Background: S100A8 and S100A9 as two subunits of heterodimeric calprotectin are identified mainly in leukocytes and are involved in inflammatory processes and several cancerous pathogens. This study was performed in order to evaluate the interaction of recombinant calprotectin subunits and to estimate calprotectin’s tertiary and secondary structures.

Objectives: The aim of this study was to investigate the effects of calcium in calprotectin dimerization as a cancer biomarker.

Materials and Methods: Heterodimeric calprotectin was formed with incubation of recombinant S100A8 and S100A9 subunits in the presence of Ca (1 mM), at 25˚C for 15 minutes. Tertiary and secondary structures of S100A8, S100A9 and their complex were investigated, using fluorescence and circular dichroism (CD) spectroscopy, respectively.

Results: Interaction of S100A8 and S100A9 in the presence of Ca²⁺ were revealed by decreasing the emission intensity of intrinsic fluorescence and increasing of the external fluorescence and also changes in the CD spectra of subunits after Ca²⁺ interactions.

Conclusions: The expression of recombinant calprotectin, as an effective protein, can help in diagnosis or treatment of inflammatory and cancer processes in the future. Furthermore, Ca²⁺ induced a partial change in secondary and tertiary structure of calprotectin subunits and this change is probably necessary for protein dimerization.

Calprotectin is a member of the S100 family of proteins, and is a marker of inflammation and a calcium and zinc-binding protein. Expression of calprotectin has been reported mainly in neutrophils (30-60% in the cytosol), followed by monocytes and macrophages (mainly associated with membranes), and to a lesser extent in other cells. Expression of S100A8 and S100A9 and hence calprotectin are induced following recruitment of macrophages to inflammatory sites; calprotectin is not stored in tissue macrophages (1). The calprotectin structure is comprised of a hetero-dimer with two calcium-binding chains and two calcium-binding sites per chain. The heavy chain is a 14 KD protein, also known as MRP14/S100A9/P14/L1H and the light chain is an 8 KD protein, also known as MRP8/S100A8/L1L/P8 (1–3). The chains bind non-covalently in the presence of calcium. Other compounds namely, hetero- or homo-dimer of the two chains, tetrameric or more monomers per polymer chain have also been identified. Twenty-one S100 genes, including those for calprotectin, are clustered on human chromosome 1q21. Until now homo-dimer of S100 proteins including S100A8 and S100A9 have been reported; the primer functional form was reported to be heterodimeric consisting of antiparallel arrangement of S100A8/S100A9 known as calprotectin, which is induced in the presence of calcium. S100A8 and S100A9 are produced primarily in myeloid cells and cells triggered by inflammation of myeloid lineage with the exception of lymphocytes. S100A9 gene deletion leads to the loss of S100A8. Expression of S100A9 and S100A8 proteins in phagocytes are associated with a set of actions in the innate immune system. The expression of these proteins occur during differentiation of macrophages and dendritic cells; both proteins can be simultaneously expressed in monocytes, endothelial cells, keratinocytes and epithelial cells by several mediators such as interleukin (IL1)-alpha, IL1-beta, IL10, IL22, tumor necrosis factor (TNF) alpha and lipoteichoic acid (LPS) (4). Different roles have been reported for calprotectin, including; anti-microbial, cytotoxicity, cytokine-like activity, anti-proliferation, induction of apoptosis, chemotactic effects, leukocyte-endothelium interaction, cell adhesion, immune regulation, inflammation and coagulation responses. Levels of calprotectin were found increase following infections and inflammatory disease states (1, 3). Normally, calprotectin has been reported to be at a concentration of about 5 mg in plasma and 2 mg in stool, with a maximum of 10 mg per liter. S100A8 and S100A9 are soluble mediators, which are involved in cancer processes; they are damage associated molecular patterns (DAMP), involved in tumor progression and malignancy. As DAMP ligands for cell surface receptors, they trigger signaling cascades mediating cellular responses to cytokine and chemokines (4). Calprotectin secretion occurs as a result of a pathological attack when the leukocytes increase in tissues and this occurrence can be traced in the plasma, cerebrospinal fluid, urine or feces (5). Calprotectin is produced as an early inflammatory response protein and has reported to be increased in many different human cancers (3). S100A9 is reported to be involved in de- differentiation of cells by making changes in cytoskeleton in a calcium-dependent manner, and by signaling to normal cells, they can lead changes in neoplasms (6). Genomic changes in S100A8/S100A9 loci in tumors with portions removed, double displacement and condensation have also been reported which may be associated with malignancy. The question is whether calprotectin or its subunits as a diagnostic marker can predict disease progression or metastasis in some cancers? The aim of the present study was to investigate the interactions of calprotectin’s subunits with calcium. There are many evidences that indicate calprotectin is increased in inflammatory diseases as well as many cancers including skin, breast, stomach, prostate and colon (2, 7).

The present study aimed to evaluate the effects of calcium on calprotectin subunits dimerization.

