The effects of a truncated form of Staphylococcus aureus protein A (SpA) on the expression of cytokines of autoimmune patients and healthy individuals

doi:10.21203/rs.3.rs-1635617/v1

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The effects of a truncated form of Staphylococcus aureus protein A (SpA) on the expression of cytokines of autoimmune patients and healthy individuals

https://doi.org/10.21203/rs.3.rs-1635617/v1

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Background: The recombinant Staphylococcal protein A (SpA) is widely used in biotechnology to purify polyclonal and monoclonal IgG antibodies. At very low concentrations, the highly-purified form of the protein A can down-regulate the activation of human B-lymphocytes and macrophages which are the key cells in determining autoimmune diseases.

Methods: In the present study, the efficiency of three different forms of protein A, including native SpA, the recombinant full-length SpA, and a truncated form of SpA on the reduction of 4 inflammatory cytokines, including IL-8, IL-1β, TNF-α, and IL-6 by peripheral blood mononuclear cell (PBMCs) were studied and compared to an anti-rheumatoid arthritis commercial drug, Enbrel. The recombinant proteins were expressed in E. coli expression vector and the native form of SpA was commercially provided. PBMCs were obtained from adult patients with active rheumatoid arthritis (RA) and healthy control donors. Then, the effect of different doses of the three pure forms of SpA in comparison with Enbrel was investigated by analyzing the expression of selected cytokines using ELISA.

Results: The results showed that the recombinant truncated form of SpA significantly reduced the expression of the cytokines more effectively than other full-length formulations as well as the commercial drug Enbrel. In silico analysis suggests that in the truncated protein, by increasing the radius of gyration, the IgG-binding domains find a more open structure and are more exposed to IgG.

Conclusion: In conclusion, our results showed that the truncated form of protein A is the most potent form of SpA that reduces the secretion of the evaluated cytokines from PBMCs in vitro.

Autoimmune diseases

Cytokines

Protein A

Recombinant drug

Rheumatoid arthritis

Due to the lack of an all-encompassing effective treatment for autoimmune diseases, the prevention of long-term complications of such diseases requires finding a pharmacological treatment [1]. The main medications are the disease-modifying anti-rheumatic drugs (DMARDs) including methotrexate, injectable or oral gold compounds, penicillamine, azathioprine, chloroquine, hydroxychloroquine, sulfasalazine, and biologically modified agents, such as etanercept, infliximab, adalimumab, and anakinra. Besides, these drugs are expensive and associated with many dangerous complications, such as infection and cancer [1].

According to a report, protein A is used as a medication for autoimmune diseases [2]. This protein directly binds to monocytes and a subset of B cells, which are involved in the growth and progression of various autoimmune diseases, and controls diseases such as Rheumatoid arthritis (RA) and Idiopathic thrombocytopenic purpura (ITP) [2].

RA is a dangerous autoimmune disease, in which the immune system’s antibodies mistakenly attack the surface of the joints, creating inflammation and pain [3]. The ITP is a rare disorder that occurs when the blood doesn't clot properly [4]. This happens when a person's immune system recognizes the blood platelets as a foreign agent and gears up for an attack against it [4].

Protein A immunotherapy aims to restore the normal function of the immune system by binding directly to the surface of the monocytes and a B cell subset that is potentially involved in the growth and progression of autoimmune diseases [2]. This mechanism enables protein A to alter the function of these cells and restore the balance to the immune system of the patients [2].

Compared to other medications, significantly lower doses of protein A can be effective and has a long-term effect, which alleviates the need to use it over the long term.

