Iron Sequestration by Murine Calprotectin Induces Starvation Responses in Pseudomonas aeruginosa.

Pathogen sensing by the mammalian host induces a pro-inflammatory response that involves release of the antimicrobial metal-sequestering protein calprotectin (CP, S100A8/S100A9 heterooligomer, MRP8/MRP14 heterooligomer) from neutrophils. Biochemical investigations on human CP (hCP) have informed the molecular basis of how this protein sequesters metal ions. Murine models of infection have provided invaluable insights into the ability of murine CP (mCP) to compete with bacterial pathogens for essential metal nutrients. Despite this extensive work, our knowledge of how mCP sequesters metals from bacterial pathogens and its impacts on bacterial physiology is limited. Moreover, whether mCP sequesters iron and induces iron-starvation responses in bacterial pathogens has not been evaluated. Here, we examine the ability of mCP to withhold iron from Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen that causes severe infections in immunocompromised individuals and cystic fibrosis patients. We demonstrate that mCP prevents iron uptake and induces iron-starvation responses in P. aeruginosa laboratory strains PA14 and PAO1 and the JSRI-1 clinical isolate from a cystic fibrosis patient. We also show that mCP prevents iron uptake and induces an iron-starvation response in the Gram-positive bacterial pathogen Staphylococcus aureus. The His6 site of mCP is the iron-sequestering site; it exhibits Ca(II)-dependent Fe(II) affinity and binds Fe(II) with subpicomolar affinity in the presence of excess Ca(II) ions. This work is important for understanding the structure, function, and physiological consequences of mCP and how the mammalian host and bacterial pathogens compete for essential metal nutrients.

Spectroscopic and computational investigations of Cobalt(II) binding to the innate immune protein human calprotectin.

Human calprotectin (CP) is an innate immune protein that participates in the metal-withholding response to infection by sequestering essential metal nutrients from invading microbial pathogens. CP is comprised of S100A8 (α subunit, 10.8 kDa) and S100A9 (ß subunit, 13.2 kDa). Two transition-metal binding sites of CP form at the S100A8/S100A9 dimer interface. Site 1 is a His3Asp motif comprised of His83 and His87 from the S100A8 subunit and His20 and Asp30 from the S100A9 subunit. Site 2 is an unusual hexahistidine motif composed of S100A8 residues His17 and His27 and S100A9 residues His91, His95, His103, and His105. In the present study, the His3Asp and His6 sites of CP were further characterized by utilizing Co2+ as a spectroscopic probe. Magnetic circular dichroism spectroscopy was employed in conjunction with electron paramagnetic resonance spectroscopy and density functional theory computations to characterize the Co2+-bound S100A8(C42S)/S100A9(C3S) CP-Ser variant and six site variants that allowed the His3Asp and His6 sites to be further probed. Our results provide new insight into the metal-binding sites of CP-Ser and the effect of amino acid substitutions on the structure of site 2.

Calprotectin as new potential clinical marker for multiple myeloma.

Increased levels of inflammatory cytokines in multiple myeloma (MM) patients and the role of inflammation in disease pathogenesis, have recently been considered. The aim of this study was to quantitatively evaluation of fecal calprotectin (CP) as a non-invasive biomarker for the evaluation of inflammation in patients with multiple myeloma. This study is a hospital-based case control study. MM patients referred to patients referred to medical centers of Tehran province, Iran, were identified and classified into two groups of new MM patients (n = 40) and patients undergoing treatment (n = 28). Healthy individuals were included in the study as healthy control (n = 25). Morning stool samples were collected and CP was extracted immediately. After collecting the samples, CP was measured according to ELISA method and was determined in µg/g of feces. Values ​​above 50 µg/g of feces are positive and indicate inflammation. The results revealed that there is a significant difference between groups in terms if CP mean (p = 0.001). The mean of CP among new cases, under treatment and control groups were 301.3 (SD: 141.0), 165.1 (SD: 153.9) and 36.9 (SD: 13.5), respectively. Then the groups were compared in pairs, the results showed that the new case group was significantly different from the under-treatment group (p = 0.001), and also the control group showed a significant difference with the new case group (p = 0.001) and the under-treatment group (p = 0.001) that the amount of CP in the control group was significantly lower than the other two groups. In addition, the results of the study showed a significant correlation between age and plasma cells with CP value, so that with increasing age and plasma cells, CP value also showed a significant increase. The results indicate that quantitative evaluation of CP as a non-invasive laboratory biomarker has a high potential as a clinical marker in patients with multiple myeloma and inflammation should considered as a hallmark of cancer. Further diagnostic studies are recommended.

