Calcium stabilizes fibrillin-1 against proteolytic degradation.
The calcium-binding epidermal growth factor (cbEGF)-like domain is a structural motif that is present in many matrix proteins throughout the animal kingdom from invertebrates to mammals. This module has been demonstrated to bind calcium in the micromolar range. However, little is known about the functional consequences of calcium binding to proteins that contain this structural element. We used fibrillin-1, an extracellular matrix protein consisting of approximately 60% cbEGF-like motifs, as a model system to study stabilizing effects of calcium in protease degradation assays. Authentic human fibrillin-1 and recombinant human fibrillin-1 subdomains, spanning the whole molecule, showed significantly slower proteolytic degradation in the presence of CaCl2 than in the presence of EDTA, demonstrating that calcium stabilizes the structure of fibrillin-1 and protects the molecule against proteolytic degradation. Information about cleavage sites protected by calcium was obtained with a new recombinant subdomain, rF17 (Asp 952-Val 1527), comprising the longest stretch of cbEGF-like motifs in the center of the fibrillin-1 molecule. The most sensitive sites for trypsin and endoproteinase Glu-C were observed in cbEGF-like motifs 11 (Met 1034 and Asn 1046), 12 (Ser 1103), and 17 (Thr 1318). Since most of the currently known mutations in fibrillin-1 are found within cbEGF-like motifs and are predicted to disrupt calcium binding, we suggest that these mutations render fibrillin-1 more susceptible to proteolytic cleavage, and this might be one of the reasons why these mutations result in Marfan's syndrome.
Reinhardt
D P
DP
Shriners Hospital for Children, Portland, Oregon 97201, USA. dpr@shcc.org
Ono
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RN
Sakai
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LY
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004330:Drosophila; D004815:Epidermal Growth Factor / Q000737:chemistry*; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D000071838:Fibrillin-1; D000071837:Fibrillins; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D054392:Adipokines
D002118:Calcium; D004815:Epidermal Growth Factor; D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; C000605722:FBN1 protein, human; D054392:Adipokines