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Different requirements for proteolytic processing of bone morphogenetic protein 5/6/7/8 ligands in Drosophila melanogaster.

Fritsch C, Sawala A, Harris R, Maartens A, Sutcliffe C, Ashe HL, Ray RP - J. Biol. Chem. (2011)

Bottom Line: The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies.Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality.Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Falmer Brighton BN1 9QG, United Kingdom.

ABSTRACT
Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.

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Related in: MedlinePlus

Processing and function of BMP7 in Drosophila.A, diagrams of the BMP7 protein, as described for Gbb in the Fig. 2 legend, are shown. B, anti-FLAG Western blot of BMP7 proteins secreted into the media of transfected S2R+ cells are shown. M, main site mutation. (C) The gbb=BMP7 ligand domain chimera (gbb=Gbb-BMP7LD) fully rescues the gbb4 cross-vein defect (D) as do the full-length BMP7 lines that partially rescue the lethality of gbb s. (E) Full-length BMP7 lines that do not rescue lethality of gbb s partially rescue the gbb4 cross-vein defect (F), whereas the Main site mutant (BMP7R-292G) does not rescue.
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Figure 5: Processing and function of BMP7 in Drosophila.A, diagrams of the BMP7 protein, as described for Gbb in the Fig. 2 legend, are shown. B, anti-FLAG Western blot of BMP7 proteins secreted into the media of transfected S2R+ cells are shown. M, main site mutation. (C) The gbb=BMP7 ligand domain chimera (gbb=Gbb-BMP7LD) fully rescues the gbb4 cross-vein defect (D) as do the full-length BMP7 lines that partially rescue the lethality of gbb s. (E) Full-length BMP7 lines that do not rescue lethality of gbb s partially rescue the gbb4 cross-vein defect (F), whereas the Main site mutant (BMP7R-292G) does not rescue.

Mentions: Consistent with studies in mammalian tissue culture cells (31, 32), in S2R+ cells BMP7 is cleaved at the single Main site but is secreted as two isoforms (Fig. 5B). The two forms arise from differential glycosylation, as peptide N-glycosidase treatment reduces the doublet to a single 19-kDa band (supplemental Fig. S3). Mutation of the Main site results in the loss of both mature forms, and instead the 50-kDa BMP7 proprotein is secreted into the medium (Fig. 5B).


Different requirements for proteolytic processing of bone morphogenetic protein 5/6/7/8 ligands in Drosophila melanogaster.

Fritsch C, Sawala A, Harris R, Maartens A, Sutcliffe C, Ashe HL, Ray RP - J. Biol. Chem. (2011)

Processing and function of BMP7 in Drosophila.A, diagrams of the BMP7 protein, as described for Gbb in the Fig. 2 legend, are shown. B, anti-FLAG Western blot of BMP7 proteins secreted into the media of transfected S2R+ cells are shown. M, main site mutation. (C) The gbb=BMP7 ligand domain chimera (gbb=Gbb-BMP7LD) fully rescues the gbb4 cross-vein defect (D) as do the full-length BMP7 lines that partially rescue the lethality of gbb s. (E) Full-length BMP7 lines that do not rescue lethality of gbb s partially rescue the gbb4 cross-vein defect (F), whereas the Main site mutant (BMP7R-292G) does not rescue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285362&req=5

Figure 5: Processing and function of BMP7 in Drosophila.A, diagrams of the BMP7 protein, as described for Gbb in the Fig. 2 legend, are shown. B, anti-FLAG Western blot of BMP7 proteins secreted into the media of transfected S2R+ cells are shown. M, main site mutation. (C) The gbb=BMP7 ligand domain chimera (gbb=Gbb-BMP7LD) fully rescues the gbb4 cross-vein defect (D) as do the full-length BMP7 lines that partially rescue the lethality of gbb s. (E) Full-length BMP7 lines that do not rescue lethality of gbb s partially rescue the gbb4 cross-vein defect (F), whereas the Main site mutant (BMP7R-292G) does not rescue.
Mentions: Consistent with studies in mammalian tissue culture cells (31, 32), in S2R+ cells BMP7 is cleaved at the single Main site but is secreted as two isoforms (Fig. 5B). The two forms arise from differential glycosylation, as peptide N-glycosidase treatment reduces the doublet to a single 19-kDa band (supplemental Fig. S3). Mutation of the Main site results in the loss of both mature forms, and instead the 50-kDa BMP7 proprotein is secreted into the medium (Fig. 5B).

Bottom Line: The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies.Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality.Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Falmer Brighton BN1 9QG, United Kingdom.

ABSTRACT
Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.

Show MeSH
Related in: MedlinePlus