姜茜 张震 李颀 肖萍 苏琳 李龙.EMA3C/SEMA3D基因错义突变对蛋白稳定性和受体亲合力的影响[J].,2015,15(15):2806-2810 |
EMA3C/SEMA3D基因错义突变对蛋白稳定性和受体亲合力的影响 |
Effect of Missense Mutation in EMA3C/SEMA3D Gene on Semaphorin 3Protein Stability and Receptor Binding Affinity |
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DOI: |
中文关键词: 先天性巨结肠 Semaphorin 3 基因 错义突变 蛋白质模型 受体亲合力 |
英文关键词: Hirschsprung disease Semaphorin 3 gene Missense mutation modeling Receptor binding affinity |
基金项目:国家自然科学基金青年科学基金项目(81300266);北京市自然科学基金面上项目(7142029);
北京市优秀人才培养个人项目(2013D003034000007) |
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中文摘要: |
目的:明确先天性巨结肠患者携带的5 个基因错义突变对Semaphorin 3(Sema3)蛋白自身稳定性和受
体亲合力的影响作用。方法:构建Sema3-Neuropilin-Plexin 配体-受体复合物蛋白质模型,对全部5个错义突变进行定位,通过计
算标准能量功能赋值(驻驻G)和复合物界面值(驻I_sc)预测突变对Sema3 的影响作用。将野生型和突变型AP-tagged Sema3 质粒分
别转染HEK293T 细胞,72 h后收集含有融合蛋白的细胞培养液上清并与分别表达Neuropilin 1(Nrp-1)或Neuropilin 2(Nrp-2)的
COS-7 细胞孵育,洗脱未结合的蛋白后加入碱性磷酸酶底物显色拍片,或提取细胞总蛋白,利用融合蛋白N- 末端含有的碱性磷
酸酶在底物PNPP 存在时可以发生颜色变化的特性,对与受体结合的野生型和突变型AP-Sema3 蛋白进行定量。结果:5 个错义突
变中的4 个都会不同程度地影响相应Semaphorin 3蛋白与其受体Neuropilin 的结合(与Nrp-1 的结合:SEMA3C S329G,V337M,
SEMA3D H424Q,V457I,P615T 分别与野生型相比:1.12± 0.15,0.37± 0.03,0.56± 0.07,0.51± 0.05,0.66± 0.05;与Nrp-2 的结合:
SEMA3C S329G,V337M,SEMA3D H424Q,V457I,P615T 分别与野生型相比:1.18 ± 0.09,0.37 ± 0.03,0.76 ± 0.01,0.65 ±
0.06,0.85± 0.03,n=3,单因素方差分析,差异有统计学意义),说明它们可能通过严重影响分子通路的信号转导而妨碍蛋白功能的
正常行使。结论:先天性巨结肠患者携带的基因错义突变可不同程度影响蛋白与其受体的结合,提示
Semaphorin 3这类经典的神经元轴突导向因子在功能失常的情况下可能参与先天性巨结肠的发生。 |
英文摘要: |
Objective:To dissect the effect of 5 missense mutation in gene detected in Hirschsprung disease
(HSCR) patients on stability and receptor binding affinity of Semaphorin 3 protein.Methods:We predicted the effect of each variant on
semaphorin stability or receptor binding affinity by constructing computational homology modeling of the Sema3-Neuropilin-Plexin complex.
驻I_sc and 驻驻G values that signify the effects of the substitutions were estimated. Wild-type and mutant AP-Sema3 fusion protein
constructs were transiently transfected into HEK293T cells. AP-Sema3 containing media (quantified by measuring AP activity) was collected
and applied to COS-7 cells over-expressing Neuropilin 1 or 2. Binding ligands were enzymatically detected by adding substrate of
the alkaline phosphatase. Concentrations of the binding Sema3 ligands were also quantified with colorimetric assays.Results:Four (SEMA3C:
V337M; SEMA3D: H424Q, V457I, P615T) out of five HSCR variants showed reduced binding affinity of the cognate Sema3
protein to both Neuropilin 1 and Neuropilin 2, but to different extents. Specifically, S329G and V337M in SEMA3C exhibited fold
changes in binding affinity to Nrp-1 as of 1.12 ± 0.15 and 0.37 ± 0.03; while H424Q, V457I and P615T in SEMA3D showed fold
changes of 0.56± 0.07, 0.51± 0.05 and 0.66± 0.05, respectively. With respect to Nrp-2, these cognate Sema3 proteins displayed binding
affinity changes as of 1.18± 0.09, 0.37± 0.03, 0.76± 0.01, 0.65± 0.06 and 0.85± 0.03 respectively, relative to the wild-type construct
(n=3, one-way ANOVA).Conclusion:The missense mutation harbored by HSCR patients may damage protein function to different ex--
tents, indicating that loss-of-function of Semaphorin 3 family members may exert pathological roles in predisposing people to HSCR. |
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