薄基底膜肾病基因点突变后蛋白空间二级结构的改变及与表型的关系

目的 分析薄基底膜肾病（TBMN）患者COL4A4基因点突变后导致编码蛋白中甘氨酸被其它类型氨基酸替代后的结构；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1例常染色体显性连锁遗传型TBMN并发局灶节段性肾小球硬化症（FSGS）患者为研究对象，该患者症状较重，临床表现为血尿、大量蛋白尿，基因检测确定为COL4A4中的g. 1214G>A导致p．G405E。对照选取1例健康人和1例文献报道的单纯TBMN患者（基因检测确定为COL4A4中的g. 1550G>A导致p．G448S）。应用E. coli分别表达患者α4（Ⅳ）链的含有突变位点的结构域及对照α4（Ⅳ）链的相同结构域，圆二色谱检测并比较它们二级结构的差异。 结果 TBMN并发FSGS患者重组蛋白的圆二色谱最低峰所在的波长由正常对照的208 nm变为约220 nm处，而且峰度降低。单纯TBMN对照重组蛋白的检测结果与健康对照相比改变较轻微，最低峰所在的波长不变，峰度仅轻度降低。二级结构分析显示，来自健康对照的重组蛋白中α螺旋、β折叠、转角和无规卷曲均存在，其中前二者各占约1/4。与健康对照蛋白相比，来自TBMN并发FSGS患者的重组蛋白中α螺旋结构增多，约占1/3，无β折叠结构；单纯TBMN对照的重组蛋白与健康对照相似，α螺旋的比例下降而β折叠增多。 结论 位于α4(Ⅳ)链相邻结构域的两个不同位置的甘氨酸被不同的氨基酸替代，它们的临床表型不同，α4(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临床表型的严重性一致。

Proteins structure change of COL4A4 gene point mutation and its association with phenotype in thin basement membrane nephropathy

Objective To detect the proteins structure encoded by COL4A4 gene with different missense mutations of thin basement membrane nephropathy (TBMN) and to analyze the effect of gene mutation on the secondary structure of α4 (Ⅳ) chain and its association with phenotype. Methods A COL4A4-linked TBMN patient with FSGS by a missense mutation (g. 1214G>A resulting in p. G405E) diagnosed by clinical manifestations, family history and renal biopsy examination, as well as two controls (one healthy, one pure TBMN carrying a g. 1550G>A mutation resulting in p. G448S) were enrolled in this study. The fragments of cDNA with the two mutations and that of corresponding cDNA from the healthy control were expressed in E. coll. The secondary structures of recombinant polypeptides were analyzed by circular dichroism (CD) spectroscopy. Results CD spectra of healthy control exhibited a negative peak near 208 nm whereas that of TBMN patient with FSGS exhibited a negative peak near 220 nm. Furthermore, the magnitude of the negative peak of this patient decreased as compared with that of healthy control. CD spectra of pure TBMN control was slightly changed with the negative peak remaining near 208 run and the magnitude slightly decreased as compared with that of healthy control. In addition, the secondary structure of pelypeptide from healthy control was composed of about 1/4 α-helix and 1/4 β-sheet, whereas that from the patient presented about 1/3 α-helix without any β-sheet. The secondary structure of polypeptide from pure TBMN control was almost the same as the healthy control, except a shght reduction of α-helix and a slight increase of β-sheet. Conclusions Although the glycine substitutions exists in the nearby domain of α4 (Ⅳ)chain, the TBMN patient complicating FSGS with severe phenotype and g. 1214G>A mutation and the pure TBMN control with the mild phenotype and g. 1550G>A mutation are revealed with different secondary structures of α4 (Ⅳ)chain. Moreover, the secondary structure change of α4 (Ⅳ) chain is consistent with their corresponding phenotype severity.