Characterization and engineering of S100A12-heparan sulfate interactions.

S100A12, an EF-hand calcium-binding protein, can be secreted by a variety of cell types and plays proinflammatory roles in a number of pathological conditions. Although S100A12 has been shown to interact with heparan sulfate (HS), the molecular detail of the interaction remains unclear. Here we investigate the structural basis of S100A12-HS interaction and how the interaction is regulated by the availability of divalent cations and the oligomeric states of S100A12. We discovered that S100A12-HS interaction requires calcium, while zinc can further enhance binding by inducing S100A12 hexamerization. In contrast, the apo form and zinc-induced tetramer form were unable to bind HS. Guided by the crystal structures of S100A12, we have identified the HS-binding site of S100A12 by site-directed mutagenesis. Characterization of the HS-binding site of S100A12 allowed us to convert the non-HS-binding apo and tetramer forms of S100A12 into a high affinity HS-binding variant by engineering a single-point mutation. Using a HS oligosaccharide microarray, we demonstrated that the N43K mutant displayed markedly enhanced selectivity toward longer HS oligosaccharides compared to the WT S100A12, likely due to the expanded dimension of the reengineered HS-binding site in the mutant. This unexpected finding strongly suggests that HS-binding sites of proteins might be amenable for engineering.

Calcium Regulates S100A12 Zinc Sequestration by Limiting Structural Variations.

Antimicrobial proteins such as S100A12 and S100A8/A9 are highly expressed and secreted by neutrophils during infection and participate in human immune response by sequestering transition metals. At neutral pH, S100A12 sequesters Zn2+ with nanomolar affinity, which is further enhanced upon calcium binding. We investigated the pH dependence of human S100A12 zinc sequestration by using Co2+ as a surrogate. Apo-S100A12 exhibits strong Co2+ binding between pH 7.0 and 10.0 that progressively diminishes as the pH is decreased to 5.3. Ca2+ -S100A12 can retain nanomolar Co2+ binding up to pH 5.7. NMR spectroscopic measurements revealed that calcium binding does not alter the side-chain protonation of the Co2+ /Zn2+ binding histidine residues. Instead, the calcium-mediated modulation is achieved by restraining pH-dependent conformational changes to EF loop 1, which contains Co2+ /Zn2+ binding Asp25. This calcium-induced enhancement of Co2+ /Zn2+ binding might assist in the promotion of antimicrobial activities in humans by S100 proteins during neutrophil activation under subneutral pH conditions.

Ca(II) and Zn(II) Cooperate To Modulate the Structure and Self-Assembly of S100A12.

S100A12 is a member of the Ca2+ binding S100 family of proteins that functions within the human innate immune system. Zinc sequestration by S100A12 confers antimicrobial activity when the protein is secreted by neutrophils. Here, we demonstrate that Ca2+ binding to S100A12's EF-hand motifs and Zn2+ binding to its dimeric interface cooperate to induce reversible self-assembly of the protein. Solution and magic angle spinning nuclear magnetic resonance spectroscopy on apo-, Ca2+-, Zn2+-, and Ca2+,Zn2+-S100A12 shows that significant metal binding-induced chemical shift perturbations, indicative of conformational changes, occur throughout the polypeptide chain. These perturbations do not originate from changes in the secondary structure of the protein, which remains largely preserved. While the overall structure of S100A12 is dominated by Ca2+ binding, Zn2+ binding to Ca2+-S100A12 introduces additional structural changes to helix II and the hinge domain (residues 38-53). The hinge domain of S100A12 is involved in the molecular interactions that promote chemotaxis for human monocyte, acute inflammatory responses and generates edema. In Ca2+-S100A12, helix II and the hinge domain participate in binding with the C-type immunoglobulin domain of the receptor for advanced glycation products (RAGE). We discuss how the additional conformational changes introduced to these domains upon Zn2+ binding may also impact the interaction of S100A12 and target proteins such as RAGE.

The localization of CD3, CD79a, CD68 and S100 protein immunoreactive cells in hemal nodes of Saanen goat (Capra hircus).