Preliminary research has shown that protein A can be used as a suitable treatment for RA. Preclinical studies on this drug have shown that very low doses are as effective in treating RA as the commercial drug Enbrel and cause fewer side effects [2]. In addition, its clinical effects on collagen-induced autoimmune arthritis have been demonstrated in a mouse model [5]. Protein A is known as a new medication because of its ability to limit blood platelet phagocytosis in patients with ITP [6, 7]. Protein A which was used as a drug in these experiments was secreted to the culture medium by Staphylococcus aureus strain, A676, and was highly pure that was [2]. Based on data from a clinical trial and treatment of patients for several years, protein A has been effective in treating patients with autoimmune diseases, including RA and ITP with platelet, which counts less than 100,000 per cubic millimeter of blood [7, 8].

RA and ITP are associated with proinflammatory cytokines, which are a group of signaling molecules that are stimulated by inflammatory lymphocytes (T), macrophages, and some other inflammatory cells and intensify the inflammatory response (7). Decreased biological activity of inflammatory cytokines decreases the damage caused by resulting diseases [9]. Blockage of interleukin-1 and tumor necrosis factor-alpha has been successful in the treatment of patients with rheumatoid arthritis, inflammatory bowel diseases [10]. Rituximab, Etanercept (Enbrel), remicid, and Homira is currently used to treat patients with rheumatoid arthritis [2]. Enbrel, under the brand name Etanercept, is a bio-drug that treats autoimmune diseases by limiting the inflammatory cytokine (Tumor Necrosis Factor (TNF) [11]. It is approved by the Food and Drug Administration (FDA) for the treatment of rheumatoid arthritis. It is a fusion protein with a human TNFα receptor extracellular portion 75 kDa attached to the Fc portion of human IgG1 immunoglobulin (TNFR: FC) [11]. This protein is a large molecule with a molecular weight of 150 kDa that binds to TNFα and inhibits autoimmune diseases [12]. However, these treatments cause many problems and side effects for patients. When a patient is treated with Rituximab, it takes a year for the patient's B cells to be replaced and normal immune function to be restored and beneficial antibodies to be produced [13]. The use of biological drugs, such as Etanercept (Enbrel), Remicade, and Humira, is also limited because they reduce the patient's immunity [14]. In our previous studies, various native and recombinant full-length forms and a truncated form of SpA with only 5 IgG domains without additional sequences were expressed as secretory forms in E. coli and [15–23] then purified [24, 25].

This study was an in vitro investigation on the therapeutic effects of the three pure recombinant forms of protein A, the full-length, the truncated form, and the natural forms (produced in Staphylococcus aureus), on the immune system. In vitro effects of these three pure forms of SpA on the reduction of inflammatory cytokines, IL-8, TNF-α, IL-1ß, and IL-6 from PBMCs of RA patients and healthy individuals were studied.

Chemicals, enzymes, cells and kits

Native protein A was purchased from EMD Millipore. Commercial drug Enbrel (Enbrels, Amgen Inc., Thousand Oaks, CA) was used as a control. Lipopolysaccharide from Escherichia coli 0111:B4 (Sigma, USA) and chemicals such as Dulbecco's modified Eagle's medium (DMEM, Sigma, St Louis, MO, USA), dimethylsulfoxide (DMSO) (Merck Co., Darmstadt, West Germany), FBS- Fetal Bovine Serum (Gibco, USA), Penicillin/Streptomycin 100X – (EuroClone, Italy), and TNFα, IL-8, IL-1β, IL-6 Human ELISA kits (Bender Med Systems, Vienna Austria) were used in this study.

Preparation of protein A

The required proteins were prepared during previous studies in our laboratory. In this section, a summary of how to prepare these proteins is provided by mentioning the relevant references published by us. In the previous studies expression and secretion of the full-length and truncated recombinant forms of protein A in Escherichia coli BL21 (DE3) were optimized in the pET Expression System [17, 18, 20, 23]. The truncated form contained only 5 immunoglobulin binding dominoes [20]. Optimization was carried out using response surface methodology (RSM) and statistical estimations were done [20]. The function of these two proteins was studied using ELISA, which showed a higher activity for the truncated form for binding to IgG, compared to the full-length protein [15, 20]. In another study, these two forms of recombinant produced proteins were purified using the hydrophilic interaction liquid chromatography (HILIC) with desired bioactivity [24]. A highly-purified native form of protein A obtained from Staphylococcus aureus bacteria purchased from EMD Millipore Company as a standard. A commercial drug called Enbrel (Enbrels, Amgen Inc., Thousand Oaks, CA), which is prescribed to patients with Rheumatoid arthritis, was purchased as a control. In addition, all the properties of these proteins produced have already been mentioned and published in detail in a review article [19].

