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

•Patients with RDEB lack functional Type VII collagen (C7).•The Inversa subtype (RDEB-I) affects body areas of higher temperature.•The structural and functional abnormalities of RDEB-I C7 are temperature-dependent.•The abnormal RDEB-I fibroblast phenotype is correctable at lower temperatures. 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. 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. 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 37 °C (the temperature of skin folds) and 30 °C (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. 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 37 °C. All of these aberrancies were corrected by reducing the temperature to 30 °C. C7s generated from severe-RDEB mutations (non-Inversa) did not display temperature-dependent perturbations. 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.