title: 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.
authors.affiliation: Department of Clinical Chemistry, Institute of Laboratory Medicine, University of Lund, University Hospital, Sweden.
authors.forename: B
authors.initials: B
authors.lastname: Gerhartz
authors.forename: I
authors.initials: I
authors.lastname: Ekiel
authors.forename: M
authors.initials: M
authors.lastname: Abrahamson
mesh_terms: D016657:Cerebral Amyloid Angiopathy / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D055316:Cystatin C; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D014644:Genetic Variation; D005973:Glutamine / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding
chemical_list: C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D005973:Glutamine; D007930:Leucine