Isolation of peripheral blood mononuclear cells (PBMC)

The peripheral blood mononuclear cells (PBMC) were isolated through the density gradient of Ficoll, as described by Ulmer, A. J., et al. [26]. Briefly, bleeding was done from patients with Rheumatoid arthritis (RA) and healthy individuals. 14 μL/ mL of heparin was added to the blood to prevent blood clotting. 5 mL of blood samples were diluted 1:1 with PBS and added to Ficoll-Hypaque solution and centrifuged at 900 × g for 20 min at 20 ºC. The mononuclear lymphocyte cell layer was transferred to a clean centrifuge tube. The cells were washed by the addition of HBSS and centrifuged at 550 × g for 10 min at 20 ºC. The supernatant was discarded and mononuclear lymphocyte cells were suspended 2 × 10⁶ cells/mL in a complete RPMI-1640 medium.

Evaluation of the proteins cytotoxicity

Following the preparation of the proteins, their cytotoxicity was evaluated by MTT assay [27]. For this analysis, a Sigma® cell culture 96 wells plate was used. 3 × 10⁴ mononuclear cells were seeded in 100 µL of the culture medium (RPMI-1640 containing 10% FBS) and the mixture was added to the wells and the plate was kept in an incubator for an overnight (37 ºC, 5% CO2). Different concentrations of each protein (0, 12.5 pg/ mL, 50 pg/ mL, 200 pg/ mL, 800 pg/ mL, 2 ng/ mL, 3 ng/ mL, 12.8 ng/ mL, 51.2 ng/ mL, 204 ng/ mL, 819 ng/ mL, 3.277 µg/ mL, 13.1 µg/ mL and 15 µg/ mL) were added to each well. After 24 h, 48 h, and 72 h, 20 µL of MTT stock solution (5 mg/mL) was added to each well and the plate was incubated at 37 ºC for 4 h. Then, the medium was carefully removed and 200 µL of MTT solvent (DMSO) was added to wells. The plate was agitated for 15 min on an orbital shaker at room temperature. At last, the absorbance at 570 nm was read by a plate reader. The absorbance correlated with percentage cell viability. All experiments were done in triplicates and data are presented as means ± standard deviations.

Treatment of PBMCs with optimum concentration of SpA and stimulation with LPS for immunological examinations

To investigate the effect of proteins on PBMCs, these cells were cultured in Sigma® cell culture plate size 24 wells. For this aim, in each well, 2 × 10⁵ cells/mL were seeded. After 24 hours of incubation at 37 °C, 15 μg/mL of the three forms of protein A, native protein (EMD millipore), truncated protein, and full-length recombinant protein, was added to separate wells. The commercial drug, Enbrel, at a concentration of 1 mg/mL was also used as the control. After 24h incubation at 37 °C, LPS (1 ug/mL) was added to the cells. The plate was incubated at 37 °C for 24h and then, the supernatant was collected and used for the enzyme-linked immunosorbent assay (ELISA) experiment [28].

