Two Stable Unfolding Intermediates of the Disease-Causing L68Q Variant of Human Cystatin C
详细信息 查看全文
- 作者：Bernd Gerhartz ; Irena Ekiel ; and Magnus Abrahamson
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：December 8, 1998
- 年：1998
- 卷：37
- 期：49
- 页码：17309 - 17317
- 全文大小：105K
- 年卷期：v.37,no.49(December 8, 1998)
- ISSN：1520-4995
文摘
In hereditary cystatin C amyloid angiopathy (HCCAA), presence of the Leu68 
Glnsubstitution in cystatin C is coupled to a decreased concentration of this major cysteine proteinase inhibitorin cerebrospinal fluid and leads to its amyloid deposition in the brain. We established a high-yieldexpression system for L68Q cystatin C in Escherichia coli resulting in inclusion body accumulation at alevel of 40% of the total cellular protein. Refolding of protein from purified inclusion bodies yielded apure, almost completely monomeric and active inhibitor. CD and NMR spectroscopy demonstrated thatso produced L68Q cystatin C is folded, conformationally homogeneous, and structurally very similar towild-type cystatin C. Incubation at pH 7.0-5.5 caused the cystatin C variant to dimerize rapidly. Themolecular form present at pH 6.0 displayed a slightly increased amount of hydrophobic parts on thesurface as measured by 1-anilinonaphthalene-8-sulfonic acid (ANS) binding. NMR results showed thatthe dimer has a structure similar to that of the wild-type cystatin C dimer formed as a result of slightdenaturation. Under more acidic conditions, at pH 4.5, another stable unfolding intermediate of L68Qcystatin C was identified. This molecular form exists in a monomeric state, is characterized by changesin secondary structure according to far UV CD spectroscopy, and shows an altered ANS binding resemblingthat of a molten globule state. The acidic pH also caused an almost complete monomerization of preformeddimers. The state of denaturation of L68Q cystatin C in vivo is thus a critical factor for the concentrationof active cysteine proteinase inhibitor in cerebrospinal fluid and likely also for the development ofamyloidosis, in HCCAA patients.
Glnsubstitution in cystatin C is coupled to a decreased concentration of this major cysteine proteinase inhibitorin cerebrospinal fluid and leads to its amyloid deposition in the brain. We established a high-yieldexpression system for L68Q cystatin C in Escherichia coli resulting in inclusion body accumulation at alevel of 40% of the total cellular protein. Refolding of protein from purified inclusion bodies yielded apure, almost completely monomeric and active inhibitor. CD and NMR spectroscopy demonstrated thatso produced L68Q cystatin C is folded, conformationally homogeneous, and structurally very similar towild-type cystatin C. Incubation at pH 7.0-5.5 caused the cystatin C variant to dimerize rapidly. Themolecular form present at pH 6.0 displayed a slightly increased amount of hydrophobic parts on thesurface as measured by 1-anilinonaphthalene-8-sulfonic acid (ANS) binding. NMR results showed thatthe dimer has a structure similar to that of the wild-type cystatin C dimer formed as a result of slightdenaturation. Under more acidic conditions, at pH 4.5, another stable unfolding intermediate of L68Qcystatin C was identified. This molecular form exists in a monomeric state, is characterized by changesin secondary structure according to far UV CD spectroscopy, and shows an altered ANS binding resemblingthat of a molten globule state. The acidic pH also caused an almost complete monomerization of preformeddimers. The state of denaturation of L68Q cystatin C in vivo is thus a critical factor for the concentrationof active cysteine proteinase inhibitor in cerebrospinal fluid and likely also for the development ofamyloidosis, in HCCAA patients.