Effects of mutations in the reactive center loop of alpha-1-antitrypsin on inhibitory activity against neutrophil elastase

Alpha-1-antitrypsin (AAT) is an abundant plasma protein and a critical inhibitor of serine proteases, such as neutrophil elastase (NE) and proteinase-3, which are released by neutrophils at sites of inflammation. The inhibition mechanism is initiated by proteolytic cleavage of a pseudo-substrate in the Reactive Center Loop (RCL) of AAT, followed by a conformational change of AAT that irreversibly inactivates the protease. Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder caused by mutations in the SERPINA1 gene encoding AAT, leading to a diminished AAT concentration in the plasma. Patients with severe AATD exhibit uncontrolled elastase activity particularly in the lungs, resulting in an elevated risk of emphysema and chronic obstructive pulmonary disease (COPD). Furthermore, a percentage of AATD patients develop liver diseases due to the accumulation of specific AAT mutants as polymeric chains in the endoplasmic reticulum (ER) of hepatocytes, which are the main source of AAT production. The most prevalent pathogenic variants of AAT are the severely deficient Z-AAT (E342K) and the milder S variant (E264V), but many ultrarare variants have been also reported to cause AATD, generally in heterozygous association with either Z or S-AAT. While most of these variants result in plasma deficiency, the Iners AAT variant (G349R), characterized by a single amino acid substitution in the RCL region, shows a rather unique behavior: it is normally secreted, but it is completely inactive as protease inhibitor. Mutations such as Iners can cause a severe AAT functional deficiency, despite their normal circulating levels. Most of this thesis has been dedicated to study the functional effects of amino acid variations mapping in the RCL region directly involved in the interaction with NE. We selected twenty-three variations, which can arise from single nucleotide polymorphisms, in the P5-P4'peptide of the RCL flanking the pseudo-substrate cleavage site. We also included in our analysis some naturally occurring variations identified in genome-wide screenings and annotated in genomic databases. The AAT variants were transiently produced in the HEK293T cell line, along with the wild-type AAT, the Z-AAT polymerogenic mutant and the dysfunctional Iners variant. We then investigated the secretory efficiency and the specific anti-elastase activity of each variant. As expected for mutations occurring in the surface exposed RCL region, all the investigated variants were properly folded and secreted by transfected cells. The inhibitory activity of each variant was investigated either by immunoblot analysis of the AAT-NE complexes or by NE enzymatic assays in the presence of chromogenic substrates. As expected, the wild-type AAT was totally converted into an AAT-NE complex and so did most of the investigated variants. However, a few variants showed dysfunctional behavior: the M358K, M358R and S359P failed to interact with NE, while Iners, A355D, A355P, P357R and P357S behaved as substrates rather than inhibitors of the protease. A natural RCL variant (M351T), identified in in a 20-year-old male suffering from asthma, was similarly characterized and found to behave as benign in our cellular model. Another aim of the thesis was the development and the characterization of a polymeric AAT standard to be applied in an ELISA method, developed by our group, to quantify Z-AAT polymers in plasma samples. Indeed, a small fraction of the polymeric chains formed within the ER of hepatocytes is secreted into the bloodstream. Since polymers are incapable of inhibiting proteases, the different levels of Z-AAT polymers in the plasma may explain the different severity of lung disease observed in ZZ homozygote patients. The polymer standard was prepared by heating purified AAT under different experimental conditions. This preparation is now being applied for quantification of polymers in plasma samples collected from a cohort of AATD patients.