Bone Morphogenetic Protein-1 (BMP-1)

IDENTIFICATION OF THE MINIMAL DOMAIN STRUCTURE FOR PROCOLLAGEN C-PROTEINASE ACTIVITY*

Nichola Hartigan, Laure Garrigue-Antar, and Karl E. KadlerDagger

From the Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Stopford Building 2.205, Oxford Road, Manchester M13 9PT, United Kingdom

Received for publication, November 10, 2002, and in revised form, February 17, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 EGF-like 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 beta 4-beta 5 loop of CUB2 is essential for C-proteinase 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 procollagens (6, 7), which implies that BMP-1 contains the minimal sequence requirement for procollagen C-proteinase activity (PCP). Indeed, studies in which human recombinant Tld was subjected to limited proteolysis by alpha -chymotrypsin showed that the procollagen binding function of human Tld was contained within the first (most N-terminal) three CUB domains (15).

In this study we wanted to know if all the CUB domains of BMP-1 are required for PCP activity. Our approach was to generate truncated and domain-swap mutants of BMP-1, to express and purify the variant proteins, and to examine the proteins in assays of procollagen C-proteinase. We show that BMP-1 molecules lacking the C-terminal EGF-like and CUB3 domains exhibit full PCP activity in vitro. In contrast, BMP-1 lacking the CUB2 domain is a poor C-proteinase. Furthermore, BMP-1 lacking CUB2, EGF-like, and CUB3 domains exhibit no PCP activity. We subsequently went on to identify residues in the CUB2 domain that are essential for PCP activity. We also show that the CUB1 domain is absolutely required for secretion of BMP-1 and, moreover, that this domain needs to be located immediately C-terminal of the metalloproteinase. The results show that the minimal sequence for PCP activity is shorter than the sequences encoded by BMP-1 and includes sequences N-terminal of the EGF-like domain, i.e. the EGF-like and CUB3 domains are not important for C-proteinase activity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Source of Materials-- Full-length BMP-1 cDNA (GenBankTM accession number P13497) was cloned from a human placental cDNA library. A FLAG tag amino acid sequence (DYKDDDDK) recognized by a mouse monoclonal anti-FLAG M2 antibody (Sigma) was introduced into the BMP-1 sequence (BMP-1-FLAG) immediately 5' of the stop codon. The cDNA encoding FLAG-tagged BMP1 was subcloned into the episomal expression vector pCEP4 (Invitrogen) and pcDNA3 for heterologous protein expression studies in cultured cells.

Site-directed Mutagenesis-- For deletion, rearrangement, and truncation of the CUB and EGF-like domains, NotI restriction enzyme sites were inserted into the junctions of individual domains by PCR using Pfx polymerase (Invitrogen). A two-step strategy was undertaken using the following primers: CUB1a, 5'-TGCCCAGCCGCGGCCGCTTGTGGAGAGA-3'; CUB1b, 5'-GGGTCTCTCCACAAGCGGCCGCGGCTGGGCACTT-3'; CUB1c, 5'-GTCTACGAAGCCATCGCGGCCGCTTGCGGGGGTGAT-'3; CUB1d, 5'-CATCACCCCCGCAAGCGGCCGCGATGGCTTCGTA-3'; CUB2c, 5'-AACTTTTTCAAAGCGGCCGCTGAGGTGGA-3'; CUB2d, 5'-TCCACCTCAGCGGCCGCTTTGAAAAAGTT-3'; EGF1c, 5'-TGTGAGGCTGCTGCGGCCGCTTGTGGCGGATT-3'; EGF1d, 5'-AATCCGCCACAAGCGGCCGCAGCAGCCTCA-3'; CUB3c, 5'-ACTTCTTCTCAGCGGCCGCTGAAAAGAGGCCA-3'; and CUB3d, 5'-TGGCCTCTTTTCAGCGGCCGCTGAGAAGAA-3' (A and B inserted sites into the sense and antisense strands at the N termini and C and D at the C termini of the respective domains; NotI sites are underlined). The up- and down-stream wild type primers Seq5-sense (5'-TCGTGAGAACATCCAGCCAGGGCA-3') and pCEP4-antisense (5'-TCTAGTTGTGGTTTGTCCAAACT-3') were used with the appropriate mutant primers. PCR products were purified and inserted into wild type cDNA by restriction enzyme digestion. Individual domains were excised by digestion with NotI, and the remaining BMP-1-FLAG pCEP4 cDNA were religated.

