Heteropolymerization of S, I, and Z alpha1-antitrypsin and liver cirrhosis

Abstract

The association between Z alpha1-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 alpha1-antitrypsin. Four percent of the northern European Caucasian population are heterozygotes for the Z variant, but even more common is S alpha1-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 alpha1-antitrypsin and another conformationally unstable variant (I alpha1-antitrypsin; 39Arg-->Cys) identified in a 34-year-old man with cirrhosis related to alpha1-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 alpha1-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.

Figure 1

IZ α₁-antitrypsin accumulation in hepatocytes resulting in liver damage and cirrhosis. The liver biopsy from the propositus showed cirrhosis when stained with hematoxylin and eosin (a) and evidence of α₁-antitrypsin retention when immunostained with polyclonal antibodies to human α₁-antitrypsin (b).

Figure 2

Elution of α₁-antitrypsin from the anion exchange column allowed the separation of early eluting Z from late eluting I α₁-antitrypsin. Nondenaturing PAGE (7.5–15% wt/vol). All lanes contain 4 μg protein. Lane 1, M α₁-antitrypsin control; lane 2, 1:1 mixture of control M and Z α₁-antitrypsin; lane 3, Z α₁-antitrypsin control, lane 4, Z α₁-antitrypsin from the plasma of the propositus; lane 5, a mixture of I and Z α₁-antitrypsin isolated from the central fractions of the α₁-antitrypsin peak from the anion exchange column; lane 6, I α₁-antitrypsin from the plasma of the propositus; lane 7, M α₁-antitrypsin control cleaved at the reactive center loop with Staphylococcus aureus V8 proteinase. The difference in migration between Z α₁-antitrypsin from the propositus (lane 4) and the control (lane 3) is due to NH₂-terminal cleavage.

Figure 3

Nondenaturing PAGE (7.5–15% wt/vol) showing that I α₁-antitrypsin polymerized more readily than M α₁-antitrypsin after incubation at 37°C at 2 mg/ml in 50 mM Tris, 50 mM KCl (pH 7.4). All lanes contain 10 μg protein. (a) M α₁-antitrypsin 0 days, 2 days, 3 days, 6 days, and 12 days at 37°C. (c) I α₁-antitrypsin 0 days, 2 days, 3 days, 6 days, and 12 days at 37°C. The electron micrograph of M (b) and I (d) α₁-antitrypsin incubated at 2 mg/ml and 37°C for 12 days confirmed that I α₁-antitrypsin formed chains of polymers, but M α₁-antitrypsin remained monomeric when incubated under identical conditions. The electron micrographs were stained with 1% wt/vol uranyl acetate. Scale bar: 100 nm.

Figure 4

(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.

Figure 5

Secretion of M, Z, and I α₁-antitrypsin by the Xenopus oocyte. The results are the average values (and SE) obtained by injection into batches of 20 oocytes on at least four separate occasions.

Figure 6

(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.

Figure 7

(a) The crystal structures of α₁-antitrypsin (32, 33) demonstrated the availability of ²³²Cys and the position of the I (³⁹Arg→Cys) mutation in helix A at the back of the molecule. The reactive center loop is shown in red; the A β-sheet, which must open to allow polymer formation, is illustrated in green. S α₁-antitrypsin mediates its effect by breaking a hydrogen bond with ³⁸Tyr in the shutter domain (20), which controls A β-sheet mobility. (b) Reactive loop/A-sheet polymerization with an open helical conformation (32) places the cysteine residues over 60 Å apart (right), but a closed helical conformation predictably brings the cysteine residues of different α₁-antitrypsin molecules close to each other and therefore available for resonance energy transfer (left). In this model, the α₁-antitrypsin molecules are ordered blue, green, and red (from bottom to top), with RET predictably occurring between the labeled cysteines of molecules of the same color.