Two stable unfolding intermediates of the disease-causing L68Q variant of human cystatin C
(1998) In Biochemistry 37(49). p.17309-17317- 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... (More)
- 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. (Less)
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https://lup.lub.lu.se/record/1113930
- author
- Gerhartz, Bernd ; Ekiel, I and Abrahamson, Magnus LU
- organization
- publishing date
- 1998
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biochemistry
- volume
- 37
- issue
- 49
- pages
- 17309 - 17317
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- scopus:0032497910
- ISSN
- 0006-2960
- DOI
- 10.1021/bi980873u
- language
- English
- LU publication?
- yes
- id
- c28a30c8-39ec-4c4a-9530-5ba69be76318 (old id 1113930)
- date added to LUP
- 2016-04-01 11:59:58
- date last changed
- 2022-01-26 21:22:48
@article{c28a30c8-39ec-4c4a-9530-5ba69be76318,
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.}},
author = {{Gerhartz, Bernd and Ekiel, I and Abrahamson, Magnus}},
issn = {{0006-2960}},
language = {{eng}},
number = {{49}},
pages = {{17309--17317}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Biochemistry}},
title = {{Two stable unfolding intermediates of the disease-causing L68Q variant of human cystatin C}},
url = {{http://dx.doi.org/10.1021/bi980873u}},
doi = {{10.1021/bi980873u}},
volume = {{37}},
year = {{1998}},
}