The E483K point mutation was introduced into the CUB2 domain of a recombinant BMP-1-FLAG cDNA using a strategy similar to that used above. Primers encoding the desired point mutation (underlined in the primer sequence) were used with either sense or antisense wild type primers: E483K-sense, 5'-CCTTTGAGATTAAGCGCCACGAC-3'; E483K-antisense, 5'-GTCGTGGCGCTTAATCTCAAAGG-3'. The up- and down-stream wild type primers were: Seq5-sense, 5'-TCGTGAGAACATCCAGCCAGGGCA-3'; and pCEP4-antisense, 5'-TCTAGTTGTGGTTTGTCCAAACT-3'. The PCR products were purified and digested with the enzymes BamHI and XhoI and inserted into the BMP-1FLAG pcDNA3 cDNA. To allow for stable expression in 293-EBNA cells the constructs were transferred into the pCEP4 expression vector using the restriction enzymes KpnI and XhoI.

Protein Expression-- 293-EBNA cells (European Collection of Cell Cultures, ECACC 85120602) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen) (complete DMEM) and 0.25 mg/ml Geneticin (G418, Invitrogen) in a 37 °C incubator with 5% CO2. Two µg of wild type or mutant plasmid was incubated with the transfection agent Lipofectin (Invitrogen) and then added to the 293-EBNA cells. After 48 h selection was initiated by the addition of 0.25 mg/ml hygromycin B (Invitrogen). Once the cells had become confluent the serum content of the medium was reduced to 2.5% fetal calf serum in which the expressing cells were then maintained. Once a week the cells were starved in medium containing 0% fetal calf serum, and the medium was used as a source of protein.

Preparation of Medium and Cell Lysates-- The medium was centrifuged for 5 min at 1,600 × g to clear any cell debris, and then 1 ml of 1 M Tris, pH 7.4, was added. For preparation of cell lysates, plates were rinsed in phosphate-buffered saline and incubated on ice with 500 µl of radioimmune precipitation buffer (150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 10 mM Tris, pH 7.4) containing 10 µl of 0.5 M EDTA for 15 min with occasional shaking. The cells were scraped, sonicated on ice, and then centrifuged at 13,000 × g for 5 min.

SDS-PAGE and Western Blot Analysis-- SDS-PAGE was carried out according to standard procedures in 10% polyacrylamide gels for Western blotting and 7% for procollagen assays. Cell lysate and supernatant samples were run under reducing conditions and subjected to Western immunoblotting using the mouse monoclonal M2 antibody (Sigma) directed against the FLAG tag. Secondary antibody (anti-mouse peroxidase-conjugated IgG (Sigma)) was detected by the enhanced chemiluminescence method (SuperSignal West Dura extended duration, Pierce). The levels of BMP-1-FLAG were quantified by laser densitometry of enhanced chemiluminescence fluorograms exposed to preflashed films.

Procollagen Assay-- Recombinant BMP1 was assayed for procollagen C-proteinase activity using human U-L-14C-labeled type I procollagen substrate (0.4 µg) as described (16). Analysis of the cleavage products on SDS gels (7% separating, 3.5% stacking) was performed as described, and the cleaved products were visualized by exposing dried gels to a phosphorimaging plate (Fuji, type BAS III) in a Fujix BAS 2000 Phosphor-imager. Bands corresponding to the proalpha 1(I) and pNalpha 2(I) chains of type I procollagen and type I pN collagen (an intermediate in the conversion of procollagen to collagen containing the N propeptides but not the C propeptides), respectively, were quantified using AIDA version 2.0 software. The percent cleavage was calculated by multiplying the intensity of the pNalpha 2(I), corrected for molecular mass, by the initial concentration of procollagen.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Deletion Mutants Lacking CUB1 Are Not Secreted from 293-EBNA Cells-- To determine the contribution of each CUB domain and the EGF-like domain of BMP-1 to the PCP activity of the molecule, we generated a series of deletion mutants that lacked each of these domains and expressed the proteins in 293-EBNA cells. The strategy for generating deletion, rearranged, and truncated variants of BMP-1 was to engineer NotI sites at the junctions of individual domains. Once the NotI sites were positioned in the desired place, domains could be excised and flanking sequences re-ligated. This cassette system simplified the synthesis of BMP-1 variants. However, the NotI sites introduced three consecutive alanine residues into the protein sequence. Control experiments were carried out to determine the influence of the three alanine residues on secretion of BMP-1. The test molecule was a wild type BMP-1 sequence in which a NotI site was introduced between the metalloproteinase and CUB1 domain. Consequently, the resultant protein had three alanine residues inserted between the metalloproteinase domain and CUB1. The results showed that the NotI-derived alanine residues had no effect on secretion of the protein (data not shown). Subsequent work (see below) showed that they have no influence on PCP activity of BMP-1 when inserted at domain junctions. The recombinant proteins contained a FLAG-peptide epitope at the C terminus to aid detection by Western blot analysis (5).

