Gerhartz B, Ekiel I, Abrahamson M
Department of Clinical Chemistry, Institute of Laboratory Medicine, University of Lund, University Hospital, Sweden.
Biochemistry. 1998 Dec 8;37(49):17309-17. doi: 10.1021/bi980873u.
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.
在遗传性胱抑素C淀粉样血管病（HCCAA）中，胱抑素C中Leu68→Gln的替换与脑脊液中这种主要半胱氨酸蛋白酶抑制剂浓度降低相关联，并导致其在大脑中淀粉样沉积。我们在大肠杆菌中建立了L68Q胱抑素C的高产表达系统，结果包涵体积累量达到总细胞蛋白的40%。从纯化的包涵体中重折叠蛋白质得到了一种纯的、几乎完全单体且有活性的抑制剂。圆二色光谱（CD）和核磁共振光谱（NMR）表明，如此产生的L68Q胱抑素C已折叠，构象均一，且结构与野生型胱抑素C非常相似。在pH 7.0 - 5.5下孵育导致胱抑素C变体迅速二聚化。通过1 - 苯胺基萘 - 8 - 磺酸（ANS）结合测量，在pH 6.0时存在的分子形式在表面显示出略多的疏水部分。NMR结果表明，该二聚体的结构与因轻微变性而形成的野生型胱抑素C二聚体的结构相似。在更酸性条件下，pH 4.5时，鉴定出L68Q胱抑素C的另一种稳定的去折叠中间体。这种分子形式以单体状态存在，根据远紫外CD光谱其二级结构发生变化，并显示出类似于熔球态的改变的ANS结合。酸性pH还导致预先形成的二聚体几乎完全单体化。因此，L68Q胱抑素C在体内的变性状态是脑脊液中活性半胱氨酸蛋白酶抑制剂浓度的关键因素，并且可能也是HCCAA患者淀粉样变性发展的关键因素。
