Enzymatic regulation of pattern: BMP4 binds CUB domains of Tolloids and inhibits proteinase activity.
In Xenopus embryos, a dorsal-ventral patterning gradient is generated by diffusing Chordin/bone morphogenetic protein (BMP) complexes cleaved by BMP1/Tolloid metalloproteinases in the ventral side. We developed a new BMP1/Tolloid assay using a fluorogenic Chordin peptide substrate and identified an unexpected negative feedback loop for BMP4, in which BMP4 inhibits Tolloid enzyme activity noncompetitively. BMP4 binds directly to the CUB (Complement 1r/s, Uegf [a sea urchin embryonic protein] and BMP1) domains of BMP1 and Drosophila Tolloid with high affinity. Binding to CUB domains inhibits BMP4 signaling. These findings provide a molecular explanation for a long-standing genetical puzzle in which antimorphic Drosophila tolloid mutant alleles displayed anti-BMP effects. The extensive Drosophila genetics available supports the relevance of the interaction described here at endogenous physiological levels. Many extracellular proteins contain CUB domains; the binding of CUB domains to BMP4 suggests a possible general function in binding transforming growth factor-beta (TGF-beta) superfamily members. Mathematical modeling indicates that feedback inhibition by BMP ligands acts on the ventral side, while on the dorsal side the main regulator of BMP1/Tolloid enzymatic activity is the binding to its substrate, Chordin.
PubMed ID: 19884260
PMC ID: PMC2779747
Article link: Genes Dev.
Grant support: HD21502-23 NICHD NIH HHS , Howard Hughes Medical Institute , R01 HD021502-23 NICHD NIH HHS , R01 HD021502 NICHD NIH HHS
Genes referenced: bmp1 bmp4 bmp7.1 bmper chrd.1 dspp fst gtf2ird1 nog otx2 pclo sox2 tbx2 tgfb1 tll1 tll2
Morpholinos referenced: chrd MO1
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|Figure 1. BMP1 CUB domains dorsalize ventral half-embryos independently of Chordin. (A) Diagram of the generic Tolloid family metalloproteinase primary structure, BMP1, and the BMP1 CUB1/2/3 construct. (B) Xenopus laevis embryos were bisected into dorsal and ventral halves at late blastula/early gastrula stage using a surgical blade. (C,C′) Bisected wild-type embryos result in the dorsal half-embryo that self-regulates into a relatively normal embryo and a ventral belly piece lacking all neural structures marked by the pan-neural marker Sox2. (D,D′) BMP1 CUB1/2/3 mRNA (1 ng per embryo injected at the two-cell stage) dorsalizes the ventral half-embryo (n = 86, 96% positive for Sox2). (E,E′) Microinjection of morpholino oligos against Chordin (ChdMO) reduced neural structures in the dorsal half. (F,F′) The dorsalizing effect of BMP1 CUB1/2/3 mRNA in the ventral half-embryo is not affected by Chordin depletion (n = 82, 95% positive for Sox2).|
|Figure 2. A fluorometric assay for the Chordinase activity of Tolloids shows that BMP4 is a specific inhibitor of the reaction. (A) A new fluorogenic peptide substrate for Tolloids containing the sequence of the Xenopus Chordin cleavage site. (B) The Chd peptide substrate is efficiently digested by BMP1 enzyme, a reaction that is competitively inhibited by 60 nM full-length Chordin proteins. The Ki (17 nM) is similar to the Km of tolloids for full-length chordin (Lee et al. 2006). (C) BMP4, but not other regulators of D–V patterning, inhibits BMP1 metalloproteinase activity. All proteins were added at 120 nM final concentration.|
|Figure 3. BMP4 is a noncompetitive inhibitor of the BMP1 enzyme. (A) BMP4 inhibits BMP1 activity in a dose-dependent manner. (B) BMP4 inhibition cannot be competed by increasing amounts of substrate, as shown by the difference in maximal velocity with or without BMP4. This result is consistent with that expected of a noncompetitive enzyme inhibitor. (C) BMP4 affects the maximal velocity (Vmax), but not the Michaelis constant (Km) of the BMP1 enzyme. (D) Effects of different concentrations of inhibitor at two different substrate concentrations. The Dixon plot shows that BMP4 inhibited BMP1 with an inhibition constant (Ki) of 40 nM. (E) BMP4 does not exhibit cooperativity when inhibiting BMP1, indicating one-to-one binding is required for inhibition of catalytic activity.|
|Figure 4. BMP1 CUB domains bind BMP4 growth factor. (A) Diagram of BMP1 and BMP1 CUB1/2 constructs and their mutations. (B) Mimicking an antimorph revertant mutant in BMP1 (DN-BMP1WR) or BMP1 CUB1/2 impairs protein secretion. (C–E) Overexpression of DN-BMP1 dorsalized embryos (note the enlarged head marked by Otx2), but the second site revertant mutation (DN-BMP1WR) had no effect. (F) BMP4 binds to full-length BMP1 on a BIAcore sensor chip. (G) Purified BMP1 CUB1 and CUB2 domains are sufficient for BMP4 binding. (H,I) Embryos injected with BMP1 CUB1/2 RNA were dorsalized, as indicated by the expanded Otx-2 expression domain (n = 83, 81% dorsalized). (J) Dorsalization (anti-BMP) effect was also observed after injection of BMP1 CUB1/2 purified protein into the blastula cavity (n = 85, 82% dorsalized).|
