Identification and characterization of antibodies elicited by human cystatin C fragment.

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.

Characteristics of C-terminal, ß-amyloid peptide binding fragment of neuroprotective protease inhibitor, cystatin C.

Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aß and the hCC-induced inhibition of Aß fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C-terminal fragment hCC(101-117) was identified as the Aß-binding region. The fragment of Aß peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aß(17-24). Structures and affinities of both Aß and hCC binding sites were characterized by the enzyme-linked immunosorbent assay-like assay, by surface plasmon resonance, and by nano-ESI-FTICR MS of the hCC-Aß-binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101-117), revealed the highest relative inhibitory effect toward Aß-fibril formation. Herein, we present further studies on molecular details of the hCC-Aß complex. With Ala substitution, affinity experiments, and enzyme-linked immunosorbent assay-like assays for the Aß-binding fragment, hCC(101-117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101-117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd.

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

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.

Isolation and characterization of autoantibodies against human cystatin C.

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.

Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties.

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 ß2 and ß3 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 ß sheet fragments make the hCC structure rigid and unable to undergo the swapping process.

Structural studies of the C-terminal 19-peptide of serum amyloid A and its Pro → Ala variants interacting with human cystatin C.

Serum amyloid A (SAA) is a multifunctional acute-phase protein whose concentration in serum increases markedly following a number of chronic inflammatory and neoplastic diseases. Prolonged high SAA level may give rise to reactive systemic amyloid A (AA) amyloidosis, where the N-terminal segment of SAA is deposited as amyloid fibrils. Besides, recently, well-documented association of SAA with high-density lipoprotein or glycosaminoglycans, in particular heparin/heparin sulfate (HS), and specific interaction between SAA and human cystatin C (hCC), the ubiquitous inhibitor of cysteine proteases, was proved. Using a combination of selective proteolytic excision and high-resolution mass spectrometry, a hCC binding site in the SAA sequence was determined as SAA(86-104). The role of this SAA C-terminal fragment as a ligand-binding locus is still not clear. It was postulated important in native SAA folding and in pathogenesis of AA amyloidosis. In the search of conformational details of this SAA fragment, we did its structure and affinity studies, including its selected double/triple Pro → Ala variants. Our results clearly show that the SAA(86-104) 19-peptide has rather unordered structure with bends in its C-terminal part, which is consistent with the previous results relating to the whole protein. The results of affinity chromatography, fluorescent ELISA-like test, CD and NMR studies point to an importance of proline residues on structure of SAA(86-104). Conformational details of SAA fragment, responsible for hCC binding, may help to understand the objective of hCC-SAA complex formation and its importance for pathogenesis of reactive amyloid A amyloidosis.

Interaction of serum amyloid A with human cystatin C--assessment of amino acid residues crucial for hCC-SAA formation (part II).

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life-threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute-phase-reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases--human cystatin C (hCC)--has been proved. Using a combination of selective proteolytic excision and high-resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86-104) and hCC(96-102), respectively. Here, we report further details concerning the hCC-SAA interaction. With the use of affinity tests and florescent ELISA-like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser-98 and Tyr-102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC-SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins.

Identification of the epitope for anti-cystatin C antibody.

Human cystatin C (hCC), like many other amyloidogenic proteins, has been shown to form dimers by exchange of subdomains of the monomeric protein. Considering the model of hCC fibrillogenesis by propagated domain swapping, it seems possible that inhibition of this process should also suppress the entire process of dimerization and fibrillogenesis which leads to specific amyloidosis (hereditary cystatin C amyloid angiopathy (HCCAA)). It was reported that exogenous agents like monoclonal antibody against cystatin C are able to suppress formation of cystatin C dimers. In the effort to find a way of controlling the cystatin fibrillization process, the interactions between monoclonal antibody Cyst-13 and cystatin C were studied in detail. The present work describes the determination of the epitope of hCC to a monoclonal antibody raised against cystatin C, Cyst-13, by MALDI mass spectrometry, using proteolytic excision of the immune complex. The shortest epitope sequence was determined as hCC(107-114). Affinity studies of synthetic peptides revealed that the octapeptide with epitope sequence does not have binding ability to Cyst-13, whereas its longer counterpart, hCC(105-114), binds the studied antibody. The secondary structure of the peptides with epitope sequence was studied using circular dichroism and NMR spectroscopy.

FTIR spectroscopic studies on aggregation process of the beta-amyloid 11-28 fragment and its variants.

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of the Abeta assembly process would facilitate the targeting and design of fibrillogenesis inhibitors. Here, conformational studies using FTIR spectroscopy are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural behavior of the peptide during aggregation provoked by the addition of water to Abeta(11-28) solution in hexafluoroisopropanol was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). The results showed that the aggregation of the peptides proceeds via a helical intermediate, and it is possible that the formation of alpha-helical structures is preceded by creation of 3(10)-helix/3(10)-turn structures.

The Arctic mutation alters helix length and type in the 11-28 beta-amyloid peptide monomer-CD, NMR and MD studies in an SDS micelle.

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease, and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial Alzheimer's-like diseases with extensive cerebrovascular pathology. It has been demonstrated that such mutations alter the aggregation ability of Abeta and its neurotoxicity. Among the five mutations at positions 21-23 there is one with distinct clinical characteristics and a potentially distinct pathogenic mechanism-the Arctic (E22G) mutation. We have examined the structures of fragment 11-28 of the native peptide and its E22G variant. This fragment was chosen because it has been shown to be a good model for conformational and aggregation studies as it contains the hydrophobic core responsible for aggregation and the residues critical to alpha-secretase cleavage of APP. The detailed structure of the two peptides was determined using CD, 2D NMR and molecular dynamics techniques under water-SDS micelle conditions. Our studies indicated the existence of partially alpha- and 3(10)-helical conformations in the native and mutated peptide, respectively.

Conformational solution studies of the SDS micelle-bound 11-28 fragment of two Alzheimer's beta-amyloid variants (E22K and A21G) using CD, NMR, and MD techniques.

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease (AD) and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial AD-like diseases with extensive cerebrovascular pathology. It was proved that the mutations alter the aggregation ability of Abeta and its neurotoxicity. Among five mutations at positions 21-23 there are two mutations with distinct clinical characteristics and potentially distinct pathogenic mechanism-the Italian (E22K) and the Flemish (A21G) mutations. In our studies we have examined the structures of the 11-28 fragment of the Italian and Flemish Abeta variants. The fragment was chosen because it has been shown to be the most important for amyloid fibril formation. The detailed structure of both variants Abeta(11-28) was determined using CD, 2D NMR, and molecular dynamics techniques under water-SDS micelle conditions. The NMR analysis revealed two distinct sets of proton resonances for the peptides. The studies of both peptides pointed out the existence of well-defined alpha-helical conformation in the Italian mutant, whereas the Flemish was found to be unstructured with the possibility of a bent structure in the central part of the peptide.

Circular dichroism and aggregation studies of amyloid beta (11-8) fragment and its variants.

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Here comparative conformational and aggregation studies using CD spectroscopy and thioflavine T fluorescence assay are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural and aggregational behaviour of the peptide was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). In HFIP (hexafluoro-2-propanol), a strong alpha-helix inducer, the CD spectra revealed an unexpectedly high amount of beta-sheet conformation. The aggregation process of Abeta(11-28) variants provoked by water addition to HFIP was found to be consistent with a model of an alpha-helix-containing intermediate. The aggregation propensity of all Abeta(11-28) variants was also compared and discussed.