The Carboxyl Terminus of Type VII Collagen Mediates Antiparallel Dimer Formation and Constitutes a New Antigenic Epitope for Epidermolysis Bullosa Acquisita Autoantibodies

Abstract

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC2 domain has been implicated in catalyzing the antiparallel dimer formation of type VII procollagen. In this study, we produced the entire 161 amino acids of the NC2 domain plus 186 amino acids of adjacent collagenous domain (NC2/COL) and purified large quantities of the recombinant NC2/COL protein. Recombinant NC2/ COL readily formed disulfide-bonded hexamers, each representing one antiparallel dimer of collagen VII. Removal of the collagenous helical domain from NC2/COL by collagenase digestion abolished the antiparallel dimer formation. Using site-directed mutagenesis, we found that mutation of either cysteine 2802 or cysteine 2804 alone within the NC2 domain blocked antiparallel dimer formation. In contrast, a single cysteine mutation, 2634, within the collagenous helical domain had no effect. A generated methionine to lysine substitution, M2798K, that is associated 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 collagen VII. Epidermolysis bullosa acquisita autoantibodies bound to this domain may destabilize anchoring fibrils by interfering with antiparallel dimer assembly leading to epidermal-dermal disadherence.Type VII collagen, a genetically distinct member of the collagen family, is found within the basement membrane zone beneath stratified squamous epithelium (1, 2). Type VII collagen is a major component of anchoring fibrils, attachment structures within the basement membrane between the epidermis and dermis of human skin (3, 4). In inherited forms of dystrophic epidermolysis bullosa (DEB), 1 anchoring fibrils are diminutive and/or reduced in number (5-7). In addition to inherited DEB, epidermolysis bullosa acquisita (EBA) is an acquired autoimmune form of epidermolysis bullosa. EBA is characterized by circulating and tissue-bound IgG autoantibodies to type VII collagen (8, 9). Like DEB, ultrastructural studies have demonstrated a dramatic paucity of anchoring fibrils in EBA skin (10). These observations suggest that type VII collagen plays an important role in maintaining epidermal-dermal adherence. Type VII collagen has been cloned, and a genetic linkage has been established between inherited DEB and mutations in the gene that encodes for type VII collagen, COL7A1 (11-14). There have been over 100 distinct COL7A1 gene mutations identified in patients with DEB, and these mutations have occurred within NC1, NC2, and the helical domain (15,16).Type VII collagen is composed of three identical ␣ chains, each consisting of a 145-kDa central collagenous triple-helical segment, flanked by a large 145-kDa amino-terminal non-collagenous domain (NC1), and a smaller 34-kDa carboxyl-terminal non-collagen...

The bone morphogenetic protein 1/Tolloid-like metalloproteinases

“…Synthesized and secreted by epidermal keratinocytes, type VII procollagen monomers self-assemble into antiparallel dimers, which then associate laterally to form anchoring fibrils Morris et al, 1986). Initial dimer formation and stabilization through disulfide bonding seems to require retention of the type VII procollagen C-propeptide, also known as the NC2 domain (Chen et al, 2001;Lunstrum et al, 1987). However, proteolytic removal of NC2 seems necessary to anchoring fibril formation, as a mutation that eliminates the cleavage site results in the severe blistering disease dystrophic epidermolysis bullosa (Bruckner-Tuderman et al, 1995).…”

Characterization of Molecular Mechanisms Underlying Mutations in Dystrophic Epidermolysis Bullosa Using Site-directed Mutagenesis

“…Therefore, structural alterations in C7 may result in functional disruption of its interactions with ECM components, leading to the epidermal-dermal separation seen in DEB. We also showed that the NC2 domain and its adjacent triplehelical domain initiate the triple-helical assembly of C7 and catalyze the antiparallel dimer formation of the procollagen C7 (17).…”

“…The antiparallel dimers then aggregate laterally to form anchoring fibrils which interact with microfibrils, collagen fibrils, and micro-thread-like fibers within the papillary dermis. These interactions are thought to secure the epidermis and its underlying basement membrane zone to the underlying papillary dermis.We have expressed and purified large quantities of recombinant full-length C7 as well as various domains including the NC1 and NC2 domains in human 293 cells (15)(16)(17). Using these recombinant proteins, we carried out extensive structural and functional studies and showed that recombinant C7 is identical to authentic C7 purified from skin cells (15).…”

“…We have expressed and purified large quantities of recombinant full-length C7 as well as various domains including the NC1 and NC2 domains in human 293 cells (15)(16)(17). Using these recombinant proteins, we carried out extensive structural and functional studies and showed that recombinant C7 is identical to authentic C7 purified from skin cells (15).…”

Basement membranes and human disease

“…2D). The pro-collagen type VII molecule is a homotrimer of proα1(VII)-chains and each pro-collagen molecule forms an anti-parallel dimer, with a short C-terminal overlap and disulfide-bonding (Chen et al 2001). Finally, dimers aggregate laterally to form the anchoring fibrils.…”