ELISA assay

The concentration of the selected proinflammatory cytokines, including IL-8, TNF-α, IL-1ß, and IL-6 was investigated by ELISA assay. For this aim, commercial ELISA kits (Bender, MedSystems, Vienna, Austria) were used and ELISA was performed according to the manufacturer's instructions. Briefly, the microwell strips were washed twice with Bender Med Wash Buffer with a thorough aspiration of microwell content between washes. Standard dilution was prepared on the microwell plate (Each Microwell Plate was coated with monoclonal antibody to human tested cytokines) according to the manual of the kit. 100 µl of Sample Diluent was added in duplicate to the blank wells, 50 µl of Sample Diluent to the sample wells, and 50 µl of each sample in duplicate were added to the sample wells. 50 µl of Biotin-Conjugate anti-human tested cytokines polyclonal antibody was added to all wells for each cytokine. The wells were covered with an adhesive film and incubated at room temperature (18 to 25°C) for 2 hours, on a microplate shaker set at 400 rpm. Then microwell strips were washed 4 times. 100 µl of diluted Streptavidin-HRP was added to all wells, except the blank wells, and covered with an adhesive film and incubate at room temperature (18° to 25°C) for 1 hour, on a microplate shaker set at 400 rpm. After 4 times washing of microwell strips, 100 µl of TMB Substrate Solution was pipette to all wells. The microwell strips were incubated at room temperature for about 10 min. The enzyme reaction was stopped by quickly pipetting 100 µl of Bender Med kit Stop Solution into each well. The absorbance was read at 450 nm in an ELISA microplate reader.

Statistical analysis

Statistical analysis was performed with Student paired t-test using SPSS 20 software (Chicago, IL, USA). P-values of <0.05 were considered as significant and to compare the groups, t-test was used for comparisons between groups, and data were expressed as mean±standard deviation.

In silico analysis

3D protein structure prediction

The structure of protein in complete and truncated forms was predicted through Robetta server [29]. ProSA was used to evaluate local and overall quality of the predicted 3D-models [30]. Furthermore, the quality of the models was investigated by Ramachandran plot [31].

Evaluation of model stability

Molecular dynamics (MD) simulation was done to evaluate the stability of the protein structure in full and truncated forms using GROMACS 4.6.5 suite [32]. The GROMOSE96 54A7 force field was used to generate topology files. The models were placed at the center of a dodecahedral box and solvated with simple point charge (SPC) water model. The Cl- or Na+ ions were used for neutralizing each system. The steepest descent method was applied to remove possible clashes and bad contacts. Subsequently, the equilibrations of systems were done under NVT up to 100 ps at 300 K with restraint forces of 1000 kJ/mol, followed by 100 ps under NPT at the pressure of 1 bar and with restraint forces of 1000 kJ/mol. The electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method [33]. Finally, 50 ns MD run with no restraint was performed for evaluating the stability of the system.

Preparation of the proteins

The native protein A (purchased from EMD Millipore company) and full-length (56 kDa) and truncated (32 kDa) recombinant forms expressed in Escherichia coli obtained from our previous studies were used in this research. The function of recombinant proteins was studied using ELISA, which showed a higher activity for the truncated form for binding to IgG, compared to the full-length protein [20]. In another part of previous studies, attempts were made to purify the full-length and truncated forms of protein A using the cost-effective and powerful separation technique of hydrophilic interaction liquid chromatography (HILIC) [24]. The separated proteins retained their function and activity, as confirmed by ELISA [24].

Investigation of the presence of endotoxin in purified recombinant proteins

Since the recombinant proteins were produced in a Gram-negative bacterial expression system, E. coli, the amounts of endotoxin in the recombinant proteins were evaluated by LAL kit, as described in materials and methods section. The concentration of endotoxin in the final preparations of the truncated protein and full-length recombinant protein was estimated to be 48448 EU / ml and 48421 EU / ml, respectively. This amount of endotoxin in a sample is much lower than the permissible standards [34]. This result confirms that our strategy in producing the secretion of protein A has been appropriate, and due to the use of bacterial culture supernatant and no need to break down the bacterial cell wall used in the purification of cytoplasmic proteins, the level of endotoxin is much lower than Is allowed

Cytotoxicity assay

The MTT-assay was employed to determine the viability of the cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is a yellow dye, which is reduced by cellular enzymes to the blue product formazan [35]. For the MTT assay, cells were cultured on 96-well plates. The results of triplicates of MTT assay after 72 h, are shown in fig 1. None of the three treatments had not a lethal effect on cells at applied concentrations, after 24 h, 48 h, and 72 h.

