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

Abstract

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.
Publication typeIn press (online first, early view)
Author Monika Rafalik PChM
Monika Rafalik,,
- Laboratory of Medical Chemistry
, Marta Spodzieja PChM
Marta Spodzieja,,
- Laboratory of Medical Chemistry
, Aleksandra Kołodziejczyk PChM
Aleksandra Kołodziejczyk,,
- Laboratory of Medical Chemistry
, Sylwia Rodziewicz-Motowidło PChM
Sylwia Rodziewicz-Motowidło,,
- Laboratory of Medical Chemistry
, Aneta Szymańska PChM
Aneta Szymańska,,
- Laboratory of Medical Chemistry
, A. Grubb
A. Grubb,,
-
, Paulina Czaplewska KBMiK
Paulina Czaplewska,,
- Department of Molecular and Cellular Biology
Journal seriesJournal of Proteomics, ISSN 1874-3919
Issue year2018
Noonline first
Pages1-39
Publication size in sheets1.9
Keywords in Englishhuman cystatin C (HCC), anti-hCC antibodies, epitopes, hereditary cystatin C amyloid angiopathy (HCCAA), mass spectrometry (MS), hydrogen deuterium exchange (HDX)
DOIDOI:10.1016/j.jprot.2018.04.020
URL https://doi.org/10.1016/j.jprot.2018.04.020
Languageen angielski
Score (nominal)35
ScoreMinisterial score = 35.0, 27-04-2018, ArticleFromJournal
Ministerial score (2013-2016) = 35.0, 27-04-2018, ArticleFromJournal
Publication indicators WoS Impact Factor: 2016 = 3.914 (2) - 2016=3.926 (5)
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