The Cardiomyopathy and Lens Cataract Mutation in αB-crystallin Alters Its Protein Structure, Chaperone Activity, and Interaction with Intermediate Filaments in Vitro*
- Ming Der Perng‡§,
- Paul J. Muchowski§¶,
- Paul van den IJssel‡,
- Gabrielle J. S. Wu¶,
- Aileen M. Hutcheson‡,
- John I. Clark¶‖ and
- Roy A. Quinlan‡**
- From the ‡Department of Biochemistry, Medical Science Institute, The University, Dundee DD1 5EH, United Kingdom and the Departments of ¶Biological Structure and ‖Ophthalmology, University of Washington, Seattle, Washington 98195-7420
Abstract
Desmin-related myopathy and cataract are both caused by the R120G mutation in αB-crystallin. Desmin-related myopathy is one of several diseases characterized by the coaggregation of intermediate filaments with αB-crystallin, and it identifies intermediate filaments as important physiological substrates for αB-crystallin. Using recombinant human αB-crystallin, the effects of the disease-causing mutation R120G upon the structure and the chaperone activities of αB-crystallin are reported. The secondary, tertiary, and quaternary structural features of αB-crystallin are all altered by the mutation as deduced by near- and far-UV circular dichroism spectroscopy, size exclusion chromatography, and chymotryptic digestion assays. The R120G αB-crystallin is also less stable than wild type αB-crystallin to heat-induced denaturation. These structural changes coincide with a significant reduction in thein vitro chaperone activity of the mutant αB-crystallin protein, as assessed by temperature-induced protein aggregation assays. The mutation also significantly altered the interaction of αB-crystallin with intermediate filaments. It abolished the ability of αB-crystallin to prevent those filament-filament interactions required to induce gel formation while increasing αB-crystallin binding to assembled intermediate filaments. These activities are closely correlated to the observed disease pathologies characterized by filament aggregation accompanied by αB-crystallin binding. These studies provide important insight into the mechanism of αB-crystallin-induced aggregation of intermediate filaments that causes disease.
Footnotes
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↵* This work was supported by Wellcome Trust Grant No. 46747 (to R. A. Q. and P. v. d. I.), grants from the University of Dundee and the Overseas Research Student Award Scheme (to M. D. P.), and National Institutes of Health NEI Grant EY0452 (to J. I. C.) and NEI Vision Training Grant T32 EY07031 (to P. J. M.).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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↵§ These authors contributed equally.
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↵** To whom correspondence should be addressed. Tel.: 44-1382-344752; Fax: 44-1382-201063; E-mail: raquinlan@bad.dundee.ac.uk.
- Abbreviations:
- DRM
-
desmin-related myopathy
- BB
-
binding buffer
- UVCD
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ultraviolet circular dichroism
- DTT
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dithiothreitol
- GFAP
-
glial fibrillary acidic protein
- PMSF
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phenylmethylsulfonyl fluoride
- PAGE
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polacrylamide gel electrophoresis
- sHSP
-
small heat shock protein
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- Received April 29, 1999.
- Revision received July 27, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
