Wang Yun-Feng, Ding Jie, Wang Fang, Bu Ding-Fang
Department of Pediatrics, Peking University First Hospital, Beijing, PR China.
Biochem Biophys Res Commun. 2004 Apr 16;316(4):1143-9. doi: 10.1016/j.bbrc.2004.02.168.
The phenotype variety caused by glycine substitutions in alpha5(IV) chain in X-linked Alport syndrome (XLAS) prompted the complexity of structure changes of alpha5(IV) chain that was little to know now. In this study, we expressed a domain of alpha5(IV) chain containing different glycine substitutions (G1015V and G1030S, respectively) which were revealed in two XLAS pedigrees with different phenotype severities and the corresponding domain of a control in Escherichia coli. The recombinant proteins were characterized by immunoblot and mass spectrometry and analyzed the secondary structure by using circular dichroism (CD) spectroscopy. CD analysis showed that the recombinant protein containing G1015V mutation identified in the pedigree of juvenile-onset XLAS exhibited 12.9% alpha-helix that was not found in the control recombinant protein. The spectrum of the recombinant protein containing G1030S mutation identified in the pedigree of adult-onset XLAS was slightly different from that of the control, that is, mostly with the random coil and the beta-sheet, while without alpha-helix. These results demonstrated that two kinds of glycine substitutions, although in the same domain of alpha5(IV) chain, displayed the distinctly different secondary structures. The changes of the secondary structure could explain the phenotypic diversities of XLAS, which would be hardly understood solely by analyzing genomic DNA or mRNA of alpha5(IV) chain.
X连锁遗传性肾炎（XLAS）中α5（IV）链甘氨酸替代所导致的表型多样性，提示了α5（IV）链结构变化的复杂性，而目前对此了解甚少。在本研究中，我们在大肠杆菌中表达了α5（IV）链的一个结构域，该结构域包含在两个具有不同表型严重程度的XLAS家系中发现的不同甘氨酸替代（分别为G1015V和G1030S）以及一个对照的相应结构域。通过免疫印迹和质谱对重组蛋白进行表征，并使用圆二色性（CD）光谱分析其二级结构。CD分析表明，在青少年发病型XLAS家系中鉴定出的含有G1015V突变的重组蛋白呈现出12.9%的α-螺旋，而对照重组蛋白中未发现该结构。在成人发病型XLAS家系中鉴定出的含有G1030S突变的重组蛋白的光谱与对照略有不同，即主要为无规卷曲和β-折叠，而无α-螺旋。这些结果表明，尽管两种甘氨酸替代位于α5（IV）链的同一结构域，但它们呈现出明显不同的二级结构。二级结构的变化可以解释XLAS的表型多样性，仅通过分析α5（IV）链的基因组DNA或mRNA很难理解这一点。