As shown in Fig. 1, wild type BMP-1 (i.e. containing the normal arrangement and complement of CUB and EGF-like domains) occurred as proBMP-1 (i.e. containing the prodomain) in the cell lysate and as the mature form (i.e. lacking the prodomain) in the cell medium. Analysis of Western blots showed that BMP-1 molecules lacking (singularly) CUB2, EGF-like, or CUB3 domains were readily secreted from 293-EBNA cells as the mature form. However, BMP-1 lacking the CUB1 domain occurred only in the cell lysate and as the pro-form. BMP-1 lacking the CUB1 domain was never observed in the culture medium.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 1.   CUB1 is required for secretion of BMP-1. A, schematic of wild type and mutated BMP-1 molecules in which the CUB and EGF-like domains were deleted. SP, signal peptide; P, prodomain; M, metalloproteinase domain; CUB, the CUB domains of BMP-1; EGF, the EGF-like domain of BMP-1; S, specific domain of BMP-1; Delta , deletion. B, Western blot analysis of cell lysates and cell culture medium of 293-EBNA cells stably expressing wild type and domain deleted BMP-1 molecules. The proteins were detected using an anti-FLAG antibody followed by ECL-SuperSignal reagent. The difference in migration between molecules in cell lysate and culture medium is because of the removal of the prodomain prior to secretion. The assignment of pro-forms and cleaved forms is based on the migration in SDS gels.

The Position of the CUB1 Domain Immediately C-terminal of the Metalloproteinase Domain Is a Prerequisite for Secretion of BMP-1-- In further studies of the CUB1 domain, we generated a series of mutants in which the positions of the CUB domains were shuffled. As shown in Fig. 2, when the order of the CUB1 and CUB2 domains was reversed, or when the CUB1 and CUB3 domains were swapped, the molecules were not secreted. Furthermore, a BMP-1 in which the CUB1 domain was replaced by a CUB3 domain (such that this molecule contained a prodomain, a metalloproteinase domain, two CUB3 domains, one CUB2 domain, and the EGF-like domain) was not secreted. A molecule in which the CUB3 domain was replaced by a CUB1 domain, but which retained a CUB1 domain in its normal position, was secreted.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   CUB1 has to be located at position 1 for secretion of BMP-1. A, schematic of wild type and mutated BMP-1 molecules in which the position of the CUB1 domain was varied. See Fig. 1A legend for definitions. B, Western blot analysis of cell lysates and cell culture medium of 293-EBNA cells stably expressing wild type and CUB rearranged BMP-1 molecules. The proteins were detected using an anti-FLAG antibody followed by ECL-SuperSignal reagent.

We wanted to know whether the CUB1 domain alone (i.e. in the absence of other CUB domains) was able to promote secretion of BMP-1 molecules. We generated a series of mutants in which BMP-1 was truncated stepwise from its C terminus but retained the specific domain. BMP-1 molecules containing the metalloproteinase domain and CUB1, CUB1-and-CUB3, or CUB1-and-CUB2 were secreted efficiently (Fig. 3). As shown in Fig. 4, a truncated BMP-1 that lacked all of the CUB domains and the EGF-like domain was not secreted. The conclusion drawn from these experiments was that the CUB1 domain is essential for secretion of BMP-1, and moreover, that the domain must be located immediately C-terminal of the metalloproteinase domain to ensure secretion of BMP-1.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 3.   Secretion of truncated BMP-1 molecules. A, schematic of BMP-1 molecules lacking CUB and EGF-like domains. See Fig. 1A legend for definitions. B, Western blot analysis of cell lysates and cell culture medium of 293-EBNA cells stably expressing truncated BMP-1 molecules. The proteins were detected using an anti-FLAG antibody followed by ECL-SuperSignal reagent.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   BMP-1 lacking the C-terminal domains is not secreted. Western blot analysis of the cell lysate and culture medium of 293-EBNA cells stably expressing a BMP-1 containing only the signal peptide (SP), prodomain (P), metalloproteinase domain (M), and specific domain of BMP-1 (s).

