A large bioactive BMP ligand with distinct signaling properties is produced by alternative proconvertase processing

Abstract

Dimers of conventional transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) ligands are composed of two 100- to 140-amino acid peptides that are produced through the proteolytic processing of a proprotein precursor by proconvertases, such as furin. We report the identification of an evolutionarily conserved furin processing site in the amino terminus (NS) of the Glass bottom boat (Gbb; the Drosophila ortholog of vertebrate BMP5, 6, and 7) proprotein that generates a 328-amino acid, active BMP ligand distinct from the conventional 130-amino acid ligand. Gbb38, the large ligand form of Gbb, exhibited greater signaling activity and a longer range than the shorter form Gbb15. The abundance of Gbb15 and Gbb38 varied among different tissues, raising the possibility that differential processing could account for tissue-specific behaviors of BMPs. In human populations, mutations that abolished the NS cleavage site in BMP4, BMP15, or anti-Müllerian hormone were associated with cleft lip with or without cleft palate (BMP4), premature ovarian failure (BMP15), and persistent Müllerian duct syndrome (anti-Müllerian hormone), suggesting the importance of NS processing during development. The identification of this large BMP ligand form and the functional differences between large and small ligands exemplifies the potential for differential proprotein processing to substantially affect BMP and TGF-β signaling output in different tissue and cellular contexts.

Fig. 1

Furin cleavage site mutations differentially affect Gbb signaling activity. (A) Schematic of Gbb pre-proprotein with amino acid (aa) position of furin cleavage consensus sequences. NS, S2, and S1 and disrupting mutations are noted. SP, signal peptide. (B) Schematic representation of cell-based BMP signaling assay. brkSE-lacZ reporter is activated in response to active Notch signaling (N*) through Su(H) binding sites (BS). Repression of brkSE-lacZ is a direct response to active BMP signaling (BMP*) resulting from binding of the Drosophila BMP signal transducer pMad-Med-Shn complex to brkSE. (C) Signaling activity induced by gbb cleavage mutants (gbb-CMs). In the absence of Gbb-induced signaling (−), β-galactosidase activity is set to 100%. Repression of brkSE-lacZ in response to BMP signaling is measured as reduction in β-gal activity. Horizontal lines indicate significance at P < 0.05 with the Tukey-Kramer HSD procedure for multiple comparisons. gbb^mS1 is marginally different from gbb (dotted horizontal line) on the basis of its significant difference using Hsu’s MCB test but not at P < 0.05 with Tukey-Kramer. Each bar represents the average of three independent experiments; error bars indicate SE. (D) Induction of phosphorylated Mad (pMad) measured in S2 cells transfected with Mad-Flag and treated with 0.1× and 1× conditioned media from S2 cells expressing gbb, gbb^mS1, or empty vector (pAc).

Fig. 2

Large and small ligand forms are produced by differential processing of Gbb. (A) Extracts from gbb null (gbb¹/gbb² and gbb¹/gbb³) and wild-type (+/+) third instar larvae (left) and S2 cells (right) probed for cross-reactivity to a Gbb monoclonal antibody (α-Gbb). In addition to Gbb proprotein (proGbb) and expected ligand (Gbb15), a 42-kD protein specific to Gbb was detected (arrow). Anti-actin (α-Actin) served as a loading control. (B) Extracts from gbb-1xHA– and gbb-1xHA–transfected S2 cells probed with anti-HA (α-HA). Gbb15 product is absent from gbb^mS1-1xHA extracts. (C) Extracts from wing discs expressing gbb, gbb^mNS, and gbb^mS1 under the control of hhGal4 probed with α-Gbb and α-actin. The 42-kD product is lost when the NS cleavage site is mutated (gbb^mNS). Predicted size of a product produced by processing at NS is 38 kD. Lower part of blot was exposed for 15 times longer to reveal Gbb15 in gbb^mNS extracts and its absence in gbb^mS1 extracts. (D to F) Extracts from S2 cells transfected with wild-type gbb and gbb-CM constructs analyzed for Gbb products. Media (D) and cell (E) fractions indicate secreted and cellular products, respectively. Total extracts (F). The abundance of endogenous Gbb38 and Gbb15 differs between different tissues from isolated third instar larval tissues, including brain (B), salivary glands (SG), fat body (FB), and wing discs (WD), which were probed with α-Gbb and α-actin (G). Actin is the loading control. The bottom panel of each Western blot was exposed twice as long as the top panel to visualize the lower–molecular weight products.

Fig. 3

NS cleavage sites are conserved in the TGF-β superfamily. (A) Phylogenetic tree of TGF-β family members from bilateria [Drosophila melanogaster (Dm, protostome), Strongylocentrotus purpuratus (Sp), and Homo sapiens (Hs; deuterostomes)] and radiata [Nematostella vectensis (Nv)]. Shaded proteins contain a putative NS cleavage site. (B) Specific mutations at NS sites in human BMP4, BMP15, and AMH proteins are associated with the indicated developmental abnormalities.

