Department of Basic Pathology, Federal University of Parana, Curitiba, 81531-970, Brazil.
School of Biological Sciences, Queen's University Belfast, Belfast, Northern Ireland, UK.
BMC Mol Cell Biol. 2020 Dec 7;21(1):90. doi: 10.1186/s12860-020-00335-5.
The zoonotic worm parasite Fasciola hepatica secretes an abundance of cathepsin L peptidases that are associated with virulence, invasiveness, feeding and migration. The peptidases are produced as inactive zymogens that activate at low pH by autocatalytic removal of their N-terminal pro-domain or propeptide. Propeptides bind to their cognate enzyme with high specificity. Little is known, however, about the mechanism by which the propeptide of FhCL3, a cathepsin L peptidase secreted by the infective newly excysted juveniles (NEJs), regulates the inhibition and activation of the mature enzyme before it is secreted into host tissues.
Immunolocalisation/immunoblotting studies show that the FhCL3 zymogen is produced and secreted by gastrodermal cells of the NEJs gut. A recombinant propeptide of FhCL3 (ppFhCL3) was shown to be a highly potent and selective inhibitor of native and recombinant F. hepatica FhCL3 peptidase, and other members of the cathepsin L family; inhibition constant (K) values obtained for FhCL1, FhCL2 and FhCL3 were 0.04 nM, 0.004 nM and < 0.002 nM, respectively. These values are at least 1000-fold lower than those K obtained for human cathepsin L (HsCL) and human cathepsin K (HsCK) demonstrating the selectivity of the ppFhCL3 for parasite cathepsins L. By exploiting 3-D structural data we identified key molecular interactions in the specific binding between the ppFhCL3 and FhCL3 mature domain. Using recombinant variants of ppFhCL3 we demonstrated the critical importance of a pair of propeptide residues (TyrLys) for the interaction with the propeptide binding loop (PBL) of the mature enzyme and other residues (Leu and Glu) that allow the propeptide to block the active site.
The FhCL3 peptidase involved in host invasion by F. hepatica is produced as a zymogen in the NEJs gut. Regulation of its activation involves specific binding sites within the propeptide that are interdependent and act as a "clamp-like" mechanism of inhibition. These interactions are disrupted by the low pH of the NEJs gut to initiate autocatalytic activation. Our enzyme kinetics data demonstrates high potency and selectivity of the ppFhCL3 for its cognate FhCL3 enzyme, information that could be utilised to design inhibitors of parasite cathepsin L peptidases.
人畜共患的肝片吸虫寄生虫分泌大量组织蛋白酶 L 肽酶，这些肽酶与毒力、侵袭性、摄食和迁移有关。这些肽酶以无活性的酶原形式产生，在低 pH 值下通过自催化去除其 N 端前导肽或前肽而激活。前肽与它们的同源酶具有高度特异性结合。然而，对于肝片吸虫 FhCL3 的前肽（由新生囊蚴（NEJ）分泌的一种组织蛋白酶 L 肽酶）在分泌到宿主组织之前如何调节成熟酶的抑制和激活，我们知之甚少。
免疫定位/免疫印迹研究表明，FhCL3 酶原由 NEJ 肠道的胃基质细胞产生和分泌。重组 FhCL3 前肽（ppFhCL3）被证明是天然和重组 F. hepatica FhCL3 肽酶以及组织蛋白酶 L 家族其他成员的高效且选择性抑制剂；获得的 FhCL1、FhCL2 和 FhCL3 的抑制常数（K）值分别为 0.04 nM、0.004 nM 和 <0.002 nM。这些值至少比获得的人组织蛋白酶 L（HsCL）和人组织蛋白酶 K（HsCK）的 K 值低 1000 倍，表明 ppFhCL3 对寄生虫组织蛋白酶 L 的选择性。通过利用 3D 结构数据，我们确定了 ppFhCL3 与 FhCL3 成熟结构域特异性结合的关键分子相互作用。使用 ppFhCL3 的重组变体，我们证明了一对前肽残基（TyrLys）对于与成熟酶的前肽结合环（PBL）相互作用以及允许前肽封闭活性位点的其他残基（Leu 和 Glu）的重要性。
参与肝片吸虫对宿主侵袭的 FhCL3 肽酶在 NEJ 肠道中以酶原形式产生。其激活的调节涉及前肽中的特定结合位点，这些结合位点相互依赖，并作为抑制的“夹钳样”机制。这些相互作用被 NEJ 肠道的低 pH 破坏，以启动自催化激活。我们的酶动力学数据表明，ppFhCL3 对其同源 FhCL3 酶具有高活性和选择性，这一信息可用于设计寄生虫组织蛋白酶 L 肽酶抑制剂。
