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Characterization of Molecular Mechanisms Underlying Mutations in Dystrophic Epidermolysis Bullosa Using Site-directed Mutagenesis*

机译：突变背后的分子机制的表征。 定向营养性大疱性表皮松解 诱变*

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摘要

Type VII collagen (C7) is a major component of anchoring fibrils, structures that mediate epidermal-dermal adherence. Mutations in gene COL7A1 encoding for C7 cause dystrophic epidermolysis bullosa (DEB), a genetic mechano-bullous disease. The biological consequences of specific COL7A1 mutations and the molecular mechanisms leading to DEB clinical phenotypes are unknown. In an attempt to establish genotype-phenotype relationships, we generated four individual substitution mutations that have been associated with recessive DEB, G2049E, R2063W, G2569R, and G2575R, and purified the recombinant mutant proteins. All mutant proteins were synthesized and secreted as a 290-kDa mutant C7 α chain at levels similar to wild type C7. The G2569R and G2575R glycine substitution mutations resulted in mutant C7 with increased sensitivity to protease degradation and decreased ability to form trimers. Limited proteolytic digestion of mutant G2049E and R2063W proteins yielded aberrant fragments and a triple helix with reduced stability. These two mutations next to the 39-amino acid helical interruption hinge region caused local destabilization of the triple-helix that exposed an additional highly sensitive proteolytic site within the region of the mutation. Our functional studies demonstrated that C7 is a potent pro-motility matrix for skin human keratinocyte migration and that this activity resides within the triple helical domain. Furthermore, G2049E and R2063W mutations reduced the ability of C7 to support fibroblast adhesion and keratinocyte migration. We conclude that known recessive DEB C7 mutations perturb critical functions of the C7 molecule and likely contribute to the clinical phenotypes of DEB patients.
机译：VII型胶原蛋白（C7）是锚定原纤维的主要成分，锚定原纤维是介导表皮-皮肤粘附的结构。编码C7的基因COL7A1中的突变会导致营养不良性大疱性表皮松解症（DEB）。特定的COL7A1突变的生物学后果和导致DEB临床表型的分子机制尚不清楚。为了建立基因型与表型的关系，我们产生了四个与隐性DEB，G2049E，R2063W，G2569R和G2575R相关的单个取代突变，并纯化了重组突变蛋白。合成了所有突变蛋白，并以与野生型C7相似的水平分泌为290 kDa突变C7α链。 G2569R和G2575R甘氨酸取代突变导致突变体C7对蛋白酶降解的敏感性增加，并且形成三聚体的能力下降。突变G2049E和R2063W蛋白的有限蛋白水解消化产生异常片段和稳定性降低的三重螺旋。靠近39个氨基酸的螺旋中断铰链区的这两个突变导致三螺旋的局部失稳，从而使突变区内的另一个高灵敏度蛋白水解位点暴露出来。我们的功能研究表明，C7是皮肤人角质形成细胞迁移的有效促运动基质，并且该活性位于三螺旋结构域内。此外，G2049E和R2063W突变降低了C7支持成纤维细胞粘附和角质形成细胞迁移的能力。我们得出结论，已知的隐性DEB C7突变会扰乱C7分子的关键功能，并可能有助于DEB患者的临床表型。

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