The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant

Abstract

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

Comprehensive Clinical Diagnostic Pipelines Reveal New Variants in Alpha-1 Antitrypsin Deficiency

“…Impact of the mutations on secretion efficiency was evaluated by quantifying AAT in the culture media of both HEK293T and Hepa1.6 transfected cells by sandwich ELISA (Figure 3). Consistent with previous studies (26,31,38), the Z variant was secreted significantly less than wild-type M AAT. In comparison, E98K was secreted at nearly normal levels, L383P showed the most severe reduction, a moderate reduction was observed for D341Y, while a milder but significant deficiency was observed for the other variants.…”

A novel pathological mutant reveals the role of torsional flexibility in the serpin breach in adoption of an aggregation‐prone intermediate

“…As previously described in Ronzoni et al . [44], Hepa 1.6 cells were seeded onto 2 cm 2 coverslips (Millipore Sigma, Burlington, MA, USA) and transfected as described above. After 48 h, the cells were fixed with 4% v/v paraformaldehyde, permeabilised with 0.1% v/v Triton X‐100, and immunodecorated with anti‐human AAT (Agilent Dako, Glostrup, Denmark) (at a concentration of 2.2 μg·mL −1 ) or the anti‐AAT polymer 2C1 mAb [9] (0.8 μg·mL −1 ) overnight at 4 °C.…”

“…Hepa 1.6 cells, extensively utilised for AAT studies [16,[43][44][45] had been procured from and authenticated by Merck KGaA (Darmstadt, Germany) < 3 years before the first transfection. All experiments were conducted using cells routinely screened for mycoplasma contamination.…”