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.