Tolloid-Related Metalloproteinases, Including Novel Family Member Mammalian Tolloid-Like 2, Have Differential Enzymatic Activities and Distributions of Expression Relevant to Patterning and Skeletogenesis

Developmental Biology 213, 283–300 (1999) Article ID dbio.1999.9383, available online at http://www.idealibrary.com on

Mammalian BMP-1/Tolloid-Related Metalloproteinases, Including Novel Family Member Mammalian Tolloid-Like 2, Have Differential Enzymatic Activities and Distributions of Expression Relevant to Patterning and Skeletogenesis Ian C. Scott,* Ira L. Blitz,† William N. Pappano,‡ Yasutada Imamura,* Timothy G. Clark,* Barry M. Steiglitz,‡ Christina L. Thomas,‡ Sarah A. Maas,‡ Kazuhiko Takahara,* Ken W. Y. Cho,† and Daniel S. Greenspan* ,‡ ,1 *Department of Pathology and Laboratory Medicine and ‡Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, Wisconsin 53706; and †Department of Developmental and Cell Biology and the Developmental Biology Center, University of California, Irvine, California 92697

Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal–ventral patterning by releasing TGFb-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, we identify novel family member mammalian Tolloid-like 2 (mTLL-2) and compare enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2]. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 are shown to cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and to counteract dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis. © 1999 Academic Press Key Words: astacin metalloprotease; BMP signaling; Chordin; patterning; osteogenesis.

INTRODUCTION Bone morphogenetic protein-1 (BMP-1), first identified in osteogenic extracts of bone (Wozney et al., 1988), is the prototype of a family of metalloproteases implicated in 1

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morphogenetic processes in a broad range of species (Bond and Beynon, 1995). Each family member has a highly similar structure comprising an astacin-like protease domain and CUB protein–protein interaction and EGF motifs (Bond and Beynon, 1995). Among early members of this group BMP-1 was structurally most similar to Drosophila Tolloid (TLD) (Shimell et al., 1991), although TLD contains additional EGF and CUB motifs. A single mammalian gene

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was later found to produce alternatively spliced mRNAs for BMP-1 and for a larger protein designated mammalian tolloid (mTLD), because of a domain structure identical to that of TLD (Takahara et al., 1994b). A single gene also encodes BMP-1 and mTLD homologues in Xenopus (Lin et al., 1997). BMP-1/TLD-like proteases Xenopus Xolloid (Piccolo et al., 1997) and zebrafish Tolloid (Blader et al., 1997) can exert ventralizing effects during embryonic patterning by cleaving the protein Chordin, which forms latent complexes with ventralizing TGFb-like BMPs 2 and 4, and BMP 4/7 heterodimers (Piccolo et al., 1996). Similarly, TLD affects dorsal–ventral patterning of Drosophila embryos by cleaving Chordin homologue Short Gastrulation (SOG) (Marque´s et al., 1997) which has been inferred to bind the BMP-2/4 homologue Decapentaplegic (DPP) or the BMP-5/6/7 homologue Screw (SCW) (Neul and Ferguson, 1998; Nguyen et al., 1998). Since overexpression of BMP-1 can ventralize Xenopus embryos (Goodman et al., 1998) and because BMP-1 copurifies from osteogenic bone extracts with TGFb-like BMPs 2 through 7 (Wozney et al., 1988; Celeste et al., 1990), it has been suggested that BMP-1 may also affect morphogenesis by potentiating the activities of TGFb-like BMPs. To date, however, BMP-1 and mTLD have only been demonstrated to affect morphogenesis via effects on matrix deposition. These include biosynthetic cleavage of the C-terminal propeptides of the precursor molecules procollagens I–III, to yield the major fibrillar structures of vertebrate extracellular matrix (Kessler et al., 1996; Li et al., 1996). Despite the multiple functions either proposed or demonstrated for BMP-1 and mTLD in morphogenesis, BMP-1/ mTLD-null mice, although perinatal lethal, lack gross derangements of basic morphogenetic processes (Suzuki et al., 1996). This suggested genetic redundancy and led to isolation of cDNAs for a genetically distinct protease with a domain structure and sequences highly similar to those of mTLD and, thus, designated mammalian Tolloid-like (mTLL) (Takahara et al., 1996). It has been suggested that mTLL may be the mammalian orthologue of Xolloid and TLD (Piccolo et al., 1997; Marque´s et al., 1997) and/or that it may provide the procollagen-processing activity that partially compensates for loss of BMP-1 and mTLD in BMP-1/mTLD-null mice (Takahara et al., 1996). However, although expression of mTLL-1 has been shown to be necessary for normal development of the murine heart (Clark et al., 1999), enzymatic activities have not previously been characterized for this molecule. In the present report, use of degenerate PCR primers corresponding to conserved regions of BMP-1/TLD-like metalloproteases has identified an additional mammalian member of this family. The new protease is designated mTLL-2, due to a domain structure identical to that of mTLD and mTLL. Consequently, mTLL is redesignated mTLL-1. We directly compare the enzymatic activities of the known mammalian BMP-1/TLD-like proteases and demonstrate both similarities and differences. We demon-

strate that mTLL-1 is a procollagen C-proteinase and that both mTLL-1 and BMP-1 cleave Chordin, with different levels of activity, at the same two specific sites that resemble the physiological cleavage sites of procollagen C-propeptides. Comparison of the abilities of BMP-1, mTLD, mTLL-1, and mTLL-2 to cleave Chordin in vitro and to counteract dorsalizing effects of Chordin in vivo is combined with comparisons of the expression patterns of cognate mRNAs in early mouse development and in both pre- and postnatal endochondral bone formation. Together, these data indicate BMP-1 as more likely than mTLD, mTLL-1, or mTLL-2 to act as the predominant protease influencing dorsal–ventral patterning and skeletogenesis in mammalian development. Implications of the various data are discussed.