The CUB2 Domain Is Essential for PCP Activity of BMP-1-- The efficient secretion of BMP-1 molecules lacking CUB2, CUB3, and EGF-like domains provided us with the opportunity to assay these molecules for PCP activity. The assay comprised human 14C-labeled type I procollagen and purified BMP-1 (and mutants thereof). Cleavage of proalpha -chains to pNalpha -chains was detected by SDS-PAGE and by exposing dried gels to a phosphorimaging plate. The results of these experiments (Fig. 5) show that the deletion of CUB3 and deletion of EGF-like domain mutants had no effect on the PCP activity of BMP-1. These proteinases were inhibited by EDTA, which removes the catalytic zinc ion from the active site in the metalloproteinase domain. In contrast, BMP-1 lacking the CUB2 domain was a poor C-proteinase. In further studies we showed that BMP-1 lacking both the CUB2 and EGF-like domains, or BMP-1 lacking the CUB2, EGF-like, and CUB3 domains, exhibited undetectable levels of PCP activity (see Fig. 6).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 5.   The CUB2 domain is essential for maximal C-proteinase activity. A, SDS gel showing cleavage of human type I 14C-labeled procollagen with wild type BMP-1 (BMP-1), BMP-1 lacking CUB2 (Delta CUB2), BMP-1 lacking the EGF-like domain (Delta EGF-1), and BMP-1 lacking CUB3 (Delta CUB3). B, graphical representation of the C-proteinase assay showing percent cleavage of procollagen with time. BMP-1, closed diamond; Delta CUB2, closed square; Delta EGF-1, closed triangle; Delta CUB3, closed circle.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   Deletion of EGF-like and CUB3 domains has no effect on PCP activity. The PCP activity of BMP-1 (closed square), Delta (EGF-CUB3) (closed diamond), Delta (CUB2-EGF-CUB3) (cross), and Delta (CUB2-EGF) (closed triangle) was assayed using type I 14C-labeled procollagen as substrate. The graph shows the percent cleavage of procollagen during a 6-h incubation at 37 °C.

Glu-483 Is Critical for PCP Activity of BMP-1-- Evidence from Drosophila genetic studies suggested that the CUB2 domain is important for tolloid function (17, 18). One mutation, E517K (numbering for Drosophila tolloid) resulted in an antagonistic phenotype in the fly. Sequence alignments showed that Glu-517 is highly conserved in the tolloid family of proteins (data not shown). In BMP-1 the corresponding residue is Glu-483. To examine the importance of this residue for the ability of BMP-1 to cleave procollagen, we used site-directed mutagenesis to make the E483K mutant, and then we assayed the mutated protein for PCP activity. As shown in Fig. 7, we could not detect PCP activity in the E483K mutant. In further experiments we showed that BMP-1 containing the E483K mutation and also lacking the EGF-like domain exhibited no detectable PCP activity.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   E483K abolishes PCP activity. The PCP activity of BMP-1 (closed diamond), E483K (closed triangle), and E483K-Delta EGF (closed square) was assayed using type I 14C-labeled procollagen as substrate. The graph shows the percent cleavage of procollagen during a 6-h incubation at 37 °C.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study has shown that: 1) the CUB1 domain of BMP-1 is required for secretion of the molecule; 2) CUB1 needs to be immediately C-terminal of the metalloproteinase domain for secretion of the molecule; and 3) the CUB2 domain is essential for C-proteinase activity. These observations have implications for folding and assembly of other proteins containing CUB domains, for studies of the structure-and-function of proteinases, and for the mode of action of other members of the tolloid family of proteinases.

