A Kinetic Mechanism for the Polymerization of α1-Antitrypsin*
- Timothy R. Dafforn‡§,
- Ravi Mahadeva‡¶,
- Peter R. Elliott‡¶,
- Pasupathy Sivasothy‡¶‖ and
- David A. Lomas‡¶
- From the ‡Department of Haematology and¶Respiratory Medicine Unit, Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge CB2 2XY, United Kingdom
Abstract
The mutation in the Z deficiency variant of α1-antitrypsin perturbs the structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepatocytes to form inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The process of polymer formation has been investigated here by intrinsic tryptophan fluorescence, fluorescence polarization, circular dichroic spectra and extrinsic fluorescence with 8-anilino-1-naphthalenesulfonic acid and tetramethylrhodamine-5-iodoacetamide. These biophysical techniques have demonstrated that α1-antitrypsin polymerization is a two-stage process and have allowed the calculation of rates for both of these steps. The initial fast phase is unimolecular and likely to represent temperature-induced protein unfolding, while the slow phase is bimolecular and associated with loop-sheet interaction and polymer formation. The naturally occurring Z, S, and I variants and recombinant site-directed reactive loop and shutter domain mutants of α1-antitrypsin were used to demonstrate the close association between protein stability and rate of α1-antitrypsin polymerization. Taken together, these data allow us to propose a kinetic mechanism for α1-antitrypsin polymer formation that involves the generation of an unstable intermediate, which can form polymers or generate latent protein.
Footnotes
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↵* This work was supported by the Wellcome Trust, Medical Research Council (United Kingdom), the Cystic Fibrosis Trust, and Papworth National Health Service Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed. Tel.: 44-1223-336829; Fax: 44-1223-336827; E-mail: td214{at}cam.ac.uk.
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↵‖ A Medical Research Council Training Fellow.
- Abbreviations:
- PEG
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polyethylene glycol
- PAGE
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polyacrylamide gel electrophoresis
- 5-TMRIA
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tetramethylrhodamine-5-iodoacetamide
- ANSA
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8-anilino-1-naphthalenesulfonic acid
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- Received June 25, 1998.
- Revision received January 3, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