MATERIALS AND METHODS Isolation of full-length human and mouse mTLL-2 cDNA sequences. To obtain novel human BMP-1/TLD-like sequences, degenerate primers were employed corresponding to sequences AMRHWE and HYARNTF, conserved in the protease domains of BMP1/TLD-like proteins (Bond and Benyon, 1995). Two alternative forward primers corresponding to the AMRHWE sequence were designed, differing only at the most 39 base [primer 1, 59-CA(A/ G)GC(A/C)ATG(A/C)G(A/C/G/T)CACTGGGAG-39; primer 2, 59CA(A/G)GC(A/C)ATG(A/C)G(A/C/G/T)CACTGGGAA-39]. A single reverse primer [primer 3, 59-GAA(A/G/T)GTGTT(A/C/G)C(G/T)(A/ C/G/T)GC(A/G)TA(A/G)TGC-39] corresponded to the HYARNTF sequence. All PCR for human mTLL-2 sequences used human placenta Marathon-Ready cDNA (Clontech) as template (0.5 ng). A 375-bp mTLL-2 PCR product obtained with primers 2 and 3, was used to screen 10 6 plaques of a human placenta cDNA library (Clontech), yielding one strongly hybridizing positive with a 1.6-kb insert. 59 RACE, performed with nested primers corresponding to sequences near the 59-end of the 1.6-kb fragment (primer 4, 59-TGTGGTGTCTGGGCTGCTCTCAGATGC-39; primer 5, 59-ACTGTCTGCTTGGTCCAGTCTCTGG-39) and the Marathon cDNA Amplification Kit (Clontech) produced a ;900-bp product containing the remaining 59 mTLL-2 coding sequences and ;360 bp of 59-untranslated region (UTR). PCR using two nested forward primers corresponding to sequences at the 39-end of the 1.6-kb cDNA (primer 6, 59-TACCTGGAAGTCCGGGATGGCCCCACG-39; primer 7, 59-GAGGATGTGAAATCGAGCTCCAACAGAC-39) and degenerate reverse primer 10 [59-(A/G)AA(A/C/G/T)CC(T/C)TT(T/C)TT(A/C/G/T)(A/C/G/T)(A/C/G/T)(A/G/T)AT(A/C/G/T)GTGTCATC-39], corresponding to the sequence DDTIT/N/SKKGFH, conserved in CUB5 of human and mouse mTLD and mTLL-1, produced a ;1.3-kb product, and 39 RACE performed with two nested primers corresponding to sequences near the 39-end of the 1.3-kb fragment (primer 12, 59-CAACAACTACCCGAGCGAGGCCCG-39; primer 13, 59-GAAGCCTACGACGGCTACGACAGCTC-39) produced a ;1.7-kb product encoding the remainder of CUB5 and containing 1.6 kb of 39-UTR, including a poly(A) tail. Conditions used to obtain the initial human mTLL-2 375-bp fragment (above) were used to obtain a mouse mTLL-2 375 bp product, except that the template in this and subsequent PCRs for mouse sequences was 0.5 ng of 7 days postcoitus (dpc) mouse embryo Marathon-Ready cDNA (Clontech), and the murine 375-bp mTLL-2 PCR product was obtained using primers 1 and 3. 59 RACE

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using reverse primer 3 (above) and a nested primer (primer 14, 59-GCTTTCCTCATCTGTCCTCTCTACG-39) corresponding to sequences near the 39-end of the 375-bp fragment produced a ;900-bp product containing remaining 59 mTLL-2 coding sequences plus ;200 bp of 59-UTR. 39 RACE, using forward primer 1 (above) and a nested primer (primer 15, 59-CCTGTGTGACCTTCGTAGAGAGG-39) corresponding to sequences near the 59-end of the 375-bp fragment yielded a ;1-kb mTLL-2 product, and additional 39 sequences were obtained by PCR with nested forward primers (primer 16, 59-CCCACAGAGGACAGCACCCTGATTGGC-39; primer 17, 59-CGAGAAGCCGGAGGCCGTCAAATCCAGC-39) corresponding to sequences located near the 39 end of the 1-kb product and reverse primer 10 (above), producing a ;1.2-kb product. A final 39 RACE, with two nested primers (primer 18, 59-GGCTTCCAGGCTGTGCACAG-39; primer 19, 59GCAGGCTGAAGGCTGAAGTAC-39) corresponding to sequences near the 39 end of the 1.2-kb product produced a 1.8-kb product containing the remaining mouse mTLL-2 coding sequences and ;1.4 kb of 39-UTR. Isolation of human mTLL-1 sequences. A 677-bp NdeI–Eco72I fragment of mouse mTLL-1 cDNA clone KO 3 (Takahara et al., 1996), encoding part of CUB4 and all of CUB5, was used to screen a human placenta genomic DNA library. A genomic clone was isolated containing the final three exons of the TLL1 gene. A 339-bp TaqI fragment of mouse mTLL-1 cDNA KO 7-2 (Takahara et al., 1996), encoding part of the proregion, was then used to screen the same library, yielding two clones, each containing the most 59 TLL1 exon. Genomic sequences were used to design primers 59-TCTTGCAGTCAGTTGCTTTGCTGG-39 (U91963 nucleotides 16 –39) and 59-TAGTGCGGCCGCACATTCCTTTGTGTTCTGACAGAG-39 (U91963 nucleotides 3724 –3699, plus 10 nucleotides providing a NotI site), used for RT-PCR amplification, from human fetal cartilage RNA (the kind gift of Dr. Linda Sandell, Washington University), of full-length mTLL-1 sequences. Production of recombinant proteins. A vector for expression of HA-tagged Xenopus Noggin (Piccolo et al., 1997) was the kind gift of Dr. Eddy M. De Robertis (University of California, Los Angeles). Error-free PCR products were derived using Ultma polymerase and following manufacturer’s instructions (Perkin–Elmer), corresponding to full-length human BMP-1, mTLD and mTLL-1, mTLL-2, and mouse Chordin, except for the lack of signal peptide sequences. These products were inserted between the NheI and NotI sites of the pCEP-Pu/BM40s episomal expression vector (Kohfeldt et al., 1997) such that a full-length recombinant version of each protein would be produced differing from the native protein only in replacement of the native signal peptide by the BM40 signal peptide. Constructs for Flag-tagged versions of each enzyme and mouse Chordin and for a c-Myc-tagged version of mouse Chordin were derived through PCR addition of sequences encoding DYKDDDDK-Stop and EQKLISEEDL-Stop, respectively, after the codon encoding the final amino acid of the recombinant protein. Sequence fidelity of constructs was confirmed by DNA sequencing of both strands. 293 EBNA-1 cells were maintained as described (Imamura et al., 1998). For transient expression of enzymes and c-Myc-tagged Chordin, 70 –90% confluent 293-EBNA cells (;2 3 10 6 cells) were transfected with 10 mg DNA using lipofectAMINE (Life Technologies). After 24 h, cells were washed in 3 3 10 ml phosphate-buffered saline (PBS) and transferred to serum-free Dulbecco’s modified Eagle’s medium (DMEM) containing 13 L-glutamine and 40 mg/ml soybean trypsin inhibitor (SBTI, Sigma), and the media were harvested 24 h later, with a second harvest after a further 24 h in