We investigated the structure of hemal nodes in Saanen goats using immunohistochemical staining. We examined the distribution of CD3 positive T lymphocytes, CD79a positive B lymphocytes, CD68 positive macrophages and S100 protein positive follicular dendritic cells. Hemal nodes of six healty adult female goats were used. Hemal nodes were removed from the thoracic and abdominal cavities. The oval to round hemal nodes were observed especially between the abdominal aorta and vena cava, and near the kidneys and adrenal glands. Tissue sections were stained with Crossmon's modified triple stain to demonstrate general histological structure. The avidin-biotin-peroxidase technique using anti-CD3, anti-CD79a, anti-CD68 and anti-S100 primary antibodies was used for immunohistochemistry. Many CD3 positive T lymphocytes were found in the germinal center of the lymph follicles and in the lymphatic cords of hemal nodes; CD3 positive cells also were observed in the sinuses. CD79a and CD68 positive cells were found at the germinal center of the lymph follicles. In the lymph follicles near the subcapsular sinuses, CD79a and CD68 positive cells were found especially in e areas bordering the mantle zone. S100 positive cells were found in the lymph follicles, lymphatic cords and sinuses.

A Method for Selective Depletion of Zn(II) Ions from Complex Biological Media and Evaluation of Cellular Consequences of Zn(II) Deficiency.

We describe the preparation, evaluation, and application of an S100A12 protein-conjugated solid support, hereafter the "A12-resin", that can remove 99% of Zn(II) from complex biological solutions without significantly perturbing the concentrations of other metal ions. The A12-resin can be applied to selectively deplete Zn(II) from diverse tissue culture media and from other biological fluids, including human serum. To further demonstrate the utility of this approach, we investigated metabolic, transcriptomic, and metallomic responses of HEK293 cells cultured in medium depleted of Zn(II) using S100A12. The resulting data provide insight into how cells respond to acute Zn(II) deficiency. We expect that the A12-resin will facilitate interrogation of disrupted Zn(II) homeostasis in biological settings, uncovering novel roles for Zn(II) in biology.

Consequences of Energetic Frustration on the Ligand-Coupled Folding/Dimerization Dynamics of Allosteric Protein S100A12.

Allosteric proteins are featured by energetic degeneracy of two (or more) functionally relevant conformations, therefore their energy landscapes are often locally frustrated. How such frustration affects the protein folding/binding dynamics is not well understood. Here, by using molecular simulations we study the consequences of local frustration in the dimerization dynamics of allosteric proteins based on a homodimer protein S100A12. Despite of the structural symmetry of the two EF-hand motifs in the three-dimensional structures, the S100A12 homodimer shows allosteric behaviors and local frustration only in half of its structural elements, i.e., the C-terminal EF-hand. We showed that such spatially asymmetric location of frustration leads to asymmetric dimerization pathways, in which the dimerization is dominantly initiated by the interchain binding of the minimally frustrated N-terminal EF-hands, achieving optimal balance between the requirements of rapid conformational switching and interchain assembling to the energy landscapes. We also showed that the local frustration, as represented by the double-basin topography of the energy landscape, gives rise to multiple cross-linked dimerization pathways, in which the dimerization is coupled with the allosteric motions of the C-terminal EF-hands. Binding of metal ions tends to reshape the energy landscape and modulate the dimerization pathways. In addition, by employing the frustratometer method, we showed that the highly frustrated residue-pairs in the C-terminal EF-hand are partially unfolded during the conformational transitions of the native homodimer, leading to lowing of free energy barrier. Our results revealed tight interplay between the local frustration of the energy landscape and the dimerization dynamics for allosteric proteins.

Blocking the Interactions between Calcium-Bound S100A12 Protein and the V Domain of RAGE Using Tranilast.

