Abstract
COL7A1 gene mutations cause dystrophic epidermolysis bullosa, a skin blistering disorder. The phenotypes result from defects of collagen VII, the major component of the anchoring fibrils at the dermo-epidermal junction; however, the molecular mechanisms underlying the phenotypes remain elusive. We investigated naturally occurring COL7A1 mutations and showed that some, but not all, glycine substitutions in collagen VII interfered with biosynthesis of the protein in a dominant-negative manner. Three point mutations in exon 73 caused glycine substitutions G2006D, G2034R, and G2015E in the triple helical domain of collagen VII and interfered with its folding and secretion. Confocal laser scanning studies and semiquantitative immunoblotting determined that dystrophic epidermolysis bullosa keratinocytes retained up to 2.5-fold more procollagen VII within the rough endoplasmic reticulum than controls. Limited proteolytic digestions of mutant procollagen VII produced aberrant fragments and revealed reduced stability of the triple helix. In contrast, the glycine substitution G1519D in another segment of the triple helix affected neither procollagen VII secretion nor anchoring fibril function and remained phenotypically silent. These data demonstrate that collagen VII presents a remarkable exception among collagens in that not all glycine substitutions within the triple helix exert dominant-negative interference and that the biological consequences of the substitutions probably depend on their position within the triple helix.
Highlights
COL7A1 gene mutations cause dystrophic epidermolysis bullosa, a skin blistering disorder
Effect of the Mutations on Procollagen VII Production in Keratinocytes in Vitro—Immunofluorescence staining with antibodies to collagen VII demonstrated a weak intracellular granular staining in normal control keratinocytes (Fig. 2) that colocalized with protein-disulfide isomerase, a resident protein in the endoplasmic reticulum
Since early studies on folding of fibrillar collagens with long uninterrupted triple helices, i.e. collagens I–III, had shown that the presence of a glycine in every third amino acid position in the polypeptide is a prerequisite for formation and secretion of a stable triple helix, it has been generally assumed that glycine substitutions prevent adequate folding of all collagens by dominant-negative interference
Summary
Probands—The diagnosis of DDEB [6, 7] in probands 1–3 was based on 1) the pedigree consistent with dominant inheritance; 2) history of mechanically induced skin blistering and scarring since infancy; 3) clinical observations of skin blisters, scarring, milia, and nail dystrophy at trauma-exposed body sites, such as hands, feet, and knees; 4) electron microscopic findings of dermo-epidermal tissue separation below the lamina densa of the skin basement membrane and/or paucity and altered morphology of the anchoring fibrils; 5) antigen mapping [25] of blistered skin regions that revealed structural antigens of the dermoepidermal junction at the blister roof; and 6) positive immunofluorescence staining of collagen VII using different domain-specific antibodies. In family 2, the proband was a 64-year-old male [] His father and brother were deceased, but were reported to have had similar blistering tendency. The samples were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting using domain-specific collagen VII antibodies [3]. For a limited pepsin digestion of procollagen VII, cell extracts were acidified with glacial acetic acid to a final concentration of 0.1 M, and the samples were incubated with 10 g/ml pepsin (Fluka, Deisenhofen, Germany) at 5 °C for 2 h [1, 30]. Proteins were precipitated with ethanol, redissolved in sample buffer, separated by SDS-polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose, and analyzed by immunoblotting using domain-specific collagen VII antibodies [3, 28]. Electron Microscopy—Electron microscopy of biopsy samples of intact normal appearing skin from probands 1-1 and 3-1 was performed using standard methods as described [7, 17]
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