Fibulin-3, -4, and -5 are highly susceptible to proteolysis, interact with cells and heparin, and form multimers
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2013 |
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| Erschienen: |
2013 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:288 |
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| Enthalten in: |
The Journal of biological chemistry - 288(2013), 31 vom: 02. Aug., Seite 22821-35 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Djokic, Jelena [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 21.10.2013 Date Revised 21.10.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1074/jbc.M112.439158 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM228485002 |
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| 100 | 1 | |a Djokic, Jelena |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Fibulin-3, -4, and -5 are highly susceptible to proteolysis, interact with cells and heparin, and form multimers |
| 264 | 1 | |c 2013 | |
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| 500 | |a Date Completed 21.10.2013 | ||
| 500 | |a Date Revised 21.10.2021 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Cell Adhesion | |
| 650 | 4 | |a Elastic Fiber | |
| 650 | 4 | |a Extracellular Matrix | |
| 650 | 4 | |a Extracellular Matrix Proteins | |
| 650 | 4 | |a Fibulin | |
| 650 | 4 | |a Heparin-binding Protein | |
| 650 | 4 | |a Matrix Metalloproteinase (MMP) | |
| 650 | 4 | |a Protein Multimerization | |
| 650 | 7 | |a Biopolymers |2 NLM | |
| 650 | 7 | |a DNA Primers |2 NLM | |
| 650 | 7 | |a EFEMP1 protein, human |2 NLM | |
| 650 | 7 | |a EFEMP2 protein, human |2 NLM | |
| 650 | 7 | |a Extracellular Matrix Proteins |2 NLM | |
| 650 | 7 | |a FBLN5 protein, human |2 NLM | |
| 650 | 7 | |a Heparin |2 NLM | |
| 650 | 7 | |a 9005-49-6 |2 NLM | |
| 700 | 1 | |a Fagotto-Kaufmann, Christine |e verfasserin |4 aut | |
| 700 | 1 | |a Bartels, Rainer |e verfasserin |4 aut | |
| 700 | 1 | |a Nelea, Valentin |e verfasserin |4 aut | |
| 700 | 1 | |a Reinhardt, Dieter P |e verfasserin |4 aut | |
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