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Full-Text Articles in Medicine and Health Sciences
Fluorescent Protein Markers To Tag Collagenous Proteins: The Paradigm Of Procollagen Vii, Hye J. Chung, Andrzej Steplewski, Jouni Uitto, Andrzej Fertala
Fluorescent Protein Markers To Tag Collagenous Proteins: The Paradigm Of Procollagen Vii, Hye J. Chung, Andrzej Steplewski, Jouni Uitto, Andrzej Fertala
Department of Dermatology and Cutaneous Biology Faculty Papers
Fluorescent proteins are powerful markers allowing tracking expression, intracellular localization, and translocation of tagged proteins but their effects on the structure and assembly of complex extracellular matrix proteins has not been investigated. Here, we analyzed the utility of fluorescent proteins as markers for procollagen VII, a triple-helical protein critical for the integrity of dermal-epidermal junction. DNA constructs encoding a red fluorescent protein-tagged wild type mini-procollagen VII α chain and green fluorescent protein-tagged α chains harboring selected mutations were genetically engineered. These DNA constructs were co-expressed in HEK-293 cells and the assembly of heterogeneous triple-helical mini-procollagen VII molecules was analyzed. Immunoprecipitation …
High-Affinity Binding Of The Nc1 Domain Of Collagen Vii To Laminin 5 And Collagen Iv, Raymond Brittingham, Jouni Uitto, Andrzej Fertala
High-Affinity Binding Of The Nc1 Domain Of Collagen Vii To Laminin 5 And Collagen Iv, Raymond Brittingham, Jouni Uitto, Andrzej Fertala
Department of Dermatology and Cutaneous Biology Faculty Papers
Anchoring functions of collagen VII depend on its ability to form homotypic fibrils and to bind to other macromolecules to form heterotypic complexes. Biosensor-based binding assays were employed to analyze the kinetics of the NC1 domain-mediated binding of collagen VII to laminin 5, collagen IV, and collagen I. We showed that collagen VII interacts with laminin 5 and collagen IV with Kd values of 10-9 M. In contrast, the NC1-mediated binding to collagen I was weak with a Kd value of 10-6 M. Binding assays also showed that the NC1 domain utilizes the same region to bind to both laminin …