Abstract
Alpha-1 antitrypsin (a1AT) deficiency, in its classical form, is an autosomal recessive disease associated with an increased risk of liver disease in adults and children, and with lung disease in adults. The vast majority of liver disease is associated with homozygosity for the Z mutant allele, also called PiZZ. This homozygous allele synthesizes large quantities of a1AT mutant Z protein in the liver, but the mutant protein also folds improperly during biogenesis. As a result, approximately 85% of the molecules are retained within the hepatocytes instead of being appropriately secreted. The resulting low, or “deficient,” serum level leaves the lungs vulnerable to inflammatory injury from uninhibited neutrophil proteases. Most of the mutant Z protein retained within hepatocytes is directed into intracellular proteolysis pathways, but some molecules remain in the endoplasmic reticulum for long periods of time and others adopt an unusual aggregated or “polymerized” conformation. It is thought that these intracellular polymers trigger a cascade of intracellular injury which can lead to end organ liver injury including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. It is widely accepted that the disease causing factor in mutant Z-alpha-1 antitrypsin deficiency (AATD-Z) is the toxic build-up of the mutant Z protein. Since misfolding of some but not all of the Z protein during its maturation leads to homopolymerization, an assay to assess the amount of normally folded ATZ and accumulated polymeric ATZ would be very useful. Here we describe a method to semiquantitatively assess these two fractions in a tissue or cell culture source.
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References
Dul JL, Davis DP, Williamson EK, Stevens FJ, Argon Y (2001) Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains. J Cell Biol 152(4):705–716
Lin L, Schmidt B, Teckman J, Perlmutter DH (2001) A naturally occurring nonpolymerogenic mutant of alpha 1-antitrypsin characterized by prolonged retention in the endoplasmic reticulum. J Biol Chem 276(36):33893–33898
An JK, Blomenkamp K, Lindblad D, Teckman JH (2005) Quantitative isolation of alphalAT mutant Z protein polymers from human and mouse livers and the effect of heat. Hepatology 41(1):160–167
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Blomenkamp, K.S., Teckman, J.H. (2017). Semiquantitation of Monomer and Polymer Alpha-1 Antitrypsin by Centrifugal Separation and Assay by Western Blot of Soluble and Insoluble Components. In: Borel, F., Mueller, C. (eds) Alpha-1 Antitrypsin Deficiency . Methods in Molecular Biology, vol 1639. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7163-3_23
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DOI: https://doi.org/10.1007/978-1-4939-7163-3_23
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7163-3
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