S100A12 promotes Mn(II) binding to pneumococcal PsaA and staphylococcal MntC by Zn(II) sequestration.

Human S100A12 (calgranulin C, EN-RAGE) is a Zn(II)-sequestering host-defense protein that contributes to the metal-withholding innate immune response against microbial pathogens. S100A12 coordinates Zn(II) ions at two His3Asp sites with high affinity. A similar His3Asp site found in calprotectin (S100A8/S100A9, calgranulin A/B), a closely related human S100 protein, can sequester divalent metal ions from the solute-binding proteins (SBPs) pneumococcal PsaA (pneumococcal surface protein A) and staphylococcal MntC (manganese transport protein C). Both SBPs are components of Mn(II) transporters and capture extracellular Mn(II) ions for subsequent delivery into the bacterial cytosol. Nevertheless, PsaA and MntC exhibit a thermodynamic preference for Zn(II) over Mn(II), and Zn(II) binding can interfere with Mn(II) acquisition. In this work, we have used a biotinylated variant of S100A12 to show that S100A12 can sequester Zn(II) ions from PsaA and MntC. Moreover, electron paramagnetic resonance (EPR) spectroscopy indicates that by sequestering Zn(II) from Zn(II)-bound PsaA and MntC, S100A12 promotes Mn(II) binding to the SBPs. These results inform the function of S100A12 in Zn(II) sequestration, and further suggest that Zn(II)-sequestering S100 proteins may inadvertently protect bacterial pathogens during infection.

TdfH selectively binds metal-loaded tetrameric calprotectin for zinc import.

To combat nutritional immunity, N. gonorrhoeae has evolved systems to hijack zinc and other metals directly from host metal-binding proteins such as calprotectin (CP). Here, we report the 6.1 Å cryoEM structure of the gonococcal surface receptor TdfH in complex with a zinc-bound CP tetramer. We further show that TdfH can also interact with CP in the presence of copper and manganese, but not with cobalt.

Metal sequestration by S100 proteins in chemically diverse environments.

During infection, the mammalian host initiates a metal-withholding response against invading microbial pathogens to inhibit their growth and survival, a process often termed 'nutritional immunity'. The host-defense S100 proteins calprotectin (CP) (S100A8/S100A9 oligomer), S100A12, and S100A7 play key roles in the innate immune response by sequestrating essential transition metal nutrients from microbes in the extracellular space. Accumulating evidence suggests that the antimicrobial activity of these proteins varies between infection sites and may be affected by the local chemical environment. Herein, we discuss the interplay between host metal-withholding proteins and microbial pathogens in the context of the chemical complexity of infection sites and highlight recent advances in our understanding of how chemically diverse conditions affect the properties and functions of S100 proteins.

Metal Sequestration and Antimicrobial Activity of Human Calprotectin Are pH-Dependent.

