Mammalian tolloid metalloproteinase,and not matrix metalloprotease 2 or membrane type 1 metalloprotease,processes laminin-5 in keratinocytes and skin

Laminin-5, a major adhesive ligand for epithelial cells, undergoes processing of its gamma2 and alpha3 chains. This study investigated the mechanism of laminin-5 processing by keratinocytes. BI-1 (BMP-1 isoenzyme inhibitor-1), a selective inhibitor of a small group of astacin-like metalloproteinases, which includes bone morphogenetic protein 1 (BMP-1), mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mammalian Tolloid-like 2 (mTLL-2), inhibited the processing of laminin-5 gamma2 and alpha3 chains in keratinocyte cultures in a dose-dependent manner. In a proteinase survey, all BMP-1 isoenzymes processed human laminin-5 gamma2 and alpha3 chains to 105- and 165-kDa fragments, respectively. In contrast, MT1-MMP and MMP-2 did not cleave the gamma2 chain of human laminin-5 but processed the rat laminin gamma2 chain to an 80-kDa fragment. An immunoblot and quantitative PCR survey of the BMP-1 isoenzymes revealed expression of mTLD in primary keratinocyte cultures but little or no expression of BMP-1, mTLL-1, or mTLL-2. mTLD was shown to cleave the gamma2 chain at the same site as the previously identified BMP-1 cleavage site. In addition, mTLD/BMP-1 null mice were shown to have deficient laminin-5 processing. Together, these data identify laminin-5 as a substrate for mTLD, suggesting a role for laminin-5 processing by mTLD in the skin.     

Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.     

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

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 processes probiglycan

Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays important roles in regulating the deposition of fibrous extracellular matrix in vertebrates, including provision of the procollagen C-proteinase activity that processes the major fibrillar collagens I-III. Biglycan, a small leucine-rich proteoglycan, is a nonfibrillar extracellular matrix component with functions that include the positive regulation of bone formation. Biglycan is synthesized as a precursor with an NH(2)-terminal propeptide that is cleaved to yield the mature form found in vertebrate tissues. Here, we show that BMP-1 cleaves probiglycan at a single site, removing the propeptide and producing a biglycan molecule with an NH(2) terminus identical to that of the mature form found in tissues. BMP-1-related proteases mammalian Tolloid and mammalian Tolloid-like 1 (mTLL-1) are shown to have low but detectable levels of probiglycan-cleaving activity. Comparison shows that wild type mouse embryo fibroblasts (MEFs) produce only fully processed biglycan, whereas MEFs derived from embryos homozygous null for the Bmp1 gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and MEFs homozygous null for both the Bmp1 gene and the mTLL-1 gene Tll1 produce only unprocessed probiglycan. Thus, all detectable probiglycan-processing activity in MEFs is accounted for by the products of these two genes.     

The mammalian Tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart.

Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. To investigate possible roles for mTLL-1 in mammalian development, we have used gene targeting in ES cells to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants were embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that were apparently confined solely to the heart. Constant features were incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression was specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c. embryos, respectively. Tll1 expression was also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, was not observed. Cardiac structures that were not affected in Tll1-/- embryos either showed no Tll1 expression (atrio-ventricular cushions) or showed overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum.     

Identification of the minimal domain structure of bone morphogenetic protein-1 (BMP-1) for chordinase activity: chordinase activity is not enhanced by procollagen C-proteinase enhancer-1 (PCPE-1)