|Figure 5. dTld binds BMP4 and BMP7. (A) Diagram of dTld and the dTld CUB1/2 and CUB4/5 constructs. (B) dTld CUB1/2ΔL mutant is not secreted, and CUB4/5SR mutant has impaired secretion in transfected 293T cells. Multiple bands are presumably due to glycosylation. (C) dTld CUB1/2 binds BMP4 within the physiological range (KD ≈ 17 nM). (D) When BMP7 is placed on the sensor chip, dTld CUB1/2 protein is able to bind to it (KD ≈ 26 nM). (E,F) Both dTld CUB4/5 and CUB4/5SR bind BMP4 with similar KD. (G–J) dTld CUB1/2 (n = 81, 78% dorsalized) and CUB4/5 (n = 79, 77% dorsalized) have anti-BMP (dorsalizing) activity in a Xenopus assay, while dTld CUB4/5SR did not show any anti-BMP activity (n = 52).|
|Figure 6. Mathematical modeling of the effects of the BMP negative feedback loop on BMP1/Tolloid (Tld) activity. (A) Partial differential equation describing the temporal evolution of free Tld concentration. The terms accounting for the inhibition of Tld by BMP (reaction 10 in Supplemental Fig. 1) are boxed. The reactions involving Tld and their corresponding parameters are described in Supplemental Figure 2. (B) Schematic diagram of the negative feedback loop between Tld, BMP, and Chd, which regulates the D–V gradient of pSmad1 activity. (C,D) Effect of BMP negative feedback on the temporal evolution of free Tld concentration profile. D and V indicate the position of the dorsal and ventral sides. Each concentration profile is labeled from t0 (0 h) to t4 (2 h) to indicate their evolution over time. The arrows indicate the final level of free Tld on the ventral side without (C) or with (D) BMP4 feedback. Note that the model predicts that free (active) Tld is low in the dorsal midline due to the large amounts of Chd substrate secreted by the dorsal side (Supplemental Fig. 3).|
|Figure 7. Enzymatic regulation of Xenopus D–V patterning. This diagram depicts the extracellular network of biochemical protein–protein interactions that establishes the embryonic D–V axis through protein–protein interactions (black arrows), BMP-dependent transcription (blue arrows), and the flux of Chordin/BMP complexes from more dorsal regions to the ventral side, where it is bound by CV2 (red arrows). Tolloid and CV2 act as sinks for Chordin in the ventral side, where BMPs made in more dorsal regions can be released by Tolloid to reach peak signaling levels. The system is self-regulating because transcription of dorsal genes is repressed by BMP signals, while ventral genes are under the opposite transcriptional regulation (Reversade and De Robertis 2005; Lee et al. 2006; Ambrosio et al. 2008; Ben-Zvi et al. 2008). The two new reactions reported in this study are the inhibitory black arrows from BMP4/7 to Tolloid in the ventral side (enzyme activity inhibition) and from Tolloid to BMP4/7 (inhibition through binding and sequestration of the growth factor). Other important regulators of D–V patterning, such as Noggin and Follistatin (Khokha et al. 2005), are not included in this simplified model.|
|Lee et al. Supplemental Figure 1. Alignment of CUB domain protein sequences. CUB domains 1 and 2 from Xenopus laevis Xolloid-related, Drosophila melanogaster Tolloid, and human BMP1 were aligned using the Multalin algorithm (bioinfo.genotoul.fr/multalin/multalin.html). Red denotes high-consensus amino acids. Drosophila second-site mutations that suppress tolloid antimorphs are shown.|
|Lee et al. Supplemental Figure 2. The 14 Biochemical Reactions Included in the Mathematical Model. For each reaction the name and value of its biochemical parameters are shown. We estimated the D-V circumference of the Xenopus gastrula embryo to be 4400 μm and Chordin to be expressed in the dorsal 30% of the embryo (700 μm). BMP4 and Tld/BMP1 were assumed to be expressed uniformly in the early embryo. The diffusion constant for Chd (DChd), BMP (DBMP), and ChdBMP (DChdBMP) was set to D=102 μm2.s-1, and to 0 μm2.s-1 for the other molecular species; relevant references for these parameters are listed in the figure. The initial concentrations of the different components were set between 0.3 and 1.0 nM for Tld, between 5 and 10 nM for the BMP receptor complex (BMPR), and at 0 nM for the rest. The concentrations of each molecular species were allowed to evolve for 2 hours, which represents the time taken by the Xenopus embryo to develop from late blastula to early gastrula.|
|Lee et al. Supplemental Figure 3. Free Tld Levels Decrease on the Dorsal Side as the Concentration of its Substrate Chordin Increases. Free Tld decreases as free Chd increases, because Chordin binds to the enzyme that normally degrades it. Our mathematical modeling unexpectedly revealed that a gradient of Tld activity is formed by the action of free Chordin, which limits the availability of free Tld for other substrates such as Chordin/BMP4 complexes. Vertebrate BMP1/tolloids digest free Chordin protein (Piccolo et al. 1997; Wardle et al. 1999), although cleavage is more efficient in the presence of BMP4 (our unpublished observations). Drosophila Tolloid enzyme cleaves Sog complexed with Dpp efficiently, but uncomplexed Sog very inefficiently or not at all (Marqués et al. 1997). It is not known at present whether Tolloid would bind to free Sog in Drosophila embryos, but if it did it would act as an even stronger competitive inhibitor than its vertebrate counterpart because it would not be cleaved efficiently. The curves labeled from t0 to t4 indicate the evolution over time of the free Tld and free Chd gradients. Note that the final level of free Tld on the ventral side (arrows) differs without or with BMP4 feedback inhibition, but that the levels of free Tld or Chd on the dorsal midline are not affected.|