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

Abstract
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.
AuthorsB Gerhartz, I Ekiel, M Abrahamson
JournalBiochemistry (Biochemistry) Vol. 37 Issue 49 Pg. 17309-17 (Dec 08 1998) ISSN: 0006-2960 [Print] United States
PMID9860845 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • CST3 protein, human
  • Cystatin C
  • Cystatins
  • Glutamine
  • Leucine
Topics
  • Cerebral Amyloid Angiopathy (genetics, metabolism)
  • Circular Dichroism
  • Cystatin C
  • Cystatins (chemistry, genetics)
  • Dimerization
  • Genetic Variation
  • Glutamine (genetics)
  • Humans
  • Hydrogen-Ion Concentration
  • Leucine (genetics)
  • Magnetic Resonance Spectroscopy
  • Protein Folding

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