 |
INTRODUCTION |
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
-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 |
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 pro
1(I) and
pN
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 pN
2(I), corrected
for molecular mass, by the initial concentration of procollagen.
 |
RESULTS |
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;
, 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 pro
-chains to pN
-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 ( CUB2),
BMP-1 lacking the EGF-like domain ( EGF-1), and BMP-1
lacking CUB3 ( CUB3). B, graphical
representation of the C-proteinase assay showing percent cleavage of
procollagen with time. BMP-1, closed diamond; CUB2,
closed square; EGF-1, closed triangle;
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), (EGF-CUB3) (closed diamond),
(CUB2-EGF-CUB3) (cross), and (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- 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 |
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
-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
-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.