Dominant-negative Effects of COL7A1 Mutations Can be Rescued by Controlled Overexpression of Normal Collagen VII

Abstract

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°C, and of WT collagen VII 41°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°C for G1776R-collagen VII and to 39°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.Mutations in the collagen VII gene, COL7A1, cause dystrophic epidermolysis bullosa (DEB), 3 a heritable skin fragility disorder characterized by mechanically induced blistering of the skin and mucosa, and excessive scarring (1). DEB is classified into clinical subtypes with dominant or recessive inheritance (2), and so far more than 400 different COL7A1 mutations are known, which underlie a broad spectrum of clinical presentations. Collagen VII is the major molecular constituent of anchoring fibrils in the skin. These centro-symmetrically banded fibrils extend from the epidermal basement membrane into the underlying dermal stroma and connect the epidermis to the dermis. Collagen VII is synthesized as three identical pro-␣1(VII) polypeptide chains, which are hydroxylated and glycosylated in a coordinated manner and then fold into triple-helical procollagen VII in the endoplasmic reticulum (ER). The procollagen, which contains a central collagenous triple-helix flanked by two non-collagenous domains, NC-1 and NC-2, is secreted into the extracellular space, where the C-terminal NC-2 propeptide is proteolytically removed by bone morphogenetic protein-1 (3). Subsequently, mature collagen VII undergoes a multistep fibril polymerization process to form the anchoring fibrils (4).The pathology in DDEB has been thought to result from negative ...

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Development of Allele-Specific Therapeutic siRNA for Keratin 5 Mutations in Epidermolysis Bullosa Simplex

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