Human calprotectin (CP, S100A8/S100A9 oligomer) is an abundant innate immune protein that sequesters transition metal ions in the extracellular space to limit nutrient availability and the growth of invading microbial pathogens. Our current understanding of the metal-sequestering ability of CP is based on biochemical and functional studies performed at neutral or near-neutral pH. Nevertheless, CP can be present throughout the human body and is expressed at infection and inflammation sites that tend to be acidic. Here, we evaluate the metal binding and antimicrobial properties of CP in the pH range of 5.0-7.0. We show that Ca(II)-induced tetramerization, an important process for the extracellular functions of CP, is perturbed by acidic conditions. Moreover, a low pH impairs the antimicrobial activity of CP against some bacterial pathogens, including Staphylococcus aureus and Salmonella enterica serovar Typhimurium. At a mildly acidic pH, CP loses the ability to deplete Mn from microbial growth medium, indicating that Mn(II) sequestration is attenuated under acidic conditions. Evaluation of the Mn(II) binding properties of CP at pH 5.0-7.0 indicates that mildly acidic conditions decrease the Mn(II) binding affinity of the His6 site. Lastly, CP is less effective at preventing capture of Mn(II) by the bacterial solute-binding proteins MntC and PsaA at low pH. These results indicate that acidic conditions compromise the ability of CP to sequester Mn(II) and starve microbial pathogens of this nutrient. This work highlights the importance of considering the local pH of biological sites when describing the interplay between CP and microbes in host-pathogen interactions.

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry.

Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients.

Effects of unsaturated fatty acids (Arachidonic/Oleic Acids) on stability and structural properties of Calprotectin using molecular docking and molecular dynamics simulation approach.

Calprotectin is a heterodimeric protein complex with two subunits called S100A8/A9. The protein has an essential role in inflammation process and various human diseases. It has the ability to bind to unsaturated fatty acids including Arachidonic acid, Oleic acid and etc., which could be considered as a major carrier for fatty acids. In this study we aimed to appraise the thermodynamics and structural changes of Calprotectin in presence of Arachidonic acid/Oleic acid) using docking and molecular dynami simulation method. To create the best conformation of Calprotectin-Oleic acid/Arachidonic acid complexes, the docking process was performed. The complexes with the best binding energy were selected as the models for molecular dynamics simulation process. Furthermore, the structural and thermodynamics properties of the complexes were evaluated too. The Root Mean Square Deviation and Root Mean Square Fluctuation results showed that the binding of Arachidonic acid/Oleic acid to Calprotectin can cause the protein structural changes which was confirmed by Define Secondary Structure of Proteins results. Accordingly, the binding free energy results verified that binding of Oleic acid to Calprotectin leads to instability of S100A8/A9 subunits in the protein. Moreover, the electrostatic energy contribution of the complexes (Calprotectin-Oleic acid/Arachidonic acid) was remarkably higher than van der Waals energy. Thus, the outcome of this study confirm that Oleic acid has a stronger interaction with Calprotectin in comparison with Arachidonic acid. Our findings indicated that binding of unsaturated fatty acids to Calprotectin leads to structural changes of the S100A8/A9 subunits which could be beneficial to play a biological role in inflammation process.

Avian MRP126 Restricts Microbial Growth through Ca(II)-Dependent Zn(II) Sequestration.

The calgranulins form a class of S100 proteins in higher vertebrates that innate-immune cells release in abundance at infection sites. These proteins function by binding transition metal ions to prevent microbial pathogens from obtaining those essential nutrients. Mammals express three distinct members of this family: S100A8 (calgranulin A), S100A9 (calgranulin B, which heterooligomerizes with S100A8 to form calprotectin), and S100A12 (calgranulin C), that exhibit Ca(II)-dependent transition metal binding properties. Human calprotectin effectively sequesters Mn(II), Fe(II), Ni(II), and Zn(II), whereas human S100A12 selectively sequesters Zn(II) over these other metal ions. Birds and reptiles express a single calgranulin homologue named MRP126, which we reasoned could have properties more similar to those of either calprotectin or S100A12. Here we present the purification and biophysical characterization of recombinant chicken MRP126 and, to the best of our knowledge, provide the first assessment of the metal binding and antimicrobial properties of an avian MRP126. We show that MRP126 is a homodimer that selectively sequesters Zn(II) and restricts the growth of certain microbes. MRP126 binds Zn(II) at two canonical His3Asp sites. The presence of excess Ca(II) increases the affinity of the His3Asp sites from the low-nanomolar to the low-picomolar range, thereby enhancing antimicrobial activity. Chicken MRP126 also binds additional Zn(II) equivalents with low-nanomolar affinity at two nonconserved dicysteine sites and with high-nanomolar affinity using a histidine-rich C-terminal tail that is a hallmark of this clade of calgranulins. Our results with chicken MRP126 suggest that Ca(II)-dependent Zn(II) sequestration was a role of the last common ancestor of calgranulin proteins, with mammalian calprotectin subsequently evolving a broader metal binding repertoire.

