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

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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. 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.

Detaljer

Författare
  • M. Rafalik
  • M. Spodzieja
  • A. S. Kołodziejczyk
  • S. Rodziewicz-Motowidło
  • A. Szymańska
  • A. Grubb
  • P. Czaplewska
Enheter & grupper
Externa organisationer
  • University of Gdansk
  • Skåne University Hospital
  • Medical University of Gdansk
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Läkemedelskemi

Nyckelord

Originalspråkengelska
Sidor (från-till)58-67
TidskriftJournal of Proteomics
Volym191
Tidigt onlinedatum2018 jan 1
StatusPublished - 2019
PublikationskategoriForskning
Peer review utfördJa