Pro-sterol carrier protein-2 - Role of the N-terminal presequence in structure, function, and peroxisomal targeting
Pro-sterol carrier protein-2 - Role of the N-terminal presequence in structure, function, and peroxisomal targeting
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DOI：
10.1074/jbc.m000431200
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发表时间：
2000-08-18
影响因子：
4.8
通讯作者：
Kier, AB
中科院分区：
文献类型：
--
作者：
Schroeder, F;Frolov, A;Kier, AB
尽管存在于15-kDa前固醇载体蛋白-2（pro-SCP-2，成熟13-kDa SCP-2的前体）中的20个氨基酸前序列改变了SCP-2在脂质代谢中的功能，但这种作用的分子基础尚未得到解决。通过圆二色谱、质谱和抗体可及性测定，前序列显著改变了SCP-2的结构，使得pro-SCP-2的α-螺旋减少了3倍，p-结构增加了7倍，C末端对羧肽酶A的反应性增加了6倍，抗SCP-2的结合减少了2倍，并且没有增强质膜的固醇转移。这些差异不是由于蛋白质的稳定性，因为（i）50%解折叠需要相同浓度的盐酸胍，和（ii）配体结合位点显示出相同的高亲和力（纳摩尔Kd值），顺序为：胆固醇远大于直链脂肪酸>扭结链脂肪酸。激光扫描共聚焦显微镜和双重免疫荧光证实pro-SCP-2更有效地靶向过氧化物酶体。用编码pro-SCP-2的cDNA转染L细胞或McAR 7777肝癌细胞，分别导致45%和59%的SCP-2与过氧化物酶体标记物PMP 70共定位。相反，用编码SCP-2的cDNA转染的L-细胞显示SCP-2与PMP 70的共定位低3倍。总之，这些数据首次表明，pro-SCP-2的20个氨基酸前序列改变了SCP-2的结构，以促进由C末端SKL过氧化物酶体靶向序列介导的过氧化物酶体靶向。
Although the 20-amino acid presequence present in 15-kDa pro-sterol carrier protein-2 (pro-SCP-2, the precursor of the mature 13-kDa SCP-2) alters the function of SCP-2 in lipid metabolism, the molecular basis for this effect is unresolved. The presequence dramatically altered SCP-2 structure as determined by circular dichroism, mass spectroscopy, and antibody accessibility such that pro-SCP-2 had 3-fold less alpha-helix, 7-fold more p-structure, 6-fold more reactive C terminus to carboxypeptidase A, 2-fold less binding of anti-SCP-2, and did not enhance sterol transfer from plasma membranes. These differences were not due to protein stability since (i) the same concentration of guanidine hydrochloride was required for 50% unfolding, and (ii) the ligand binding sites displayed the same high affinity (nanomolar K-d values) in the order: cholesterol much greater than straight chain fatty acid > kinked chain fatty acid. Laser scanning confocal microscopy and double immunofluorescence demonstrated that pro-SCP-2 was more efficiently targeted to peroxisomes. Transfection of L-cells or McAR7777 hepatoma cells with cDNA encoding pro-SCP-2 resulted in 45% and 59% of SCP-2, respectively, colocalizing with the peroxisomal marker PMP70. In contrast, L-cells transfected with cDNA encoding SCP-2 exhibited 3-fold lower colocalization of SCP-2 with PMP70. In summary, the data suggest for the first time that the 20-amino acid presequence of pro-SCP-2 alters SCP-2 structure to facilitate peroxisomal targeting mediated by the C-terminal SKL peroxisomal targeting sequence.