Alternative cleavage of the bone morphogenetic protein (BMP), Gbb, produces ligands with distinct developmental functions and receptor preferences

Abstract

The family of TGF-β and bone morphogenetic protein (BMP) signaling proteins has numerous developmental and physiological roles. They are made as proprotein dimers and then cleaved by proprotein convertases to release the C-terminal domain as an active ligand dimer. Multiple proteolytic processing sites in Glass bottom boat (Gbb), the Drosophila BMP7 ortholog, can produce distinct ligand forms. Cleavage at the S1 or atypical S0 site in Gbb produces Gbb15, the conventional small BMP ligand, whereas NS site cleavage produces a larger Gbb38 ligand. We hypothesized that the Gbb prodomain is involved not only in regulating the production of specific ligands but also their signaling output. We found that blocking NS cleavage increased association of the full-length prodomain with Gbb15, resulting in a concomitant decrease in signaling activity. Moreover, NS cleavage was required in vivo for Gbb-Decapentaplegic (Dpp) heterodimer-mediated wing vein patterning but not for Gbb15-Dpp heterodimer activity in cell culture. Gbb NS cleavage was also required for viability through its regulation of pupal ecdysis in a type II receptor Wishful thinking (Wit)-dependent manner. In fact, Gbb38-mediated signaling exhibits a preference for Wit over the other type II receptor Punt. Finally, we discovered that Gbb38 is produced when processing at the S1/S0 site is blocked by O-linked glycosylation in third instar larvae. Our findings demonstrate that BMP prodomain cleavage ensures that the mature ligand is not inhibited by the prodomain. Furthermore, alternative processing of BMP proproteins produces ligands that signal through different receptors and exhibit specific developmental functions.

Keywords: Drosophila; O-GlcNAcylation; Glass bottom boat (Gbb); bone morphogenetic protein (BMP); cell signaling; prodomain; proprotein convertase; protein processing; transforming growth factor β (TGF-β).

Figure 1.

Gbb prodomain cleavage affects ligand abundance and activity. A, Gbb protein schematic, showing domains, signal peptide (SP), cleavage sites, mutations, and location of antibody epitopes. Furin cleavage motifs at NS and S1 are underlined, with the downward arrow indicating the cleaved bond. The atypical S0 cleavage site is underlined with a dashed line. B and C, reducing Western blots (WB) of Gbb cleavage products secreted by S2 cells expressing gbb cleavage mutants, using antibodies that recognize specific epitopes in the Gbb prodomain (B) or the C-terminal ligand domain (C). D, quantification of secreted ligand abundance for representative Western blot shown in C. For mutant constructs that produce multiple forms of secreted Gbb ligand, the summed total of both forms is shown. E, steady-state signaling activity produced by gbb cleavage site mutant constructs when co-expressed with the BrkSE-lacZ reporter in S2 cells. Expression of BrkSE-lacZ reporter is repressed by BMP signaling and is shown here as opposite log-transformed β-Gal activity, with the activity of empty vector (EV) transfected cells set to 0. D and E, bars indicate the mean and 95% CI. * indicates p < 0.05 compared with EV; #, p < 0.05 compared with WT gbb, using GLHT for multiple comparison testing.

Figure 2.

Activity and kinetics of Gbb cleavage mutant ligands. A, Western blots of phosphorylated Mad–FLAG (pMad) in lysates of S2 cells treated with gbb-conditioned media for 2 h. B, quantification of Mad–FLAG Western blots shown in A, also showing time course of gbb-induced pMad from 0.5 to 10 h. Bars indicate mean and 95% CI. At each time point, *, p < 0.05 compared with EV; #, p < 0.05 compared with WT gbb, using the GLHT multiple comparison test. C, summary of the cleavage products and activity present in each conditioned medium.

Figure 3.

Cleavage of the NS site reduces Gbb15–prodomain association. A, reducing Western blots (WB) of co-immunoprecipitating Gbb cleavage products. Conditioned medium from S2 cells expressing gbb-HA cleavage mutants was immunoprecipitated with α-proGbb and α-CoreGbb to directly pull down prodomain cleavage products and α-HA to pull down C-terminal cleavage products. Immunoprecipitate was analyzed by Western blotting using α-proGbb, α-CoreGbb, and α-GbbC to identify each cleavage product. B, summary of relative co-IP efficiencies between cleavage products for WT and cleavage mutant gbb-HA.

Figure 4.

Receptor-specific activity of Gbb cleavage mutants. A, type II receptor-dependent pMad induced by Gbb cleavage mutants. S2 cells expressing Mad–FLAG and punt or wit were treated with gbb-conditioned media for 2 h, and pMad was measured by Western blotting. B, steady-state BrkSE-lacZ signaling assay of gbb co-expressed with either the type I receptors tkv or sax, and the type II receptors punt or wit. Bars indicate mean and 95% CI. *, p < 0.05 compared with EV; #, p < 0.05 compared with WT gbb co-expressed with a given receptor, using GLHT multiple comparison test. Western blot panels show expression of FLAG- or HA-tagged receptors in representative sample lysates.

Figure 5.

Dpp signaling activity is enhanced by forming heterodimers with Gbb cleavage mutants. A, non-reducing Western blots (WB) of media from cells co-expressing dpp-HA and gbb-HA, showing secreted homodimers and heterodimers (indicated with †). B and C, steady-state BrkSE-lacZ signaling assay, measuring activity of gbb-HA cleavage mutants co-expressed with EV (B) or dpp-HA (C). All signaling experiments were performed in parallel and are presented separately to emphasize the relative effects of gbb-HA expression. Bars indicate mean and 95% CI. * indicates p < 0.05 compared with EV; # indicates p < 0.05 compared with WT gbb-HA, using GLHT multiple comparison test.

Figure 6.

dpp wing phenotypes are enhanced by mutation of the gbb NS cleavage site. A, representative images of wings showing phenotypes resulting from genetic interactions between gbbR^mNS and dpp^hr4 or dpp^d12. Inset images show the distal tip of L2 with ectopic veins (arrowhead) and the PCV with spurs (arrow). B, proportion of L2 ectopic veins. Phenotype is scored 0 for WT, 1 for a single ectopic vein spot, and 2 for multiple ectopic vein spots. C, proportion of PCV phenotypes. *, p < 0.05; ns indicates p > = 0.05 using Fisher's exact test and FDR multiple comparison adjustment.

Figure 7.

Blocking NS cleavage causes pupal ecdysis defects and a loss of Gbb38 in vivo. A, representative images of pupae 4 days after pupariation. gbbR^mNSmS gbb¹ pupal phenotypes include failure to evert anterior spiracles (arrowhead), reduced leg extension (dashed lines and bars), partial or complete failure of pupal ecdysis, and developmental arrest. B, quantification of pupal leg extension, measured as fraction of pharate body length. *, p < 0.001 using Wilcoxon rank-sum test. C, immunoprecipitation of HA-tagged Gbb from wandering 3rd instar larvae, showing that blocking NS cleavage in gbbR^mNSmS-HA gbb¹ causes a loss of Gbb38. WB, Western blot.

Figure 8.

Gbb NS cleavage is required for expression of the ecdysis-regulating hormone CCAP. A, schematic of CCAP-expressing peptidergic neurons in the larval VNC, adapted from Veverytsa and Allan (55). Prior to the 3rd larval instar wandering stage, CCAP is expressed in interneurons (IN) that lack nuclear pMad and Dac (CCAP-IN) and in efferent neurons that have nuclear pMad and Dac (CCAP-EN). At later stages, CCAP expression is activated in additional pMad+, Dac+ efferent neurons (ENL) and pMad+ posterior lateral neurons (PL). B and C, representative confocal image stacks of VNC from wandering 3rd instar larvae. CCAP > GFP is used as a reporter of CCAP expression, and nuclear Dac and pMad serve as markers and indicators of BMP signaling. Insets, CCAP neurons with pMad+ nuclei are indicated with arrowheads and dashed outline. D, number of late CCAP neurons per VNC. *, p < 0.05 using Wilcoxon rank-sum test and FDR multiple comparison adjustment. E, quantification of mean cell body CCAP > GFP signal intensity for each VNC and neuron class. F, quantification of mean nuclear pMad signal intensity for each VNC and neuron class. E and F, bars indicate mean and 95% CI; *, p < 0.05 using GLHT multiple comparison test. D–F, n = 8, VNCs for gbbR gbb¹, n = 7, VNCs for gbbR^mNSmS gbb¹.

Figure 9.

O-Linked glycosylation blocks S1/S0 cleavage and Gbb15 production. A, predicted O-linked glycosylation sites on Ser/Thr residues in Gbb. Residues with NetOGlyc 4.0 scores > = 0.5 indicate predicted O-linked glycosylation, and scores <0.5 (gray) indicate no predicted glycosylation. PC cleavage sites are indicated with solid red lines, and dashed red lines indicate ±3 residues. B, in vitro cleavage of Gbb-HA immunoprecipitated from gbbR^HA gbb¹ 3rd instar larvae. Immunoprecipitate was treated with the indicated combinations of recombinant O-glycosidase, β-glucuronidase (β-Glcase), β-N-acetylhexosaminidase (β-HexNAcase), or furin and analyzed on a non-reducing Western blot (WB).

Figure 10.

Model of Gbb processing, prodomain–ligand complexes, and activity. Gbb expressed in S2 cells is cleaved by a proprotein convertase (PC) at both the NS and S1/S0 site, and the resulting ligand (Gbb15) and prodomain cleavage products (NH₃-NS and NS-S1) are secreted as a loosely associated complex that has signaling activity mediated by Wit or Punt. When NS cleavage is blocked by mutation of the NS site (mNS), the uncleaved prodomain (NH₃-S1) is tightly associated with Gbb15, and Punt-mediated signaling is reduced. When S1/S0 cleavage is blocked, by mutation of the S1/S0 site (mS1mS0), the ligand Gbb38 is secreted in complex with the N-terminal prodomain cleavage fragment (NH₃-NS), and Punt-mediated signaling is greatly reduced. In the 3rd larval instar, cleavage of the S1/S0 site is blocked by O-glycosylation, depicted here as hypothetical extended Core 1 O-glycan. Thus, Gbb15 production is blocked, and Gbb38 is produced as a ligand that is required for Wit-mediated regulation of pupal ecdysis.