The role of fibulin-4 in development and homeostasis of elastic components of the skeletal connective tissue
Final Report Abstract
Fibulins are extracellular glycoproteins associated with elastic fibers and basement membranes. The unique structural feature of this protein is a tandem array of calcium-binding epidermal growth factor (cbEGF)-like modules and a fibulin-type C-terminal (FC) domain. Mutations in the fibulin-4gene FBLN4have been identified in patients suffering from vascular abnormality and/or cutis laxa, arachnodactyly, pectus excavatum and joint laxity.A major goal of this project was to elucidate the consequences of the reported FBLN4 mutations on protein structure, stability, synthesis, extracellular assembly, and ability to interact with other matrix proteins and regulatory factors, in order to pave the way for further investigations on the genotype- phenotype relationship of heritable disorders caused by fibulin-4 mutations. A complete model of the modular structure of fibulin-4 was calculated by molecular dynamic simulationwhich illustrated the changes in structure and molecular dynamicscaused by the D203A mutation. We report that several mutations such as E126K and C267Y affected secretion of fibulin-4, thus causing fibulin-4 deficieny.Other mutations, in particular those affecting calcium binding sites in the EGF modules of fibulin, resulted in enhanced susceptibility to proteases and in impaired interactions withmatrix components of elastic fibers includingfibrillin, elastin, and collagen IV, and with factors regulating TGFβ activity such as LTBPs. One mutation (A397T) caused additional N-and O- glycosylation. Interestingly, several mutations also affected the binding to lysyloxidases, which are essential for the formation of collagen and elastin crosslinks. This result corroborated our observations of impaired lysyloxidase activity and collagen crosslinking in fibulin-4 deficient mice and undermined the importance of fibulin-4 in crosslink formation and stability of the extracellular matrix of elastic tissues. Further information of the role of fibulin-4 in connective tissue structure and function was recruited from the analysis of a fibulin-4 null mouse which was generated by Dr. Mon-Li Chu, Philadelphia. Fbln4-/-deficient mice did not survive the first postanatal day, probably due to vascular failure, but showed otherwise no obvious phenotypic alteration in the musculoskeletal system except conspicuous contractures of the forelimb. By electromicroscopy, however, an unusual appearance of irregular sized collagen fibrils with numerous fibrils larger than 70 nm wasobserved in Fbln4-/-deficient bone which was never seen in wild type nor heterozygote mice. Most strikingly, collagen crosslinking was strongly impaired in bones ofFbln4-/- mice as evidenced by enhanced extractability of collagen and impaired lysyloxidase activity in comparison to bones ofwildtype littermates. These findings demonstrate the importance of fibulin-4 as a regulatory element in crosslink formation and extracellular assembly of collagens in bone and, as recently published, also in other connective tissues including skin and tendon. Our studies should provide useful information for further investigations of the complex molecular interactions of fibulin-4 with other matrix constituents, growth factors, and enzymes involved in the fabrication, stability and function of connective tissue. Moreover, our findings will contribute to our understanding of the genotype-phenotype relationship in hereditary disorders such as cutis laxa, aneurysm, joint laxity or arachnodactyly associated with fibulin-4 mutations.
Publications
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Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities. J Biol Chem. 2015 Aug28;290(35):21443-59
Igoucheva O, Alexeev V, Halabi CM, Adams SM, Stoilov I, Sasaki T, Arita M, Donahue A, Mecham RP, Birk DE, Chu ML
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Forelimb Contractures and Abnormal Tendon Collagen Fibrillogenesis in Fibulin-4 Null Mice. Cell and Tissue Research
June 2016, Volume 364, Issue 3, pp 637–646
Markova D, Pan Te-Cheng, Zhang RZ, Zhang G, Sasaki T, Arita M, Birk D E, Chu Mon-Li
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Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa. Matrix Biology
Volume 56, December 2016, Pages 132-149
Takako Sasaki, Franz-Georg Hanisch, Rainer Deutzmann, Lynn Y Sakai, Tetsushi Sakuma, Tatsuo Miyamoto, Takashi Yamamoto, Ewald Hannappel, Mon-Li Chu, Harald Lanig, Klaus von der Mark
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Loss of fibulin-4 results in abnormal collagen fibril assembly in bone, caused by impaired lysyl oxidase processing and collagen cross-linking. Matrix Biology, Volume 50, March 2016, Pages 53-66
Takako Sasaki, Reinout Stoop, Takao Sakai, Andreas Hess, Rainer Deutzmann,Ursula Schlötzer-Schrehardt, Mon-Li Chu and Klaus von der Mark
