The identification of discontinuous epitope in the human cystatin C - monoclonal antibody HCC3 complex
Monika Rafalik , Marta Spodzieja , Aleksandra Kołodziejczyk , Sylwia Rodziewicz-Motowidło , Aneta Szymańska , A. Grubb , Paulina Czaplewska
AbstractHuman 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.
|Journal series||Journal of Proteomics, ISSN 1874-3919, (A 35 pkt)|
|Publication size in sheets||1.9|
|Keywords in English||human cystatin C (HCC), anti-hCC antibodies, epitopes, hereditary cystatin C amyloid angiopathy (HCCAA), mass spectrometry (MS), hydrogen deuterium exchange (HDX)|
|Score||= 35.0, 24-07-2019, ArticleFromJournal|
|Publication indicators||= 0; = 0; : 2017 = 0.982; : 2017 = 3.722 (2) - 2017=3.725 (5)|
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