Investigating the cytokine release following the PBMCs' treatment with proteins

Previous studies have reported that overproduction of cytokines is associated with increased production of proinflammatory mediators such as IL-6, IL-8, in cell types [36]. Among these, IL-6 and IL-8 are likely to act as main stimuli for RA inflammation, as deletion of their genes leads to protection against rheumatoid arthritis. To investigate the effect of the different forms of protein A, recombinant truncated and full-length, and natural form (derived from S. aureus), on the secretion of 4 cytokines, IL-8, IL-1Ɓ, TNF-α, and IL-6 from PBMCs, the cells were cultured, stimulated and treated according to the methods mentioned in Materials and Methods section. At the end of each incubation period, the supernatant was collected and the amount of these cytokines were measured using commercial ELISA kits (Bender Med).

The effect of proteins on IL-8 secretion

The results showed that the commercial drug, Enbrel, significantly reduced the secretion of IL-8 cytokine in comparison to the negative control (untreated cells) (P<0.01). In comparison to Enbrel, protein A reduced the secretion of this cytokine significantly (P<0.01). No significant difference was seen between the reduction effect of full-length recombinant protein A and natural protein A on IL-8 secretion. Interestingly, the truncated form of the recombinant protein A decreased the secretion of IL-8 in comparison to all other forms (full-length recombinant protein A, natural protein A, and Enbrel). The results were true and compatible in both healthy donors and patients (Fig 2 A).

The effect of proteins on IL-1β secretion

The results of the effect of different forms of protein A and Enbrel on the secretion of IL-1β by PBMC cells are presented in fig 2 B. As can be seen, like IL-8, the truncated form of protein A has the highest efficiency in the lowering of IL-1β secretion from PBMCs.

While the natural form of protein A and recombinant full-length protein A had no significant difference in the decrease of IL-1β (P>0.05), they reduced the secretion of this interleukin from PBMCs significantly higher than Enbrel (P<0.01). Again, the results were true and compatible in both healthy donors and patients.

The effect of proteins on TNF-α secretion

Following the treatment of PBMCs with 4 formulations, the same pattern as of IL-8 and IL-1β was observed for TNF-α, i.e. the highest reduction of TNF-α secretion was observed when the truncated form of protein A. There was no significant difference between the native and the full-length recombinant forms of the SpA, however, these two forms were significantly more potent in reducing TNF-α than Enbrel (Fig 2 C).

The effect of proteins on IL-6 secretion

Finally, the measurement of IL-6 secretion from PBMCs following the treatment of these cells with different formulations including the recombinant truncated and full-length and the native form of the protein A and Enbrel showed that the truncated form, full-length of protein A and Enbrel had the highest effect on the secretion of IL-6 by PBMCs (Fig 2 D).

In silico analysis

3D Protein structure prediction

To investigate why the truncated form of protein A could reduce the secretion of cytokines in the culture model of RA more effectively than the recombinant full-length and native forms, 3D protein structures were predicted using the Robetta server (http://robetta.bakerlab.org) [14, 29]. In both predicted models five IgG binding domains with a three-helix bundle structure were observed. Furthermore, the full-length protein was contained an XR domain. The protein structures have been shown in fig 3. The 5 IgG-binding domains in both proteins have been depicted with different colors. As shown in fig 3, domains are more accessible in the truncated form of the protein.

Evaluation of model stability

The protein structure evaluation through the ProSA server showed that the z-score of the proteins is in the range of the scores of native proteins (Figs 4 A, C).

In addition, the analysis of the Ramachandran plot indicated that in both of the proteins more than 99 % of residues are within the most favored and allowed regions (Figs 4 B, D).

