02122nas a2200169   4500000000100000008004100001100001400042700001100056700001100067700001600078700001300094700001200107700001100119245007800130520173000208022001401938       2019                            d1 aRastogi R1 aKaur G1 aMaan P1 aBhatnagar A1 aNarang T1 aDogra S1 aKaur J00aMolecular characterization and immunogenic function of ML1899 (LipG) of .3 a<p>. ML1899 is conserved in all mycobacterium sp. and is a middle member of operon involved in mycolic acid modification.. In the present study attempts were made to characterize ML1899 in detail.. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.. analysis predicted ML1899 as a member of / hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM min catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. , and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min ml and 27 U mg, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.</p>
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