serum-free medium. Cells reproducibly yielded ;0.2 mg/ml BMP-1 and mTLD, ;0.4 mg/ml mTLL-2, and ;1.0 mg/ml mTLL-1 for both Flag-tagged and non-Flag-tagged versions of each enzyme, over the course of several transfections. A stable line producing Flag-tagged mouse Chordin (;0.4 mg/ml medium) was created by selecting transfected cells in 5 mg/ml puromycin as described (Imamura et al., 1998). Conditioned media were harvested as above, and all harvested media were made 10 mg/ml leupeptin, 0.4 mM PMSF and stored at 270°C. Purification of Flag-tagged proteins was essentially as in Piccolo et al. (1997). Purification of c-Myc-tagged mouse Chordin was with a High S column (Bio-Rad) as described for c-Myc-tagged Xenopus Chordin by Piccolo et al. (1997) except that, upon elution with a linear gradient of 80 –1200 mM NaCl in 10 mM Hepes (pH 7.6), mouse Chordin eluted at 0.4 M NaCl. Concentrations of purified proteins were calculated by comparing intensities of Coomassie-stained bands from serial dilutions of each sample to those of serially diluted protein standards of known concentrations. Immunoblotting. Subsequent to SDS–PAGE, proteins were transferred to Immobilon-P membranes (Millipore) as described (Lee et al., 1997). Blots were blocked in PBS containing 0.1% Tween 20 (T-PBS) and incubated 15 h with primary antibody diluted in T-PBS containing 1% BSA. After washing three times with T-PBS, blots were incubated with SuperSignal CL-HRP substrate (Pierce), exposed to film, or, where indicated, incubated 1 h with secondary antibody. After incubation with secondary antibody, blots were washed four times with T-PBS, incubated with SuperSignal CL-HRP, and exposed to film. Apparent molecular weights of bands were estimated by comparison to electrophoretic mobilities of prestained standards (Bio-Rad). Amino acid sequence analysis. Proteins were subjected to SDS–PAGE and electrotransferred, as above, to Sequi-Blot polyvinylidene difluoride membrane (Bio-Rad). N-terminal amino acid sequences were determined by automated Edman degradation on a Perkin–Elmer/Applied Biosystems Division Procise 494 HT Protein Sequencing System at the Harvard University Microchemistry Facility. Preparation of procollagen substrates and in vitro procollagen cleavage assays. Type I procollagen was prepared from human fibroblast cultures and purified by DEAE-cellulose chromatography as described (Imamura et al., 1998) except that cells were incubated 24 h in serum-free DMEM with 50 mg/ml ascorbate, 100 mg/ml SBTI, 32 mg/ml b-aminopropionitrile fumarate, and 7 mCi/ml [2,3- 3H]proline (DuPont). Recombinant type II procollagen was prepared from HT1080 human kidney cells stably transformed with an expression construct (Fertala et al., 1994) (the kind gift of Dr. Darwin Prockop, Allegheny University, Philadelphia, PA). Confluent cells were placed in DMEM with 40 mg/ml SBTI, 50 mg/ml ascorbate, and 20 mCi/ml [2,3- 3H]proline. After 24 h, medium was harvested, and type II procollagen was isolated essentially as described (Fertalla et al., 1994). Purified type I procollagen (500 ng) or type II procollagen (200 ng) was combined with 15 ng purified Flag-tagged BMP-1, mTLD, mTLL-1, or mTLL-2 in 50 ml final volume buffer A (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 5 mM CaCl 2). After 18 h at 37°C reactions were stopped by adding 103 SDS–PAGE loading buffer and boiling 5 min. Samples were subjected to SDS–PAGE on 5% acrylamide gels, which were treated with EN 3HANCE (DuPont) and exposed to Film. In vitro chordin and noggin cleavage assays. Purified mouse c-Myc-tagged Chordin was dialyzed against 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, and ;100 ng was incubated 12 h at 37°C with 15 ng purified Flag-tagged BMP-1, mTLD, mTLL-1, or mTLL-2 in a

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FIG. 1. (A) Alignment of the deduced amino acid residues of human and mouse mTLL-2 with those of human mTLD and mTLL-1. Mouse mTLL-2 (mmTLL-2), human mTLL-2 (hmTLL-2), human mTLD (hmTLD), and human mTLL-1 (hmTLL-1) deduced amino acid sequences are aligned beneath a schematic of the domain structure common to all four proteases. Stippled, darkly shaded, white, lightly shaded, black, and striped boxes represent signal peptide, proregion, metalloprotease, CUB, EGF, and C-terminal domains, respectively. Mouse mTLL-2 sequences are shown only where they differ from the human. The human mTLL-2 signal peptide cleavage site was predicted as described by Nielsen et al. (1997). Alignment was performed using the PILEUP program (Genetics Computer Group). Cysteines are boxed and the metalloendopeptidase active site motif HEXXH (Bond and Beynon, 1995) is enclosed by a dashed box. Human mTLD sequences are from Wozney et al. (1988) and Takahara et al. (1994b). Full-length human mTLL-1 and human and mouse mTLL-2 sequences, derived in the current study (see Materials and Methods), have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. U91963, AF059516, and AF073526, respectively. (B) Percentages of similarity and percentages of identity (parentheses) between the various proteases were obtained by alignment using the GAP program (Genetics Computer Group). Alignments above or below the diagonal were performed for full-length proteins or for proteins minus signal peptides and proregions, respectively.

final volume of 50 ml buffer A. Purified Flag-tagged Chordin (5 mg) was incubated 24 h at 37°C with 75 ng BMP-1 or 150 ng mTLL-1 in a 200-ml final volume buffer A. Reactions were stopped by adding 103 SDS–PAGE loading buffer and boiling 5 min. Assay samples were subjected to SDS–PAGE on 4 –15% gradient gels (Bio-Rad) and analyzed by western blot (for c-Myc-tagged Chordin) using antiMyc primary antibodies (Invitrogen) diluted 1:5000 and peroxidaselinked anti-mouse Ig secondary antibodies (Amersham) at 1:5000 or by zinc staining (for Flag-tagged Chordin) using the GelCode E-Zinc Reversible Stain Kit (Pierce). Conditioned medium containing HA-tagged Xenopus Noggin was dialyzed against 50 mM Tris–HCl, pH 7.5, 150 mM NaCl and concentrated ;20-fold in a Centricon concentrator (Amicon). Concentrated medium containing ;200 ng Noggin-HA was incubated with 45 ng purified Flag-tagged BMP-1, mTLD, mTLL-1, or mTLL-2 in a 50-ml final volume of buffer A containing 0.4 mM PMSF, 10 mg/ml leupeptin and 40 mg/ml SBTI. Reactions were incubated 6 h at 37°C and stopped by adding 103 SDS–PAGE loading buffer and boiling 5 min. Assay samples were subjected to SDS–PAGE on a 10 –20% gradient gel and analyzed by Western blotting with peroxidase-conjugated anti-HA antisera (Boehringer-Mannheim) diluted 1:1000. Xenopus embryo manipulations and RNA microinjections. Embryos were obtained from in vitro fertilized eggs, dejellied, and cultivated as described (Cho et al., 1991). Chordin RNA was

prepared from plasmid pSP35-chd (kindly provided by Dr. Eddy M. De Robertis) (Sasai et al., 1994), linearized with XbaI. Xenopus BMP-1 (XBMP-1) and mTLD (XTLD) RNAs were prepared from constructs containing full-length cDNAs obtained from a Xenopus oocyte cDNA library and blunt-end ligated into the StuI site of pCS21 (Rupp et al., 1994). To prepare human BMP-1, mTLD, mTLL-1, and mTLL-2 RNAs, coding sequences for each protease fused to the BM40 signal peptide were excised as AflII–XhoI fragments from the pCEP-Pu/BM40s mammalian expression vectors described above and inserted between the EcoRI and XhoI sites of pCS21 after partial filling of EcoRI ends with adenosines and AflII ends with thymidines. The pCS21 constructs containing Xenopus and human sequences were linearized with NotI or BstXI, respectively. RNA for a dominant negative BMP type I receptor was prepared from plasmid DxTGR11 (Suzuki et al., 1994) (a kind gift of Dr. Naoto Ueno, Hokkaido University, Sapporo, Japan) and RNA for a BMP-7 dominant negative cleavage mutant was prepared from plasmid Cm-XBMP-7 (Hawley et al., 1995), linearized with EcoRI and XhoI, respectively. Sense-strand-capped RNAs were prepared by in vitro transcription using the mMessage mMachine SP6 kit (Ambion). Capped mRNA’s were injected into a single blastomere at the ventral equatorial region of two- to four-cell-stage embryos. Dorsal–ventral polarity of two- to four-cell-stage embryos was determined as previously described (Klein, 1987; Cho et al., 1991). Injected embryos were cultivated in 0.13 Barth’s saline at room temperature until tailbud stages (Nieuwkoop and Faber, 1967). In situ hybridization and histological analysis. mTLL-2 riboprobes were prepared from a 1.8-kb 39 RACE product, containing the complete 39-UTR (see above) inserted into pBluescript II KS1 (Stratagene). This template was linearized with HindIII and transcribed with polymerase T7 (antisense) or linearized with NotI and transcribed with polymerase T3 (sense). Preparation of riboprobes for BMP-1, mTLD, and mTLL-1 was as described (Takahara et al., 1994b; Takahara et al., 1996). Template for Chordin riboprobes was a 1377-bp cDNA containing the Chordin 39-UTR (Pappano et al., 1998), linearized with SpeI and transcribed with T7 (antisense) or linearized with NcoI and transcribed with SP6 (sense). Template for PCPE was a 1327-bp full-length mouse PCPE cDNA (Takahara et al., 1994a) linerized with XhoI and transcribed with SP6 (antisense) or linearized with BamHI and transcribed with T7 (sense). Uniform labeling of riboprobes with [ 35S]UTP, tissue preparation, and in situ hybridization were performed as described (Takahara et al., 1994b), except that sections were 5 mm thick and mounted two to six/slide. Bone sections were fixed overnight in 4% paraformaldehyde, washed 3 min in deionized water, demineralized in Immunocal (Decal Chemical Corp) overnight, washed 3 min in running deionized water, and then dehydrated and embedded as for nonmineralized tissues. For histological analysis, sections were deparaffinized in xylenes, rehydrated, stained 20 min in 0.3% alcian blue, pH 2.5, immersed 10 min in 0.3% sodium carbonate, and stained with hematoxylin and eosin, but without a final acid–alcohol wash. Slides were analyzed using light- and dark-field optics of a Zeiss Axiophot 2 microscope.

