title: Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa.
abstract: Fibulin-4 is a 60kDa calcium binding glycoprotein that has an important role in development and integrity of extracellular matrices. It interacts with elastin, fibrillin-1 and collagen IV as well as with lysyl oxidases and is involved in elastogenesis and cross-link formation. To date, several mutations in the fibulin-4 gene (FBLN4/EFEMP2) are known in patients whose major symptoms are vascular deformities, aneurysm, cutis laxa, joint laxity, or arachnodactyly. The pathogenetic mechanisms how these mutations translate into the clinical phenotype are, however, poorly understood. In order to elucidate these mechanisms, we expressed fibulin-4 mutants recombinantly in HEK293 cells, purified the proteins in native forms and analyzed alterations in protein synthesis, secretion, matrix assembly, and interaction with other proteins in relation to wild type fibulin-4. Our studies show that different mutations affect these properties in multiple ways, resulting in fibulin-4 deficiency and/or impaired ability to form elastic fibers. The substitutions E126K and C267Y impaired secretion of the protein, but not mRNA synthesis. Furthermore, the E126K mutant showed less resistance to proteases, reduced binding to collagen IV and fibrillin-1, as well as to LTBP1s and LTBP4s. The A397T mutation introduced an extra O-glycosylation site and deleted binding to LTBP1s. We show that fibulin-4 binds stronger than fibulin-3 and -5 to LTBP1s, 3, and 4s, and to the lysyl oxidases LOX and LOXL1; the binding of fibulin-4 to the LOX propeptide was strongly reduced by the mutation E57K. These findings show that different mutations in the fibulin-4 gene result in different molecular defects affecting secretion rates, protein stability, LOX-induced cross-linking, or binding to other ECM components and molecules of the TGF-β pathway, and thus illustrate the complex role of fibulin-4 in connective tissue assembly.
authors.affiliation: Department of Experimental Medicine I, Nikolaus-Fiebiger Center of Molecular Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany; Department of Biochemistry II, Faculty of Medicine, Oita University, Oita 879-5593, Japan. Electronic address: tsasaki@oita-u.ac.jp.
authors.forename: Takako
authors.initials: T
authors.lastname: Sasaki
authors.affiliation: Institute for Biochemistry II, Medical Faculty, Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany.
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
authors.affiliation: Institute of Biochemistry, Microbiology and Genetics, University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Deutzmann
authors.affiliation: Shriners Hospital for Children, Portland Research Center, Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239, USA.
authors.forename: Lynn Y
authors.initials: LY
authors.lastname: Sakai
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Miyamoto
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Institut für Biochemie, Emil-Fischer-Zentrum, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Ewald
authors.initials: E
authors.lastname: Hannappel
authors.affiliation: Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: Mon-Li
authors.initials: ML
authors.lastname: Chu
authors.affiliation: Central Institute for Scientific Computing (ZISC), University of Erlangen-Nürnberg, 91058 Erlangen, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Lanig
authors.affiliation: Department of Experimental Medicine I, Nikolaus-Fiebiger Center of Molecular Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: von der Mark
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D003483:Cutis Laxa / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D056726:Genetic Association Studies; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008822:Mice, Transgenic; D008907:Mink; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D011485:Protein Binding; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D008249:Protein-Lysine 6-Oxidase / Q000378:metabolism; D059748:Proteolysis; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000502:physiology
chemical_list: C403672:EFEMP2 protein, human; D016326:Extracellular Matrix Proteins; D016212:Transforming Growth Factor beta; D008249:Protein-Lysine 6-Oxidase
keywords: Elastogenesis; Extracellular matrix protein; Fibulin-4; Matrix assembly; Mutations; Protein folding