The CUB domain is an extracellular domain of ~110 residues organized into five beta -pleated sheets with two disulfide bonds that stabilize the structure. The domain is found in at least 300 proteins listed on the InterPro site at the European Bioinformatics Institute on-line data bases and at NCBI. Proteins that contain CUB domains exhibit a wide range of biologically different and regulatory functions (19). For example, in addition to BMP-1, CUB domains occur in complement subcomponents C1s/C1r, which form part of the calcium-dependent complex C1; serine protease Casp, which cleaves type I and IV collagen and fibronectin in the presence of calcium ions; enteropeptidase, which activates trypsinogen; neuropilin, a cell adhesion molecule that functions during the formation of certain neuronal circuits; and spermadhesins. The function of CUB domains is poorly understood, although they are thought to be involved mostly in protein-protein interactions. In this study we showed that each CUB domain in BMP-1 has a different function. The CUB1 domain is critical for secretion of BMP-1, and moreover, the domain needs to be positioned immediately C-terminal of the metalloproteinase domain. The replacement of CUB1 with either CUB2 or CUB3, even when CUB1 was positioned downstream of these domains, resulted in retention of BMP-1. Therefore, only CUB1 in the first position could facilitate secretion of BMP-1. This was an unexpected result given the fact that the three CUB domains of BMP-1 are 38% identical. The main differences occur in the loop regions, and this may indicate different functions and protein binding specificities. The fact that the CUB1 domain is necessary and sufficient for secretion of the metalloproteinase domain suggests that there are specific sequences, perhaps in the loop regions of the domain, which bind the metalloproteinase domain and stabilize its three-dimensional structure. Previous studies have shown that BMP-1 lacking the two N-linked glycans on CUB1 is secreted (20). Thus, retention of CUB1 deletion mutants was because the CUB1 sequences per se were absent and not because of the underglycosylated state of the protein. These observations have implications for other proteins with CUB domains, particularly proteinases that have the CUB domain adjacent to the proteinase domain, such as the serine proteases in complement components. Mutations in these CUB domains might be expected to result in intracellular retention or misfolding of the proteins.

Early work on Drosophila tolloid revealed a number of natural mutations that affected dorsal-ventral patterning (17, 18). Two such mutations, M487K and E517K (numbering for Drosophila tolloid), occurred in the CUB2 domain. Glu-517 is conserved in the CUB2 domain of all mammalian tolloids and is equivalent to residue Glu-483 in BMP-1. Mapping of the mTld CUB domains onto the crystal structure of the CUB domain from the porcine sperm protein PSPI/PSPII shows that Glu-483 is situated in a loop region between beta -strands 4 and 5 (21). We hypothesized that Glu-483 could have a role in the PCP activity of BMP-1. The results of site-directed mutagenesis experiments showed that the E483K mutation abolished the C-proteinase activity of BMP-1. The role of Glu-483 in C-proteinase activity of BMP-1 is unknown but might include disruption of the domain fold or destruction of a procollagen binding site.

A surprising observation was that the EGF-like and CUB3 domains are not required for PCP of BMP-1 in vitro. Thus, the minimal sequences for PCP activity are contained within the metalloproteinase domain and the first two CUB domains. A mutant that contained the metalloproteinase domain and CUB1 exhibited weak PCP activity, which further demonstrated the importance of the CUB2 domain in PCP activity of BMP-1. Careful structure modeling of the CUB domains as well as type I procollagen binding experiments to fragments of human Tld containing CUB2 and CUB3 have been performed (15). These studies suggest that CUB2 and CUB3 present several sites for specific interactions. In particular, well defined hydrophobic sites within these domains are postulated to be sites of interaction with substrates, including type I procollagen.

    ACKNOWLEDGEMENT

We thank Elizabeth Canty for helpful discussions.

    FOOTNOTES

* These studies were supported by grants from the Wellcome Trust (to K. E. K.) and by a Wellcome Trust prize studentship (to N. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 44-161-275-5086; Fax: 44-161-275-1505; E-mail: karl.kadler@man.ac.uk.

Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M211448200

    ABBREVIATIONS

The abbreviations used are: BMP-1, bone morphogenetic protein-1; mTld, mammalian tolloid; PCP, procollagen C-proteinase; EGF, epidermal growth factor; CUB domain, a protein domain first found in the complement components C1r/C1s, the sea urchin protein Uegf, and BMP-1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Urist, M. R. (1965) Science 150, 893-899[Medline] [Order article via Infotrieve]
2. Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M., and Wang, E. A. (1988) Science 242, 1528-1534[Medline] [Order article via Infotrieve]
3. Bond, J. S., and Beynon, R. J. (1995) Protein Sci. 4, 1247-1261[Abstract/Free Full Text]
4. Takahara, K., Lyons, G. E., and Greenspan, D. S. (1994) J. Biol. Chem. 269, 32572-32578[Abstract/Free Full Text]
5. Scott, I. C., Blitz, I. L., Pappano, W. N., Imamura, Y., Clark, T., Steiglitz, B., Maas, S. A., and Greenspan, D. S. (1999) Dev. Biol. 213, 283-300[CrossRef][Medline] [Order article via Infotrieve]
6. Kessler, E., Takahara, K., Biniaminov, L., Brusel, M., and Greenspan, D. S. (1996) Science 271, 360-362[Abstract]
7. Shi-Wu-Li, Sieron, A. L., Fertala, A., Hojima, Y., Arnold, W., and Prockop, D. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5127-5130[Abstract/Free Full Text]
8. Scott, I. C., Imamura, Y., Pappano, W. M., Troedel, J. M., Recklies, A. D., Roughley, P. J., and Greenspan, D. S. (2000) J. Biol. Chem. 275, 30504-30511[Abstract/Free Full Text]
9. Rattenholl, A., Pappano, W. M., Koch, M., Keene, D., Kadler, K., Sasaki, T., Timpl, R., Burgeson, R. E., Greenspan, D. S., and Bruckner-Tuderman, L. (2002) J. Biol. Chem. 277, 26372-26378[Abstract/Free Full Text]
10. Panchenko, M. V., Stetler-Stevenson, W. G., Trubetskoy, O. V., Gacheru, S. N., and Kagan, H. M. (1996) J. Biol. Chem. 271, 7113-7119[Abstract/Free Full Text]
11. Uzel, M. I., Scott, I. C., Babakhanlou-Chase, H., Palamakumbura, A. H., Pappano, W. N., Hong, H. H., Greenspan, D. S., and Trackman, P. C. (2001) J. Biol. Chem. 276, 22537-22543[Abstract/Free Full Text]
12. Amano, S., Scott, I. C., Takahara, K., Koch, M., Champliaud, M. F., Gerecke, D., Keene, D., Hudson, D. L., Nishiyama, T., Lee, S., Greenspan, D. S., and Burgeson, R. E. (2000) J. Biol. Chem. 275, 22728-22735[Abstract/Free Full Text]
13. Veitch, D. P., Nokelainen, P., McGowan, K. A., Nguyen, T. T., Nguyen, N. E., Stephenson, R., Pappano, W. N., Keene, D. R., Spong, S. M., Greenspan, D. S., Findell, P. R., and Marinkovich, M. P. (2003) J. Biol. Chem. 278, 15661-15668[Abstract/Free Full Text]
14. Suzuki, N., Labosky, P. A., Furuta, Y., Hargett, L., Dunn, R., Fogo, A. B., Takahara, K., Peters, D. M., Greenspan, D. S., and Hogan, B. L. M. (1996) Development 122, 3587-3595[Abstract/Free Full Text]
15. Sieron, A. L., Tretiakova, A., Jameson, B. A., Segall, L. M., Lund-Katz, S., Shan, M. T., and Stocker, W. (2000) Biochemistry 39, 3231-3239[CrossRef][Medline] [Order article via Infotrieve]
16. Garrigue-Antar, L., Barker, C., and Kadler, K. E. (2001) J. Biol. Chem. 276, 26237-26242[Abstract/Free Full Text]
17. Childs, S. R., and O'Connor, M. B. (1994) Dev. Biol. 162, 209-220[CrossRef][Medline] [Order article via Infotrieve]
18. Finelli, A. L., Bossie, C. A., Xie, T., and Padgett, R. W. (1994) Development 120, 861-870[Abstract/Free Full Text]
19. Bork, P., and Beckmann, G. (1993) J. Mol. Biol. 17, 565-574
20. Garrigue-Antar, L, Hartigan, N, and Kadler, K. E. (2002) J. Biol. Chem. 277, 43327-43334[Abstract/Free Full Text]
21. Romao, M. J., Kolln, I., Dias, J. M., Carvalho, A. L., Romero, A., Varela, P. F., Sanz, L, Topfer-Petersen, E., and Calvete, J. J. (1997) J. Mol. Biol. 274, 650-660[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.