Grothe Heather L, Little Morgan R, Sjogren Phayvanh P, Chang Angela A, Nelson Elizabeth F, Yuan Ching
Department of Ophthalmology and Visual Neurosciences, University of Minnesota Medical School, Minneapolis, MN, USA.
Mol Vis. 2013;19:593-603. Epub 2013 Mar 20.
Transforming growth factor beta-induced protein (TGFBIp) is a widely expressed extracellular matrix protein that plays roles in cell adhesion and migration, differentiation, apoptosis, bone morphogenesis, and carcinogenesis. Mutations of TGFBIp have been linked to stromal corneal dystrophies, a group of protein conformational diseases characterized by abnormal protein aggregations in the cornea. However, the underlying pathogenic mechanism remains elusive due to a lack of insight into the molecular properties of the disease-causing mutants. In the current study, we applied spectroscopic tools to compare the conformation and protein stability of recombinant wild-type (WT) TGFBIp to two dystrophic mutants, R124C and R555W.
A serum-free expression system was used to produce the recombinant TGFBIp proteins. Fluorescence and far-ultraviolet circular dichroism spectroscopies were used to compare WT and dystrophic mutants under various conditions.
Our results showed that dystrophic mutants were processed differentially by the expressing cells and produced different proteolytic fragment patterns by proteolysis. Intrinsic tryptophan fluorescence studies revealed moderate shifts in the emission maxima and increased quenching by iodide ion of mutant TGFBIp, suggesting a different conformation than WT protein. Denaturation experiments indicated a difference in protein stability between WT and mutant proteins. Under oxidizing conditions, the mutants produced higher 1-anilinonaphthalene-8-sulfonic acid and thioflavin T fluorescence signals than the WT, indicating increased protein unfolding and fibril formation, respectively. Finally, far-ultraviolet circular dichroism spectroscopy revealed that WT TGFBIp undergoes concentration-dependent conformational changes; similar experiments were not possible on mutant TGFBIp, which remained soluble only at low concentrations.
Our study provides new evidence for the pathogenic mechanism of dystrophic mutants. Although mutant TGFBIp has moderate but consistent structural perturbations, other factors such as oxidation or degradation may be required to cause the phenotypic abnormal aggregations.
转化生长因子β诱导蛋白（TGFBIp）是一种广泛表达的细胞外基质蛋白，在细胞黏附与迁移、分化、凋亡、骨形态发生及致癌作用中发挥作用。TGFBIp的突变与角膜基质营养不良有关，这是一组蛋白质构象疾病，其特征为角膜中出现异常蛋白质聚集。然而，由于对致病突变体的分子特性缺乏深入了解，其潜在致病机制仍不清楚。在本研究中，我们应用光谱学工具比较重组野生型（WT）TGFBIp与两种营养不良突变体R124C和R555W的构象和蛋白质稳定性。
使用无血清表达系统生产重组TGFBIp蛋白。利用荧光和远紫外圆二色光谱在不同条件下比较WT和营养不良突变体。
我们的结果表明，表达细胞对营养不良突变体的处理方式不同，且通过蛋白水解产生不同的蛋白水解片段模式。内在色氨酸荧光研究显示，突变型TGFBIp的发射最大值有适度偏移，且碘离子淬灭增加，这表明其构象与WT蛋白不同。变性实验表明WT和突变蛋白之间的蛋白质稳定性存在差异。在氧化条件下，突变体产生的1-苯胺基萘-8-磺酸和硫黄素T荧光信号高于WT，分别表明蛋白质解折叠增加和原纤维形成增加。最后，远紫外圆二色光谱显示WT TGFBIp经历浓度依赖性构象变化；对突变型TGFBIp无法进行类似实验，因为它仅在低浓度下可溶。
我们的研究为营养不良突变体的致病机制提供了新证据。尽管突变型TGFBIp有适度但一致的结构扰动，但可能还需要其他因素如氧化或降解来导致表型异常聚集。
