Giblin Sean Patrick, McKenna Sophie, Matthews Stephen, Sriskandan Shiranee, Pease James Edward
National Heart and Lung Institute, Imperial College London, London, United Kingdom.
Department of Life Sciences, Imperial College London, London, United Kingdom.
J Biol Chem. 2025 Mar 25;301(5):108448. doi: 10.1016/j.jbc.2025.108448.
Streptococcus pyogenes (Group A Streptococcus or GAS) is a major human pathogen for which an effective vaccine is highly desirable. Invasive S. pyogenes strains evade the host immune response in part by producing a cell envelope protease, SpyCEP. This neutralizes chemokines containing an N-terminal Glu-Leu-Arg motif (ELR chemokines) by cleavage at a distal C-terminal site within the chemokine. SpyCEP is a component of several S. pyogenes vaccines, yet the molecular determinants underlying substrate selectivity are poorly understood. We hypothesized that chemokine recognition and cleavage is a multistep process involving distinct domains of both substrate and enzyme. We generated a panel of recombinant CXCL8 variants where domains of the chemokine were exchanged or mutated. Chemokine degradation by SpyCEP was assessed by SDS-PAGE, Western blot, and ELISA. Extension of the CXCL8 N-terminus was found to inhibit chemokine cleavage. Reciprocal exchanges of the N-termini of CXCL8 with that of the ELR chemokine CXCL4 resulted in the generation of loss of function and gain of function substrates. This suggested a key role for the ELR motif in substrate recognition, which was supported directly by alanine substitution of the ELR motif of CXCL8, impairing the parameters, K, V, and Kcat in kinetic assays with SpyCEP. Collectively, our findings identify the N-terminal ELR motif as a major determinant for recognition by SpyCEP and expose a vulnerability in the mechanism by which the protease recognises its substrates. This likely presents potential avenues for therapeutic intervention via targeted vaccine design and small molecule inhibition.
化脓性链球菌（A组链球菌或GAS）是一种主要的人类病原体，非常需要一种有效的疫苗。侵袭性化脓性链球菌菌株部分通过产生一种细胞包膜蛋白酶SpyCEP来逃避宿主免疫反应。它通过在趋化因子远端的C末端位点切割来中和含有N末端Glu-Leu-Arg基序的趋化因子（ELR趋化因子）。SpyCEP是几种化脓性链球菌疫苗的组成部分，但对底物选择性的分子决定因素了解甚少。我们假设趋化因子的识别和切割是一个多步骤过程，涉及底物和酶的不同结构域。我们构建了一组重组CXCL8变体，其中趋化因子的结构域被交换或突变。通过SDS-PAGE、蛋白质免疫印迹和酶联免疫吸附测定评估SpyCEP对趋化因子的降解。发现CXCL8 N末端的延伸会抑制趋化因子的切割。将CXCL8的N末端与ELR趋化因子CXCL4的N末端相互交换，导致功能丧失和功能获得底物的产生。这表明ELR基序在底物识别中起关键作用，CXCL8的ELR基序的丙氨酸替代直接支持了这一点，在与SpyCEP的动力学测定中损害了参数K、V和催化常数。总的来说，我们的研究结果确定N末端ELR基序是SpyCEP识别的主要决定因素，并揭示了蛋白酶识别其底物机制中的一个弱点。这可能为通过靶向疫苗设计和小分子抑制进行治疗干预提供潜在途径。
