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The identification of discontinuous epitope in the human cystatin C – Monoclonal antibody HCC3 complex

Rafalik, M.; Spodzieja, M.; Kołodziejczyk, A. S.; Rodziewicz-Motowidło, S.; Szymańska, A.; Grubb, A. LU and Czaplewska, P. (2019) In Journal of Proteomics 191. p.58-67
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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Anti-hCC antibodies, Epitopes, Hereditary cystatin C amyloid angiopathy (HCCAA), Human cystatin C (hCC), Hydrogen deuterium exchange (HDX), Mass spectrometry (MS)
in
Journal of Proteomics
volume
191
pages
58 - 67
publisher
Elsevier
external identifiers
  • scopus:85046629004
ISSN
1874-3919
DOI
10.1016/j.jprot.2018.04.020
language
English
LU publication?
yes
id
fa035be2-7699-43f9-85d9-cf4be16339b9
date added to LUP
2018-05-25 14:11:35
date last changed
2019-02-20 11:18:15
@article{fa035be2-7699-43f9-85d9-cf4be16339b9,
  abstract     = {<p>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.</p>},
  author       = {Rafalik, M. and Spodzieja, M. and Kołodziejczyk, A. S. and Rodziewicz-Motowidło, S. and Szymańska, A. and Grubb, A. and Czaplewska, P.},
  issn         = {1874-3919},
  keyword      = {Anti-hCC antibodies,Epitopes,Hereditary cystatin C amyloid angiopathy (HCCAA),Human cystatin C (hCC),Hydrogen deuterium exchange (HDX),Mass spectrometry (MS)},
  language     = {eng},
  pages        = {58--67},
  publisher    = {Elsevier},
  series       = {Journal of Proteomics},
  title        = {The identification of discontinuous epitope in the human cystatin C – Monoclonal antibody HCC3 complex},
  url          = {http://dx.doi.org/10.1016/j.jprot.2018.04.020},
  volume       = {191},
  year         = {2019},
}