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Two stable unfolding intermediates of the disease-causing L68Q variant of human cystatin C

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Author

Summary, in English

In hereditary cystatin C amyloid angiopathy (HCCAA), presence of the Leu68 Gln substitution in cystatin C is coupled to a decreased concentration of this major cysteine proteinase inhibitor in cerebrospinal fluid and leads to its amyloid deposition in the brain. We established a high-yield expression system for L68Q cystatin C in Escherichia coli resulting in inclusion body accumulation at a level of 40% of the total cellular protein. Refolding of protein from purified inclusion bodies yielded a pure, almost completely monomeric and active inhibitor. CD and NMR spectroscopy demonstrated that so produced L68Q cystatin C is folded, conformationally homogeneous, and structurally very similar to wild-type cystatin C. Incubation at pH 7.0-5.5 caused the cystatin C variant to dimerize rapidly. The molecular form present at pH 6.0 displayed a slightly increased amount of hydrophobic parts on the surface as measured by 1-anilinonaphthalene-8-sulfonic acid (ANS) binding. NMR results showed that the dimer has a structure similar to that of the wild-type cystatin C dimer formed as a result of slight denaturation. Under more acidic conditions, at pH 4.5, another stable unfolding intermediate of L68Q cystatin C was identified. This molecular form exists in a monomeric state, is characterized by changes in secondary structure according to far UV CD spectroscopy, and shows an altered ANS binding resembling that of a molten globule state. The acidic pH also caused an almost complete monomerization of preformed dimers. The state of denaturation of L68Q cystatin C in vivo is thus a critical factor for the concentration of active cysteine proteinase inhibitor in cerebrospinal fluid and likely also for the development of amyloidosis, in HCCAA patients.

Publishing year

1998

Language

English

Pages

17309-17317

Publication/Series

Biochemistry

Volume

37

Issue

49

Document type

Journal article

Publisher

The American Chemical Society (ACS)

Topic

  • Biochemistry and Molecular Biology

Status

Published

ISBN/ISSN/Other

  • ISSN: 0006-2960