Alport综合征基因点突变后蛋白结构的改变及与表型的关系

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

Proteins structure changes of COL4A5 gene point mutations and structure-phenotype relations in Alport syndrome

OBJECTIVE: Alport syndrome (AS) is a progressive hereditary nephritis presented with hematuria and renal failure, frequently associated with sensorineural deafness and ocular lesions. So far, more than 300 gene mutations in AS have been identified which provides a better way to analyze the association between genotype and phenotype. It is hard to understand all the phenotype according to the gene mutations, because the structure and function changes of the relevant protein, alpha5(IV) chain, encoded by mutated COL4A5 gene are rare to know. This study aimed to detect the proteins structure encoded by COL4A5 gene with different missense mutations and to analyze the effect of gene mutations on the secondary structure of alpha5(IV) chain and structure-phenotype relations. METHODS: Two X-linked AS patients with different missense mutations (g.3246G > T resulting in p.G1015V and g.3290G > A resulting in p.G1030S, respectively) diagnosed by clinical manifestations, family history and skin or renal biopsy examinations, as well as a control were included in this study. The fragments of cDNA with the two mutations, respectively, and that of corresponding cDNA from the control were expressed in E. coli. The secondary structure of the recombinant polypeptides were analyzed by using circular dichroism (CD) spectroscopy. RESULTS: CD spectra of the control exhibited a negative peak near 200 nm whereas that of the patient 1 exhibited a negative peak near 220 nm. Furthermore, the magnitude of the negative peak of patient 1 decreased from -9000 deg x cm2 x dmol(-1) to -4000 deg x cm2 x dmol(-1) as compared with that of the control. CD spectra of the patient 2 were slightly changed with the negative peak remaining near 220 nm but the magnitude increasing from -9000 deg x cm2 x dmol(-1) to -11000 deg x cm2 x dmol(-1) as compared with that of the control. In addition, the secondary structure of the control polypeptide was mainly composed of beta-sheet and random coil without alpha-helix, whereas that of the patient 1 presented 12.9% alpha-helix. Although the secondary structure of polypeptide of the patient 2 was also mainly composed of beta-sheet and random coil, the composition of beta-sheet reduced and random coil increased. CONCLUSION: Although the glycine substitutions existed in the same domain of alpha5(IV) chain, the patient 1 with the severe AS phenotype and g.3246G > T mutation, and patient 2 with the mild AS phenotype and g.3290G > A mutation were revealed with different secondary structures of alpha5(IV) chain. Moreover, the secondary structure changes of alpha5(IV) chain were consistent with their corresponding phenotype severity.