In silico assessment of human Calprotectin subunits (S100A8/A9) in presence of sodium and calcium ions using Molecular Dynamics simulation approach.

Calprotectin is a heterodimeric protein complex which consists of two subunits including S100A8 and S100A9. This protein has a major role in different inflammatory disease and various types of cancers. In current study we aimed to evaluate the structural and thermodynamic changes of the subunits and the complex in presence of sodium and calcium ions using molecular dynamics (MD) simulation. Therefore, the residue interaction network (RIN) was visualized in Cytoscape program. In next step, to measure the binding free energy, the potential of mean force (PMF) method was performed. Finally, the molecular mechanics Poisson-Boltzmann surface area (MMPBSA) method was applied as an effective tool to calculate the molecular model affinities. The MD simulation results of the subunits represented their structural changes in presence of Ca2+. Moreover, the RIN and Hydrogen bond analysis demonstrated that cluster interactions between Calprotectin subunits in presence of Ca2+ were greater in comparison with Na+. Our findings indicated that the binding free energy of the subunits in presence of Ca2+ was significantly greater than Na+. The results revealed that Ca2+ has the ability to induce structural changes in subunits in comparison with Na+ which lead to create stronger interactions between. Hence, studying the physical characteristics of the human proteins could be considered as a powerful tool in theranostics and drug design purposes.

Exploring Iron Withholding by the Innate Immune Protein Human Calprotectin.

Calprotectin (CP) is a versatile player in the metal-withholding innate immune response, a process termed "nutritional immunity." CP is a heterooligomer of the polypeptides S100A8 and S100A9 and houses two transition-metal-binding sites at its S100A8/S100A9 heterodimer interface. During infection, CP is released from host cells and sequesters "bioavailable" transition metal ions in the extracellular space, thereby preventing microbial acquisition of these essential nutrients. For many years, the role of CP in nutritional immunity was interpreted in the contexts of Mn(II) and Zn(II) limitation, but recent work has broadened our understanding of its contributions to this process. We uncovered that CP provides a form of nutritional immunity that has previously received little attention: the battle between host and microbe for ferrous iron (Fe(II)). In this Account, we present our current understanding of Fe(II) coordination by CP and its role in Fe(II) withholding as well as considerations for future discovery. Nutritional immunity was first described in the context of host-microbe competition for ferric iron (Fe(III)). The battle for Fe(II) has received comparably little attention because the abundance of Fe(II) at infection sites and the importance of Fe(II) acquisition for microbial pathogenesis were recognized only recently. Several years ago, we discovered that human CP sequesters Fe(II) at its His6 site with subpicomolar affinity and thus hypothesized that it provides a means for Fe(II) limitation by the host during microbial infection. Fe(II) coordination by CP is unprecedented in biology because of its novel hexahistidine coordination sphere and its high-affinity binding, which surpasses that of other known Fe(II)-binding proteins. CP is also capable of shifting the Fe redox equilibrium by stabilizing Fe(II) in aerobic solution and can thereby sequester Fe in both reducing and nonreducing environments. These coordination chemistry studies allowed us to hypothesize that CP provides a means for Fe(II) limitation by the host during microbial infection. While investigating this putative Fe(II)-sequestering function, we discovered that CP withholds Fe from diverse bacterial pathogens. Recent studies by our lab and others of the bacterial pathogens Pseudomonas aeruginosa and Acinetobacter baumannii have shown that, by preventing sufficient Fe acquisition, CP induces Fe starvation responses in these organisms. As a result, CP affects bacterial virulence and metabolism. We also elucidated a complex interplay between CP and secondary metabolites produced by P. aeruginosa during the competition for Fe. Our work provides a foundation for understanding how CP affects Fe homeostasis during microbial infection. We believe that understanding how bacterial physiology is altered when challenged with Fe(II) withholding by CP will likely reveal crucial determinants of bacterial survival within the host.

