A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells
A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells
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DOI：
10.1042/bj20021226
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发表时间：
2003-01-01
影响因子：
4.1
通讯作者：
Braulke, T
中科院分区：
文献类型：
--
作者：
Partanen, S;Storch, S;Braulke, T
溶酶体蛋白组织蛋白酶D(CTSD)中天冬氨酸残基被天冬酰胺取代，导致一种新型的神经元型蜡样脂褐素沉着症的酶活性丧失和严重的大脑皮层萎缩[Tyynela，Sohar，Sleat，Gin，Donnelly，Baumann，Haltia和Lobel(2000)EMBO J.19,2786-2792]。在本研究中，我们将天冬氨酸残基293位天冬氨酸替换为天冬酰胺(D293N)的相应突变引入到小鼠CTSD的cDNA中，以分析其对转基因HEK-293细胞合成、转运和稳定性的影响。一种新开发的荧光定量系统证实了突变的D293N小鼠CTSD完全失活。此外，在所使用的异源过表达系统中，突变体D293N小鼠CTSD在分泌途径的早期步骤中明显不稳定和蛋白水解性修饰，导致质量损失约1 kDa。在发病的绵羊体内，内源突变酶是稳定的，但其分子质量也发生了变化。在HEK-293细胞中，突变的D293N小鼠CTSD向溶酶体的转运延迟，与野生型CTSD相比分泌速度较慢。这些数据表明，突变可能导致构象变化，从而影响酶的稳定性、加工和运输。
The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynela, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19,2786-2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1 kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme.