title: Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils.
abstract: Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.
authors.affiliation: Department of Medical Molecular Biology, University of Lübeck, Germany.
authors.forename: Guoqing
authors.initials: G
authors.lastname: Lin
authors.forename: Kerstin
authors.initials: K
authors.lastname: Tiedemann
authors.forename: Tillman
authors.initials: T
authors.lastname: Vollbrandt
authors.forename: Hannelore
authors.initials: H
authors.lastname: Peters
authors.forename: Boris
authors.initials: B
authors.lastname: Batge
authors.forename: Jurgen
authors.initials: J
authors.lastname: Brinckmann
authors.forename: Dieter P
authors.initials: DP
authors.lastname: Reinhardt
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics; D002460:Cell Line; D002942:Circular Dichroism; D017931:DNA Primers; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D000071838:Fibrillin-1; D000071840:Fibrillin-2; D000071837:Fibrillins; D006801:Humans; D008382:Marfan Syndrome / Q000235:genetics; D020894:Microfibrils / Q000473:pathology / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D054392:Adipokines
chemical_list: D002135:Calcium-Binding Proteins; D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071840:Fibrillin-2; D000071837:Fibrillins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C000605722:FBN1 protein, human; D054392:Adipokines; C000605723:FBN2 protein, human