Designing a less immunogenic nattokinase from Bacillus subtilis subsp. natto: a computational mutagenesis.

Nattokinase is an enzyme produced by Bacillus subtilis subsp. natto that contains strong fibrinolytic activity. It has potential to treat cardiovascular diseases. In silico analysis revealed that nattokinase is considered as an antigen, thus hindering its application for injectable therapeutic protein. Various web servers were used to predict B-cell epitopes of nattokinase both continuously and discontinuously to determine which amino acid residues had been responsible for the immunogenicity. With the exclusion of the predicted conserved amino acids, four amino acids such as S18, Q19, T242, and Q245 were allowed for mutation. Substitution mutation was done to lower the immunogenicity of native nattokinase. Through the stability of the mutated protein with the help of Gibbs free energy difference, the proposed mutein was S18D, Q19I, T242Y, and Q245W. The 3D model of the mutated nattokinase was modeled and validated with various tools. Physicochemical properties and stability analysis of the protein indicated that the mutation brought higher stability without causing any changes in the catalytic site of nattokinase. Molecular dynamics simulation implied that the mutation indicated similar stability, conformation, and behavior compared to the native nattokinase. These results are highly likely to contribute to the wet lab experiment to develop safer nattokinase.

In silico mutagenesis: decreasing the immunogenicity of botulinum toxin type A.

Botulinum toxin serotype A is a prominent therapeutic enzyme, for both clinical and cosmetic uses. Since this protein is produced by bacteria, it exhibits an allergenic effect when subjected to human therapy. Protein mutagenesis is one method to improve the characteristics of protein. However, in silico study is needed to give suggestion of which amino acid should be mutated. Hence, a lot of money and time can be saved. This study initially screened which residue of the Botulinum toxin serotype A is B-cell epitopes both linearly and conformationally. By overlapping the B-cell epitopes with the excluded conserve sequence, seven residues were allowed to be mutated. There were two proposed muteins showing a reduction in the antigenicity probability: ΔE147, E510F, T1062F, ΔE1080, N1089M and ΔQ1090; and ΔE147, E510F, T1062F, E1080W, N1089M and ΔQ1090. Molecular dynamics simulation of the 3D proposed muteins indicated an increase of flexibility in both muteins compared to that in the native protein. Both muteins have lower antigenicity. In addition, they are similar in structure, stability and functionality compared to the native protein.

Formation of osteoclast-like cells from peripheral blood of periodontitis patients occurs without supplementation of macrophage colony-stimulating factor.

AIM: To determine whether peripheral blood mononuclear cells (PBMCs) from chronic periodontitis patients differ from PBMCs from matched control patients in their capacity to form osteoclast-like cells. MATERIAL AND METHODS: PBMCs from 10 subjects with severe chronic periodontitis and their matched controls were cultured on plastic or on bone slices without or with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL). The number of tartrate-resistant acid phosphatase-positive (TRACP(+)) multinucleated cells (MNCs) and bone resorption were assessed. RESULTS: TRACP(+) MNCs were formed under all culture conditions, in patient and control cultures. In periodontitis patients, the formation of TRACP(+) MNC was similar for all three culture conditions; thus supplementation of the cytokines was not needed to induce MNC formation. In control cultures, however, M-CSF or M-CSF/RANKL resulted in higher numbers compared with cultures without cytokines. Upregulations of osteoclast marker mRNA cathepsin K and carbonic anhydrase II confirmed the osteoclastic character. Bone resorption was only observed when PBMCs were cultured in the presence of M-CSF and RANKL. CONCLUSION: Our data indicate that PBMCs from periodontitis patients do not need priming by M-CSF to become osteoclast-like cells, suggesting that PBMCs from periodontitis patients are present in the circulation in a different state of activity.