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

Abstract

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.

Identification and characterization of antibodies elicited by human cystatin C fragment

“…The results obtained earlier proved the ability of monoclonal anti‐hCC antibodies to suppress the dimerization process . In the most potent in dimerization reduction monoclonal antibody (Cyst28), one of the immunogenic determinants was found to be located on the third beta strand (β3) . In theoretical epitope predictions, this fragment was also predicted as immunogenic one .…”

“…This site coincides in part with the interaction site of hCC with the Aβ peptide . The epitope for clone 10 is located in the loops L1 and L2 …”

“…Additionally, the interactions in the immunocomplex with hCC are much stronger for monoclonal anti‐hCC antibodies than for polyclonal rAbs directed against hCC(60‐70) (data not shown). From our previously obtained data, it is known that engagement of loop L1 with 3 β‐strands (β2‐β4) sequences in the interactions with monoclonal antibody Cyst28 resulted in significant inhibition of the dimerization process …”

“…In our previous studies on commercial mAb and hCC autoantibodies, this fragment was present in epitope extraction/excision results for clone 10 (proposed as part of the conformational epitope). HDX experiments performed for the mentioned antibodies revealed that fragment 65‐73 is possibly a part of the epitope in the case of Cyst28 and autoantibodies . The results of theoretical predictions of epitopes for hCC pointed on longer fragments: 60‐81 (ElliPro prediction, linear or part of a discontinuous epitope) or fragment 44‐71 (based on Antigenicity prediction), as a potential immunogenic part of hCC .…”

“…The subject of our long‐lasting interest—human cystatin C (hCC)—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 .…”