突变型COL4A5基因编码蛋白片段的表达及圆二色谱结构分析

Expression of the partial protein encoded by mutated COL4A5 gene and analysis of the structure by circular dichroism

目的 :通过分析COL4A5基因点突变后蛋白结构 ,探讨基因突变对编码蛋白的基本结构及可能的二级结构的影响。方法 :以已确诊的X连锁显性遗传型Alport综合征 (Alportsyndrome ,AS)病人的α5 (Ⅳ )链为研究对象 ,其COL4A5的点突变 g .32 4 6G >T使编码蛋白的一个甘氨酸被缬氨酸替代 p .G1 0 1 5V。用E .coli 分别表达该病人α5 (Ⅳ )链的含有突变位点的结构域及对照α5 (Ⅳ )链的同一结构域 ,圆二色谱检测并比较它们二级结构的差异。结果 :病人的圆二色谱最低峰所在的波长由对照的 2 0 0nm左右 ,变为 2 2 0nm左右 ,而且峰度降低。二级结构分析显示 ,来自对照的融合蛋白主要以 β折叠和无规卷曲为主 ,无α螺旋结构 ,而来自病人的融合蛋白中出现了约占1 /8的α螺旋结构。结论 :AS时胶原区中的一个甘氨酸被缬氨酸替代后 ,不仅使发生替代处局部的二级结构发生变化 ,而且使整个α5 (Ⅳ )链的折叠动力学发生改变。异常折叠的肽链会影响链间的相互作用 ,或者使得三螺旋分子不能正常形成 ,或者使形成的三螺旋分子结构松散而易被蛋白酶降解破坏 ,最终使肾小球基底膜出现特征性的病理改变。

Objective: To analyze the protein structure encoded by COL4A5 gene with a missense mutation and to discuss the effect of gene mutation on basic structure and predicted secondary structure of the encoded protein. Methods: A fragment of cDNA with a g.3246G>T mutation resulting in p.G1015V from an X linked Alport patient, and that of corresponding cDNA from a control were expressed in E.coli. The recombinant and mutant polypeptide was a fragment of COL4A5 , containing 158 amino acid residues with a glycine to valine substitution mutation in it. The secondary structure of the two recombinant proteins was analyzed using circular dichroism(CD) spectroscopy. Results: CD spectra of the control exhibited a negative peak near 200 nm whereas that of the patient exhibited a negative peak near 220 nm. The magnitude of the negative peak of the patient decreased as compared with that of the control. Furthermore, secondary structure of the control polypeptide was mainly composed of β sheet and random coil without α helix, whereas that of the patient presented 12.9% α helix. Conclusion: Not only local structure of the substitution site but also folding kinetics of the entire α5 chain may be changed due to Gly→Val substitution in Alport syndrome. We speculate that the abnormally folded polypeptide chain may not be assembled into the triple helix and the network of type Ⅳ collagen, or may be assembled into loosen triple helix then degraded easily, resulting in the pathognomonic ultrastructural changes of the glomerular basement membrane.