Bone Morphogenetic Protein-1 (BMP-1)

Abstract

Bone morphogenetic protein-1 (BMP-1) is a shorter spliced variant of mammalian tolloid (mTld), both of which cleave the C-propeptides of type I procollagen during the synthesis of extracellular matrix collagen fibrils. The fact that BMP-1 and mTld both exhibit procollagen C-proteinase (PCP) activity and that BMP-1 is the smaller variant might indicate that BMP-1 comprises the minimal required sequences for PCP activity. BMP-1 comprises a metalloproteinase domain, three CUB domains, and an epidermal growth factor (EGF)-like domain, which is located between the second and third CUB (complement components C1r/C1s, the sea urchin protein Uegf, and BMP-1) domains. In this study we showed the following. 1) The CUB1 domain is required for secretion of the molecule. Domain swapping experiments, in which CUB1 and other CUB domains were interchanged, resulted in retention of the proteins by cells. Therefore, CUB1 and its location immediately adjacent to the metalloproteinase domain are essential for secretion of the protein. 2) Mutants lacking the EGFlike and CUB3 domains exhibited full C-proteinase activity. In contrast, mutants lacking the CUB2 domain were poor C-proteinases. 3) Further studies showed that Glu-483 on the ␤4-␤5 loop of CUB2 is essential for Cproteinase activity of BMP-1. In conclusion, the study showed that the minimal domain structure for PCP activity is considerably shorter than expected and comprises the metalloproteinase domain and the CUB1 and CUB2 domains of BMP-1. BMP-11 was isolated in the latter half of the 1980s from osteogenic fragments of bone and was shown to induce cartilage formation in vivo (1, 2). Unlike other BMPs, BMP-1 is a metalloproteinase with a proteinase domain that is homologous to the crayfish enzyme astacin (3) and a C-terminal domain comprising three CUB domains and one EGF-like domain. Subsequent work showed that BMP-1 is a smaller spliced variant of mTld (4). The functions of BMP-1 remained unknown until it was shown that it can cleave chordin, an antagonist of BMP-2, which can direct bone and cartilage formation (5).BMP-1 and mTld exhibit similar substrate specificity in vitro in that they can cleave precursors of extracellular matrix proteins including fibrillar procollagens (6, 7), biglycan (8), type VII procollagen (9), prolysyl oxidase (10, 11), and chains of laminin (12, 13). Evidence from gene knock-out studies in mice in which the mouse tolloid gene was mutated showed that BMP-1 and/or mTld are essential for normal assembly of extracellular matrix (14). Electron microscopy of the skin detected the presence of abnormal collagen fibrils in the knock-out mouse, which showed that BMP-1 and/or mTld are essential for normal cleavage of procollagen.BMP-1 is encoded by mtld, which also gives rise to mammalian tolloid (mTld or vertebrate Tld). mTld contains five CUB domains and two EGF-like domains at its C terminus, whereas BMP-1 lacks the most C-terminal two CUBs and one EGF-like domain. Interestingly, both BMP-1 and mTld can cleave the C-propeptides of fibril-forming proc...

Deletion of Epidermal Growth Factor-like Domains Converts Mammalian Tolloid into a Chordinase and Effective Procollagen C-proteinase

“…NotI sites were introduced by PCR at the borders of the EGF domain of the cDNA clone, and restriction enzyme digestion was used to delete individual domains. Previous work showed that the NotI-derived alanine residues had no effect on the procollagen C-proteinase activity of BMP-1 when inserted at the domain junctions (20). The recombinant proteins contained a V5-peptide epitope at the C terminus to facilitate Western blot analysis.…”

“…Site-directed Mutagenesis-Deletion of the EGF-like domain 1 was as described (20). The ⌬EGF1-BMP-1 clone was used to generate the His-tagged mTld-⌬EGF1 mutant, by excising and re-cloning the fragment comprising the deletion by BamHI (position 1390) and AviII (position 1954).…”

“…Both wild-type BMP-1 and BMP-1 minimal structure for C-proteinase activity (i.e. BMP-1⌬EC3, lacking EGF and CUB3 (20)) were His 6 -tagged. Mammalian tolloid (mTld) (GenBank TM accession number U50330) was obtained by synthesis of the sequence from position 1883 to the stop codon (Genscript).…”

“…The ⌬EGF1-BMP-1 clone was used to generate the His-tagged mTld-⌬EGF1 mutant, by excising and re-cloning the fragment comprising the deletion by BamHI (position 1390) and AviII (position 1954). For the deletion of the EGF-like domain 2 of mTld, NotI restriction enzyme sites were inserted at the borders of the EGF2 domain by PCR, using Pfx polymerase (Invitrogen) as described (20). A two-step strategy was undertaken using the mTld tail in pUC18 as a template and the primers M13pUC18Forward (5Ј-CCCAGTCACGAC-GTTGTAAAACG) and E2NotaReverse (5Ј-ACTCGTCCTTGTCAGCGG-CCGCTGAGAAGAAGT) on the one hand and E2NotbForward (5Ј-GA-AGCCGGCGCGGCCGCTTGTGACCACAA) and pUC18DraIIReverse (5Ј-GGAGTAAAGGTCCTTGGTCTT) on the other hand.…”

“…Calcium ion binding occurs in domains containing the consensus sequence (D/N)X(D/N)(E/Q)(X) m (D/ N)*(X) n (Y/F), where X is any amino acid; m and n are integers; and * indicates possible ␤-hydroxylation (19). In a previous study (20), we showed that the EGF-like and CUB3 domains of BMP-1 are not required for cleavage of type I procollagen. Thus the minimal domain structure for procollagen C-proteinase activity is as follows: metalloproteinase-CUB1-CUB2-specific domain (BMP-1⌬EC3).…”