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 . 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) . The separated proteins retained their function and activity, as confirmed by ELISA .
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 . 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
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 . 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 . 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.