RESULTS Isolation of cDNAs Encoding a Novel Mammalian BMP-1/TLD-like Protease: Mammalian Tolloid-like 2 (mTLL-2) In a screen for novel mammalian BMP-1/TLD-like proteases, degenerate primers corresponding to sequences con-

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served in the catalytic domains of known BMP-1/TLD-like proteases from various species (Bond and Benyon, 1995) were employed for PCR amplification, using human placenta cDNA or mouse 7, 13.5, and 17.5 dpc whole embryo cDNA as template. Novel BMP-1/TLD-like sequences were obtained from the human placenta and mouse 7 dpc cDNA templates, but not from mouse 13.5 or 17.5 dpc embryo cDNA. All novel sequences corresponded to the mouse or human version of a single gene product. Since assembly of full-length cDNA sequences for the new protease showed it to have a domain structure identical to that of mTLD and mTLL (Fig. 1A), it was designated mammalian Tolloid-like 2 (mTLL-2), and mTLL was redesignated mTLL-1. All other amplified BMP-1/TLD-like sequences obtained in the screen corresponded to previously identified mammalian proteases BMP-1, mTLD (Takahara et al., 1994b), and mTLL-1 (Takahara et al., 1996). Comparison of the full-length amino acid sequence of human mTLL-2 with those of human mTLD and mTLL-1 and of the other known vertebrate BMP-1/TLD-like proteases, Xenopus Xolloid (XLD) (Goodman et al., 1998) and zebrafish Tolloid (zTLD) (Blader et al., 1997), shows high sequence similarity between the various family members, especially when sequences in the proregion, which is proteolytically removed from the mature active protease, are not included in the comparison (Fig. 1B). Mature mTLL-2 was found to be most similar to XLD (89% similar, 83% identical). In fact, these two proteases have greater sequence similarity than do known human and Xenopus orthologues mTLD and xTLD (88% similar, 82% identical). Nevertheless, the high sequence similarity between mTLL-2 and XLD does not appear to reflect an orthologous relationship, as the two enzymes have quite different enzymatic activities (see below). Similarly, mTLL-1 and zTLD, the two most similar proteases in the comparisons of Fig. 1B (91% similar, 86% identical), may not be orthologues, as the two enzymes appear to have different distributions of expression in the gastrulating embryos of mouse and zebrafish (Clark et al., 1999; Blader et al., 1997; and see below).

Production of Recombinant BMP-1, mTLD, mTLL-1, and mTLL-2 To compare the enzymatic activities of mammalian BMP-1/TLD-like proteases, Flag-tagged and non-Flag-tagged versions of recombinant human BMP-1, mTLD, mTLL-1, and mTLL-2 were produced in transfected 293-EBNA human embryonic kidney cells. Recombinant proteases appeared as single bands on zinc- or Coomassie-stained SDS– PAGE gels and corresponded in size (BMP-1, mTLD, mTLL-1, and mTLL-2 were ;88, 130, 115, and 120 kDa, respectively) to processed forms from which proregions had been proteolytically removed (Fig. 2A). This is important, as the proregions of astacin-like proteases must be removed for activation of enzymatic activity (Bond and Beynon, 1995). Automated Edman degradation of the mTLL-1 and

mTLL-2 bands generated N-terminal sequences of AATSR and ATTSR, respectively, confirming these as processed active forms of the respective enzymes. In fact, these data are the first experimental evidence that BMP-1/TLD-like enzymes are actually processed at predicted dibasic cleavage sites. BMP-1 and mTLD bands were confirmed as processed active forms by use of immunoblots in which these proteins were recognized by antibodies specific for the C-terminus of each protease, but not by proregion-specific antibodies, as described in Lee et al.(1997) (data not shown). Incubation of conditioned media containing recombinant proteases with the various substrates analyzed in this study showed Flag-tagged and non-Flag-tagged versions of each enzyme to have the same levels of activity against a given substrate (data not shown). Thus, since C-terminal Flag tags appeared to have no effect on enzymatic activities, purified Flag-tagged versions of each enzyme were used for the assays presented below, as this allowed cleavage assays to be performed in the absence of contaminating proteins found in conditioned media and also facilitated quantitation of the enzymes.