To evaluate the stability of the truncated and full-length protein A through molecular dynamics simulation, the RMSD and RMSF plot were generated. RMSD plot shows a deviation of 2 structures from initial structures during simulations. As shown in plots, the RMSD of full-length protein A increase up to 1.2 nm at 10 ns and after reaching about 0.75 nm remained stables up to the end of the simulation. While in the truncated protein, the amount of RMSD reached 1.2 nm at 40 ns and remained stable after that. Overall, the RMSD plots proved the stability of both proteins (Fig 5 A, B).

Furthermore, the structures were evaluated through the RMSF plot (Figs 6 A, B). This plot evaluates the flexibility of structure during simulation. High RMSF values show more fluctuation, but the lower value shows limited fluctuation. As shown in figs 3 A, B, no unusual fluctuation was observed for both proteins which indicate both proteins are stable. In other words, these results account for the stability of coordination of three-helix bundle structures

Based on the results of the experimental section, the truncated recombinant protein A reduced the secretion of cytokines more efficiently than the full-length protein.

To find out the reason at the molecular structure level following the compactness of the same regions of the two forms of the recombinant proteins were evaluated through the radius of gyration (Rg) plot. Rg is a parameter to show the change in the protein structure compactness during a simulation and show how regular secondary structures are compactly placed in the three-dimensional structure of proteins. The results have been shown in figs 7 A, B.

This analysis showed that the radius of gyration of truncated protein is larger than full-length form. The value of Rg after 20 ns remained stable in both proteins and did not change up to the end of the simulation. Overal, all 3 plots, RMSD-RMSF-Rg, proved the structure stability of both forms of protein.

Rheumatoid arthritis is the most frequent disease among chronic inflammatory joint diseases. 0.5-1% of people in developed countries suffer from rheumatoid arthritis [3]. It causes disability and, if not properly treated, premature death. Because of the high imposed costs to people and societies, it is of great importance to be properly treated [1]. There are different strategies and therapies for RA treatment, which are reviewed in some valuable papers. Various drugs are used to treat rheumatoid arthritis. The type of medication that the doctor prescribes depends on the severity of the symptoms and the duration of the disease: NSAIDs: Nonsteroidal anti-inflammatory drugs (NSAIDs) can relieve pain and reduce inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) include ibuprofen and naproxen sodium [37]. Steroids include corticosteroid drugs such as prednisone reduce inflammation and pain and slow down the process of joint damage [38]. Disease-modifying and anti-rheumatic drugs (DMARDs) can slow the process of rheumatoid arthritis and prevent permanent damage to joints and other tissues [39]. Common drugs in this category include methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine [39]. Biological agents, also known as biological response modifiers, are a new generation of DMARDs. These drugs can target parts of the immune system that cause inflammation and damage joints and tissues.

In the present study, we evaluated the effect of three different forms of protein A on the reduction of 4 inflammatory cytokines, including IL-8, IL-1β, TNF-α, and IL-6 by PBMCs, and compared the results with a commercially available drug, Enbrel. Our results showed that the truncated form of protein A is the most potent form that reduces the secretion of the evaluated cytokines from PBMCs in vitro.

One of the main applications of protein A is in biotechnology for the purification of antibodies, but a recent study has identified it as a drug against autoimmune diseases [2, 40]. In this study, it was shown that this protein binds directly to monocytes and a subset of B cells that are involved in the development and progression of various autoimmune diseases and controls the disease. There have been few studies that have shown the applicability of protein A in the treatment of autoimmune diseases, including RA [1, 2]. In comparison to RA chemical drugs, which extensively suppress the immune system, protein A restores immune system activity by selectively limiting the activity of B cells responsible for producing pathological antibodies without adversely affecting the immune system [2].

