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.
Mass spectrometry Human cystatin C Epitope Monoclonal antibodies
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This work has been supported by the grant from National Science Centre, Preludium 2011/01/N/ST5/05642 (Izabela Behrendt). We also would like to acknowledge the support of DS 530-8440-D379-13 National Science Centre Sonata Bis1 based on the Decision No. DEC-2012/05/E/ST5/03796 (grant to dr Paulina Czaplewska). The project was also supported by Mobi4Health EU project which allowed us to use high quality mass spectrometers. Mobi4Helath project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 316094 and from the Ministry of Science and Higher Education. We would like to thank NanoTemper Technologies GmbH Company for the opportunity to use the Monolith NT.115 system for the measurement of the dissociation constant of the complex hCC-Cyst10/28.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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