Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal-ventral patterning by releasing TGFbeta-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, we identify novel family member mammalian Tolloid-like 2 (mTLL-2) and compare enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2]. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 are shown to cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and to counteract dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis.

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.Bone morphogenetic protein 1 (BMP-1), first identified as a protein of unknown function that copurifies with transforming growth factor beta (TGF-␤)-like BMPs from osteogenic extracts of bone (38), is the prototype of a family of structurally similar metalloproteinases involved in morphogenesis in a broad range of species (4). Surprisingly, despite its association with TGF-␤-like molecules, the first described role for BMP-1 was as a procollagen C-proteinase (pCP) (14, 17): the activity necessary for cleaving C-propeptides from type I to III procollagen precursors to produce mature monomers capable of forming the major collagen fibrils of vertebrate extracellular matrix (ECM) (25). A single gene encodes alternatively spliced RNAs for BMP-1 and for a second protease, mammalian Tolloid (mTLD) (35), that has been shown to have pCP activity in vitro (29, 17). Nevertheless, although embryos homozygous null for the Bmp1 gene, which encodes both proteases, have abnormal collagen fibrils, mouse embryo fibroblasts (MEFs) derived from these embryos have residual pCP activity that is ϳ40% of that of the wild type (33), suggesting the existence of at least one additional mammalian pCP. Searches for additional mammalian BMP-1-related proteases identified two novel, genetically d...

The procollagen COOH-terminal proteinase enhancer (PCPE) is a glycoprotein that binds the COOH-terminal propeptide of type I procollagen and potentiates its cleavage by procollagen C-proteinases, such as bone morphogenetic protein-1 (BMP-1). Recently, sequencing of a human expressed sequence tag, which maps near the primary open angle glaucoma region on chromosome 3q21, showed it to encode a novel protein with only 43% identity with PCPE but with a similar domain structure. Here we show this novel protein to be a functional procollagen COOH-terminal proteinase enhancer with activity comparable with that of PCPE and thus propose the designations PCPE2 and PCPE1, respectively. PCPE2 is shown to have a much more limited distribution of expression than does PCPE1, with strong expression primarily in nonossified cartilage in developing tissues and at high levels in the adult heart. PCPE2 is shown to be a glycoprotein that differs markedly in the nature of its glycosylation from that of PCPE1. PCPE2 is also shown to have markedly stronger affinity for heparin than PCPE1, which may account for higher affinities for cell layers. Unexpectedly, both PCPE1 and PCPE2 were found to be collagen-binding proteins, capable of binding at multiple sites on the triple helical portions of fibrillar collagens and also capable of competing for such binding with procollagen C-proteinases. The latter observations may provide insights into the ways PCPEs affect the kinetics of the C-proteinase reaction and into the physical interactions that occur between procollagen C-proteinases and their substrates.Procollagen precursors of the major fibrillar collagens I-III contain N-and C-propeptides 1 that are cleaved to yield the mature triple helical monomers capable of forming fibrils (1). The C-propeptides are cleaved by procollagen C-proteinase (PCP) activity provided by bone morphogenetic protein-1 (BMP-1) (2, 3) and by other closely related metalloproteinases 2 (3, 4). PCP activity is potentiated by the 55-kDa glycoprotein procollagen C-proteinase enhancer (PCPE) and by 36-and 34-kDa proteolytic fragments of PCPE (5, 6). PCPE contains two NH 2 -terminal CUB domains (7), motifs thought to be involved in protein-protein interactions and found in various proteins with roles in development (8). PCPE also contains a COOHterminal NTR domain (7) that has homology with NH 2 -terminal domains of tissue inhibitors of metalloproteinases and with COOH-terminal domains of netrins, complement components C3, C4, and C5, and secreted frizzled-related proteins (9). The 36-and 34-kDa PCPE fragments retain full PCP-enhancing activity and, like the full-length 55-kDa form, bind type I procollagen C-propeptides (5, 6). Since the 36-and 34-kDa forms contain little or no sequences other than the two CUB domains (7), such abilities appear to reside exclusively in these motifs. Indeed, it has been suggested that the CUB-containing 34-and 36-kDa fragments may possess higher levels of PCPenhancing activity than full-length PCPE (6). In contrast to activities provided...

