Department of Chemical Sciences, Bernal Institute, University of Limerick, Limerick V94 T9PX, Ireland.
SSPC SFI Research Centre for Pharmaceuticals, University of Limerick, Limerick V94 T9PX, Ireland.
Mol Pharm. 2023 Aug 7;20(8):4041-4049. doi: 10.1021/acs.molpharmaceut.3c00207. Epub 2023 Jul 5.
Posttranslational modifications of proteins can impact their therapeutic efficacy, stability, and potential for pharmaceutical development. The Group AC5a peptidase (ScpA) is a multi-domain protein composed of an N-terminal signal peptide, a catalytic domain (including propeptide), three fibronectin domains, and cell membrane-associated domains. It is one of several proteins produced by Group Aknown to cleave components of the human complement system. After signal peptide removal, ScpA undergoes autoproteolysis and cleaves its propeptide for full maturation. The exact location and mechanism of the propeptide cleavage, and the impact of this cleavage on stability and activity, are not clearly understood, and the exact primary sequence of the final enzyme is not known. A form of ScpA with no autoproteolysis fragments of propeptide present may be more desirable for pharmaceutical development from a regulatory and a biocompatibility in the body perspective. The current study describes an in-depth structural and functional characterization of propeptide truncated variants of ScpA expressed incells. All three purified ScpA variants, ScpA, 79ΔPro, and 92ΔPro, starting with N32, D79, and A92 positions, respectively, showed similar activity against C5a, which suggests a propeptide-independent activity profile of ScpA. CE-SDS and MALDI top-down sequencing analyses highlight a time-dependent propeptide autoproteolysis of ScpA at 37 °C with a distinct end point at A92 and/or D93. In comparison, all three variants of ScpA exhibit similar stability, melting temperatures, and secondary structure orientation. In summary, this work not only highlights propeptide localization but also provides a strategy to recombinantly produce a final mature and active form of ScpA without any propeptide-related fragments.
蛋白质的翻译后修饰会影响其治疗效果、稳定性和药物开发潜力。AC5a 肽酶组（ScpA）是一种由 N 端信号肽、催化结构域（包括前肽）、三个纤维连接蛋白结构域和细胞膜相关结构域组成的多功能蛋白。它是 A 群细菌中几种能够切割人补体系统成分的蛋白质之一。在去除信号肽后，ScpA 经历自动蛋白水解，并切割其前肽以完全成熟。前肽切割的确切位置和机制，以及这种切割对稳定性和活性的影响，尚不清楚，也不知道最终酶的精确原始序列。从监管和体内生物相容性的角度来看，没有前肽自动水解片段的 ScpA 形式可能更适合药物开发。本研究描述了在细胞中表达的 ScpA 前肽截断变体的深入结构和功能表征。所有三种纯化的 ScpA 变体，ScpA、79ΔPro 和 92ΔPro，分别从 N32、D79 和 A92 位置开始，均显示出对 C5a 的相似活性，这表明 ScpA 具有前肽非依赖性的活性特征。CE-SDS 和 MALDI 自上而下测序分析突出了 ScpA 在 37°C 时的前肽自动蛋白水解，其终点在 A92 和/或 D93 处明显。相比之下，ScpA 的所有三种变体均表现出相似的稳定性、熔点和二级结构取向。总之，这项工作不仅突出了前肽的定位，还提供了一种策略，可以重组产生没有任何前肽相关片段的最终成熟和活性形式的 ScpA。
