The Trypanosoma cruzi Virulence Factor Oligopeptidase B (OPBTc) Assembles into an Active and Stable Dimer

Oligopeptidase B, a processing enzyme of the prolyl oligopeptidase family, is considered as an important virulence factor in trypanosomiasis. Trypanosoma cruzi oligopeptidase B (OPBTc) is involved in host cell invasion by generating a Ca2+-agonist necessary for recruitment and fusion of host lysosomes at the site of parasite attachment. The underlying mechanism remains unknown and further structural and functional characterization of OPBTc may help clarify its physiological function and lead to the development of new therapeutic molecules to treat Chagas disease. In the present work, size exclusion chromatography and analytical ultracentrifugation experiments demonstrate that OPBTc is a dimer in solution, an association salt and pH-resistant and independent of intermolecular disulfide bonds. The enzyme retains its dimeric structure and is fully active up to 42°C. OPBTc is inactivated and its tertiary, but not secondary, structure is disrupted at higher temperatures, as monitored by circular dichroism and fluorescence spectroscopy. It has a highly stable secondary structure over a broad range of pH, undergoes subtle tertiary structure changes at low pH and is less stable under moderate ionic strength conditions. These results bring new insights into the structural properties of OPBTc, contributing to future studies on the rational design of OPBTc inhibitors as a promising strategy for Chagas disease chemotherapy.

寡肽酶B（Oligopeptidase B）属于脯氨酰寡肽酶家族的加工酶，被认为是锥虫病的重要毒力因子。克氏锥虫寡肽酶B（Trypanosoma cruzi oligopeptidase B，以下简称OPBTc）可通过生成用于招募并融合宿主溶酶体至寄生虫附着位点的钙离子激动剂，参与宿主细胞入侵过程。目前其潜在作用机制仍未阐明，对OPBTc开展进一步的结构与功能表征，或有助于阐明其生理功能，并推动开发用于治疗恰加斯病的新型治疗性分子。本研究通过尺寸排阻色谱与分析超速离心实验证实，OPBTc在溶液中以二聚体形式存在，该二聚体的缔合过程不受盐浓度与pH值影响，且不依赖分子间二硫键。该酶可维持其二聚体结构，且在42℃以下仍可保持完全催化活性。通过圆二色谱与荧光光谱监测发现，当温度高于42℃时，OPBTc会发生失活，其三级结构（而非二级结构）被破坏。OPBTc的二级结构在宽泛的pH范围内稳定性极高，仅在低pH条件下发生细微的三级结构变化，而在中等离子强度条件下稳定性有所下降。上述研究结果为OPBTc的结构特性提供了新的见解，可为未来以合理设计OPBTc抑制剂作为恰加斯病化疗潜在策略的相关研究提供有力支撑。