Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations

Abstract

S100A8 and S100A9 are two proinflammatory molecules belonging to the S100 family of calcium-binding proteins. Common to all S100 proteins S100A8 and S100A9 form non-covalently associated complexes which have been shown to exhibit different functional properties. Besides dimerization, recent research is focused on the importance of higher oligomeric structures of S100 proteins induced by bivalent cations. While S100A8/S100A9-heterodimers are formed in the absence of calcium, tetramerization is strictly calcium-dependent. Heterodimer formation is not a simple process and our biophysical analyses (FRET, ESI-MS) demonstrate that simply mixing both subunits is not sufficient to induce complex formation. Steps of denaturation/renaturation are necessary for the recombinant complex to show identical biophysical properties as S100A8/S100A9 obtained from granulocytes. In addition to calcium both proteins are able to bind zinc with high affinity. Here we demonstrate for the first time by different biophysical methods (MALDI-MS, ESI-MS, fluorescence spectroscopy) that zinc-binding, like calcium, induces (S100A8/S100A9)(2)-tetramers. Using mass spectrometric investigations we demonstrate that zinc triggers the formation of (S100A8/S100A9)(2)-tetramers by zinc-specific binding sites rather than by interactions with calcium-specific EF-hands. The zinc-induced tetramer is structurally very similar to the calcium-induced tetramer. Thus, like calcium, zinc acts as a regulatory factor in S100A8/S100A9-dependent signaling pathways.

Serum and urinary concentrations of calprotectin as markers of insulin resistance and type 2 diabetes

“…Calgranulin monomers (calgranulin A and B) show a strong preference to form a calcium-dependent heterodimer called calprotectin and is regarded as the functional relevant form at circulating levels (24). This complex has been suggested as a biomarker for inflammation and of utility in the monitoring of disease activity (25).…”

“…At 20 min, regular insulin (0.03 U/kg; Actrapid, Novo, Hellerup, Denmark) was injected as a bolus. Additional samples were obtained from a contralateral antecubital vein at times 1, 2, 3, 4, 5,6,7,8,10,12,14,16,19,20,22,23,24,25,27,30,40, 50, 60, 70, 80, 90, 100, 120, 140, 160, and 180 min. Samples were rapidly collected via a three-way stopcock connected to the butterfly needle.…”

Global Gene Expression Profiling Unveils S100A8/A9 as Candidate Markers in H-Ras-Mediated Human Breast Epithelial Cell Invasion

“…Recent studies showed that S100 proteins form tetramers or even higher-order oligomers in a calcium-dependent manner, and that oligomerization is an essential prerequisite for their biological functions (16,17). To further investigate the significance of S100A8/S100A9 in H-Ras -mediated cell invasion/migration, we temporarily depleted intracellular calcium in H-Ras MCF10A cells using BAPTA/AM, an intracellular calcium chelator (18).…”

Transcriptome Assessment Reveals a Dominant Role for TLR4 in the Activation of Human Monocytes by the Alarmin MRP8

“…MRP8 was expressed in E. coli BL21(DE3) cells and purified as previously described (9,30). Briefly, pET11/20 expression vector containing human or murine MRP8 cDNA was used to transform E. coli BL21(DE3) bacteria.…”