Identification and characterization of antibodies elicited by
human cystatin C fragment
Aleksandra S. Kołodziejczyk
Susanna L. Lundström
Roman A. Zubarev
Faculty of Chemistry, University of Gdańsk,
Intercollegiate Faculty of Biotechnology,
University of Gdańsk and Medical University
of Gdańsk, Gdańsk, Poland
Department of Medical Biochemistry and
Biophysics, Division of Physiological
Chemistry, Karolinska Institutet, Stockholm,
Sylwia Rodziewicz‐Motowidło, Faculty of
Chemistry of the University of Gdańsk, Wita
Stwosza 63, 80‐308, Gdańsk, Poland.
National Science Centre, Grant/Award Num-
ber: 2011/01/N/ST5/05642 (Izabela
Amyloid formation is associated with a number of neurodegenerative diseases that affect the
independence and quality of life of aging populations. One of rather atypical, occurring at a young
age amyloidosis is hereditary cystatin C amyloid angiopathy (HCCAA) related to aggregation of
L68Q variant of human cystatin C (hCC). Human cystatin C plays a very important role in many
aspects of human health; however, its amyloidogenic properties manifested in HCCAA present a real,
lethal threat to some populations and any work on factors that can affect possible influencing hCC
aggregation is not to overestimate. It was proved that interaction of hCC with monoclonal antibodies
suppresses significantly hCC dimerization process. Therefore, immunotherapy seems to be the right
approach toward possible HCCAA treatment. In this work, the hCC fragment encompassing residue
60‐70 (in 2 variants: linear peptide and multiple antigenic peptide) was used as an immunogen in
rabbit immunization. As a result, specific anti‐hCC antibodies were found in both rabbit sera.
Surprisingly, rabbit antibodies were obtained after immunization with only a short peptide. The
obtained antibodies were characterized, and their influence on the aggregation propensity of the
hCC molecules was evaluated. The antibodies turned out not to have any significant influence on
the cystatin C dimerization process. Nevertheless, we hope that antibodies elicited in rabbits by other
hCC fragments could lead to elaboration of effective treatment against HCCAA.
epitope identification, human cystatin C, mass spectrometry, multiple antigenic peptide, polyclonal
Amyloidoses are a group of diseases in which the main role is played by
extracellular buildup of abnormally folded proteins or peptides. This
process, occurring simultaneously with normal physiological folding
or as an alternative to it, generates insoluble, toxic protein aggregates,
which accumulate in body tissues in the form of amyloid fibrils.
Generation of amyloid deposits by many different peptides and pro-
teins is associated with pathological conditions, such as Alzheimer's
disease, Parkinson's disease, and many other neurological disorders.
Some of these diseases are typical for older people, and as life
expectancy all over the world tends to increase, fighting these diseases
constitutes a very significant social
and economic problem.
The subject of our long‐lasting interest—human cystatin C
—is also involved in in vivo fibrillogenesis as the commonly
occurring wild‐type variant and as a rare, but extremely amyloidogenic
L68Q cystatin C variant. Amyloid deposits formed in cerebral arteries
by the hCC variant are the cause of hereditary cystatin C amyloid
angiopathy (HCCAA), which occurs in genetically predisposed young
adults and is a main cause of their early death.
Cystatins are a
group of inhibitors of cysteine proteases (family C1 according to
) like, among others, plant papain; mammalian
cathepsin B, C, H, K, L, and S; and the family of legumains.
The work was performed at the University of Gdańsk (Poland) and at the
Karolinska Institutet (Sweden).
Received: 26 September 2017 Revised: 23 October 2017 Accepted: 28 October 2017
J Mol Recognit. 2018;31:e2689.
Copyright © 2017 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/jmr 1of13