BMP-1, mTLD, and mTLL-1, but not mTLL-2, are Procollagen C-Proteinases When similar amounts of purified recombinant BMP-1, mTLD, mTLL-1, or mTLL-2 were incubated with human type I procollagen, strong procollagen C-proteinase (PCP) activity was observed for BMP-1 and lesser levels of PCP activity were observed for mTLD (Fig. 2B). PCP activity for both of these proteins is consistent with previous reports (Kessler et al., 1996; Li et al., 1996), although relative levels of PCP activity of the two forms have not previously been ascertained. In addition, mTLL-1 was observed to cleave type I procollagen, producing products similar in size to those produced by BMP-1 and mTLD, with the apparent absence of nonspecific cleavages. No cleavage products of type I procollagen, beyond those already existing in starting material, were observed after incubation with mTLL-2. As is more apparent from a gel run longer to better separate cleavage products (Fig. 2C), mTLL-1 cleaves the proa1(I) and proa2(I) chains of type I procollagen to produce pNa1(I) and pNa2(I) chains (processing intermediates which retain N-propeptides, but from which the C-propeptides have been removed) with electrophoretic mobilities indistinguishable from those produced by cleavage of type I procollagen by BMP-1. These results were consistent with the possibility that specific cleavage of type I procollagen C-propeptides by mTLL-1 occurred at the same sites cleaved by BMP-1. To determine whether this was the case, automated Edman degradation was performed on the C-propeptide C1 and C2 subunits (Fig. 2D) released by recombinant mTLL-1 from the proa1(I) and proa2(I) chains, respectively. The N-terminal amino acid sequences of DDANVVRDRD and DQPRSAPSLR obtained for C1 and C2, respectively, indicate that mTLL-1 cleaves type I pro-

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FIG. 2. Compared enzymatic activities of recombinant mammalian BMP-1/TLD-like enzymes. (A) Purified Flag-tagged versions of BMP-1, mTLD, mTLL-1, and mTLL-2, used in the various assays, were visualized by SDS–PAGE on a 7.5% acrylamide gel and zinc staining. (B–E) Autofluorograms are shown of purified human type I procollagen (B,C) or type II procollagen (E) incubated in the absence of (Procol I and Procol II) or in the presence of (1BMP-1, 1mTLD, 1mTLL-1, and 1mTLL-2) enzymes and analyzed by SDS–PAGE. pNa1(I) and pNa2(I) are processing intermediates of the proa1 and proa2 chains of type I procollagen (a heterotrimer), and pNa1(II) is a processing intermediate of the proa1(II) chain of type II procollagen (a homotrimer), which retain the N-propeptide, but from which the C-propeptide has been removed. The gel in (C) was run longer than that in (B) to separate the pNa1(I) and proa2(I) chains, which electrophorese with similar mobilities. A schematic of type I procollagen (D) illustrates the relative positions of the N-propeptide and C-propeptide cleavage sites (open and closed arrows, respectively) and the C1 and C2 C-propeptide subunits released by cleavage from the two proa1(I) chains (closed lines) and single proa2(I) chain (open line), respectively. (F,G) Immunoblots are shown of c-Myc-tagged Chordin (F) or HA-tagged Noggin (G) incubated in the absence of (Chordin-MYC and Noggin-HA) or in the presence of (1BMP-1, 1mTLD, 1mTLL-1, and 1mTLL-2) enzymes and analyzed by SDS–PAGE. Bands in immunoblots were detected with anti-Myc (F) or anti-HA (G) antibodies, respectively.

collagen at the physiological PCP sites previously demonstrated to be used by BMP-1 (Kessler et al., 1996). In addition to being expressed in various tissues in which type I collagen is the major fibrillar collagen, BMP-1, mTLD, and mTLL-1 are also expressed in cartilage (see below), in which type II is the major fibrillar collagen. To ascertain whether relative levels of PCP activity of BMP-1,

mTLD, mTLL-1, and mTLL-2 for type II procollagen might differ from the relative levels of PCP activity for these four proteases against type I procollagen, Flag-tagged BMP-1, mTLD, mTLL-1, and mTLL-2 were incubated with human type II procollagen substrate. As is shown (Fig. 2E), relative levels of PCP activity against type II were similar to relative levels of PCP activity against type I procollagen (Fig. 2B).

