The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy
The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy
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DOI：
10.1093/hmg/10.1.47
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发表时间：
2001-01-01
影响因子：
3.5
通讯作者：
Warren, MJ
中科院分区：
文献类型：
--
作者：
Newbold, RJ;Deery, EC;Warren, MJ
鸟苷酸环化酶激活蛋白-1（GCAP 1）是激活视网膜鸟苷酸环化酶-1（RetGC 1）所必需的，RetGC 1是感光细胞恢复到黑暗状态所必需的。在这篇论文中，实验观察报告有助于解释为什么在人类GCAP 1的位置50处的脯氨酸->亮氨酸突变导致在携带该突变的家族中的锥杆营养不良。使用定点诱变突变野生型GCAP 1的一级氨基酸序列以在位置50处产生亮氨酸。此外，丝氨酸取代位置6处的谷氨酸残基以促进N-末端肉豆蔻酰化，产生构建体GCAP 1 E6 S/P50 L。该酶在大肠杆菌细胞中过表达，分离和纯化后用于RetGC 1测定，其特征在于圆二色性（CD）光谱，并研究了蛋白酶抗性和热稳定性。在E6 S/P50 L存在下，RetGC 1由鸟苷三磷酸合成环鸟苷一磷酸（cGMP）的分析表明，E6 S/P50 L可以激活RetGC 1，并且显示出与野生型GCAP 1相似的钙敏感性。此外，E6 S/P50 L和野生型GCAP 1具有相似的CD光谱，但是，如cGMP测定和CD光谱所观察到的，E6 S/P50 L对蛋白酶降解的敏感性显著增加，并且热稳定性也降低。因此表明，尽管GCAP 1 E6 S/P50 L具有与野生型GCAP 1相似的活性和钙依赖性特征，但其较低的稳定性可能降低其细胞浓度，这反过来会改变[Ca 2 +]并导致细胞死亡。
Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. in this paper, experimentally derived observations are reported that help in explaining why a proline-->leucine mutation at position 50 of human GCAP1 results in cone-rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50, In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1, In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra, However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.