Murine Calprotectin Coordinates Mn(II) at a Hexahistidine Site with Ca(II)-Dependent Affinity.

Manganese is an essential metal ion that bacterial pathogens need to acquire from the vertebrate host during infection. In the mammalian nutritional immunity strategy to combat bacterial infection, the host restricts bacterial access to Mn(II) by sequestering this metal nutrient using the protein calprotectin (CP). The role of murine calprotectin (mCP) in Mn(II) sequestration has been demonstrated in vivo, but the molecular basis of this function has not been evaluated. Herein, biochemical assays and electron paramagnetic resonance (EPR) spectroscopy are employed to characterize the Mn(II) binding properties of mCP. We report that mCP has one high-affinity Mn(II) binding site. This site is a His6 site composed of His17 and His27 of mS100A8 and His92, His97, His105, and His107 of mS100A9. Similar to the human ortholog (hCP), Ca(II) binding to the EF-hand domains of mCP enhances the Mn(II) affinity of the protein; however, this effect requires ≈10-fold more Ca(II) than was previously observed for hCP. Mn(II) coordination to the His6 site also promotes self-association of two mCP heterodimers to form a heterotetramer. Low-temperature X-band EPR spectroscopy revealed a nearly octahedral Mn(II) coordination sphere for the Mn(II)-His6 site characterized by the zero-field splitting parameters D = 525 MHz and E/D = 0.3. Further electron-nuclear double resonance studies with globally 15N-labeled mCP provided hyperfine couplings from the coordinating ε-nitrogen atoms of the His ligands (aiso = 4.3 MHz) as well as the distal δ-nitrogen atoms (aiso = 0.25 MHz). Mn(II) competition assays between mCP and two bacterial Mn(II) solute-binding proteins, staphylococcal MntC and streptococcal PsaA, showed that mCP outcompetes both proteins for Mn(II) under conditions of excess Ca(II). In total, this work provides the first coordination chemistry study of mCP and reveals striking similarities in the Mn(II) coordination sphere as well as notable differences in the Ca(II) sensitivity and oligomerization behavior between hCP and mCP.

Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis.

Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 µM, cross-linked calprotectin (5 µM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 µM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9)2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.

Faecal calprotectin to detect inflammatory bowel disease: a systematic review and exploratory meta-analysis of test accuracy.