Bone morphogenetic protein 1 (BMP-1), which is a tolloid member of the astacin-like family of zinc metalloproteinases, is a highly effective procollagen C-proteinase (PCP) and chordinase. On the other hand, mammalian tolloid like-2 (mTLL-2) does not cleave chordin or procollagen; procollagen is cleaved by mTLL-2 in the presence of high levels of procollagen C-proteinase enhancer-1 (PCPE-1), for reasons that are unknown. We used these differences in activity between BMP-1 and mTLL-2 to narrow in on the domains in BMP-1 that specify PCP and chordinase activity. Using a domain swap approach, we showed that: 1) the metalloproteinase and CUB2 domains of BMP-1 are absolutely required for PCP activity; swaps with either of the corresponding domains in BMP-1 and mTLL-2 did not result in procollagen cleavage and 2) the proteinase domain of mTLL-2 can cleave chordin if coupled to the CUB1 domain of BMP-1. Therefore, the minimal structure for chordinase activity comprises a metalloproteinase domain (either from BMP-1 or from mTLL-2) and the CUB1 domain of BMP-1 (the CUB1 domain of mTLL-2 cannot substitute for the CUB1 domain of BMP-1). We showed that the minimal procollagen C-proteinase (BMP-1 lacking the EGF and CUB3 domain) was enhanced by PCPE-1 but not as well as BMP-1 retaining the CUB3 domain. Further studies showed that PCPE-1 had no effect on the ability of BMP-1 to cleave chordin. The data support a previously suggested mechanism of PCPE-1 whereby PCPE-1 interacts with procollagen, but in addition, the CUB3 domain of BMP-1 appears to augment the interaction.     

bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis.     

A complete domain structure of Drosophila tolloid is required for cleavage of short gastrulation

Drosophila tolloid (TLD) is a member of a family of proteinases that play important roles in development and includes mammalian tolloid (mTLD) and bone morphogenetic protein (BMP)-1. TLD accentuates the activity of decapentaplegic (DPP), a transforming growth factor beta superfamily growth factor, by cleaving its antagonist Short gastrulation (Sog). Similarly, the activity of BMP-2/4 (vertebrate homologues of DPP) is augmented by cleavage of chordin. However, whereas TLD is an effective Sogase, mTLD is a poor chordinase and is functionally replaced by its smaller splice variant BMP-1, which lacks the most C-terminal epidermal growth factor (EGF)-like and CUB domains of mTLD. Moreover, the minimal chordinase activity resides in the N-terminal half of BMP-1. This study showed that the proteolytic activity of TLD is considerably enhanced by Ca2+ and tested the hypothesis that the Sogase activity of TLD resides in the N-terminal half of the proteinase. Unexpectedly, it was found that TLD lacking the CUB4 and CUB5 domains and/or the EGF-like domains was unable to cleave Sog. Loss of function mutations have been reported in the tld gene that result in amino acid substitutions at E835K (in CUB4), S915L (in CUB5), and N760I (in EGF2) in TLD. The CUB mutants were found to be ineffective Sogases, but the activity of the EGF2 mutant was unchanged. The results show that substrate recognition and cleavage by Drosophila tolloid and mTLD are different despite their identical domain structure and homologous functions in patterning. The result that the N760I mutant has full Sogase activity suggests that novel substrates for TLD exist.     

Proteinases of the bone morphogenetic protein-1 family convert procollagen VII to mature anchoring fibril collagen

Collagen VII is the major structural component of the anchoring fibrils at the dermal-epidermal junction in the skin. It is secreted by keratinocytes as a precursor, procollagen VII, and processed into mature collagen during polymerization of the anchoring fibrils. We show that bone morphogenetic protein-1 (BMP-1), which exhibits procollagen C-proteinase activity, cleaves the C-terminal propeptide from human procollagen VII. The cleavage occurs at the BMP-1 consensus cleavage site SYAA/DTAG within the NC-2 domain. Mammalian tolloid-like (mTLL)-1 and -2, two other proteases of the astacin enzyme family, were able to process procollagen VII at the same site in vitro. Immunohistochemical and genetic evidence supported the involvement of these enzymes in cleaving type VII procollagen in vivo. Both BMP-1 and mTLL-1 are expressed in the skin and in cultured cutaneous cells. A naturally occurring deletion in the human COL7A1 gene, 8523del14, which is associated with dystrophic epidermolysis bullosa and eliminates the BMP-1 consensus sequence, abolished processing of procollagen VII, and in mutant skin procollagen VII accumulated at the dermal-epidermal junction. On the other hand, deficiency of BMP-1 in the skin of knockout mouse embryos did not prevent processing of procollagen VII to mature collagen, suggesting that mTLL-1 and/or mTLL-2 can substitute for BMP-1 in the processing of procollagen VII in situ.     