Bone morphogenetic protein-1 (BMP-1)/Tolloid-like metalloproteinases play key roles in formation of mammalian extracellular matrix (ECM), through the biosynthetic conversion of precursor proteins into their mature functional forms. These proteinases probably play a further role in formation of bone through activation of transforming growth factor ␤-like BMPs. Dentin matrix protein-1 (DMP1), deposited into the ECM during assembly and involved in initiating mineralization of bones and teeth, is thought to undergo proteolysis in vivo to generate functional cleavage fragments found in extracts of mineralized tissues. Here, we have generated recombinant DMP1 and demonstrate that it is cleaved, to varying extents, by all four mammalian BMP-1/ Tolloid-like proteinases, to generate fragments similar in size to those previously isolated from bone. Consistent with possible roles for the BMP-1/Tolloid-like proteinases in the physiological processing of DMP1, NH 2 -terminal sequences of products generated by BMP-1 cleavage of DMP1 match those predicted from processing at the predicted DMP1 site that shows greatest cross-species conservation of sequences. Moreover, fibroblasts derived from mouse embryos homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-like proteinases appear to be deficient in processing of DMP1. Thus, a further role for BMP-1-Tolloidlike proteinases in formation of mineralized tissues is indicated, via proteolytic processing of DMP1.Bone morphogenetic protein-1 (BMP-1) 1 is the prototype of a family of metalloproteinases involved in morphogenesis in a broad range of species (1). These proteinases mediate morphogenetic effects in part by biosynthetic processing of precursors into the mature functional forms of proteins necessary to formation of the extracellular matrix. For example, they provide the procollagen C-proteinase activity that excises the carboxylterminal propeptides of procollagens I-III, to yield the major fibril-forming components of the extracellular matrix (ECM) (2-6). They also participate in the biosynthetic processing of the minor fibrillar collagens V and XI (6 -8), which in turn further regulate the physical properties of types I and II collagen fibers (9, 10). The BMP-1/Tolloid-like proteinases have also been shown to process a precursor to produce the small leucinerich proteoglycan biglycan (11), a molecule that positively regulates bone growth, influences type I collagen fibril morphology, and also may influence dentin mineralization (12-14). The BMP-1/Tolloid-like proteinases have also been implicated in the biosynthetic processing of laminin 5 (15, 16) and type VII collagen (17) and shown to proteolytically activate lysyl oxidase (18), an enzyme required for covalent cross-linking of collagen and elastin fibers. These metalloproteinases may thus be central regulators in the formation of ECM.Type I collagen is the major organic component of mineralized ECM and serves as the template upon which mineral is deposited in tissues such as bone and dentin. Noncol...

Bone morphogenetic protein-1 (BMP-1) plays key roles in regulating the deposition of vertebrate extracellular matrix; it is the procollagen C-proteinase that processes the major fibrillar collagen types I-III, and it may process prolysyl oxidase to the mature enzyme necessary to the formation of covalent cross-links in collagen and elastic fibers. Type V collagen is a fibrillar collagen of low abundance that is incorporated into and helps regulate the shape and diameter of type I collagen fibrils. Here we show that, in contrast to its action on procollagens I-III, BMP-1 does not cleave the C-propeptide of pro-␣1(V) homotrimers. Instead, the single BMP-1-specific cleavage site within pro-␣1(V) chains, lies within the large globular N-propeptide. This cleavage site is immediately upstream of a glutamine, thus redefining the specificity of cleavage for BMP-1-like enzymes. It also produces an NH 2 terminus that corresponds to an equivalent NH 2 terminus on the processed matrix form of the similar ␣1(XI) chain, thus suggesting physiological significance. Cleavage of the C-propeptide occurs efficiently in recombinant pro-␣1(V) homotrimers produced in 293-EBNA human embryonic kidney cells, and this cleavage is shown to occur immediately downstream of the sequence RTRR. This is similar to sites cleaved by subtilisin-like proprotein/prohormone convertases and is shown to be specifically cleaved by the recombinant subtilisin-like proprotein/prohormone convertase furin.Collagen types I-III, the major fibrous components of vertebrate matrix, are synthesized as procollagens, precursors with N-and C-propeptides 1 that are cleaved to yield mature monomers capable of forming fibrils (1-3). In particular, failure to remove the C-propeptide seems incompatible with fibrillogenesis (4). The C-propeptides of procollagens I-III are cleaved by procollagen C-proteinase (5-7), an activity of bone morphogenetic protein-1 (BMP-1) and mammalian tolloid (mTld), two proteins encoded by alternatively spliced mRNAs of the BMP1 gene (8 -10). BMP-1 is the prototype of a subfamily of astacinlike proteases involved in embryogenetic patterning in diverse organisms (11), in some cases by liberating transforming growth factor-␤-like morphogens from latent complexes (12)(13)(14). Thus, identification of BMP-1 as procollagen C-proteinase provided a link between enzymes involved in matrix deposition and genes involved in pattern formation and suggested that such enzymes may be involved in coordinating various molecular events underlying morphogenesis. Monomers of the low abundance or minor fibrillar collagen types V and XI are incorporated into the fibrils of the much more abundant collagen types I and II, respectively, and act as regulators of the sizes and shapes of the resultant heterotypic fibrils (15-21). Type V collagen is most widely distributed in tissues as a heterotrimer of the chain composition ␣1(V) 2 ␣2(V) (22) but is also found, almost exclusively in placenta, as the heterotrimer ␣1(V)␣2(V)␣3(V) (23), and in certain cell types and tissues ...