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Human BMP-1 and mTLL-1, but Not mTLD or mTLL-2, Cleave Chordin in Vitro Although the ability to cleave Chordin/SOG appears to be an important function of Xenopus XLD (Piccolo et al., 1997), zebrafish TLD (Blader et al., 1997), and Drosophila TLD (Marque´s et al., 1997), it has not previously been determined which mammalian BMP-1/TLD-like proteases might be capable of this activity. To compare the abilities of BMP-1, mTLD, mTLL-1, and mTLL-2 to cleave Chordin, similar amounts of purified Flag-tagged versions of each enzyme were incubated with purified recombinant, c-Myctagged mouse Chordin. Under conditions of the assay, the purified c-Myc-tagged Chordin was completely stable in the absence of added enzyme (Fig. 2F). In contrast, the Chordin substrate was completely cleaved by BMP-1, while mTLL-1 cleaved Chordin to a somewhat lesser extent. Surprisingly, both mTLD and mTLL-2 showed little or no apparent Chordin-cleaving activity. Cleavage of Chordin in Fig. 2F was marked by disappearance of c-Myc-tagged Chordin on Western blots, without the appearance of cleavage products recognized by the anti-Myc antibody. An absence of distinct cleavage products would be in contrast to previous reports that XLD cleaves frog Chordin at two sites, to produce three distinct fragments (Piccolo et al., 1997), and that Drosophila TLD cleaves the Chordin orthologue SOG at three sites, to produce four distinct fragments (Marque´s et al., 1997). It seemed possible that distinct Chordin cleavage products were present in samples such as those shown in Fig. 2F, but that these were not detected by anti-Myc antibodies due to loss or inaccessibility of the C-terminal c-Myc epitope. To investigate this possibility, a stable cell line was established that constitutively produces recombinant Flag-tagged Chordin, enabling the purification of enough Chordin such that Chordin and Chordin-derived cleavage products would be visible on SDS–PAGE gels via protein stains. As is shown in Fig. 3A, cleavage of the ;104-kDa Flag-tagged mouse Chordin by either BMP-1 or mTLL-1 produced the same ;83-, ;15-, and ;13-kDa bands, similar in size to the protein fragments described by Piccolo et al. (1997) upon cleavage of frog Chordin by XLD. The sites at which BMP-1/TLD-like proteases cleave Chordin or SOG have not previously been characterized. In order to characterize the nature of such sites, the cleavage products derived from Chordin incubated with either BMP-1 or mTLL-1 were isolated from an SDS–PAGE gel and subjected to automated Edman degradation for determination of N-terminal amino acid sequences. As is shown in Fig. 3B, BMP-1 and mTLL-1 each cleaved Chordin at the same two sites, thus showing identical specificity of the two enzymes for Chordin. In addition, both Chordin cleavage sites occur upstream of aspartate residues. This is similar to the physiological procollagen C-propeptide cleavage sites characterized for BMP-1 (Kessler et al., 1996) and mTLL-1 (above) and to the cleavage site previously shown to be used by procollagen C-proteinase in the proteolytic

activation of prolysyl oxidase (Panchenko et al., 1996), an enzyme necessary for formation of covalent cross-links in collagen fibers. The N-terminal Chordin cleavage site occurs downstream of the first of four cysteine-rich repeats found in Chordin, and the C-terminal cleavage occurs between the third and fourth cysteine-rich repeats (Fig. 3C). Aspartates and additional residues found at the sites of cleavage of mouse Chordin are conserved at the same positions in human, chick, zebrafish, and Xenopus Chordin (Fig. 3D). However, alignment of Chordin with SOG did not reveal conserved SOG aspartates and other residues that corresponded to the Chordin cleavage sites in an obvious way (not shown). Thus, the specificity for cleavage of SOG by TLD may be somewhat different than that for cleavage of Chordin by vertebrate BMP-1/TLD-like enzymes. As with Drosophila TLD (Marque´s et al., 1997) and Xenopus XLD (Piccolo et al., 1997), none of the mammalian BMP-1/TLD-like proteases cleaved Noggin (Fig. 2G), another secreted protein which, like Chordin, appears to affect dorsal–ventral patterning through the binding of TGFb-like BMPs into latent complexes (Zimmerman et al., 1996). Importantly, although the purified recombinant mTLL-2 used in the present report did not cleave the substrates presented in Fig. 2, preliminary results show it to have proteolytic activity against other substrates such as prolysyl oxidase (I. C. Scott, M. Uzel, P. C. Trackman, and D. S. Greenspan, unpublished results). Thus, inability of the recombinant mTLL-2 to cleave Chordin or procollagen appears to reflect the lack of specificity of mTLL-2 for these substrates, rather than a lack of activity in the enzyme prep. This conclusion is supported by the in vivo studies described below.

Human BMP-1 and mTLL-1, but not mTLD or mTLL-2, Counteract the Dorsalizing Effects of Chordin in Vivo Overexpression of Chordin, through microinjection of mRNA, dorsalizes Xenopus embryos, inducing formation of secondary dorsal axes (Sasai et al., 1994). This Chordininduced dorsalization can be countered by coinjection with mRNA for Chordin-cleaving proteases XLD (Piccolo et al., 1997) or Drosophila TLD (Marque´s et al., 1997). Thus, we employed the same assay to ascertain which mammalian BMP-1/TLD-like protease(s) might counteract the dorsalizing effects of Chordin in vivo. Toward this end, Chordin mRNA was injected into a single ventral blastomere of twoto four-cell-stage embryos either alone or together with mRNA for BMP-1, mTLD, mTLL-1, or mTLL-2. Results of this biological assay (Fig. 4) were wholly consistent with those of the in vitro cleavage assays of Fig. 2F. Injected BMP-1 mRNA was highly effective at blocking induction of secondary axes by Chordin mRNA, while mTLL-1 achieved a somewhat lower level of inhibition of secondary axis induction, and both mTLD and mTLL-2 demonstrated essentially no effect in counteracting Chordin-induced secondary axis formation. Thus, both in vitro and independent

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FIG. 3. Characterization of the sites at which BMP-1 and mTLL-1 cleave mouse Chordin. (A) Electrophoretic patterns are compared for purified Flag-tagged mouse Chordin incubated for 24 h in the absence of enzyme (lane 1) or in the presence of BMP-1 (lane 2) or mTLL-1 (lane 3). Samples were run under reducing conditions on a 4 –15% acrylamide SDS–PAGE gel and visualized by staining with zinc. (B) Alignment of Chordin cleavage sites with the physiological cleavage sites for removal of the C-propeptides of fibrillar procollagens I and III (Kessler et al., 1996) and for proteolytic activation of prolysyl oxidase (Panchenko et al., 1996). (C) Positions of cleavage sites are shown in relation to the four cysteine-rich (CR) domains in a schematic of mouse Chordin. (D) Alignment of human (Pappano et al., 1999), chick (Streit et al., 1998), zebrafish (Schulte-Merker et al., 1997), and Xenopus (Sasai et al., 1994) Chordin sequences in regions corresponding to the mouse Chordin sites cleaved by BMP-1 and mTLL-1.

