title: Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties.
abstract: Human cystatin C (hCC), like many other amyloidogenic proteins, dimerizes and possibly makes aggregates by subdomain swapping. Inhibition of the process should suppress the fibrillogenesis leading to a specific amyloidosis (hereditary cystatin C amyloid angiopathy, HCCAA). It has been reported that exogenous agents like monoclonal antibodies against cystatin C are able to suppress formation of cystatin C dimers and presumably control the neurodegenerative disease. We have studied in detail two monoclonal antibodies (mAbs) representing very different aggregation inhibitory potency, Cyst10 and Cyst28, to find binding sites in hCC sequence responsible for the immunocomplex formation and pave the way for possible immunotherapy of HCCAA. We used the epitope extraction/excision mass spectrometry approach with the use of different enzymes complemented by affinity studies with synthetic hCC fragments as a basic technique for epitope identification. The results were analyzed in the context of hCC structure allowing us to discuss the binding sites for both antibodies. Epitopic sequences for clone Cyst28 which is a highly potent dimerization inhibitor were found in N-terminus, loop 1 and 2 (L1, L2) and fragments of β2 and β3 strands. The crucial difference between conformational epitope sequences found for both mAbs seems to be the lack of interactions with hCC via N-terminus and the loop 1 in the case of mAb Cyst10. Presumably the interactions of mAbs with hCC via L1 and β sheet fragments make the hCC structure rigid and unable to undergo the swapping process.
authors.affiliation: Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952, Gdansk, Poland.
authors.forename: Izabela
authors.initials: I
authors.lastname: Behrendt
authors.affiliation: Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952, Gdansk, Poland.
authors.forename: Martyna
authors.initials: M
authors.lastname: Prądzińska
authors.affiliation: Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952, Gdansk, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Spodzieja
authors.affiliation: Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952, Gdansk, Poland.
authors.forename: Aleksandra S
authors.initials: AS
authors.lastname: Kołodziejczyk
authors.affiliation: Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952, Gdansk, Poland.
authors.forename: Sylwia
authors.initials: S
authors.lastname: Rodziewicz-Motowidło
authors.affiliation: Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952, Gdansk, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Szymańska
authors.affiliation: Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland. pczaplewska@gmail.com.
authors.forename: Paulina
authors.initials: P
authors.lastname: Czaplewska
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry* / Q000276:immunology; D055316:Cystatin C / Q000737:chemistry* / Q000276:immunology; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D051379:Mice; D017433:Protein Structure, Secondary
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; C525030:CST3 protein, human; D055316:Cystatin C; D000939:Epitopes
keywords: Epitope; Human cystatin C; Mass spectrometry; Monoclonal antibodies