Kutz Wendy E, Wang Lauren W, Dagoneau Nathalie, Odrcic Kazimir J, Cormier-Daire Valerie, Traboulsi Elias I, Apte Suneel S
Department of Biomedical Engineering and Orthopaedic Research Center, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Hum Mutat. 2008 Dec;29(12):1425-34. doi: 10.1002/humu.20797.
We report the identification and functional analysis of the first missense ADAMTS10 mutation (c.73G>A; p.Ala25Thr) causing recessive Weill-Marchesani syndrome (WMS). The Ala25 residue affected by the missense mutation is at the -1 position relative to the ADAMTS10 signal peptidase cleavage site. p.Ala25Thr substituted full-length ADAMTS10 showed consistent and significantly diminished secretion in both HEK293F and Cos-1 cells. However, a C-terminally truncated construct lacking the ancillary domain and containing only the signal peptide, the propeptide and the catalytic domain (p.Ala25Thr Pro-Cat) was efficiently secreted in both HEK293F cells and Cos-1 cells. Edman degradation of purified p.Ala25Thr Pro-Cat and p.Ala25Thr substituted full-length ADAMTS10 from HEK293F cells demonstrated correct signal peptide processing. Thus, the p.Ala25Thr substitution hinders secretion of full-length ADAMTS10, but not Pro-Cat from cells, yet permits signal peptide removal. We infer that folding of the complex C-terminal ancillary domain is the rate-limiting step in biosynthesis of ADAMTS10, and that it (but not Pro-Cat) is sensitive to subtle changes in efficiency of signal peptide cleavage. These observations represent an unprecedented effect of a signal peptide mutation and support a model in which the initial cotranslational processing events during protein biosynthesis can have long-range effects on protein folding and secretion.
我们报告了首例导致隐性韦尔-马切萨尼综合征（WMS）的错义ADAMTS10突变（c.73G>A；p.Ala25Thr）的鉴定及功能分析。受错义突变影响的Ala25残基相对于ADAMTS10信号肽酶切割位点位于-1位。p.Ala25Thr替代的全长ADAMTS10在HEK293F和Cos-1细胞中均表现出持续且显著减少的分泌。然而，一个C末端截短的构建体，其缺少辅助结构域，仅包含信号肽、前肽和催化结构域（p.Ala25Thr Pro-Cat），在HEK293F细胞和Cos-1细胞中均能有效分泌。对从HEK293F细胞中纯化的p.Ala25Thr Pro-Cat和p.Ala25Thr替代的全长ADAMTS10进行埃德曼降解，证明信号肽加工正确。因此，p.Ala25Thr替代阻碍全长ADAMTS10从细胞中分泌，但不影响Pro-Cat的分泌，且允许信号肽切除。我们推断复杂的C末端辅助结构域的折叠是ADAMTS10生物合成中的限速步骤，并且它（而非Pro-Cat）对信号肽切割效率的细微变化敏感。这些观察结果代表了信号肽突变前所未有的效应，并支持一种模型，即蛋白质生物合成过程中最初的共翻译加工事件可对蛋白质折叠和分泌产生远距离影响。