Structural basis for the substrate specificity of bone morphogenetic protein 1/tolloid-like metalloproteases

Procollagen C-peptidase, also known as bone morphogenetic protein 1 (BMP-1), is a multidomain, zinc endopeptidase of the astacin M12A family. BMP-1 is the prototype of a small group of proteases that have key roles in extracellular matrix formation and morphogenesis. BMP-1, its splice form mTLD, and the related proteases TLL-1 and TLL-2 are considered as promising drug targets for the treatment of excessive fibrosis and muscle wasting. We report here the crystal structures of the protease domains of human BMP-1 and the closely related Tolloid-like protease 1 (TLL-1). The crystal structures reveal an unexpected conformation of a cysteine-rich loop within the active site, and suggest that a flap movement is required in order to allow substrate binding. On the basis of these substantial differences between the BMP-1 and astacin active sites, a structural basis for their differing substrate specificities is proposed.     

Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer

The processing of the fibrillar procollagen precursors to mature collagens is an essential requirement for fibril formation. The enzymes involved in these events are known as the procollagen N and C proteinases. The latter, which cleaves the C-propeptides of the fibrillar procollagens I-III, is identical to the previously described bone morphogenetic protein-1 (BMP-1). Surprisingly, unlike the other fibrillar collagens, the processing of the C-propeptide domain of the procollagen V homotrimer was found to be mediated by furin rather than BMP-1. However, the presence of putative BMP-1 cleavage sites in the alpha1(V) C-propeptide sequence prompted us to reconsider the procollagen V C-propeptide cleavage by BMP-1. Using a recombinant system to produce substantial amounts of the proalpha1(V) homotrimer, we have previously shown that the C-propeptide is spontaneously released in the culture medium. The trimeric C-propeptide fragment, resulting from the furin cleavage, still encompassed the predicted BMP-1 cleavage sites. It was purified and tested as a substrate for BMP-1. In parallel, the release of the C-propeptide in the culture medium was inhibited by the addition of a specific furin inhibitor, allowing the re-examination of BMP-1 activity on the intact molecule. We showed that BMP-1 does cleave both substrates at one of the two predicted C-proteinase cleavage sites. Our results favor a role for PCP/BMP-1 in physiological C-terminal processing of procollagen V and imply a general mechanism for fibrillar collagen C-terminal processing.     

Biosynthetic processing of the pro-alpha 1(V)2pro-alpha 2(V) collagen heterotrimer by bone morphogenetic protein-1 and furin-like proprotein convertases.

The low abundance fibrillar collagen type V is incorporated into and regulates the diameters of type I collagen fibrils. Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays key roles in regulating formation of vertebrate extracellular matrix; it cleaves the C-propeptides of the major fibrillar procollagens I-III and processes precursors to produce the mature forms of the cross-linking enzyme prolysyl oxidase, the proteoglycan biglycan, and the basement membrane protein laminin 5. Here we have successfully produced recombinant pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, and we have used these to characterize biosynthetic processing of the most prevalent in vivo form of type V procollagen. In addition, we have compared the processing of endogenous pro-alpha1(V) chains by wild type mouse embryo fibroblasts and by fibroblasts derived from embryos doubly homozygous null for the Bmp-1 gene and for a gene encoding the closely related metalloprotease mammalian Tolloid-like 1. Together, results presented herein indicate that within pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, pro-alpha1(V) N-propeptides and pro-alpha2(V) C-propeptides are processed by BMP-1-like enzymes, and pro-alpha1(V) C-propeptides are processed by furin-like proprotein convertases in vivo.     

