑制剂实验表明，高活性抗凝肽 KABP1 对两个关键凝血因子 Xa 和 XIa 具有明显的抑制活性，这进一步通过拉下实验得到证实，表明 KABP1 可以直接与凝血因子 Xa 和 XIa 结合。五个 Kunitz 型结构域肽的结构-功能关系分析表明，三个新型细菌抗凝剂 KABP1、KABP4 和 KABP5 的 P1 位的精氨酸可能是其抗凝活性的关键残基。总之，通过生物信息学分析、肽重组和功能评价，我们首次发现了具有选择性抑制内源性凝血途径活性的细菌外毒素衍生的 Kunitz 型丝氨酸蛋白酶抑制剂，并强调了致病菌与人体凝血系统之间的新相互作用。
