Human cystatin C (hCC) is a small but very intriguing protein. Produced by all nucleated cells is found in almost all tissues and body fluids where, at physiological conditions, plays a role of a very potent inhibitor of cysteine proteases. Biologically active hCC is a monomeric protein but during cellular trafficking it forms dimers, transiently losing its inhibitory activity. In vitro, dimerization of cystatin C was observed for the mature protein during crystallization trials, revealing that the mechanism of this process is based on the three dimensional swapping of the protein domains. In our work we have focused on the impact of two proposed “hot spots” in cystatin C structure on its conformational stability. Encouraged by promising results of the theoretical calculations, we designed and produced several hCC hinge region point mutation variants that display a variety of conformational stability and propensity for dimerization and aggregation. A similar approach, i.e., rational mutagenesis, has been also applied to study the amyloidogenic L68Q variant to determine the contribution of hydrophobic interactions and steric effect on the stability of monomeric cystatin C. In this overview we would like to summarize the results of our studies. The impact of a particular mutation on the properties of the studied proteins will be presented in the context of their thermal and mechanical stability, in vitro dimerization tendency as well as the outcome of crystallization. Better understanding of the mechanism and, especially, factors affecting conformational stability of cystatin C and access to stable monomeric and dimeric versions of the protein opens new perspectives in explaining the role of dimers and the domain swapping process in hCC oligomerization, as well as designing potential inhibitors of this process.

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.

Human cystatin C (hCC) is a small cysteine protease inhibitor whose oligomerization by propagated domain swapping is linked to certain neurological disorders. One of the ways to prevent hCC dimerization and fibrillogenesis is to enable its interaction with a proper antibody. Herein, the sites of interaction of hCC with dimer-preventing mouse monoclonal anti-hCC antibodies Cyst28 are studied and compared with the binding sites found for mAb Cyst10 that has almost no effect on hCC dimerization. In addition, hCC epitopes in complexes with native polyclonal antibodies extracted from human serum were studied. The results obtained with hydrogen–deuterium exchange mass spectrometry (HDX MS) were compared with the previous findings made using the excision/extraction MS approach. The main results from the two complementary MS-based approaches are found to be in agreement with each other, with some differences being attributed to the specificity of each method. The findings of the current studies may be important for future design of hCC dimerization inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-016-2316-y) contains supplementary material, which is available to authorized users.

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.

Poster Sessions C310 exogenous recombinant 'truncated' Galectin-3 (analogous to the MMPprocessed form), but not full length Galectin-3, dramatically increases migration of the human breast cancer cell line BT-549. These results suggest that the MMP-cleaved Galectin-3 and the resulting structural changes are responsible for pro-metastatic properties of Galectin-3. We have obtained crystallographic data for CRD of Galectin-3 in complex with a pentasaccharide to which Galectin-3 has higher binding affinity compared to other galectin family members. This structural information may be utilized in the design of Galectin-3 specific inhibitors targeting the carbohydrate-binding site. We have also explored the structural differences resulting from MMP cleavage of Galectin-3 using SAXS.[1] F.T. Liu and G.A. Rabinovich, Nat Rev Cancer, 2005, 5, 29-41 Gdańsk, Gdańsk, (Poland). E-mail: martaorlikowska07@gmail.com Human cystatin C (hCC) is a low molecular mass protein (120 amino acid residues, 13,343 Da) that belongs to a family of single chain, reversible inhibitors of papain-like (C1 family) and legumainrelated (C13 family) cysteine proteases [1]. In pathophysiological processes, which nature of is not understood, hCC is codeposited in the amyloid plaques of Alzheimer's disease or Down's syndrome. The amyloidogenic properties of HCC are greatly increased in a naturally occurring L68Q variant, resulting in fatal cerebral amyloid angiopathy in early adult life [2]. At physiological conditions wild-type hCC is a monomeric protein, but under crystallization conditions (pH 4.8) forms a domain-swapped dimer [3]. The dimerization process is facilitated by the presence in the hCC structure of a flexible region created by the loop L1 (55-59, QIVAG) connecting protein subdomains undergoing the exchange process. This loop is the only part of hCC which undergoes significant structural changes during the dimerization process and, according to experimental [4], [5] and theoretical [6], [7] studies, these changes are driven by the conformational constraints attributed to the located near the top of the loop Val residue (Val57 for hCC).With the aim to check implications of grater or decreased stability of this loop on dimerization and aggregation propensity of human cystatin C, we designed and constructed hCC L1 mutants with Val57 residue replaced by Asp, Asn [8], Gly (residues favored in this position of β-turns) or Pro, respectively. By applying this rational mutagenesis approach we were able to obtain hCC variants stable in the monomeric form both in solution and in the crystal (V57G, Figure 1), monomeric in solution but dimeric in the crystal (V57D) and dimeric in solution and oligomeric in the crystal (V57P). The results of structural studies of hCC L1 mutants will be presented.