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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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Rescue of the cross-vein defect of gbb4 homozygotes by Gbb cleavage mutants.A, shown is a wild type wing indicating the posterior cross-vein. The region of the wing shown in panels B–D is boxed. B, gbb4 homozygotes are semi-viable, and adults show loss of the posterior cross-vein. C, rescue of the gbb4 cross-vein defect by the main/shadow double mutant. D, partial rescue by the pro/main double mutant results in a crossvein fragment in the L4-L5 intervein (arrow). E, survival of gbb4 homozygotes in the presence (gray) or absence (black) of one copy of the pro/main double mutant or the pro/main/shadow triple mutant is shown. F, mis-expression of the triple mutant with the Act5C-Gal4 driver is shown which phenocopies the gbb4 cross-veinless phenotype.
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Figure 3: Rescue of the cross-vein defect of gbb4 homozygotes by Gbb cleavage mutants.A, shown is a wild type wing indicating the posterior cross-vein. The region of the wing shown in panels B–D is boxed. B, gbb4 homozygotes are semi-viable, and adults show loss of the posterior cross-vein. C, rescue of the gbb4 cross-vein defect by the main/shadow double mutant. D, partial rescue by the pro/main double mutant results in a crossvein fragment in the L4-L5 intervein (arrow). E, survival of gbb4 homozygotes in the presence (gray) or absence (black) of one copy of the pro/main double mutant or the pro/main/shadow triple mutant is shown. F, mis-expression of the triple mutant with the Act5C-Gal4 driver is shown which phenocopies the gbb4 cross-veinless phenotype.

Mentions: Comparing the rescue data with our tissue culture data, our results suggest that the presence of either the 19-kDa fully processed form or the 45-kDa partially processed form is sufficient to rescue the lethality associated with gbb mutants. If true, the inability of the Pro/Main double mutant to rescue gbb lethality (Table 1) could be explained by the low level of mature ligand produced by this mutant (Fig. 2B, lane 7). To test this, we determined whether the Pro/Main double mutant could rescue defects associated with a hypomorphic allele, gbb4. gbb4 homozygotes are semi-viable, and the adult escapers have venation defects that include loss of the posterior cross-vein (21). It has previously been shown that a duplication of dpp, Dp(2;2)DTD48, rescues the lethality, but not the cross-vein defect, of gbb4 homozygotes (Ref. 20; Fig. 3B) and that this venation defect is particularly sensitive to even low levels of functional Gbb. Consistent with our rescue data with the allele, the Pro, Main, Shadow, and Main/Shadow cleavage mutant constructs completely rescue the gbb4 cross-vein defect (Fig. 3C, data not shown), and the Pro-Main double mutant, which cannot rescue gbb s, shows partial rescue of the gbb4 cross-vein defect (Fig. 3D). Thus, consistent with the tissue culture data, which shows that the Pro/Main double mutant produces a small amount of mature ligand, this mutation does produce functional Gbb.


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)

Rescue of the cross-vein defect of gbb4 homozygotes by Gbb cleavage mutants.A, shown is a wild type wing indicating the posterior cross-vein. The region of the wing shown in panels B–D is boxed. B, gbb4 homozygotes are semi-viable, and adults show loss of the posterior cross-vein. C, rescue of the gbb4 cross-vein defect by the main/shadow double mutant. D, partial rescue by the pro/main double mutant results in a crossvein fragment in the L4-L5 intervein (arrow). E, survival of gbb4 homozygotes in the presence (gray) or absence (black) of one copy of the pro/main double mutant or the pro/main/shadow triple mutant is shown. F, mis-expression of the triple mutant with the Act5C-Gal4 driver is shown which phenocopies the gbb4 cross-veinless phenotype.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Rescue of the cross-vein defect of gbb4 homozygotes by Gbb cleavage mutants.A, shown is a wild type wing indicating the posterior cross-vein. The region of the wing shown in panels B–D is boxed. B, gbb4 homozygotes are semi-viable, and adults show loss of the posterior cross-vein. C, rescue of the gbb4 cross-vein defect by the main/shadow double mutant. D, partial rescue by the pro/main double mutant results in a crossvein fragment in the L4-L5 intervein (arrow). E, survival of gbb4 homozygotes in the presence (gray) or absence (black) of one copy of the pro/main double mutant or the pro/main/shadow triple mutant is shown. F, mis-expression of the triple mutant with the Act5C-Gal4 driver is shown which phenocopies the gbb4 cross-veinless phenotype.
Mentions: Comparing the rescue data with our tissue culture data, our results suggest that the presence of either the 19-kDa fully processed form or the 45-kDa partially processed form is sufficient to rescue the lethality associated with gbb mutants. If true, the inability of the Pro/Main double mutant to rescue gbb lethality (Table 1) could be explained by the low level of mature ligand produced by this mutant (Fig. 2B, lane 7). To test this, we determined whether the Pro/Main double mutant could rescue defects associated with a hypomorphic allele, gbb4. gbb4 homozygotes are semi-viable, and the adult escapers have venation defects that include loss of the posterior cross-vein (21). It has previously been shown that a duplication of dpp, Dp(2;2)DTD48, rescues the lethality, but not the cross-vein defect, of gbb4 homozygotes (Ref. 20; Fig. 3B) and that this venation defect is particularly sensitive to even low levels of functional Gbb. Consistent with our rescue data with the allele, the Pro, Main, Shadow, and Main/Shadow cleavage mutant constructs completely rescue the gbb4 cross-vein defect (Fig. 3C, data not shown), and the Pro-Main double mutant, which cannot rescue gbb s, shows partial rescue of the gbb4 cross-vein defect (Fig. 3D). Thus, consistent with the tissue culture data, which shows that the Pro/Main double mutant produces a small amount of mature ligand, this mutation does produce functional Gbb.

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