Molecular Determinants of Xolloid Action in Vivo

Timothy J Geach,Leslie Dale

doi:10.1074/jbc.m804232200

Abstract

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.

Highlights

Our results show that only CUB1 and CUB2 are required for the ventralizing activity of Xld and for its ability to cleave Chordin
Secretion Analysis of Xld Deletion Mutants—A series of deletion mutants were constructed for Xld, removing specific C-terminal C1s-sea urchin epidermal growth factor-BMP1 (CUB) and EGF domains (Fig. 1)
CUB1 and CUB2 Are Required for the Ventralizing Activity of Xld—To test the activity of the Xld deletion mutants we used an in vivo assay in which synthetic mRNA was injected into Xenopus embryos (Fig. 3A)

Summary

Introduction

CUB Domain Specificity of Xld injected Xenopus embryos, blocks the dorsalizing activity of co-injected Chordin, and cleaves Chordin in vitro [20, 23]. Our results show that the distal CUB and EGF domains are not required for either secretion or N-terminal processing of Xld. CUB1 and CUB2 Are Required for the Ventralizing Activity of Xld—To test the activity of the Xld deletion mutants we used an in vivo assay in which synthetic mRNA was injected into Xenopus embryos (Fig. 3A).

Results

Conclusion