Fig. 4

Differential response of wing disc cells to dorsal expression of gbb cleavage mutants.(A to D)Wings from(A)apGal4>UAS-GFP, (B) apGal4>UAS-gbb^mS1, (C) apGal4>UAS-gbb^mNS, and (D) apGal4>UAS-gbb^mNSmS1 adults. Ectopic venation, abnormal wing size, and the improper apposition of the dorsal and ventral wing surfaces are phenotypes that correlate with altered BMP signaling. (E to P) Distribution of pMad(red) in third instar larval wing discs expressing UAS-gbbCMs with inset of same disc at 0.4× shows UAS-GFP (green) under the control of apGal4 in the (E) dorsal compartment (d). v, ventral compartment; N, notum; H, hinge; WP, wing pouch. (H to J and N to P) pMad quantification [in arbitrary units (a.u.)] across the wing pouch (WP) in the dorsal (red) and ventral (black) compartments of multiple discs for each genotype as indicated by open boxes in (E). Tracing is the average intensity from multiple discs with SEs shown (vertical lines). n = number of discs. (F and I) apGal4> UAS-GFP, UAS-gbb (n=5); (G and J) apGal4>UAS-GFP, UAS-gbb^mS2 (n = 5); (K and N) apGal4>UAS-GFP, UAS-gbb^mNS(n=3); (L and O) apGal4>UAS-GFP, UAS-gbb^mS1 (n = 4); (M and P) apGal4>UAS-GFP, UAS-gbb^mNSmS1 (n= 4). Tracing of wild-type endogenous pMad gradient characterized by two peaks of higher pMad separated by a trough of lower accumulation is shown for comparison (blue, n = 3) in (I) and (J) and (N) to (P). Asterisks (*) highlight regions of ectopic pMad in the hinge (H) and notum (N) regions of the wing disc. All images obtained at the same magnification under identical confocal microscope settings.

Fig. 5

Long-range activity of Gbb38 is domain-dependent. BMP signaling [distribution of pMad (gray)] assessed in third instar larval wing discs expressing UAS-gbb and UAS-gbb-CMs in posterior (hhGal4) and anterior (ciGal4) compartments. Dorsal up, posterior (P), right. Insets of same disc at 0.4× show distribution of pMad (red) and GFP (green). (A) Control wild-type disc, hhGal4>UAS-GFP.(B)hhGal4>UASgbb, UAS-GFP. (C) hhGal4> UASgbb^mS1 UAS-GFP (Gbb38). (D) hhGal4> UASgbb^mNS UAS-GFP (Gbb15). All images collected at the same confocal settings. (E) pMad quantification in arbitrary units (a.u.) collected from dorsal wing pouch (open rectangle) from multiple discs. [hhGal4>UAS-GFP (black), n = 4; hhGal4> UASgbb (red), n = 6; hhGal4>UASgbb^mS1 (blue), n = 4; hhGal4> UASgbb^mNS (purple), n = 4]. SE indicated. (F to I) pMad and Sal distribution in the wing pouch of hhGal4>UAS-GFP (F), hhGal4>UASgbb (G), hhGal4>UASgbb^mS1 (H), and hhGal4>UASgbb^mNS (I) wing discs. Arrows in (G) denote expanded Sal expression. (J to M) ciGal4>UAS-gbb, UAS-GFP (J), ciGal4>UAS-gbb^mS1, UAS-GFP (Gbb38) (K), ciGal4>UAS-gbb^mNS, UAS-GFP (Gbb15) (L), schematic of sites of ectopic BMP signaling induced by gbb^mS1 (Gbb38), gbb^mNS (Gbb15), and wild-type gbb (Gbb38 + Gbb15) when expressed in the anterior compartment (gray) (M). Portion of wing pouch refractory to gbb overexpression, red. (N to P) pMad quantification in arbitrary units (a.u.) collected from dorsal wing pouch.(N)ciGal4>UAS-GFP(black), n=2; ciGal4>UAS-gbb (red), n = 3. (O) ciGal4>UAS-GFP (black), n = 2; ciGal4>UAS-gbb^mS1 (blue), n = 3. (P) ciGal4> UAS-gbb^mS1 (blue), n = 3; ciGal4>UAS-gbb^mNS (purple), n = 3. All images collected at the same confocal settings. (O and P) X marks anomaly in pMad tracing due to fold at edge of wing pouch [see also (K)]. n = number of discs.

Fig. 6

Model summarizing the signaling capacity and range of action for products produced by TGF-β and BMP family members. (Top) Linear schematic of TGF-β/BMP protein domains (prodomain, linker, and ligand domains), NS and linker cleavage sites (vertical lines, scissors), and cleavage products. (Bottom) Model schematic of structure of products based on Shi et al. (34) (color-coded to linear representations) with secretion, signaling activity, and range of action properties noted. No structure is known for the linker region, which is designated in the large ligand model.