Bone Morphogenetic Protein-1/Tolloid-like Proteinases Process Dentin Matrix Protein-1

Abstract

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...

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

“…Because of the objective of this study we have not identified the proteins or phosphorylation sites affected by a specific inhibitor. However, a comparison between the sizes of chick OPN (~60 kD), BSP (~70kD), and ON (~30kD) and the reported murine dentin matrix protein 1 (85-90 kD [48)) with the protein bands affected by phosphorylation inhibitors prompts us to speculate that phosphorylation of these proteins may have been reduced. This suggests that the inhibitors were functioning as predicted, and also that extracting with 2M guanidine hydrochloride enriched the extracts with these phosphorylated proteins.…”

Identification of Full-Length Dentin Matrix Protein 1 in Dentin and Bone

“…Bone morphogenetic protein-1/tolloid-like proteinases, which are expressed in the bone and dentin as well as a variety of other tissues/cells [23], have been shown to cleave bacteria-derived recombinant DMP1 in vitro [24]. However, the in vitro cleavage pattern of DMP1 resulting from incubation with bone morphogenetic protein-1/tol-loid-like proteinases appeared different from that found in bone and dentin [12].…”

“…The presence of the COOH-terminal fragment of DMP1 in the nuclei suggests that it might be the COOH-terminal fragment rather than the full-length form that has transcriptional activity. It should be noted that the nuclear localization sequence is present at the COOH-terminal fragment of DMP1 [8].Bone morphogenetic protein-1/tolloid-like proteinases, which are expressed in the bone and dentin as well as a variety of other tissues/cells [23], have been shown to cleave bacteria-derived recombinant DMP1 in vitro [24]. However, the in vitro cleavage pattern of DMP1 resulting from incubation with bone morphogenetic protein-1/tol-loid-like proteinases appeared different from that found in bone and dentin [12].…”

In Vitro Effects of Dentin Matrix Protein-1 on Hydroxyapatite Formation Provide Insights into in Vivo Functions

“…A recent study demonstrated that DMP1 is cleaved by bone morphogenetic protein-1, a metalloprotease important for extracellular matrix development (39). The knowledge that there is at least one and perhaps more than one enzyme linked to bone development important for DMP1 processing may explain the contradictory data relating to the cell culture effects of overexpression of DMP1, resulting in increased mineralization (13) and the time-dependent changes in the knockout in which, in most cases, excessive mineralization is seen, but in others (presumably those in which other PHEX and bone morphogenetic protein-1 substrates are in lower abundance), mineralization is deficient.…”