Abstract

The Z variant (Glu342Lys) of α₁-antitrypsin (AT) is one of the serpinopathies; it polymerises and accumulates in the hepatocyte endoplasmic reticulum (ER) resulting in neonatal hepatitis and liver cirrhosis. The secretory defect leaves the lungs vulnerable to elastolysis and early-onset emphysema. Prevention of polymerisation of Z-AT can be achieved in vitro by targeting strand 4a of the AT molecule. Here we evaluate whether an inhibitor of polymerisation; Ac-TTAI-NH₂ (4M) would inhibit Z-AT polymerisation in a cell model. HEK293 cells were transfected with Z-AT (Z-AT cells) or control M-AT (M-AT cells). ELISA demonstrated significantly reduced Z-AT secretion, 242(SEM±63) ng/ml compared to M-AT, 2449±130 ng/ml (p≤0.001), due to retention of Z-AT polymers in inclusion bodies. This was confirmed by electron microscopy demonstrating distension of the rough ER, and ELISA and Immunoblot with a polymer specific antibody (ATZII). 4M significantly reduced the amount of polymers in inclusions in a dose-dependent manner; no peptide, 2468.5±μg/ml, 5 mg, 2181.7±26.2 ng/ml (p=0.006), 10 mg, 1576±164.7 ng/ml (p=0.001), and 20 and 30 mg completely prevented polymer formation in inclusions (p≤0.001). Unrelated peptides had no effect. Elastase activity of AT in the supernatant from Z-AT cells was significantly reduced compared to M-AT cells; p≤0.001, in keeping with the secretory defect due to retention of Z-AT in inclusion bodies. The elastase activity (and AT concentration) in the supernatant from Z-AT cells was restored by 20 mg 4M, O.D. 405 nm, Z-AT vs Z-AT + 20 μg 4M, 0.129±0.009 vs 0.788±0.054 respectively, (p≤0.001), where a higher O.D. represents higher elastase activity. Functional activity of secreted AT following treatment with 4M was confirmed by its ability to form an SDS-stable complex with elastase as shown by immunoblot. RT-PCR showed that the ER accumulation of Z-AT induced cell stress; NF-κB activation, expression of protein kinase RNA (PKR)-like ER kinase (PERK), and IL-6 (100.4±16 pg/ml) and IL-8 (2592.5±575 pg/ml), all of which could be abrogated effectively by 20 mg 4M (IL-6, 45.8±28 pg/ml, p≤0.001 and IL-8, 184.3±29 pg/ml, p=0.014). These findings are the first evidence that inhibitors of Z-AT polymerisation targeting s4A can prevent its cellular accumulation and deleterious effects. Importantly, this strategy was also able to improve plasma concentration of Z-AT.