Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Poland.
Department of Clinical Chemistry, Skane University Hospital, Lund University, Lund, Sweden.
J Proteomics. 2019 Jan 16;191:58-67. doi: 10.1016/j.jprot.2018.04.020. Epub 2018 Apr 22.
Human cystatin C (hCC) is a cysteine proteinase inhibitor involved in pathophysiological processes of dimerization and amyloid formation. These processes are directly associated with a number of neurodegenerative disorders such as Alzheimer disease or hereditary cystatin C amyloid angiopathy (HCCAA). One of the ideas on how to prevent amyloid formation is to use immunotherapy. HCC3 is one of a group of antibodies binding to hCC and reducing the in vitro formation of cystatin C dimers. Therefore, identification of the binding sites in the hCC-HCC3 complex may facilitate a search of effective drugs against HCCAA as well as understanding the mechanisms of neurodegenerative disorders. In this work we present epitope identification of the hCC-HCC3 complex using methods such as affinity chromatography, epitope excision and extraction MS approach, enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry (HDX MS). Comprehensive analysis of the obtained results allowed us to identify the epitope sequence with the key fragment covering hCC L1 loop and two potential epitopic fragments - α-helical part, hCC (17-28) and β4 strand in C-terminal part of hCC. The presence of the L1 loop in the epitope sequence accounts for the significant reduction of hCC dimer formation in the presence of HCC3 antibody. SIGNIFICANCE OF THE STUDY: Deciphering the mechanism of the cystatin C aggregation process and detailed analysis of the interactions between hCC, or its pathogenic variant, and monoclonal antibodies, potentially constituting aggregation inhibitors, might be of great value as there still is a complete lack of any kind of efficient therapy for young people with the pathogenic mutation of hCC.
人半胱氨酸蛋白酶抑制剂 C（hCC）是一种半胱氨酸蛋白酶抑制剂，参与二聚体和淀粉样形成的病理生理过程。这些过程与许多神经退行性疾病直接相关，如阿尔茨海默病或遗传性半胱氨酸蛋白酶抑制剂 C 血管淀粉样变性（HCCAA）。预防淀粉样形成的一个想法是使用免疫疗法。HCC3 是一组与 hCC 结合并减少体外胱抑素 C 二聚体形成的抗体之一。因此，鉴定 hCC-HCC3 复合物中的结合位点可能有助于寻找针对 HCCAA 的有效药物，并理解神经退行性疾病的机制。在这项工作中，我们使用亲和层析、表位切除和提取 MS 方法、酶联免疫吸附测定和氢氘交换质谱（HDX MS）等方法鉴定 hCC-HCC3 复合物的表位。对获得的结果进行综合分析，使我们能够鉴定出包含 hCC L1 环的关键片段和两个潜在表位片段的表位序列 - hCC 的α螺旋部分（17-28）和 C 末端部分的β4 链。表位序列中 L1 环的存在解释了 HCC3 抗体存在时 hCC 二聚体形成的显著减少。研究意义：阐明胱抑素 C 聚集过程的机制，详细分析 hCC 或其致病变体与单克隆抗体之间的相互作用，这些单克隆抗体可能构成聚集抑制剂，这可能具有重要价值，因为目前仍然完全缺乏针对携带 hCC 致病突变的年轻人的任何有效治疗方法。
