NC1 domain of type VII collagen binds to the beta 3 chain of laminin 5 viaa unique subdomain within the fibronectin-like repeats

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous, gl obular domains, NC1 and NC2, Approximately 50% of the molecular mass of the molecule is consumed by the NC1 domain. We previously demonstrated that NC 1 binds to various extracellular matrix components including a complex of l aminin 5 and laminin 6 (Chen er al, 1997a), In this study, we examined the interaction of NC1 with laminin 5 (a component of anchoring filaments), Bot h authentic and purified recombinant NC1 bound to human and rat laminin 5 a s measured by enzyme-linked immunosorbant assay and by binding of I-125-rad iolabeled NC1 to laminin 5-coated wells, but not to laminin 1 or albumin. N C1 bound predominantly to the beta 3 chain of laminin 5, but also to the ga mma 2 chain when examined by a protein overlay assay, The binding of I-125- NC1 to laminin 5 was inhibited by a 50-fold excess of unlabeled NC1 or de-g lycosylated NC1, as well as a polyclonal antibody to laminin 5 or a monoclo nal antibody to the beta 3 chain. In contrast, the NC1-laminin 5 interactio n was not affected by a monoclonal antibody to the alpha 3 chain, Using NC1 deletion mutant recombinant proteins, a 285 AA (residues 760-1045) subdoma in of NC1 was identified as the binding site for laminin 5, IgG from an epi dermolysis bullosa acquisita serum containing autoantibodies to epitopes wi thin NC1 that colocalized with the laminin 5 binding site inhibited the bin ding of NC1 to laminin 5, Thus, perturbation of the NC1-laminin 5 interacti on may contribute to the pathogenesis of epidermolysis bullosa acquisita.