论文部分内容阅读
目的 分析COL4A5基因不同点突变后的蛋白结构,探讨基因突变对编码蛋白二级结构的影响及与表型的关系。方法 以临床确诊的2例X连锁遗传型Alport综合征患者为研究对象,他们的基因突变类型均为点突变导致的甘氨酸替代:患者1,其家系为症状较重的青少年型,基因检测确定为COL4A5中的g.3246G>T导致p.G1015V;患者2,其家系为症状较轻的成年型,基因检测确定为COL4A5中的g.3290G>A导致p.G1030S。应用E.coli分别表达患者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 与对照相比,患者1重组蛋白的圆二色谱最低峰所在的波长由200 nm,变为近220 nm处,而且峰度降低。患者2重组蛋白的检测结果与对照相比改变较轻微,最低峰所在的波长不变,但峰度增加。二级结构分析显示,来自对照的重组蛋白主要以β折叠和无规卷曲为主,无α螺旋结构。与对照蛋白相比,来自患者1的重组蛋白中出现了约占八分之一的α螺旋结构;来自患者2的重组蛋白仍然以β折叠和无规卷曲为主,但是β折叠的比例下降而无规卷曲增多。结论位于α5(Ⅳ)链同一结构域的两个不同位置的甘氨酸被不同的氨基酸替代,它们的临床表型截然不同,α5(Ⅳ)链的二级结构也存在显著差异。而且,二级结构的改变程度与相应
OBJECTIVE: To analyze the protein structure of COL4A5 gene after different point mutations and to investigate the effect of gene mutation on the secondary structure of the encoded protein and its relationship with phenotype. Methods Two clinically diagnosed patients with X-linked Alport syndrome were studied. Their gene mutation types were all the glycine substitutions caused by point mutation. Patient 1 had a pedigree with severe symptoms and the genetic test was defined as G.3246G> T in COL4A5 resulted in p.G1015V; patient 2, whose pedigree was of less symptomatic adult type, and the genetic test determined that g.3290G> A in COL4A5 resulted in p.G1030S. E.coli were used to express the domain containing mutation site of α5 (Ⅳ) chain and the same domain of control α5 (Ⅳ) chain, circular dichroism was used to detect and compare their secondary structure differences. Results Compared with the control, the lowest circular dichroism peak of patient 1 recombinant protein was found to change from 200 nm to nearly 220 nm, and the kurtosis decreased. Patients 2 recombinant protein test results compared with the control changes slightly, the minimum peak wavelength where the same, but increased kurtosis. Secondary structure analysis showed that the recombinant proteins from the control were mainly β-sheet and random coil, without α-helical structure. Approximately one-eighth of the alpha helical structures were present in the recombinant protein from Patient 1 compared to the control protein; the recombinant protein from Patient 2 was still predominantly beta-sheet and random coil, but the proportion of beta-sheet decreased Random curly increase. Conclusion The glycine at two different positions in the same domain of α5 (Ⅳ) chain is replaced by different amino acids. Their clinical phenotypes are quite different, and the secondary structure of α5 (Ⅳ) chain is also significantly different. Moreover, the degree of secondary structure changes accordingly