Teçza Malgorzata, Kagawa Todd F, Jain Monica, Cooney Jakki C
Department of Chemical Sciences, University of Limerick, Limerick, Ireland.
Department of Biological Sciences, University of Limerick, Limerick, Ireland.
Comput Struct Biotechnol J. 2021;19:2356-2365. doi: 10.1016/j.csbj.2021.04.024. Epub 2021 Apr 17.
The Streptococcal C5a peptidase (ScpA) specifically inactivates the human complement factor hC5a, a potent anaphylatoxin recently identified as a therapeutic target for treatment of COVID-19 infections. Biologics used to modulate hC5a are predominantly monoclonal antibodies. Here we present data to support an alternative therapeutic approach based on the specific inactivation of hC5a by ScpA in studies using recombinant hC5a (rhC5a). Initial characterization of ScpA confirmed activity in human serum and against rhC5a desArg (rhC5a), the predominant hC5a form in blood. A new FRET based enzyme assay showed that ScpA cleaved rhC5a at near physiological concentrations ( 185 nM). Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) studies established a high affinity ScpA-rhC5a interaction ( 34 nM, 30.8 nM). SPR analyses also showed that substrate binding is dominated (88% of ΔG°) by interactions with the bulky N-ter cleavage product (P, 'core' residues 1-67) with interactions involving the C-ter R74 contributing most of the remaining ΔG°. Furthermore, reduced binding affinity following mutation of a subset of positively charged Arginine residues of P and in the presence of higher salt concentrations, highlighted the importance of electrostatic interactions. These data provide the first in-depth study of the ScpA-C5a interaction and indicate that ScpA's ability to efficiently cleave physiological concentrations of C5a is driven by electrostatic interactions between an exosite on the enzyme and the 'core' of C5a. The results and methods described herein will facilitate engineering of ScpA to enhance its potential as a therapeutic for excessive immune response to infectious disease.
链球菌C5a肽酶（ScpA）可特异性灭活人类补体因子hC5a，hC5a是一种强效过敏毒素，最近被确定为治疗新冠病毒感染的治疗靶点。用于调节hC5a的生物制剂主要是单克隆抗体。在此，我们展示数据以支持一种基于ScpA在使用重组hC5a（rhC5a）的研究中特异性灭活hC5a的替代治疗方法。对ScpA的初步表征证实其在人血清中具有活性，并且对血液中主要的hC5a形式rhC5a desArg（rhC5a）具有活性。一种基于荧光共振能量转移（FRET）的新酶分析表明，ScpA在接近生理浓度（185 nM）时可切割rhC5a。表面等离子体共振（SPR）和等温滴定量热法（ITC）研究确定了ScpA与rhC5a之间的高亲和力相互作用（KD分别为34 nM、30.8 nM）。SPR分析还表明，底物结合主要（占ΔG°的88%）由与庞大的N端裂解产物（P，“核心”残基1 - 67）的相互作用主导，涉及C端R74的相互作用贡献了其余大部分ΔG°。此外，P中一部分带正电荷的精氨酸残基发生突变后以及在较高盐浓度存在下结合亲和力降低，突出了静电相互作用的重要性。这些数据首次对ScpA - C5a相互作用进行了深入研究，并表明ScpA有效切割生理浓度C5a的能力是由酶上的一个外部位点与C5a的“核心”之间的静电相互作用驱动的。本文所述的结果和方法将有助于对ScpA进行工程改造，以增强其作为治疗过度免疫反应性传染病的潜力。
