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

Abstract

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.

Structure of serum amyloid A suggests a mechanism for selective lipoprotein binding and functions: SAA as a hub in macromolecular interaction networks

“…Consistent with this idea, several polar/charged ligands of SAA reportedly bind at the CT residues 70–104. These ligands include heparan sulfate that binds to the basic residues from the 83–102 region in mSAA [28]; cystatin C that forms electrostatic interactions with SAA residues 86–104 [31]); and cell receptors such as LOX-1 and CD36 that bind modified lipoproteins [37] (Figs. 2, 5).…”

“…These include hydrophobic ligands such as HDL and other plasma lipoproteins [7–11], cell membranes, model liposomes [23–25], cholesterol [26] and retinol [14]; charged ligands such as metal ions [27] and heparan sulfate proteoglycans [28–30]; and numerous proteins such as cystatin C [31], extracellular matrix proteins [32, 33] and cell receptors. The latter include several G-protein coupled receptors (such as formyl peptide receptor-like 1 and toll-like receptors 2 and 4) and scavenger receptors (SR-BI, CD36, and LOX-1).…”

Serum amyloid A – a review

“…3a, b) the potential for hydrophilic domain exposure after HDL binding has suggested regions responsible for fibronectin and laminin binding (aa 39-41 and 29-33, respectively). Residues toward the C-terminus (including aa 70-104) include regions implicated in binding to heparan sulfate (aa 83-102 (Ancsin and Kisilevsky 1999)), cystatin C (11 86-104 (Spodzieja et al 2013)) and receptors LOX 1 and CD36 (Tomita et al 2015). Such interactions could permit SAA-mediated binding and possibly internalization into cells with these receptors for these species.…”

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.…”