The receptor for advanced glycation end products (RAGE), a transmembrane receptor in the immunoglobulin superfamily, is involved in several inflammatory processes. RAGE induces cellular signaling pathways upon binding with various ligands, such as advanced glycation end products (AGEs), ß-amyloids, and S100 proteins. The solution structure of S100A12 and the V ligand-binding region of RAGE have been reported previously. Using heteronuclear NMR spectroscopy to conduct 1H-15N heteronuclear single quantum coherence (HSQC) titration experiments, we identified and mapped the binding interface between S100A12 and the V domain of RAGE. The NMR chemical shift data were used as the constraints for the High Ambiguity Driven biomolecular DOCKing (HADDOCK) calculation to generate a structural model of the S100A12-V domain complex. In addition, tranilast (an anti-allergic drug) showed strong interaction with S100A12 in the 1H-15N HSQC titration, fluorescence experiments, and WST-1 assay. The results also indicated that tranilast was located at the binding site between S100A12 and the V domain, blocking interaction between these two proteins. Our results provide the mechanistic details for a structural model and reveal a potential precursor for an inhibitor for pro-inflammatory diseases, which could be useful for the development of new drugs.

Diagnostic value of amniotic fluid inflammatory biomarkers for subclinical chorioamnionitis.

OBJECTIVE: To determine the value of measuring amniotic fluid inflammatory biomarkers for diagnosis of subclinical chorioamnionitis. METHODS: A prospective study was conducted among pregnant women with cervical dilation, preterm premature rupture of membranes, threatened late abortion, or threatened premature labor who attended a tertiary care hospital in Guangzhou, China, between June 1, 2012, and January 31, 2014. Participants were divided into two groups according to the presence or absence of subclinical chorioamnionitis. Surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF-MS) was used to detect human neutrophil defensins (HNP-1 and HNP-2), calgranulins A (S100A8), and calgranulins C (S100A12) in amniocentesis samples. RESULTS: Overall, 22 patients had subclinical chorioamnionitis and 17 patients did not. Positive test results for HNP-2 were noted for more patients with subclinical chorioamnionitis than for those without for HNP-2 (19 [86%] vs 2 [12%]; P<0.001), HNP-1 (19 [86%] vs 5 [29%]; P=0.001), S100A12 (20 [91%] vs 9 [53%]; P=0.011), and S100A8 (12 [55%] vs 0; P<0.001). When three or four of these biomarkers were present, the accuracy for a diagnosis of subclinical chorioamnionitis was 89.7%. The sensitivity, specificity, positive predictive value, and negative predictive value were 81.8%, 100.0%, 100.0%, and 81.0%, respectively. CONCLUSION: Detection of inflammatory biomarkers in the amniotic fluid by SELDI-TOF-MS exhibited high diagnostic accuracy for subclinical chorioamnionitis.

Secretion of S100A8, S100A9, and S100A12 by Neutrophils Involves Reactive Oxygen Species and Potassium Efflux.

S100A8/A9 (calprotectin) and S100A12 proinflammatory mediators are found at inflammatory sites and in the serum of patients with inflammatory or autoimmune diseases. These cytoplasmic proteins are secreted by neutrophils at sites of inflammation via alternative secretion pathways of which little is known. This study examined the nature of the stimuli leading to S100A8/A9 and S100A12 secretion as well as the mechanism involved in this alternative secretion pathway. Chemotactic agents, cytokines, and particulate molecules were used to stimulate human neutrophils. MSU crystals, PMA, and H2O2 induced the release of S100A8, S100A9, and S100A12 homodimers, as well as S100A8/A9 heterodimer. High concentrations of S100A8/A9 and S100A12 were secreted in response to nanoparticles like MSU, silica, TiO2, fullerene, and single-wall carbon nanotubes as well as in response to microbe-derived molecules, such as zymosan or HKCA. However, neutrophils exposed to the chemotactic factors fMLP failed to secrete S100A8/A9 or S100A12. Secretion of S100A8/A9 was dependent on the production of reactive oxygen species and required K(+) exchanges through the ATP-sensitive K(+) channel. Altogether, these findings suggest that S100A12 and S100A8/A9 are secreted independently either via distinct mechanisms of secretion or following the activation of different signal transduction pathways.