Abstract
Alpha 1- antitrypsin (α1AT) a 54 kDa glycoprotein is a protease inhibitor. In the absence of α1AT, elastase released by lung macrophages, was not inhibited and lead to elastin breakdown and pulmonary problems such as emphysema or COPD. α1AT has three site of N-glycosylation and a characteristic reactive central loop (RCL). As small-scale medicines are preferred for pulmonary drug delivery, in this study α1ATs (1, 2, 3, 4 and 5) were engineered and shortened from the N-terminal region. In order to investigate the effect of different mutations and the deletion of 46 amino acids theoretical studies were performed. Homology modeling was performed to generate the 3D structure of α1ATs. The 10 ns Molecular Dynamic (MD) simulations were carried out to refine the models. Results from MD and protein docking showed that α1AT2 has the highest binding affinity for neutrophil elastase, provided the basis for the experimental phase in which sequences from the five α1AT constructs were inserted into the expression vector pGAPZα and expressed in the yeast Pichia pastoris. Although, the α1AT2 construct has the highest inhibitory activity even more that the native construct (α1AT5), results indicated the presence of protease inhibitory function of all the proteins' construct against elastase.
Highlights
Alpha 1- antitrypsin (α1AT) a 54 kDa glycoprotein is a protease inhibitor
It was shown that oxidation sensitivity is a regulatory process, and α1AT inactivation possibly causing lung tissue breakdown in inflammatory site through oxygen radicals released from phagocytes
It was shown that oxidation sensitivity is a regulatory process, and α1AT inactivation possibly causing lung tissue breakdown in inflammatory site through oxygen radicals released from phagocytes [14,15]
Summary
Molecular dynamics simulation The root-mean-square deviation (RMSD) One of the most frequently used measures of assessing the stability of an MD simulation over the course of time is the RMSD of the backbone atoms relative to the starting structure during the MD production phase. The RMSDs reach a stable value within the first ns, all the analyses have been carried out discarding the first three nanoseconds, i.e. over the last seven nanoseconds (Figure 1). This was done to guarantee an investigation over a well thermalized system. Temperature and density The temperature and the densities for all α1ATs were 300 K and 1.00 gr/cm, respectively that became fixed and stable, representing further proof of simulation stability (Figure 4). SDS page The culture expressing native and truncated α1AT was harvested at the following time intervals: 0, 12, 24, 48, 60, 72 and 96 hrs. Elastase Inhibitory Capacity (EIC) The inhibitory function of native and truncated α1AT showed no significant difference and both α1ATs were found to be functional (Figure 9)
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