OBJECTIVE: Test accuracy of faecal calprotectin (FC) testing in primary care is inconclusive. We aimed to assess the test accuracy of FC testing in primary care and compare it to secondary care estimates for the detection of inflammatory bowel disease (IBD). METHODS: Systematic review and meta-analysis of test accuracy using a bivariate random effects model. We searched MEDLINE, EMBASE, Cochrane Library and Web of Science until 31 May 2017 and included studies from auto alerts up until 31 January 2018. Eligible studies measured FC levels in stool samples to detect IBD in adult patients with chronic (at least 6-8 weeks) abdominal symptoms in primary or secondary care. Risk of bias and applicability were assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 criteria. We followed the protocol registered as PROSPERO CRD 42012003287. RESULTS: 38 out of 2168 studies were eligible including five from primary care. Comparison of test accuracy by setting was precluded by extensive heterogeneity. Overall, summary estimates of sensitivity and specificity were not recorded. At a threshold of 50 µg/g, sensitivity from separate meta-analysis of four assay types ranged from 0.85 (95% CI 0.75 to 0.92) to 0.94 (95% CI 0.75 to 0.90) and specificity from 0.67 (95% CI 0.56 to 0.76) to 0.88 (95% CI 0.77 to 0.94). Across three different definitions of disease, sensitivity ranged from 0.80 (95% CI 0.76 to 0.84) to 0.97 (95% CI 0.91 to 0.99) and specificity from 0.67 (95% CI 0.58 to 0.75) to 0.76 (95% CI 0.66 to 0.84). Sensitivity appears to be lower in primary care and is further reduced at a revised threshold of 100 µg/g. CONCLUSIONS: Conclusive estimates of sensitivity and specificity of FC testing in primary care for the detection of IBD are still missing. There is insufficient evidence in the published literature to support the decision to introduce FC testing in primary care. Studies evaluating FC testing in an appropriate primary care setting are needed.

Multi-metal Restriction by Calprotectin Impacts De Novo Flavin Biosynthesis in Acinetobacter baumannii.

Calprotectin (CP) inhibits bacterial viability through extracellular chelation of transition metals. However, how CP influences general metabolism remains largely unexplored. We show here that CP restricts bioavailable Zn and Fe to the pathogen Acinetobacter baumannii, inducing an extensive multi-metal perturbation of cellular physiology. Proteomics reveals severe metal starvation, and a strain lacking the candidate ZnII metallochaperone ZigA possesses altered cellular abundance of multiple essential Zn-dependent enzymes and enzymes in de novo flavin biosynthesis. The ΔzigA strain exhibits decreased cellular flavin levels during metal starvation. Flavin mononucleotide provides regulation of this biosynthesis pathway, via a 3,4-dihydroxy-2-butanone 4-phosphate synthase (RibB) fusion protein, RibBX, and authentic RibB. We propose that RibBX ensures flavin sufficiency under CP-induced Fe limitation, allowing flavodoxins to substitute for Fe-ferredoxins as cell reductants. These studies elucidate adaptation to nutritional immunity and define an intersection between metallostasis and cellular metabolism in A. baumannii.

Preparation and Iron Redox Speciation Study of the Fe(II)-Binding Antimicrobial Protein Calprotectin.

Calprotectin (CP, S100A8/S100A9 heterooligomer) is an abundant metal-sequestering host-defense protein expressed by neutrophils, other white blood cells, and epithelial cells. The apoprotein is a S100A8/S100A9 heterodimer that contains two sites for transition metal binding at the S100A8/S100A9 interface: a His3Asp motif (site 1) and a His6 motif (site 2). In this chapter, we provide a step-by-step protocol for the overexpression and purification of the human and murine orthologues of CP that affords each apo heterodimer in high yield and purity. In these procedures, the S100A8 and S100A9 subunits are overexpressed in Escherichia coli BL21(DE3), and each apo heterodimer is obtained following cell lysis, folding, column chromatography, and dialysis against Chelex resin to reduce metal contamination. Recent studies demonstrated that human CP coordinates Fe(II) and that the protein affects the redox speciation of Fe in solution. An Fe redox speciation assay employing ferrozine is described that demonstrates the ability of both the human and murine orthologues of CP to shift the redox speciation of Fe from the ferric to the ferrous oxidation state over time.

Excluding inflammatory bowel disease in the irritable bowel syndrome patient: how far to go?