3.1. Materials

The rS100A8 and rS100A9 were obtained from a previous research. Anilinonaphthalene-8-sulfonic acid (ANS), was purchased from Sigma (Sigma-Aldrich, Germany). Calcium chloride (CaCl₂), was purchased from Merck (Germany).

3.2. Complex Formation of r-S100A8/S100A9

For the preparation of r-S100A8/A9 complex, equal volumes of r-S100A8 and r-S100A9 (1 µM), were incubated with calcium chloride (1 mM) in PBS dialysis buffer for at least 15 minutes at 25˚C. Complex formation was investigated by fluorescence and circular dichroism (CD) spectroscopy.

3.3. Circular Dichroism Spectroscopy

The content of regular secondary structures of r-S100A8, r-S100A9 and r-S100A8/A9 complex were examined in the far ultraviolet (UV) region (190-260), which correspond to peptide bond absorption, using an AVIV model J810 spectropolarimeter (JASCO) to give the content of regular secondary structure of proteins. Far UV-CD spectra of 0.04 mg/mL solution of proteins in PBS buffer (pH = 6.5) were obtained with 1 mm path length quartz cell. The background was corrected against the buffer blank. The data were calculated as molar ellipticity (deg.cm²/dmol) assuming a mean residue number of 10⁷ and average molecular weight of 25 KDa for S100A8/A9 complex using the CD deconvolution software. The molar ellipticity was determined as [θ] = 100 × (MRW) × θ_obs/(cl), where θ_obs is the observed ellipticity in degrees at a given wavelength and c is the light path length in cm.

3.4. Intrinsic Fluorescence Spectroscopy

Intrinsic fluorescence of r-S100A8, r-S100A9 and r-S100A8/A9 complex, after treatment with calcium chloride (1 mM), were studied using the Cary eclipse model 100 bio spectrofluorometer equipped with a 150 W xenon lamp and a DR-3 data recorder. The excitation and emission slits were set at 5 and 5 nm, respectively. The intrinsic fluorescence was measured by exciting the protein solution with 1 cm path length cell at 280 nm in PBS dialysis buffer at pH = 6.5 and 25˚C and emission spectra were recorded at the wavelength range of 300-450 nm.

3.5. 8-Anilino-1-Naphthalene Sulfonate Fluorescence Spectroscopy

External fluorescence spectroscopy of r-S100A8/A9 complex was performed with stock solution of 8-anilino-1-naphthalene sulfonate (ANS) (10 mM). The ANS fluorescence of r-S100A8/A9 complex was treated with calcium chloride. Excitation and emission slits were set at 5 and 5 nm, respectively. Emission spectra were recorded from 400 to 650 nm with excitation at 380 nm in increments of 1nm.

4.1. Fluorescence Spectroscopy of r-S100A8/A9 Complex

Intrinsic fluorescence spectra showed changes in the tertiary structure of r-S100A8 and r-S100A9 subunits after complex formation (Figure 1). Calcium connection to the binding sites of S100A8 and S100A9, lead to the displacement of the aromatic residue from hydrophobic environment to surface of proteins. Increasing emission spectra of ANS fluorescence showed a more hydrophobic structure for r-S100A8/A9 after treatment with Ca in comparison with only r-S100A8/A9 (Figure 2).

Figure 1. Intrinsic Fluorescence Emission Spectra of r-S100A8, r-S100A9 and r-S100A8/A9 (1 μM) After Treatment With Ca2+ (1 mM)

Figure 2. 8-Anilino-1-Naphthalene Sulfonate External Fluorescence Emission Spectra of rS100A8/A9 (1μM) in the Absence and Presence of Ca2+ (1 mM)

4.2. Circular Dichroism Assessments of r-S100A8/A9 Complex

Circular dichroism is an ideal technique for monitoring the transitional switch between regular secondary structures in proteins, which can occur as a result of changes in experimental parameters such as treatment with Ca²⁺. The far UV-CD spectra characterize the secondary structures of proteins due to peptide bond absorption, thus changes in these spectra usually reflect major backbone changes in proteins. The far UV-CD spectra of r-S100A8/A9 complex indicate significant changes in the secondary structures, compared with rS100A8 and rS100A9 (Figure 3).

Figure 3. Far Ultra Violet-Circular Dichroism Spectra (Molar Ellipticity [θ]) of r-S100A8, r-S100A9 and r-S100A8/A9 Complex (1 μM) After Treatment With Ca2+ (1 mM)

