The carboxyl terminus of type VII collagen mediates antiparallel dimer formation and constitutes a new antigenic epitope for epidermolysis bullosa acquisita autoantibodies

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous dom ains, NC1 and NC2, The NC2 domain has been implicated in catalyzing the ant iparallel dimer formation of type VII procollagen, In this study, we produc ed the entire 161 amino acids of the NC2 domain plus 186 amino acids of adj acent collagenous domain (NC2/COL) and purified large quantities of the rec ombinant NC2/COL protein. Recombinant NC2/COL readily formed disulfide-bond ed hexamers, each representing one antiparallel dimer of collagen VII, Remo val of the collagenous helical domain from NC2/COL by collagenase digestion abolished the antiparallel dimer formation. Using site-directed mutagenesi s, we found that mutation of either cysteine 2802 or cysteine 2804 alone wi thin the NC2 domain blocked antiparallel dimer formation. In contrast, a si ngle cysteine mutation, 2634, within the collagenous helical domain had no effect. A generated methionine to lysine substitution, M2798K, that is asso ciated with recessive dystrophic epidermolysis bullosa, was unable to form antiparallel dimers, Furthermore, autoantibodies from epidermolysis bullosa acquisita patients also reacted with NC2/COL. We conclude that NC2 and its adjacent collagenous segment mediate antiparallel dimer formation of colla gen VII, Epidermolysis bullosa acquisita autoantibodies bound to this domai n may destabilize anchoring fibrils by interfering with antiparallel dimer assembly leading to epidermal-dermal disadherence.