The interaction of recombinant subdomains of the procollagen C-proteinase with procollagen I provides a quantitative explanation for functional differences between the two splice variants, mammalian tolloid and bone morphogenetic protein 1

The procollagen C-proteinase (PCP) is a zinc peptidase of the astacin family and the metzincin superfamily. The enzyme removes the C-terminal propeptides of fibrillar procollagens and activates other matrix proteins. Besides its catalytic protease domain, the procollagen C-proteinase contains several C-terminal CUB modules (named after complement factors C1r and C1s, the sea urchin UEGF protein, and BMP-1) and EGF-like domains. The two major splice forms of the C-proteinase differ in their overall domain composition. The longer variant, termed mammalian tolloid (mTld, i.e., PCP-2), has the protease-CUB1-CUB2-EGF1-CUB3-EGF2-CUB4-CUB5 composition, whereas the shorter form termed bone morphogenetic protein 1 (BMP-1, i.e., PCP-1) ends after the CUB3 domain. Two related genes encode proteases similar to mTld in humans and have been termed mammalian tolloid like-1 and -2 (mTll-1 and mTll-2, respectively). For mTll-1, it has been shown that it has C-proteinase activity. We demonstrate that recombinant EGF1-CUB3, CUB3, CUB3-EGF2, EGF2-CUB4, and CUB4-CUB5 modules of the procollagen C-proteinase can be expressed in bacteria and adopt a functional antiparallel beta-sheet conformation. As shown by surface plasmon resonance analysis, the modules bind to procollagen I in a 1:1 stoichiometry with dissociation constants (K(D)) ranging from 622.0 to 1.0 nM. Their binding to mature collagen I is weaker by at least 1 order of magnitude. Constructs containing EGF domains bind more strongly than those consisting of CUB domains only. This suggests that a combination of CUB and EGF domains serves as the minimal functional unit. The binding affinities of the EGF-containing modules for procollagen increase in the order EGF1-CUB3 < CUB3-EGF2 < EGF2-CUB4. In the context of the full length PCP, this implies that a given module has an affinity that continues to increase the more C-terminally the module is located within the PCP. The tightest binding module, EGF2-CUB4 (K(D) = 1.0 nM), is only present in mTld, which might provide a quantitative explanation for the different efficiencies of BMP-1 and mTld in procollagen C-proteinase activity.     

Deletion of epidermal growth factor-like domains converts mammalian tolloid into a chordinase and effective procollagen C-proteinase

Bone morphogenetic protein (BMP)-1 and mammalian tolloid (mTld) are Ca(2+)-dependent metalloproteinases that result from alternative splicing of the bmp1 gene. They have different proteinase activities, e.g. BMP-1 effectively cleaves procollagen (an extracellular matrix protein) and chordin (a BMP antagonist), whereas mTld is a poor procollagen proteinase and will not cleave chordin in the absence of twisted gastrulation. This is perplexing because mTld (being the longer variant) might be expected to cleave all substrates cleaved by BMP-1. Studies have shown that the minimal structure for procollagen proteinase activity is proteinase-CUB1-CUB2 (BMP-1DeltaEC3) and therefore lacking the epidermal growth factor (EGF)-like domain thought to account for the Ca(2+) dependence of BMP-1. In this study we generated three deletion mutants of mTld that lacked either one or both EGF-like domains (referred to as "mTld-DeltaEGF"). The mutated proteins were poorly but sufficiently secreted from 293-EBNA cells for in vitro assays of procollagen and chordin cleavage. Most surprisingly, the mTld-DeltaEGF mutants required Ca(2+) for proteolytic activity, thereby showing that the EGF-like domains do not account for the Ca(2+) dependence of BMP-1/mTld. Moreover, the mTld-DeltaEGFs are effective procollagen proteinases and cleave chordin. Furthermore, BMP-1DeltaEC3 cleaves chordin and requires Ca(2+) for activity. Studies using nondenaturing gels showed that mTld molecules lacking EGF-like domains have a loose conformation such that in the presence of Ca(2+) binding sites for chordin and procollagen on the "BMP-1-part" of the molecule are exposed. We propose that the EGF-like domains could hold CUB4/5 domains in locations that exclude substrates cleavable by BMP-1.     

