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) Nondenaturing PAGE (7.5-15% wt/vol) showing that a mixture of IZ α1-...
(a) Nondenaturing PAGE (7.5-15% wt/vol) showing that a mixture of IZ α1-antitrypsin loses protein from the monomeric band and forms high molecular mass polymers more readily than I α1-antitrypsin alone. The proteins were incubated at 2 mg/ml and 41°C in 50 mM Tris, 50 mM KCl (pH 7.4). All lanes contain 10 μg protein. Top: I α1-antitrypsin; bottom: IZ α1-antitrypsin. Lane 1, time 0; lane 2, 1 day; lane 3, 2 days; lane 4, 3 days; lane 5, 6 days; lane 6, 12 days. (b) Rate of polymerization of M, I, S, and Z α1-antitrypsin mutants at 0.1 mg/ml and 45°C determined from the measurement of intrinsic tryptophan fluorescence. The values for the rate of polymerization (Table 1) were obtained from fitting the data to Equation 1 and are the weighted mean and standard error of three (I, S, and Z α1-antitrypsin) or four (M α1-antitrypsin) experiments. The rate of polymer formation of mixtures of IZ and MZ α1-antitrypsin were calculated using a 1:1 mixture of the variants at 45°C. The concentration of each variant in the mixture was half that shown in b in order to keep the final protein concentration at 0.1 mg/ml. The curves for each component of the reaction were dissected from the final profile, and the rate of each component in the mixture was obtained by fitting the data to Equation 1. The results shown in Table 2 are the weighted mean and SE of two kinetic experiments.