PURPOSE OF REVIEW: Irritable bowel syndrome (IBS) is among the most commonly encountered conditions in primary care and gastroenterology. There is ample evidence that an IBS diagnosis based on symptom-based criteria and exclusion of alarm features that would otherwise support diagnostic testing is accurate and durable. For many clinicians, however, IBS remains a diagnosis of exclusion because of concern surrounding missed diagnoses of inflammatory bowel disease (IBD) or other organic gastrointestinal diseases. Using blood and/or fecal biomarker tests to shift the precolonoscopy probability of IBD in patients with symptoms mimicking IBS is becoming an increasingly reasonable practice with improvement in 'preliminary' tests. RECENT FINDINGS: Fecal calprotectin (FCP) testing appears to be the most sensitive preliminary test for discriminating IBD from IBS. Although both fecal lactoferrin and FCP were superior to serum C-reactive peptide (CRP) in their diagnostic accuracy, FCP is superior to fecal lactoferrin based on available literature. SUMMARY: In patients with IBS with diarrhea who have not undergone previous extensive evaluation, the ability of screening tests to detect colonic inflammation is improving. FCP and fecal lactoferrin are reliable predictors of colonic inflammation and should be considered for standard testing in patients with IBS-D symptoms to help identify those who would benefit most from colonoscopy. Although predictive, there currently are no fecal or serum tests that can definitively identify or subtype IBD.

Calprotectin (S100A8/S100A9): a key protein between inflammation and cancer.

BACKGROUND: Calprotectin (S100A8/S100A9), a heterodimeric EF-hand Ca2+ binding protein, are abundant in cytosol of neutrophils and are involved in inflammatory processes and several cancerous pathogens. OBJECTIVE: The purpose of the present systematic review is to evaluate the pro- and anti-tumorigenic functions of calprotectin and its relation to inflammation. MATERIALS AND METHODS: We conducted a review of studies published in the Medline (1966-2018), Scopus (2004-2018), ClinicalTrials.gov (2008-2018) and Google Scholar (2004-2018) databases, combined with studies found in the reference lists of the included studies. RESULTS: Elevated levels of S100A8/S100A9 were detected in inflammation, neoplastic tumor cells and various human cancers. Recent data have explained that many cancers arise from sites of infection, chronic irritation, and inflammation. The inflammatory microenvironment which largely includes calprotectin, has an essential role on high producing of inflammatory factors and then on neoplastic process and metastasis. CONCLUSION: Scientists have shown different outcomes in inflammation, malignancy and apoptosis whether the source of the aforementioned protein is extracellular or intracellular. These findings are offering new insights that anti-inflammatory therapeutic agents and anti-tumorigenic functions of calprotectin can lead to control cancer development.

Initial Biochemical and Functional Evaluation of Murine Calprotectin Reveals Ca(II)-Dependence and Its Ability to Chelate Multiple Nutrient Transition Metal Ions.

Calprotectin (CP) is an abundant host-defense protein that contributes to the metal-withholding innate immune response by sequestering nutrient metal ions from microbial pathogens in the extracellular space. Over the past decade, murine models of infectious disease have advanced understanding of the physiological functions of CP and its ability to compete with microbes for essential metal nutrients. Despite this extensive work, murine CP (mCP) has not been biochemically evaluated, and structural and biophysical understanding of CP is currently limited to the human orthologue. We present the reconstitution, purification, and characterization of mCP as well as the cysteine-null variant mCP-Ser. Apo mCP is a mS100A8/mS100A9 heterodimer, and Ca(II) binding causes two heterodimers to self-associate and form a heterotetramer. Initial metal-depletion studies demonstrate that mCP depletes multiple first-row transition metal ions, including Mn, Fe, Ni, Cu, and Zn, from complex microbial growth medium, indicating that mCP binds multiple nutrient metals with high affinity. Moreover, antibacterial activity assays show that mCP inhibits the growth of a variety of bacterial species. The metal-depletion and antibacterial activity studies also provide evidence that Ca(II) ions enhance these functional properties of mCP. This contribution provides the groundwork for understanding the similarities and differences between the human and murine orthologues of CP and for further elucidation of its biological coordination chemistry.