BMP-1/Tolloid-like metalloproteases process endorepellin, the angiostatic C-terminal fragment of perlecan

Endorepellin, the C-terminal domain of the heparan sulfate proteoglycan perlecan, possesses angiostatic activity. The terminal laminin-like globular (LG3) domain of endorepellin appears to possess most of the biological activity on endothelial cells. LG3 protein has been detected in the urine of patients with end-stage renal disease and in the amniotic fluid of pregnant women with premature rupture of fetal membranes. These findings suggest that proteolytic processing of endorepellin and the generation of LG3 might have biological significance. In this study, we have identified specific enzymes of the bone morphogenetic protein-1 (BMP-1)/Tolloid family of metalloproteases that cleave LG3 from recombinant endorepellin at the physiologically relevant site and that cleave LG3 from endogenous perlecan in cultured mouse and human cells. The BMP-1/Tolloid family of metalloproteases is thereby implicated in the processing of a major basement membrane proteoglycan and in the liberation of an anti-angiogenic factor. Using molecular modeling, site-directed mutagenesis and angiogenic assays, we further demonstrate that LG3 activity requires specific amino acids involved in Ca(2+) coordination.     

Dentin sialophosphoprotein (DSPP) is cleaved into its two natural dentin matrix products by three isoforms of bone morphogenetic protein-1 (BMP1)

The protease that cleaves the most abundant non-collagenous protein of dentin matrix, dentin sialophosphoprotein (DSPP), into its two final dentin matrix products, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), has not been directly identified. In this study, full-length recombinant mouse DSPP was made for the first time in furin-deficient mammalian LoVo cells and used to test the ability of three different isoforms of one candidate protease, bone morphogenetic protein-1 (BMP1) to cleave DSPP at the appropriate site. Furthermore, two reported enhancers of BMP1/mTLD activity (procollagen C-endopeptidase enhancer-1, PCPE-1, and secreted frizzled-related protein-2, sFRP2) were tested for their abilities to modulate BMP1-mediated processing of both DSPP and another SIBLING family member with a similar cleavage motif, dentin matrix protein-1 (DMP1). Three splice variants of BMP1 (classic BMP1, the full-length mTolloid (mTLD), and the shorter isoform lacking the CUB3 domain, BMP1-5) were all shown to cleave the recombinant DSPP in vitro although mTLD was relatively inefficient at processing both DSPP and DMP1. Mutation of the MQGDD peptide motif to IEGDD completely eliminated the ability of all three recombinant isoforms to process full-length recombinant DSPP in vitro thereby verifying the single predicted cleavage site. Furthermore when human bone marrow stromal cells (which naturally express furin-activated BMP1) were transduced with the adenovirus-encoding either wild-type or mutant DSPP, they were observed to fully cleave wild-type DSPP but failed to process the mutant DSPP^MQΔIE during biogenesis. All three BMP1 isoforms were shown to process type I procollagen as well as DSPP and DMP1 much more efficiently in low-salt buffer (≤ 50 mM NaCl) compared to commonly used normal saline buffers (150 mM NaCl). Neither PCPE-1 nor sFRP2 were able to enhance any of the three BMP1 isoforms in cleaving either DSPP or DMP1 under either low or normal saline conditions. Interestingly, we were unable to reproduce sFRP2's reported ability to enhance the processing of type I procollagen by BMP1/mTLD. In summary, three isoforms of BMP1 process both DSPP and DMP1 at the MQX/DDP motif, but the identity of a protein that can enhance the cleavage of the two SIBLING proteins remains elusive.     

Mammalian tolloid-like 1 binds procollagen C-proteinase enhancer protein 1 and differs from bone morphogenetic protein 1 in the functional roles of homologous protein domains

Bone morphogenetic protein 1 (BMP1) is the prototype of a subgroup of metalloproteinases with manifold roles in morphogenesis. Four mammalian subgroup members exist, including BMP1 and mammalian Tolloid-like 1 (mTLL1). Subgroup members have a conserved protein domain structure: an NH2-terminal astacin-like protease domain, followed by a fixed order of CUB and epidermal growth factor-like protein-protein interaction motifs. Previous structure/function studies have documented those BMP1 protein domains necessary for secretion, and activity against various substrates. Here we demonstrate that, in contradiction to previous reports, the most NH2-terminal CUB domain (CUB1) is not required for BMP1 secretion nor is the next CUB domain (CUB2) required for enzymatic activity. The same is true for mTLL1. In fact, secreted protease domains of BMP1 and mTLL1, devoid of CUB or epidermal growth factor-like domains, have procollagen C-proteinase (pCP) activity and activity for biosynthetic processing of biglycan, the latter with kinetics superior to those of the full-length proteins. Structure-function analyses herein also suggest differences in the functional roles played by some of the homologous domains in BMP1 and mTLL1. Surprisingly, although BMP1 has long been known to be Ca2+-dependent, a property previously assumed to apply to all members of the subgroup, mTLL1 is demonstrated to be independent of Ca2 levels in its ability to cleave some, but not all, substrates. We also show that pCP activities of only versions of BMP1 and mTLL1 with intact COOH termini are enhanced by the procollagen C-proteinase enhancer 1 (PCOLCE1) and that mTLL1 binds PCOLCE1, thus suggesting reappraisal of the accepted paradigm for how PCOLCE1 enhances pCP activities.     