The present study is the first report on the use of recombinant protein A in reducing inflammatory cytokines in PBMCs cells from rheumatoid arthritis patients. The anti-inflammatory effect of the Enbrel was also evaluated by the measurement of the inflammatory cytokines, including IL-8, IL-1β, TNF-α, and IL-6. The results showed a significant decrease in the secretion of these cytokines following the treatment of PBMCs with this drug (P < 0.01). Moreover, comparing the effect of different forms of protein A and Enbrel drug on cytokine secretion by PMCs showed that protein A is significantly more effective in the decrease of inflammatory cytokines by PBMCs (p < 0.01).

Comparison of the effect of full-length recombinant protein A with natural protein A showed no significant difference between these two forms. Since these two forms have the same sequence, despite their different source of native and recombinant, this result was predictable. Maybe, the most interesting result of the present study was the effect of the truncated form of protein A on the reduction of inflammatory cytokines. The recombinant truncated form of protein A reduced the secretion of these cytokines significantly more efficiently than other forms (Enbrel, recombinant full-length, and natural form of the protein A). The in silico analysis showed that this could be due to a change in the compactness of protein. SpA protein has five IgG binding domains (A,B,C,D, and E) that individually can bind to IgG with similar strength [41, 42]. However, binding studies have shown that intact full-length SpA (five IgG-binding domains and other present domains) is able to bind to IgG proteins [43].

Based on the results of in silico analysis, we hypothesized that in the truncated form of the protein A, with increasing radius of gyration and loss of compactness, more IgG-binding domains appear to be exposed than in the full-length protein to bind to IgG.

In this study, the effects of protein A, as an anti-rheumatoid arthritis drug, on the immune system in an in vitro model of the disease were evaluated using peripheral blood mononuclear cell (PBMC) from adult patients with active rheumatoid arthritis (RA) and healthy donors. The effect of different doses of three highly purified full-length and truncated forms of protein A in comparison with commercial anti-rheumatoid arthritis drugs on the expression of selected cytokines involved in rheumatoid arthritis was investigated. Protein A significantly inhibited the expression of most of the cytokines studied under serum concentration. Protein A had greater inhibitory effects on cytokine expression and secretion than the commercial drug Enbrel. Our results show that protein A can be used as a new effective drug in the treatment of autoimmune diseases such as rheumatoid arthritis. Interestingly, for the first time, our results showed that truncated protein A containing only 5 IgG-binding domains was sufficient for the anti-rheumatic effects, and that this truncated form had a greater ability to reduce cytokine expression than its full-length. Our results suggest that protein A could be used as a new effective drug in the treatment of autoimmune diseases such as rheumatoid arthritis. Interestingly, for the first time, our results showed that protein A containing only 5 domains of IgG binding is sufficient for anti-rheumatic effects and that this the truncated form has a greater ability to reduce cytokine expression than its full-length. In search of a possible cause for the reduced efficiency of the truncated form in suppressing cytokine production associated with autoimmune disease, the three-dimensional structure of full-length and truncated proteins by bioinformatics analysis using molecular dynamics showed that both forms had a stable structure. However, the radius of attenuation in the shortened form has increased further, so that IgG binding domains are likely to find a more open and accessible structure for binding to the Fc domain of immunoglobulins.

Acknowledgment

The authors would like to thank the National Institute of Genetic Engineering and Biotechnology (NIGEB) of Iran for providing the necessary equipment.

Authors' contributions

G.R. done the biological experiments and data collection. The first draft of the manuscript was written by G.R. and completed with A.H. and all authors commented on previous versions of the manuscript. G.A. as a corresponding author and G.K. as an adviser of the study, designed the whole experiments, supported the project, and analyzed data. J.Z. performed the bioinformatics analysis. All authors contributed to the study conception and design. All authors read and approved the final manuscript.

Funding

No funding was received.

Availability of data and material

All data related to this research have been included in the manuscript.

Declarations

Ethics approval and consent to participate

Not applicable

Competing interests

The authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Consent for publication

Not applicable.

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The effects of a truncated form of Staphylococcus aureus protein A (SpA) on the expression of cytokines of autoimmune patients and healthy individuals

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