BMP-binding modules in chordin: a model for signalling regulation in the extracellular space

Abstract

A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin, a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. In this study, we show that the Chordin CRs define a novel protein module for the binding and regulation of BMPs. The biological activity of Chordin resides in the CRs, especially in CR1 and CR3, which have dorsalizing activity in Xenopus embryo assays and bind BMP4 with dissociation constants in the nanomolar range. The activity of individual CRs, however, is 5- to 10-fold lower than that of full-length Chordin. These results shed light on the molecular mechanism by which Chordin/BMP complexes are regulated by the metalloprotease Xolloid, which cleaves in the vicinity of CR1 and CR3 and would release CR/BMP complexes with lower anti-BMP activity than intact Chordin. CR domains are found in other extracellular proteins such as procollagens. Full-length Xenopus procollagen IIA mRNA has dorsalizing activity in embryo microinjection assays and the CR domain is required for this activity. Similarly, a C. elegans cDNA containing five CR domains induces secondary axes in injected Xenopus embryos. These results suggest that CR modules may function in a number of extracellular proteins to regulate growth factor signalling.

Fig. 1

The biological activity of Chordin resides in the cysteine-rich repeats (CRs). Mouse chordin was divided into three parts, each with a mouse Chordin signal peptide (SP). These vectors did not contain an epitope-tag. Synthetic mRNA from these constructs was injected ventrally into Xenopus embryos (100 pg mRNA/embryo). The individual CR constructs used in subsequent experiments are shown at the top, each with a mouse Chordin signal peptide followed by a Myc epitope tag.

Fig. 2

Individual CRs, particularly CR1 and CR3, exhibit dorsalizing activity and can bind BMP4 directly. (A) Representative phenotypes after synthetic mRNA from individual CR constructs was injected ventrally into Xenopus embryos (100 pg per embryo). (B) Summary of the injection phenotypes from two independent experiments. The percentage of dorsoanteriorized embryos (short trunk, large head and cement gland) is shown in blue and that of embryos with secondary axes is indicated in red (number of embryos ranges between 35 and 83 embryos per sample). (C) RT-PCR analysis of dorsalization of ventral marginal zones (VMZs). VMZ explants were treated with 20 nM of each CR protein. α-Actin is a dorsal mesoderm marker and elongation factor-1α (EF-1α) was used as a measure of RNA recovery. (D) Western blot analysis of BMP4 (1.5 nM) bound to the individual CRs (2 nM) after immunoprecipitation with anti-Myc polyclonal antibody.

Fig. 3

Biochemical analysis of the binding of CR1 to BMP4. (A) Equilibrium binding of increasing concentrations of BMP4 to 0.5 nM CR1 protein. Two independent experiments were performed. Scatchard analysis (inset) yields a K_D of 2.4 nM. Immunoprecipitates were resolved in western blots, developed with anti-BMP4 monoclonal antibodies and quantitated with a Phosphoimager (B) The binding of CR1 to BMP4 can be competed by BMP2, but not by Activin, EGF, IGF or TGFβ1. 10 nM CR1 was incubated with 5.0 nM BMP4 and with a 10-fold molar excess of BMP2, Activin, EGF, IGF or TGFβ1. BMP binding was analyzed by immunoprecipitation with polyclonal anti-myc antibodies and western blot with a monoclonal anti-BMP4 antibody. To measure CR1 recovery after immunoprecipitation the same membrane was stripped and probed again with an anti-Myc monoclonal antibody.

Fig. 4

CR1 has less activity than full-length Chordin. (A) RT-PCR analysis of dorsalization of ventral marginal zones (VMZ). VMZ explants were treated with 10 nM Xenopus Chordin (XChd) or Mouse Chordin (MChd) protein, and 10 or 80 nM CR1 protein. α-Actin is a dorsal mesoderm marker and EF-1α was used as loading control. (B) Histogram showing the percentage of embryos with dorsalized phenotypes (either dorsalization of the entire embryo or secondary axes) after single ventral injections of equimolar amounts of synthetic mRNA for mouse chordin (open bars) or CR1 mRNA (filled bars); full-length chordin is more active than CR1. (C) Binding of BMP4 to a BMPR-Fc fusion protein is competed more effectively by full-length Xenopus Chordin than by CR1 of mouse or Xenopus (not shown) origin. cm, conditioned medium control. (D) Hypothetical model showing that full-length Chordin binds BMP4 (one dimer per Chd monomer, Piccolo et al., 1996) with higher affinity (K_D 3×10⁻¹⁰ M) than CR1 alone (K_D 2.4×10⁻⁹ M). Chordin blocks signalling via BMP receptors more effectively than the individual CR repeats. The cleavage sites of Xolloid protease on its Chordin substrate are indicated by arrows.

Fig. 5

Procollagen IIA is expressed in dorsal mesoderm (notochord and somites) at stages in which chordin expression decreases. Digoxigenin-labeled antisense chordin and type IIA procollagen probes were hybridized to embryos at stage 13 (A,A′); stage 16 (B,B′); stage 19 (C,C′) and stage 23 (D,D′). All embryos are viewed from the dorsal side.

Fig. 6

The cysteine-rich domain of Xenopus type IIA procollagen binds BMP4. (A) Western blot analysis of BMP4 (5 nM) bound to CR1, CR2 or Coll-CR (10 nM) after immunoprecipitation with an anti-Myc polyclonal antibody. In the lower panel the same membrane was probed with a monoclonal anti-Myc antibody to detect protein recovery after immunoprecipitation. (B) Immunoprecipitation assay in which the binding of BMP4 was competed with a 10-fold excess of BMP2, activin, EGF and IGF and TGFβ1; note that only BMP2 and TGFβ1 compete. (C) Sequence comparison of procollagen IIA CR to those of other secreted proteins. Coll-CR, type IIA Xenopus procollagen; CR2, murine chordin second repeat; Nel, rat nel (accession no. U48246); TSP-1, chicken thrombospondin 1 (no. M60853); Pxdasin, Drosophila peroxidasin (no. D86983); C.eleg. EST, C. elegans hypothetical protein containing five procollagen-like domains (no. CAA94866). Black boxes, identical residues present in all sequences; dark gray boxes, identical residues present in some CR domains; light gray boxes, similar amino acids. Alignments made with the GCG sequence analysis pileup program.

Fig. 7

Xenopus type IIA procollagen has anti-BMP activity. (A) Ventral injection of Xenopus procollagen IIA mRNA (400 pg) induces secondary axes (61%, n=32). Insets show that the secondary axes contain muscle stained with the MZ 12−101 mAb and also seen in the histological section. (B) Injection of a similar construct in which the CR domain was deleted to generate procollagen IIB does not induce twinning. (C-F) Dorsalization of ventral marginal zone explants. 8-cell embryos were injected twice ventrally with (C) H₂O, (D) chordin (100 pg mRNA/injection), (E) type IIA coll-CR (200 pg/injection) and, (F) full-length procollagen IIA (200 pg/injection). VMZ explants were excised at early gastrula and cultured until stage 27. (G) RT-PCR analysis of VMZ explants treated as above; the expression of the dorsal marker α-actin and EF-1α were analyzed by RT-PCR. Note that full-length collagen, but not the CR domain alone, can dorsalize mesoderm. (H) Secondary axes caused by microinjection of C. elegans CAA94886 synthetic mRNA (800 pg) encoding a protein containing multiple CR repeats (58% axes, n=31).