Site-specific interaction of bone morphogenetic protein 2 with procollagen II

Bone morphogenetic proteins (BMPs) play a critical role in embryo development, organogenesis, and regeneration of damaged tissues. Biological activity of BMPs depends on their local concentration, which is regulated by intracellular enzymatic processing of pro-BMPs, and then the binding of secreted BMPs to antagonizing extracellular proteins. It has been suggested that BMPs interact with structural proteins of the extracellular matrix, but this process is poorly understood. To study interactions of BMPs with fibrillar collagens in detail we expressed recombinant procollagen II variants in which specific domains that correspond to the D-periods were deleted. Subsequently, the procollagen II variants were used in biosensor and immuno-precipitation binding assays to map the regions of procollagen II with a high affinity for the BMP-2. Our data suggest that interaction of BMP-2 with procollagen II is site-specific, and that the high-affinity binding site is located in the D4-period of the collagen triple helix. We hypothesize that the binding of BMP-2 to collagen II reflects a general mechanism of interaction between the fibrillar collagens and morphogens that belong to the transforming growth factor (TGF)-beta superfamily.     

Bone morphogenetic protein-1/Tolloid-like proteinases process dentin matrix protein-1

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 beta-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, NH2-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-Tolloid-like proteinases in formation of mineralized tissues is indicated, via proteolytic processing of DMP1.     

Biosynthetic processing of the Pro-alpha1(V)Pro-alpha2(V)Pro-alpha3(V) procollagen heterotrimer

Type V collagen is a quantitatively minor fibrillar collagen comprised of different chain compositions in different tissues. The most widely distributed form, an alpha1(V)2alpha2(V) heterotrimer, regulates the physical properties of type I/V heterotypic collagen fibrils via partially processed NH2-terminal globular sequences. A less characterized alpha1(V)alpha2(V)alpha3(V) heterotrimer has a much more limited distribution of expression and unknown function(s). We characterized the biosynthetic processing of pro-alpha1(V)2pro-alpha2(V) procollagen previously and showed it to differ in important ways from biosynthetic processing of the major fibrillar procollagens I-III. Here we have successfully produced recombinant pro-alpha1(V)pro-alpha2(V)pro-alpha3(V) heterotrimers. We use these, and mouse embryo fibroblasts doubly homozygous null for the Bmp1 gene, which encodes the metalloproteinase bone morphogenetic protein-1 (BMP-1), and for a gene encoding the closely related metalloproteinase mammalian Tolloid-like 1, to characterize biosynthetic processing of pro-alpha1(V)pro-alpha2(V)pro-alpha3(V) heterotrimers, thus completing characterization of type V collagen biosynthetic processing. Whereas pro-alpha1(V) and pro-alpha2(V) processing in pro-alpha1(V)pro-alpha2(V)pro-alpha3(V) heterotrimers is similar to that which occurs in pro-alpha1(V)2pro-alpha2(V) heterotrimers, the processing of pro-alpha3(V) by BMP-1 occurs at an unexpected site within NH2-terminal globular sequences. We also demonstrate that, despite similarities in NH2-terminal domain structures, pro-alpha2(V) NH2-terminal globular sequences are not cleaved by ADAMTS-2, the metalloproteinase that cleaves the N-propeptides of the major fibrillar procollagen chains.