Type VII collagen is a major component of anchoring fibrils, attachment structures that mediate dermal-epidermal adherence in human skin. Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder caused by mutations in the type VII collagen gene and perturbations in anchoring fibrils. In this study, we produced recombinant human type VII collagen in stably transfected human 293 cell clones and purified large quantities of the recombinant protein from culture media. The recombinant type VII collagen was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. Purified type VII collagen bound to fibronectin, laminin-5, type I collagen, and type IV collagen and also supported human dermal fibroblast adhesion. In an attempt to establish genotype-phenotype relationships, we generated two individual substitution mutations that have been associated with recessive DEB, R2008G and G2749R, and purified the recombinant mutant proteins. The G2749R mutation resulted in mutant type VII collagen with increased sensitivity to protease degradation and decreased ability to form trimers. The R2008G mutation caused the intracellular accumulation of type VII collagen. We conclude that structural and functional studies of in vitro generated type VII collagen mutant proteins will aid in correlating genetic mutations with the clinical phenotypes of DEB patients.Type VII collagen resides within the basement membrane zone (BMZ) 1 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 and autoimmune forms of dystrophic epidermolysis bullosa (DEB) and epidermolysis bullosa acquisita, respectively, anchoring fibrils are diminutive and/or reduced in number (5-10). A genetic linkage has been established between the inherited forms of DEB and type VII collagen (11)(12)(13)(14).Type VII collagen is composed of three identical ␣ chains, each consisting of a 145-kDa central collagenous triple-helical segment characterized by repeating Gly-X-Y amino acid sequences, flanked by a large 145-kDa amino-terminal, noncollagenous domain (NC1), and a small 34-kDa carboxyl-terminal noncollagenous domain (NC2) (4, 15). Within the extracellular space, type VII collagen molecules form antiparallel, tail-to-tail dimers stabilized by disulfide bonding through a small carboxyl-terminal NC2 overlap between two type VII collagen molecules. A portion of the NC2 domain is then proteolytically removed (16, 17). The antiparallel dimers then aggregate laterally to form anchoring fibrils with large globular NC1 domains at both ends of the structure. NC1 domains have been suggested to interact at one end with BMZ components and at the other end with type IV collagen in "anchoring plaques" (2).Our recent study using recombinant NC1 demonstrated that NC1 interacts with various extracellular matrix (ECM) components including fibronectin, lami...