Heteropolymerization of S, I, and Z α1-antitrypsin and liver cirrhosis
Ravi Mahadeva, Wun-Shaing W. Chang, Timothy R. Dafforn, Diana J. Oakley, Richard C. Foreman, Jacqueline Calvin, Derek G.D. Wight, David A. Lomas
Ravi Mahadeva, Wun-Shaing W. Chang, Timothy R. Dafforn, Diana J. Oakley, Richard C. Foreman, Jacqueline Calvin, Derek G.D. Wight, David A. Lomas
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Heteropolymerization of S, I, and Z α1-antitrypsin and liver cirrhosis

Abstract

The association between Z α1-antitrypsin deficiency and juvenile cirrhosis is well-recognized, and there is now convincing evidence that the hepatic inclusions are the result of entangled polymers of mutant Z α1-antitrypsin. Four percent of the northern European Caucasian population are heterozygotes for the Z variant, but even more common is S α1-antitrypsin, which is found in up to 28% of southern Europeans. The S variant is known to have an increased susceptibility to polymerization, although this is marginal compared with the more conformationally unstable Z variant. There has been speculation that the two may interact to produce cirrhosis, but this has never been demonstrated experimentally. This hypothesis was raised again by the observation reported here of a mixed heterozygote for Z α1-antitrypsin and another conformationally unstable variant (I α1-antitrypsin; 39Arg→Cys) identified in a 34-year-old man with cirrhosis related to α1-antitrypsin deficiency. The conformational stability of the I variant has been characterized, and we have used fluorescence resonance energy transfer to demonstrate the formation of heteropolymers between S and Z α1-antitrypsin. Taken together, these results indicate that not only may mixed variants form heteropolymers, but that this can causally lead to the development of cirrhosis.

Authors

Ravi Mahadeva, Wun-Shaing W. Chang, Timothy R. Dafforn, Diana J. Oakley, Richard C. Foreman, Jacqueline Calvin, Derek G.D. Wight, David A. Lomas

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(a) Polymerization of 5-IAF– and 5-TMRIA–labeled M α1-antitrypsin (2 mg/...
(a) Polymerization of 5-IAF– and 5-TMRIA–labeled M α1-antitrypsin (2 mg/ml) at 41°C for 14 days. Nondenaturing PAGE (7.5–15% wt/vol), all lanes contain 5 μg protein. Lane 1, M α1-antitrypsin control; lane 2, 1:1 mix of M α1-antitrypsin labeled with 5-IAF and 5-TMRIA; lane 3, M α1-antitrypsin labeled with 5-IAF; lane 4, M α1-antitrypsin labeled with 5-TMRIA; lane 5, M α1-antitrypsin labeled with 5-IAF heated at 41°C for 14 days; lane 6, M α1-antitrypsin labeled with 5-TMRIA heated at 41°C for 14 days; lane 7, 1:1 mix of M α1-antitrypsin labeled with 5-IAF and 5-TMRIA heated at 41°C for 14 days. (b) A 1:1 mixture of 5-IAF– and 5-TMRIA–labeled M α1-antitrypsin incubated at 41°C for 14 days demonstrated RET (continuous line) when excited at 492 nm and fluorescence measured over a wavelength of 500–600 nm. The peak at approximately 570 nm was not seen in M α1-antitrypsin polymerized under identical conditions with either 5-IAF or 5-TMRIA alone (bold line). Labeled Z and S α1-antitrypsin both formed polymers when incubated alone and when mixed in a 1:1 ratio. The signal from the mixed Z and S α1-antitrypsin polymers excited at 492 nm shows RET (broken line), indicating the formation of heteropolymers. (c) Nondenaturing PAGE (7.5–15% wt/vol). All lanes contain 10 μg protein. Lane 1, I α1-antitrypsin labeled with 5-TMRIA heated at 41°C and 0.4 mg/ml for 12 days; lane 2, I α1-antitrypsin control heated at 41°C and 0.4 mg/ml for 12 days; lane 3, unlabeled I α1-antitrypsin control. 5-IAF, 5-iodoacetamidofluorescein; 5-TMRIA, tetramethylrhodamine-5-iodoacetamide; RET, resonance energy transfer.