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Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1.

Wohl AP, Troilo H, Collins RF, Baldock C, Sengle G - J. Biol. Chem. (2016)

Bottom Line: However, upon binding to fibrillin-1, the BMP-7 complex is rendered into a closed ring shape, which also confers latency to the growth factor, as demonstrated by bioactivity measurements.BMP-7 prodomain variants were used to map the critical epitopes for prodomain-growth factor and prodomain-prodomain binding.Together, these data show that upon prodomain binding to fibrillin-1, the BMP-7 complex undergoes a conformational change, which denies access of BMP receptors to the growth factor.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Street 52, 50931 Cologne, Germany.

No MeSH data available.


Related in: MedlinePlus

Affinity purification of recombinant proteins used in this study.A, Coomassie Brilliant Blue-stained SDS-polyacrylamide quality control gels of recombinantly expressed and affinity-purified BMP-7 PD variants and proteins representing the fibrillin-1 N terminus. B (left), size exclusion chromatogram of the BMP-7 PD-GF complex after chelating chromatography utilizing the His6 tag placed at the N terminus of the PD. The chromatogram shows the BMP-7 PD-GF complex mainly eluting in one peak. Right, Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing the purity of the peak fraction. C, Coomassie Brilliant Blue-stained SDS-polyacrylamide gels showing successful separation of the GF from the PD. The separation was performed as described previously (20). BMP-7 complex was separated into BMP-7 PD (34 kDa) and GF dimer (31 kDa) after dialysis into 8 m urea followed by chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through.
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Figure 1: Affinity purification of recombinant proteins used in this study.A, Coomassie Brilliant Blue-stained SDS-polyacrylamide quality control gels of recombinantly expressed and affinity-purified BMP-7 PD variants and proteins representing the fibrillin-1 N terminus. B (left), size exclusion chromatogram of the BMP-7 PD-GF complex after chelating chromatography utilizing the His6 tag placed at the N terminus of the PD. The chromatogram shows the BMP-7 PD-GF complex mainly eluting in one peak. Right, Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing the purity of the peak fraction. C, Coomassie Brilliant Blue-stained SDS-polyacrylamide gels showing successful separation of the GF from the PD. The separation was performed as described previously (20). BMP-7 complex was separated into BMP-7 PD (34 kDa) and GF dimer (31 kDa) after dialysis into 8 m urea followed by chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through.

Mentions: A stably transfected BMP-7 complex expressing the HEK293 EBNA cell line and an expression plasmid coding for the N-terminal half of fibrillin-1 (rF11) were kindly provided by Dr. Lynn Sakai (Shriners Hospital for Children Portland, Oregon Health and Science University, Portland, OR) (20, 21). BMP-7 complex was purified by chelating chromatography utilizing a His6 tag placed at the N terminus of the PD followed by gel filtration. BMP-7 complex purification and separation into its components were as described previously (20). For separation of the GF from the PD, purified BMP-7 complex was dialyzed into 8 m urea, 1 m NaCl and subjected to a second chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through (Fig. 1B). rF11 was overexpressed in HT1080 cells, followed by purification as described previously (21) (Fig. 1A). BMP-7 PD truncation variants were expressed in Escherichia coli with a C-terminal His6 tag and purified by chelating chromatography (10, 22) (Fig. 1A). BMP-7 PD point mutations were introduced using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). rF87 (Ser19–Ile329) and the fibrillin-1 unique N-terminal domain (FUN) including the first EGF-like domain (Ser19–His118) containing the BMP-7 PD binding site fused together with cbEGF19-22 (Asp1363–Val1527) and a tandem C-terminal Strep-tag were expressed and purified as described previously (23) (Fig. 1A).


Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1.

Wohl AP, Troilo H, Collins RF, Baldock C, Sengle G - J. Biol. Chem. (2016)

Affinity purification of recombinant proteins used in this study.A, Coomassie Brilliant Blue-stained SDS-polyacrylamide quality control gels of recombinantly expressed and affinity-purified BMP-7 PD variants and proteins representing the fibrillin-1 N terminus. B (left), size exclusion chromatogram of the BMP-7 PD-GF complex after chelating chromatography utilizing the His6 tag placed at the N terminus of the PD. The chromatogram shows the BMP-7 PD-GF complex mainly eluting in one peak. Right, Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing the purity of the peak fraction. C, Coomassie Brilliant Blue-stained SDS-polyacrylamide gels showing successful separation of the GF from the PD. The separation was performed as described previously (20). BMP-7 complex was separated into BMP-7 PD (34 kDa) and GF dimer (31 kDa) after dialysis into 8 m urea followed by chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Affinity purification of recombinant proteins used in this study.A, Coomassie Brilliant Blue-stained SDS-polyacrylamide quality control gels of recombinantly expressed and affinity-purified BMP-7 PD variants and proteins representing the fibrillin-1 N terminus. B (left), size exclusion chromatogram of the BMP-7 PD-GF complex after chelating chromatography utilizing the His6 tag placed at the N terminus of the PD. The chromatogram shows the BMP-7 PD-GF complex mainly eluting in one peak. Right, Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing the purity of the peak fraction. C, Coomassie Brilliant Blue-stained SDS-polyacrylamide gels showing successful separation of the GF from the PD. The separation was performed as described previously (20). BMP-7 complex was separated into BMP-7 PD (34 kDa) and GF dimer (31 kDa) after dialysis into 8 m urea followed by chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through.
Mentions: A stably transfected BMP-7 complex expressing the HEK293 EBNA cell line and an expression plasmid coding for the N-terminal half of fibrillin-1 (rF11) were kindly provided by Dr. Lynn Sakai (Shriners Hospital for Children Portland, Oregon Health and Science University, Portland, OR) (20, 21). BMP-7 complex was purified by chelating chromatography utilizing a His6 tag placed at the N terminus of the PD followed by gel filtration. BMP-7 complex purification and separation into its components were as described previously (20). For separation of the GF from the PD, purified BMP-7 complex was dialyzed into 8 m urea, 1 m NaCl and subjected to a second chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through (Fig. 1B). rF11 was overexpressed in HT1080 cells, followed by purification as described previously (21) (Fig. 1A). BMP-7 PD truncation variants were expressed in Escherichia coli with a C-terminal His6 tag and purified by chelating chromatography (10, 22) (Fig. 1A). BMP-7 PD point mutations were introduced using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). rF87 (Ser19–Ile329) and the fibrillin-1 unique N-terminal domain (FUN) including the first EGF-like domain (Ser19–His118) containing the BMP-7 PD binding site fused together with cbEGF19-22 (Asp1363–Val1527) and a tandem C-terminal Strep-tag were expressed and purified as described previously (23) (Fig. 1A).

Bottom Line: However, upon binding to fibrillin-1, the BMP-7 complex is rendered into a closed ring shape, which also confers latency to the growth factor, as demonstrated by bioactivity measurements.BMP-7 prodomain variants were used to map the critical epitopes for prodomain-growth factor and prodomain-prodomain binding.Together, these data show that upon prodomain binding to fibrillin-1, the BMP-7 complex undergoes a conformational change, which denies access of BMP receptors to the growth factor.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Street 52, 50931 Cologne, Germany.

No MeSH data available.


Related in: MedlinePlus