title: Different domains in nidogen-1 and nidogen-2 drive basement membrane formation in skin organotypic cocultures.
abstract: Nidogen-1 and nidogen-2 are homologous proteins found in all basement membranes (BMs). They show comparable binding activities in vitro and partially redundant functions in vivo. Previously, we showed that in skin organotypic cocultures, BM formation was prevented in the absence of nidogens and that either nidogen was able to rescue this failure. We now dissected the two nidogens to identify the domains required for BM deposition. For that purpose, HaCaT cells were grown on collagen matrices containing nidogen-deficient, murine fibroblasts. After addition of nidogen-1 or nidogen-2 protein fragments comprising different binding domains, BM deposition was analyzed by immunofluorescence and electron microscopy. We could demonstrate that the rod-G3 domain of nidogen-2 was sufficient to achieve deposition of BM components at the epidermal-collagen interface. In contrast, for nidogen-1, both the G2 and G3 domains were required. Immunoblot analysis confirmed that all BM components were present in comparable amounts under all culture conditions. This finding demonstrates that nidogens, although homologous proteins, exert their effect on BM assembly through different binding domains, which may in turn result in alterations of BM structure and functions, thus providing an explanation for the phenotypical differences observed between nidogen-1 and -2 deficient mice.
authors.affiliation: Department of Dermatology, University Hospital of Cologne, Germany.
authors.forename: Manuela
authors.initials: M
authors.lastname: Bechtel
authors.forename: Manuel V
authors.initials: MV
authors.lastname: Keller
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Bloch
authors.forename: Takako
authors.initials: T
authors.lastname: Sasaki
authors.forename: Petra
authors.initials: P
authors.lastname: Boukamp
authors.forename: Frank
authors.initials: F
authors.lastname: Zaucke
authors.forename: Mats
authors.initials: M
authors.lastname: Paulsson
authors.forename: Roswitha
authors.initials: R
authors.lastname: Nischt
mesh_terms: D001483:Base Sequence; D001485:Basement Membrane / Q000378:metabolism*; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; D002460:Cell Line; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D007797:Laminin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D016133:Polymerase Chain Reaction; D012867:Skin / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; D017077:Culture Media, Conditioned; D017931:DNA Primers; D007797:Laminin; D008562:Membrane Glycoproteins; C112846:Nid2 protein, mouse; C029967:nidogen