Hemizygosity for a glycine substitution in collagen XVII: Unfolding and degradation of the ectodomain

Defects of collagen XVII, a keratinocyte adhesion protein, are associated w ith epidermal detachment in junctional epidermolysis bullosa. Although some missense mutations in the collagen XVII gene COL17A1 have been described, the molecular mechanisms leading to disease have remained elusive in these cases. Here we assessed the biologic consequences of a missense mutation by studying the folding and stability of wild-type and mutated recombinant co llagen XVII domains. The mutation occurred in a junctional epidermolysis bu llosa patient who was compound heterozygous for the novel glycine substitut ion mutation G633D and the novel nonsense mutation R145X. Collagen XVII mRN A was significantly reduced, indicating nonsense-mediated mRNA degradation and hemizygosity of the patient for the G633D substitution. As glycine resi dues within the collagen triple helices are important for stable conformati on, the thermal stability of the wild-type and mutated eukaryotic recombina nt Col15 domain of collagen XVII was assessed. The stability of the mutated fragment was clearly reduced. The midpoint of the helix-to-coil transition , Tm, was 5 degrees C lower than that of wild-type rCol15, indicating abnor mal triple-helix folding and susceptibility to proteolysis. Consistently, i mmunoassays demonstrated reduced amounts of the full-length collagen XVII a nd absence of the soluble ectodomain in keratinocyte cultures, and lack of the ectodomain from the junctional epidermolysis bullosa skin. These observ ations show that the glycine substitution G633D in collagen XVII causes abn ormal folding and susceptibility to degradation, and thus perturbs the phys iologic adhesive functions of collagen XVII in the skin.