Serpinopathies are a protein misfolding disease caused by members of the serpin superfamily. These diseases lead to gain-of-toxicity at the site of synthesis, which results in loss-of-function. One of the most studied serpinopathies is a1-antitrypsin (a1-AT) deficiency. a1-AT targets human neutrophil elastase (HNE), controlling the serpin: protease ratio in the lungs. Deficiency in a1-AT (levels below 35% of normal) allows for HNE to degrade the lower lungs, resulting in emphysema. The current treatment to control the activity of HNE is augmentation therapy: intravenous injections of plasma purified a1-AT, aiming to restore the deficient levels in the blood. However, this treatment is cost effective and inactive a1-AT has been detected. Therefore, the use of recombinant DNA technology and protein engineering to develop an a1-AT like serpin that is stable, yet functional, could provide a better treatment for a1-AT deficiency.
Previously, consensus-designed protein engineering was used to produce a synthetic serpin, termed conserpin. Conserpin exhibits extreme thermostability and aggregation resistance, while also being functional as an inhibitor towards trypsin. This extreme thermostability and aggregation resistance makes conserpin an attractive starting molecule for engineering of an a1-AT like serpin which also exhibits increased stability.
a1-AT was engineered for stability by grafting regions of conserpin that are hypothesized to contribute to its stability, onto a1-AT. In this study, eight regions of conserpin were grafted onto a1-AT, producing a total of eight grafts. Out of the eight grafts, two grafts fold into the latent conformation, while three natively folded serpins are more thermostable than WT a1-AT. These three thermal stable grafts are active against HNE, yield more monomeric protein post refold while having a similar folding intermediate to WT a1-AT. Combining these three thermally stable grafts together produced an even more thermally stable serpin, 3stable. The 3stable graft yields more monomeric protein post refold than each of the single thermal stable grafts, while remaining active as an inhibitor. Therefore, protein engineering was successful in producing a stable a1-AT while also remaining active as an inhibitor.