Geach TJ and Dale L (2008), Molecular determinants of Xolloid action in vivo.

XB-ART-38123

J Biol Chem 2008 Oct 03;28340:27057-63. doi: 10.1074/jbc.M804232200.

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Molecular determinants of Xolloid action in vivo.

Geach TJ , Dale L .

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Xld (Xolloid) is a member of the Tolloid family of metalloproteases found in embryos of the frog Xenopus laevis. It cleaves Chordin, an inhibitory binding protein for BMP2/4, releasing fragments with reduced affinity for these important ventralizing signals. As a consequence, increasing Xld activity ventralizes Xenopus embryos. We have used this phenotype as an assay to determine the requirement for the C-terminal, nonprotease component of Xld for in vivo activity. This part of the protein is composed of five complement C1r/C1s-sea urchin epidermal growth factor-BMP1 (CUB) and two epidermal growth factor domains, which are thought to be involved in protein-protein interactions and may confer substrate specificity. Our results show that the protease coupled to CUB1 and CUB2 is the minimum domain structure required to ventralize Xenopus embryos and to block the dorsal axis-inducing activity of Chordin. Xld-CUB1-CUB2 cleaves Chordin, and a protease-inactive version co-precipitates Chordin. Our results indicate that the first and second CUB domains bind Chordin and present it to the protease domain. Protease-inactive Xld blocks the cleavage of Chordin by wild-type Xld and dorsalizes injected Xenopus embryos. We find that protease-inactive Xld-CUB1-CUB2 does not share this activity and that all of the C-terminal domains are required to generate the dorsalized phenotype.

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Species referenced: Xenopus laevis
Genes referenced: bmp1 chrd egf myc tll2

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	FIGURE 1. Domain structure of wild-type and truncated Xld. Shown are schematic diagrams of wild-type and C-terminally truncated versions of Xld used in this study. P, proregion; MP, metalloprotease domain, C1–5, CUB domains; E, EGF domains (2); M, Myc tag, 9E10 epitopes.
	FIGURE 2. Secretion analysis of Xld deletion constructs. A, experimental design. Embryos were injected with mRNAs (800 pg/blastomere) at the 2-cell stage, and animal caps were isolated from late blastulae. Caps were incubated in calcium- and magnesium-free medium (CMFM). B, Western blot analysis of injected animal caps and culture media. All mRNAs were translated, and Xld proteins were detected in animal caps, including Xld (160 kDa), XldC1 (80 kDa), XldC2 (80 kDa), XldC1-C2 (90 kDa), XldC1–C3 (130 kDa), XldC3–C5 (130 kDa), and XldC1 C3–C5 (135kDa). N-terminally processed forms (indicated with an asterisk) were also detected in animal caps expressing Xld (135 kDa), XldC2 (60 kDa), and XldC1–C3 (105 kDa). Most of these proteins were secreted by animal cap cells, with only XldC3–C5 and XldC1 C3–C5 being completely retained by the cells. N-terminally processed forms were predominant in Xld, XldC2, and XldC1–C3 media and were also detected in XldC1 (60 kDa) and XldC1-C2 (70 kDa) media.
	FIGURE 3.CUB1 and CUB2 are required for the ventralizing activity of Xld. A, experimental design. All embryos were injected with 800 pg of mRNA/blastomere. B, stage 26 control embryo; C, ventralized xld-injected embryo (n 23). Note the reduced head and enlarged ventral-posterior region, characteristic of weakly ventralized embryos. A similar phenotype was observed following injection of xldC1–C3 (n 35) (D) and xldC1-C2 (n 23) (E) mRNAs. Normal embryos were observed following injection of xldC1 (n 25) (F), xldC2 (n 37) (G), and xldC3–C5 (n 32) (H) mRNAs. Not shown are xldC1 C3–C5-injected embryos, which were also normal (n 31).
	FIGURE 4. CUB1 and CUB2 are required for Xld to block Chordin. Xenopus embryos were co-injected (two ventral blastomeres at the 4-cell stage) with 800 pg of chordin mRNA and 800 pg of xld mRNAs and incubated until sibling controls (A) had reached stage 26. B, injecting chordin (Chd) alone induces a second dorsal axis (*) that lacks a head. (58%, n 139). Axis induction by Chordin is blocked by coinjecting either xld (0%, n 77)(C), xldC1–C3 (0%, n 74) (D), or xldC1-C2 (0%, n 74) (E) mRNAs but not by coinjecting either xldC1 (45%, n 82) (F), xldC2 (69%, n 35) (G), or xldC3–C5 (28%, n 76) (H) mRNAs.
	FIGURE 5. CUB1 and CUB2 are required for Xld to cleave Chordin. A, schematic diagram of HA-tagged Chordin, showing the location of cysteine-rich repeats (R1–R4) and Xld cleavage sites (arrows). The 120-kDa full-length protein and the 96-kDa N-terminally cleaved product are shown. B, Western blot analysis of HA-tagged Chordin in culture media and cells from animal caps injected with 800 pg of chordin and 400 pg of xld mRNAs. The full-length protein was detected in all cell extracts and culture media, demonstrating efficient translation of injected chordin mRNA and secretion of Chordin protein. Chordin levels were always much lower in the medium of XldC1-expressing caps than in the media of caps expressing other Xld constructs. The 96-kDa Chordin fragment was only detected in culture media for animal caps co-expressing Xld or XldC1-C2, demonstrating that only these constructs correctly cleave Chordin at the N-terminal site. The cleaved C-terminal fragment of Chordin was not detected in these experiments.
	FIGURE 6. CUB1 and CUB2 are sufficient for Xld to co-precipitate Chordin. A, schematic diagram of protease-inactive XldY296N (labeled Xld) illustrating conversion of tyrosine 296 to asparagine. B, Western blot analysis of HA-tagged Chordin co-precipitated from embryo culture medium by Myctagged Xld, XldC1–C3, and XldC1-C2 but not XldC1 (top). Cell extracts were also analyzed for Chordin-HA (middle) and XldMyc constructs (bottom) to confirm translation of the injected mRNAs.
	FIGURE 7. All of the C-terminal domains are required for XldY296N to dorsalize Xenopus embryos. A, stage 25 uninjected embryo. B, hyperdorsalized phenotype of embryos (84%, n 113) injected with 1.6 ng of xldY296N (xld) mRNA. Embryos have an enlarged head and reduced tail. C, normal morphology of embryos injected with 1.6 ng of xldC1–C3 mRNA (n 69). D, normal morphology of embryos injected with 1.6 ng of xldC1–C4 mRNA (n 124). Normal morphology was also observed for embryos injected with xldC1-C2, xldC1, and xldC2.
	FIGURE 8. All of the C-terminal domains are required for dominant negative activity of Xld. A histogram shows frequency of induction of secondary dorsal axes following injection of 800 pg of chordin (chd), 200 pg of xld, and 400 pg of xldY296N (Xld) mRNAs. Axis induction by Chordin was blocked by coexpressing Xld, and this was in turn blocked by coexpressing XldC1–C5 but not by coexpressing either XldC1–C4, XldC1–C3, or Xld*C1-C2.