title: Fluorescent protein markers to tag collagenous proteins: the paradigm of procollagen VII.
abstract: 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 alpha chain and green fluorescent protein-tagged alpha 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 and fluorescence resonance energy transfer assays demonstrated that the presence of different fluorescent protein markers at the C-termini of individual alpha chains neither altered formation of triple-helical molecules nor affected their secretion to the extracellular space. Our study provides a basis for employing fluorescent proteins as tags for complex structural proteins of extracellular matrix.
authors.affiliation: Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Chung
authors.forename: Andrzej
authors.initials: A
authors.lastname: Steplewski
authors.forename: Jouni
authors.initials: J
authors.lastname: Uitto
authors.forename: Andrzej
authors.initials: A
authors.lastname: Fertala
mesh_terms: D002460:Cell Line; D024143:Collagen Type VII / Q000032:analysis* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000032:analysis* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D000097573:Red Fluorescent Protein
chemical_list: D024143:Collagen Type VII; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein