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

Riccardo Ronzoni,David A Lomas,Emanuela D'Acunto,Stefania Ottaviani,James A Irving,Ilaria Ferrarotti,Annamaria Fra,Alice M Balderacchi,Elena Miranda

doi:10.3390/ijms22115668

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.

Highlights

Endoplasmic reticulum storage diseases (ERSDs) are a group of genetically based disorders in which mutant proteins tend to accumulate in the endoplasmic reticulum (ER)
We report here a novel amino acid substitution identified in the shutter region of AAT, which was found in five unrelated subjects and named Bologna
The identification of a cluster of deficiency mutations of antitrypsin, antithrombin, anti-chymotrypsin and C1-inhibitor revealed the crucial role of the serpin shutter region of β-sheet A in regulating these conformational changes [27] and this has since been noted among other members of the family including neuroserpin [51,52]

Summary

Introduction

Endoplasmic reticulum storage diseases (ERSDs) are a group of genetically based disorders in which mutant proteins tend to accumulate in the endoplasmic reticulum (ER)as cytotoxic aggregates [1]. Endoplasmic reticulum storage diseases (ERSDs) are a group of genetically based disorders in which mutant proteins tend to accumulate in the endoplasmic reticulum (ER). A representative case of liver ERSD [2] is alpha-1-antitrypsin deficiency (AATD), caused by intra-hepatic polymerization of the Z (Glu342Lys) mutant of AAT [3]. Due to chronic accumulation of intracellular polymers, ZZ homozygotes show increased risk of liver disease, that may manifest as jaundice and hepatitis in infants, and more frequently in adults as fibrosis, cirrhosis, and increased risk of hepatocarcinoma [4,5,6]. AAT is the most abundant serine protease inhibitor (serpin) in the plasma and one of the most abundant proteins produced by hepatocytes. A fraction of the misfolded protein is degraded by ER-associated pathways [12,13], but a significant proportion escapes quality control and forms linear polymers by a domain swap mechanism [3,14,15]

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