Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa

Matrix Biol. 2016 Dec;56:132-149. doi: 10.1016/j.matbio.2016.06.003.

Takako Sasaki ¹, Franz-Georg Hanisch ², Rainer Deutzmann ³, Lynn Y Sakai ⁴, Tetsushi Sakuma ⁵, Tatsuo Miyamoto ⁶, Takashi Yamamoto ⁵, Ewald Hannappel ⁷, Mon-Li Chu ⁸, Harald Lanig ⁹, Klaus von der Mark ¹⁰

Affiliations

1 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.
2 Institute for Biochemistry II, Medical Faculty, Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany.
3 Institute of Biochemistry, Microbiology and Genetics, University of Regensburg, 93053 Regensburg, Germany.
4 Shriners Hospital for Children, Portland Research Center, Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239, USA.
5 Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan.
6 Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.
7 Institut für Biochemie, Emil-Fischer-Zentrum, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.
8 Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
9 Central Institute for Scientific Computing (ZISC), University of Erlangen-Nürnberg, 91058 Erlangen, Germany.
10 Department of Experimental Medicine I, Nikolaus-Fiebiger Center of Molecular Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.

PMID: 27339457 DOI: 10.1016/j.matbio.2016.06.003

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.

Keywords

Elastogenesis; Extracellular matrix protein; Fibulin-4; Matrix assembly; Mutations; Protein folding.

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