Characterization of mutant type VII collagens underlying the inversa subtype of recessive dystrophic epidermolysis bullosa

Abstract Background Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen (C7) leading to skin fragility, bullae, and erosive wounds. RDEB-Inversa (RDEB-I), a subset of RDEB, is characterized by lesions localized to body areas with higher skin temperatures such as flexures and skin folds. Objective We aimed to determine if C7 derived from RDEB-I mutations had structural and functional aberrancies that were temperature sensitive and could be reversed by lowering the temperature. Methods In this study, we generated 12 substitution mutations associated with RDEB-I via site-directed mutagenesis and purified recombinant C7 protein. These C7 mutants were evaluated for structural parameters (trimer formation and protease sensitivity) and the ability to promote keratinocyte migration at 37oC (the temperature of skin folds) and 30oC (the maximum skin temperature of arms and legs). Fibroblasts derived from RDEB-I patients were evaluated for C7 secretion and cellular migration at both temperatures. Results C7s from RDEB-I mutations exhibited decreased thermal stability, increased sensitivity to protease digestion, diminished formation of collagen trimers, and reduced ability to promote keratinocyte migration compared with wild-type C7. In addition, fibroblasts derived from RDEB-I patients demonstrated intracellular accumulation of C7 and abnormal cell migration at 37oC. All of these aberrancies were corrected by reducing the temperature to 30oC. C7s generated from severe-RDEB mutations (non-Inversa) did not display temperature-dependent perturbations. Conclusion These data demonstrate that RDEB-I mutations generate C7 aberrancies that are temperature dependent. This may explain why RDEB-I patients develop clinical lesions in areas where their skin is considerably warmer.