Isolation and characterization of autoantibodies against human cystatin C
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Hereditary cystatin C amyloid angiopathy (HCCAA) is a severe neurodegenerative disorder related to the point mutation in cystatin C gene resulting in human cystatin C (hCC) L68Q variant. One of the potential immunotherapeutic approaches to HCCAA treatment is based on naturally occurring antibodies against cystatin C. A recent growing interest in autoantibodies, especially in the context of neurodegenerative diseases, emerges from their potential use as valuable diagnostic markers and for controlling protein aggregation. In this work, we present characteristics of natural anti-hCC antibodies isolated from the IgG fraction of human serum by affinity chromatography. The electrophoresis (1-D and 2-D) results demonstrated that the isolated NAbs are a polyclonal mixture, but their electrophoretic properties did not allow to classify the new autoantibodies to any particular type of IgG. The Fc-glycan status of the studied autoantibodies was assessed using mass spectrometry analysis. For the isolated NAbs, the epitopic fragments in hCC sequence were identified by MS-assisted proteolytic excision of the immune complex and compared with the ones predicted theoretically. The knowledge of hCC fragments binding to NAbs and other ligands may contribute to the search for new diagnostic methods for amyloidosis of different types and the search for their treatment.
KeywordsAutoantibodies Human cystatin C Mass spectrometry Epitope
The project was financially supported by National Science Center, based on the decision no DEC-2012/05/E/ST5/03796. We would like to acknowledge DS 530-8440-D379-13 and MOBI4Health EU project which allowed us to use high quality mass spectrometers. MOBI4Health has received funding from the European Union’s Seventh Framework Program for research, technological development and demonstration under Grant Agreement No. 316094 and from the Ministry of Science and Higher Education.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
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