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.

Application of amide hydrogen/deuterium exchange mass spectrometry for epitope mapping in human cystatin C

“…The choice of two monoclonal antibodies Cyst10 and Cyst28 may be of special importance as they possess opposite inhibitory properties toward the dimerization of human cystatin C. HDX-MS experiments confirmed our earlier results (Śladewska et al 2011), indicating that the epitopes for both antibodies are of discontinuous type and are located in the middle part as well as in the C-terminal part of the cystatin C molecule. It was found that the Cyst10 antibody recognizes two fragments which are located in two loops of the hCC structure (53–61—loop L1, 101–112—loop L2, see Figs.…”

“…The hCC epitopes recognized by Cyst10 and Cyst28 previously defined by MS-coupled limited proteolysis are of the discontinuous kind. The present results from the HDX-MS approach are mostly in accordance with the epitopic sequences obtained by proteolytic excision/extraction (Śladewska et al 2011). In addition, we made an attempt to localize with the HDX-MS approach the epitopes for natural, polyclonal anti-hCC autoantibodies (NAbs) isolated from human IgG fraction, and compare them with our previous results obtained using an MS-assisted proteolysis approach (Johnstone and Thorpe 1996).…”

“…In our previous report, identification of the epitopes for the same monoclonal antibodies with the use of epitope excision and extraction procedures combined with mass spectrometry was presented (Śladewska et al 2011). The comparison of epitopes identified by two methods, excision/extraction of epitope and HDX-MS, is given in Table 1.…”

“…For the Cyst10 antibody, the fragment corresponding to the sequence of loop L2 (101–112) was identified using both methods, and in the ELISA experiment, the fragment presented the highest affinity to the Cyst10 clone (Śladewska et al 2011). The differences between the epitopic sequence locations found using both methods exist in the fragment located in the middle of human cystatin C. The fragment 60–70, identified in the extraction/excision experiments, is a part of the β3-strand, while the 53–61 fragment, localized using the HDX-MS approach, covers the amino acids sequence in loop L1.…”

“…The hCC binding sites (epitopes) for two antibodies with opposite antiaggregational potential, Cyst10 and Cyst28, have already been identified using MS-assisted limited proteolysis (epitope excision or extraction procedures) (Behrendt et al 2016). The same method has been used in our previous work on epitope identification for the monoclonal anti-cystatin antibody Cyst13 (Hager-Braun and Tomer 2005), which represents moderate inhibitory properties (Östner et al 2011).…”