Differential tolerance of 'pseudo-pathogenic' tryptophan residues in calcium-binding EGF domains of short fibulin proteins
Differential tolerance of 'pseudo-pathogenic' tryptophan residues in calcium-binding EGF domains of short fibulin proteins
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DOI：
10.1016/j.exer.2014.12.002
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发表时间：
2015-01-01
影响因子：
3.4
通讯作者：
Hulleman, John D.
中科院分区：
文献类型：
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作者：
Annie Nguyen;Hulleman, John D.
Arg 345 Trp（R345 W）突变在最后一个典型的钙结合表皮生长因子（cbEGF）结构域的fibulin-3（F3）导致罕见的黄斑营养不良，Malattia Leventinese（ML）。在细胞培养研究中，这种突变导致F3分泌效率低下和细胞内稳态水平较高，可能是由于F3二硫键和/或蛋白质折叠问题。然而，R345 W突变实际上是如何导致ML的，在很大程度上仍然未知。在此，我们测试了在其它cbEGF结构域中的130个半胱氨酸（B（n+1））之后立即引入类似的“假致病性”色氨酸突变是否也引起蛋白质折叠/分泌挑战。我们发现，引入色氨酸突变到其他四个F3典型的cbEGF结构域的每一个引起的蛋白质分泌的显着减少，范围从2.7%至56%的野生型（WT）F3水平。令人惊讶的是，在F3的第一个典型cbEGF结构域中的R185 W突变在F3色氨酸突变体中产生了最高量的分泌，并且在生长温度降低后，其分泌缺陷可以被挽救到接近WT水平（95%）。有趣的是，当类似位置的色氨酸突变被引入到任何典型的cbEGF结构域的高度同源的蛋白质，fibulin-5（F5），有没有对分泌的影响。为了使F3耐受色氨酸残基（如F5），我们通过删除F3中存在的三个插入区而不是F5中存在的三个插入区，对F3进行基因工程改造，使其与F5具有更高的序列同源性。然而，这些区域中的一个或多个的缺失对R345 W F3分泌没有有益的影响。总之，这些结果表明，在B（n+1）位置引入色氨酸残基并不普遍地破坏cbEGF结构域的折叠和分泌，但它们的作用是环境依赖性的，并且在这种情况下，独特地破坏F3而不是F5的典型cbEGF结构域的折叠。(C)2014爱思唯尔有限公司版权所有。
An Arg345Trp (R345W) mutation in the last canonical calcium-binding epidermal growth factor (cbEGF) domain of fibulin-3 (F3) causes the rare macular dystrophy, Malattia Leventinese (ML). In cell culture studies, this mutation leads to inefficient F3 secretion and higher intracellular steady state levels, likely due to F3 disulfide bonding and/or protein folding problems. However, how the R345W mutation actually causes ML is still largely unknown. Herein we tested whether the introduction of analogous, 'pseudo-pathogenic' tryptophan mutations immediately after the 130 cysteine (b(n+1)) in other cbEGF domains also caused protein folding/secretion challenges. We found that introduction of tryptophan mutations into each of the four other F3 canonical cbEGF domains caused a significant reduction in protein secretion ranging from 2.7 to 56% of wild-type (WT) F3 levels. Surprisingly, an R185W mutation in the first canonical cbEGF domain of F3 yielded the highest amount of secretion among the F3 tryptophan mutants, and its secretion defect could be rescued to near WT levels (95%) after growth temperature reduction. Interestingly, when similarly positioned tryptophan mutations were introduced into any of the canonical cbEGF domains of the highly homologous protein, fibulin-5 (F5), there was no effect on secretion. In an attempt to make F3 tolerant of tryptophan residues (like F5), we genetically engineered F3 to have a higher sequence homology with F5 by deleting three insert regions present in F3, but not F5. However, deletion of one or more of these regions did not have a beneficial effect on R345W F3 secretion. Overall, these results demonstrate that the introduction of tryptophan residues at the b(n+1) position does not universally disrupt cbEGF domain folding and secretion, but that their effect is context dependent, and in this case, uniquely disrupt the folding of canonical cbEGF domains of F3, but not F5. (C) 2014 Elsevier Ltd. All rights reserved.