Use of Bmp1/Tll1 Doubly Homozygous Null Mice and Proteomics To Identify and Validate In Vivo Substrates of Bone Morphogenetic Protein 1/Tolloid-Like Metalloproteinases

Abstract

Bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD), two proteinases encoded by Bmp1, provide procollagen C-proteinase (pCP) activity that converts procollagens I to III into the major fibrous components of mammalian extracellular matrix (ECM). Yet, although Bmp1 ^−/− mice have aberrant collagen fibrils, they have residual pCP activity, indicative of genetic redundancy. Mammals possess two additional proteinases structurally similar to BMP-1 and mTLD: the genetically distinct mammalian Tolloid-like 1 (mTLL-1) and mTLL-2. Mice lacking the mTLL-1 gene Tll1 are embryonic lethal but have pCP activity levels similar to those of the wild type, suggesting that mTLL-1 might not be an in vivo pCP. In vitro studies have shown BMP-1 and mTLL-1 capable of cleaving Chordin, an extracellular antagonist of BMP signaling, suggesting that these proteases might also serve to modulate BMP signaling and to coordinate the latter with ECM formation. However, in vivo evidence of roles for BMP-1 and mTLL-1 in BMP signaling in mammals is lacking. To remove functional redundancy obscuring the in vivo functions of BMP-1-related proteases in mammals, we here characterize Bmp1 Tll1 doubly null mouse embryos. Although these appear morphologically indistinguishable from Tll1 ^−/− embryos, biochemical analysis of cells derived from doubly null embryos shows functional redundancy removed to an extent enabling us to demonstrate that (i) products of Bmp1 and Tll1 are responsible for in vivo cleavage of Chordin in mammals and (ii) mTLL-1 is an in vivo pCP that provides residual activity observed in Bmp1 ^−/− embryos. Removal of functional redundancy also enabled use of Bmp1 ^−/− Tll1 ^−/− cells in a proteomics approach for identifying novel substrates of Bmp1 and Tll1 products.

ACKNOWLEDGMENTS

We thank Nick Morris (Shriners Hospital for Children, Portland, Ore.) and Larry Fisher (National Institute of Dental and Craniofacial Research) for generously providing antibodies against type XI and type I collagens, respectively. We also thank Brigid L. M. Hogan for provision of heterozygous Bmp1-null mice and Cian Leahy, Melvin Ayala, and Sara Tufa for excellent technical assistance.

This work was supported by funds provided by the National Institutes of Health to D.S.G. (GM63471 and AR47746), by a predoctoral fellowship from the American Heart Association awarded to W.N.P., and by National Institutes of Health Predoctoral Training Grant T32 GM07215 in Molecular Biosciences to B.M.S.