In this study, the interaction of calprotectin monomers in the presence of Ca²⁺ was investigated. The existence of the heterodimeric calprotectin was confirmed by decreasing intrinsic fluorescence, increasing ANS external fluorescence and change in CD spectra of rS100A8 and A9 after their interaction with Ca²⁺. The role of calcium has been demonstrated in calprotectin function (8). The same structural changes have been reported in the presence of excess Ca²⁺, which in turn increases the propensity of calprotectin to form protein aggregates (9). Spectroscopic techniques were used to verify protein–protein interaction, this required the induction of change in the spectroscopic parameters following complex formation, i.e., change in fluorescence intensity, wavelength maximum or polarization, fluorescence resonance energy transfer efficiency, circular dichroism or nuclear magnetic resonance (NMR) chemical shift or intensity (10). High levels of S100A8 and S100A9 occur during inflammatory processes. Also, there is a close relationship between inflammation and carcinogenesis, while chronic inflammation can increase the risk of tumorigenesis. Even in the absence of inflammation as a causative factor, tumor formation can be due to genetic changes associated with immune cells triggering inflammation. S100A8/S100A9 secretion can also be provoked by tumor cell necrosis followed by hypoxia-induced tumor growth. They can induce tumor formation as a result of the inflammatory process or elicit inflammatory response. They can mediate or prompt tumor formation and/or anti-tumor responses. As an anti-tumor, S100A8/S100A9, act to induce cytotoxicity and apoptosis in tumor cells. Promotion of growth signals, blocking growth-inhibitors, apoptosis inhibition, potential uncontrolled proliferation, initiation of angiogenesis, tumor invasion and metastasis, are the essentials for malignant tumor (4). However, in spite of the anti-tumor properties of S100A8/S100A9 and the possibility of their use as tools for cancer therapy (still not proven in vivo), this complex triggers a number of responses leading to tumor formation. The effective dose of S100A8/S100A9 for tumor cell apoptosis is 20-25 micrograms per milliliter, whereas lower concentrations of S100A8/S100A9 cause proliferation of tumor cells. Pro-apoptotic effects of S100A8/S100A9 ensue receptors for advanced glycation end products (RAGE), also, effects on growth promotion follow RAGE-induced signaling pathways by phosphorylation of mitogen-activated protein kinase (MAPK) and the activity of NF-KP (involved in cell signaling pathways) (11–14). Toll-like receptors (TLR) are membrane receptors associated with innate immune inflammatory response against pathogens. S100A8/S100A9 boost inflammatory responses by TLR4 and recently, the important role of TLR in carcinogenesis has been identified (4). The molecular pathways mediated by S100A8/S100A9 are potential targets for development of new cancer treatments and detection of cancer biomarkers for early diagnosis and treatment processes (Table 1). In colon cancer, the abnormal increase in stool calprotectin, which is stable against enzymatic degradation, may be used as a biomarker for screening patients with acute colorectal cancer from healthy individuals (15). However, calprotectin lacks the required efficiency for screening patients with inflammatory bowel disease or polyps, and neoplasms. Furthermore, since calprotectin levels are increased to some extent in these subjects, stool calprotectin testing, could help isolate patients suspected of having colon cancer to be subjected to colonoscopy for determination of their health or disease progression (16). Also, levels of S100A9 expression in the stool can be a marker for diagnosis of metastasis and follow-up treatment in colorectal cancer. Using a combination of stool blood and calprotectin can yield more accurate diagnosis of colorectal cancer (17). Overexpression of S100A8 and S100A9 occur in breast cancer, which are associated with poor cell differentiation and mitotic activity of the tumor cells respectively, and therefore, may be used as a marker for diagnosis, monitoring therapy as well as detecting metastasis (18). Also, they may be used as drug targets (19). S100A9 expression increases in prostate cancer, which may be used to differentiate patients with benign tumors from those with malignant tumors. Moreover, it can be used as a marker for the onset of metastasis (20, 21). Overexpression of calprotectin, S100A8 and S100A9 in ovarian cancer, may be used as a diagnostic marker to detect malignant tumors and in endometrial cancer this overexpression can act as a diagnostic marker, and monitor therapy or disease progression (22, 23). Overexpression of calprotectin and S100A8 have been reported in endometrial cancer, which can be used as a diagnostic marker and for monitoring therapy or disease progression (7). High levels of S100A8/S100A9 expression may be used as a marker for the diagnosis of severe inflammation. S100A9 increases in thyroid cancer cells, and thus may be used as a marker for predicting the disease process and perhaps can be one of the drug targets in the future (6). S100A8 and S100A9 may be used for the diagnosis of esophageal cancer (24) and gastric cancer and metastasis (25). Increased expression of S100A9 in lung cancer macrophages is a risk factor for the onset of metastasis and predicts weak recovery for patients; this increased expression can also act as a diagnostic marker for liver and larynx cancer (26). Therefore, calprotectin or its subunits may be used as a cheap, non-invasive, readily available and easy marker. The structure and function of this protein and its subunits showed a narrow dependency to Ca²⁺. In this study the structural change of calprotectin and its subunits were confirmed by fluorescence and CD techniques. It seemed that Ca²⁺ induced a partial change in secondary and tertiary structure of subunits and was probably necessary for protein dimerization.

Table 1. S100A8/A9 and Calprotectin as Biomarkers in Different Cancers

The authors appreciate the financial support of the Research Council of Qazvin University of Medical Sciences.