Dominant-negative Effects of COL7A1 Mutations Can be Rescued by Controlled Overexpression of Normal Collagen VII
Dominant-negative Effects of COL7A1 Mutations Can be Rescued by Controlled Overexpression of Normal Collagen VII
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DOI：
10.1074/jbc.m109.045294
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发表时间：
2009-10-30
影响因子：
4.8
通讯作者：
Bruckner-Tuderman, Leena
中科院分区：
文献类型：
--
作者：
Fritsch, Anja;Spassov, Sashko;Bruckner-Tuderman, Leena
COL7A1 基因中甘氨酸取代突变的显性负干扰会导致显性营养不良性大疱性表皮松解症 (DDEB)，这是一种机械引起水疱的皮肤脆弱性疾病。尽管 COL7A1 基因产物 VII 型胶原蛋白的定性和定量改变是 DDEB 的基础，但突变与临床表型之间缺乏直接相关性，使得 DDEB 不太适合治疗靶向。为了描述 DDEB 的分子机制，我们使用野生型 (WT) 和突变型 VII 胶原蛋白的重组表达来表征三螺旋分子，其中突变型 VII 胶原蛋白含有天然发生的 COL7A1 突变、G1776R、G2006D 或 G2015E。亲和纯化和质谱分析证明，突变体与 WT 等量共表达，并且可以形成异源三聚体杂合三螺旋。突变体分子的热稳定性大大降低，这从它们对胰蛋白酶消化的敏感性中可以看出。突变分子的螺旋到螺旋转变 T-m 为 31-34 摄氏度，WT 胶原蛋白 VII 为 41 摄氏度。WT 与 G1776R-或 G2006D-胶原蛋白 VII 的共表达导致胶原蛋白部分保留在细胞内，并且突变型胶原蛋白 VII 支持细胞粘附的能力降低。有趣的是，WT VII 胶原蛋白的受控过度表达逐渐改善了 VII 胶原蛋白分子集合的热稳定性。以 90% WT:10% 突变体的比例共表达，使 G1776R-VII 胶原蛋白的 T-m 增加至 41 摄氏度，对 G2006D-和 G2015E-VII 胶原蛋白的 T-m 增加至 39 摄氏度。因此，增加 DDEB 患者皮肤中 WT VII 型胶原的表达可被认为是一种有效的治疗方法。
Dominant-negative interference by glycine substitution mutations in the COL7A1 gene causes dominant dystrophic epidermolysis bullosa (DDEB), a skin fragility disorder with mechanically induced blistering. Although qualitative and quantitative alterations of the COL7A1 gene product, collagen VII, underlie DDEB, the lack of direct correlation between mutations and the clinical phenotype has rendered DDEB less amenable to therapeutic targeting. To delineate the molecular mechanisms of DDEB, we used recombinant expression of wildtype (WT) and mutant collagen VII, which contained a naturally occurring COL7A1 mutation, G1776R, G2006D, or G2015E, for characterization of the triple helical molecules. The mutants were co-expressed with WT in equal amounts and could form heterotrimeric hybrid triple helices, as demonstrated by affinity purification and mass spectrometry. The thermal stability of the mutant molecules was strongly decreased, as evident in their sensitivity to trypsin digestion. The helix-to-coil transition, T-m, of the mutant molecules was 31-34 degrees C, and of WT collagen VII 41 degrees C. Co-expression of WT with G1776R- or G2006D-collagen VII resulted in partial intracellular retention of the collagen, and mutant collagen VII had reduced ability to support cell adhesion. Intriguingly, controlled overexpression of WT collagen VII gradually improved the thermal stability of the collective of collagen VII molecules. Co-expression in a ratio of 90% WT:10% mutant increased the T-m to 41 degrees C for G1776R- collagen VII and to 39 degrees C for G2006D- and G2015E-collagen VII. Therefore, increasing the expression of WT collagen VII in the skin of patients with DDEB can be considered a valid therapeutic approach.