Interaction of serum amyloid A with human cystatin C—identification of binding sites

Abstract

Serum amyloid A (SAA) is a multifunctional acute-phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N-terminal 76-amino acid residue-long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases--human cystatin C (hCC)--has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA-hCC complex, using a combination of selective proteolytic excision and high-resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86-104), whereas the binding site in hCC sequence was identified as hCC(96-102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA-hCC complex.

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

“…Homology modeling studies performed by Stevens (Stevens, ) indicated that approximately 80% of the molecule may consist of a helical bundle with the remaining portion of the C‐terminal part of SAA likely disordered. Our ab initio structure prediction, using UNRES program (Liwo et al, ), corroborated this hypothesis (Spodzieja et al, ) giving a result also in an elementary agreement with the crystal structure, refer to the preceding texts.…”

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

“…A relative deficiency of circulating hCC during a systemic inflammation in rheumatoid arthritis is probably the result of hCC elimination by the complex created by SAA and hCC. The formation of this complex was proved in our group using affinity chromatography experiments, and its details were characterized (Spodzieja et al ., ).…”

Exploring the ‘aggregation-prone’ core of human Cystatin C: A structural study

“…Consecutive structural experiments were performed using selective proteolytic excision and high resolution mass spectrometry (Juszczyk et al, 2009;Maftei et al, 2012;Tian et al, 2007) in order to identify the crucial binding 'hotspots' both in Ab and SAA. Surprisingly, the identified interaction site of Fb is located in the 101 IYAVPWQGTMTLSKSTC 117 hCC fragment (Juszczyk et al, 2009), whereas the identified epitope on SAA is located in 96 FCSFQIY 102 fragment (Spodzieja et al, 2013;Spodzieja et al, 2012). These studies highlight the importance of CysC3 peptide, which according to our experimental results exhibits the tendency to self assemble into amyloid-like fibrils.…”