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      Fibulin-3, -4, and -5 are highly susceptible to proteolysis, interact with cells and heparin, and form multimers.

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          Abstract

          Extracellular short fibulins, fibulin-3, -4, and -5, are components of the elastic fiber/microfibril system and are implicated in the formation and homeostasis of elastic tissues. In this study, we report new structural and functional properties of the short fibulins. Full-length human short fibulins were recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. All three fibulins showed various levels of degradation after the purification procedure. N-terminal sequencing revealed that all three fibulins are highly susceptible to proteolysis within the N-terminal linker region of the first calcium-binding epidermal growth factor domain. Proteolytic susceptibility of the linker correlated with its length. Exposure of these fibulins to matrix metalloproteinase (MMP)-1, -2, -3, -7, -9, and -12 resulted in similar proteolytic fragments with MMP-7 and -12 being the most potent proteases. Fibulin-3 proteolysis was almost completely inhibited in cell culture by the addition of 25 μm doxycycline (a broad spectrum MMP inhibitor). Reducible fibulin-4 dimerization and multimerization were consistently observed by SDS-PAGE, Western blotting, and mass spectrometry. Atomic force microscopy identified monomers, dimers, and multimers in purified fibulin-4 preparations with sizes of ∼10-15, ∼20-25, and ∼30-50 nm, respectively. All short fibulins strongly adhered to human fibroblasts and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate.

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          Author and article information

          Journal
          J Biol Chem
          The Journal of biological chemistry
          American Society for Biochemistry & Molecular Biology (ASBMB)
          1083-351X
          0021-9258
          Aug 02 2013
          : 288
          : 31
          Affiliations
          [1 ] Faculty of Dentistry, Division of Biomedical Sciences, McGill University, Montreal, Quebec H3A 0C7, Canada.
          Article
          S0021-9258(20)45442-0
          10.1074/jbc.M112.439158
          3829366
          23782690
          73bd3c47-b66d-4e70-99eb-0426172a1a86
          History

          Cell Adhesion,Elastic Fiber,Extracellular Matrix,Extracellular Matrix Proteins,Fibulin,Heparin-binding Protein,Matrix Metalloproteinase (MMP),Protein Multimerization

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