in vivo evidence indicate that BMP-1 has strong Chordincleaving activity, that mTLL-1 has somewhat less Chordincleaving activity, and that mTLD and mTLL-2 lack detect-

able levels of Chordin-cleaving activity under conditions of the two assays employed. Like mammals, Xenopus has a single gene that encodes

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FIG. 4. BMP-1 and mTLL-1 inhibit Chordin-mediated secondary axis formation, but mTLD and mTLL-2 do not. (A) A schematic depicts the route of injection of RNA into the single blastomere at the ventral equatorial region of four-cell-stage embryos (An, Vg, V, and D denote animal and vegetal poles and ventral and dorsal aspects of the embryo, respectively). Representative examples are shown below of embryos injected with mRNA for Chordin alone, or injected with mRNA for Chordin plus mRNA for human BMP-1 (1huBMP-1), human mTLD (1huTLD), human mTLL-1 (1huTLL-1), or human mTLL-2 (1huTLL-2). Note that some embryos coinjected with mRNAs for Chordin and mTLL-1 retained secondary axes, although the majority of these were much reduced in size. Representatives of such “weak” residual axes are denoted by arrows in the 1huTLL-1 panel. (B) Histogram showing the induction of secondary axes by Chordin, Noggin, a dominant negative BMP type I receptor (DN-BR), or a “cleavage mutant” BMP-7 (Cm-XBMP7) in the presence or absence of human or Xenopus mTLD or BMP-1 (huTLD, XTLD, huBMP-1, or XBMP-1, respectively), human mTLL-2 (huTLL-2), or human mTLL-1 (huTLL-1). The frequency of induction of secondary axes was calculated from the total number (“n,” above the bars) of injected embryos. Human and Xenopus BMP-1 strongly inhibit secondary axis induction by Chordin, whereas mRNA encoding the alternatively spliced product huTLD or XTLD does not. Human mTLL-1 had intermediate effects, in that 37% of embryos retained secondary axes, although the majority of these were greatly reduced in size, as illustrated in (A). The lack of effect of BMP-1 or mTLL-1 on secondary axes induced by noggin or by BMP pathway inhibitors DN-BR or Cm-BMP7 shows that these enzymes specifically act through effects on Chordin.

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alternatively spliced RNAs for BMP-1 (XBMP-1) and for a longer protein (XTLD) corresponding to mTLD (Lin et al., 1997). Interestingly, we found XBMP-1 to counteract the dorsalizing effects of Chordin to the same extent as did mammalian BMP-1, while XTLD, like mTLD, showed a virtual absence of anti-dorsalizing activity (Fig. 4B). The nearly identical results obtained with BMP-1 and XBMP-1 or with mTLD and XTLD, indicate a conservation in the difference in Chordin-cleaving activity of these two alternatively spliced products of the same gene in mammals and frog. Overexpression of Noggin also induces secondary axis formation in Xenopus embryos, but this induction is not countered by overexpression of XLD (Piccolo et al., 1997) or Drosophila TLD (Marque´s et al., 1997), which seem specific for cleaving Chordin. Similarly, we found neither BMP-1 nor mTLL-1 to be capable of rescuing embryos from Noggin-induced dorsalization (Fig. 4B). These latter results are also in agreement with the cleavage assays of Fig. 2 and indicate that BMP-1 and mTLL-1, like XLD and Drosophila TLD, cleave some secreted BMP antagonists, but not others. Neither BMP-1 nor mTLL-1 rescued embryos in which secondary dorsal axes had been induced by injection of mRNA for a dominant negative BMP type I receptor (Fig. 4B), supporting the interpretation that the ventralizing actions of BMP-1 and mTLL-1 occur within the BMP pathway, upstream of the BMP receptor. Also consistent with this interpretation was the inability of BMP-1 and mTLL-1 to rescue embryos dorsalized with a dominant negative BMP-7 cleavage mutant (Hawley et al., 1995), thought to block BMP signaling by the formation of nonfunctional heterodimers with endogenous TGFb-like BMPs (Fig. 4B). Various in vitro (Figs. 2F and 3) and in vivo (Fig. 4) data thus indicate that BMP-1 and mTLL-1, but not mTLD or mTLL-2, are capable of affecting morphogenetic events by specifically cleaving Chordin to release TGFb-like BMPs.

Differential Expression Patterns of BMP-1, mTLD, mTLL-1, mTLL-2, and Chordin in 7.5 dpc Mouse Embryos The interaction of SOG/Chordin with BMP-1/TLD-like enzymes has been shown to be important in formation of the dorsal–ventral axis in flies (Marque´s et al., 1997), frogs (Piccolo et al., 1997), and fish (Blader et al., 1997). Thus, it was of interest to examine the expression domains of

BMP-1, mTLD, mTLL-1, and mTLL-2 in relation to the distribution of expression of Chordin at the time of dorsal– ventral axis formation in mouse. Toward this end, in situ hybridization was performed to determine the expression domains of BMP-1, mTLD, mTLL-1, mTLL-2, and Chordin in 7.5 dpc neural plate stage mouse embryos. Both BMP-1 (e.g., Figs. 5A and 5B) and mTLD (not shown) were found to be broadly expressed at high levels throughout the mouse gastrula and in extraembryonic tissues. This is similar to the broad distribution of BMP-1 expression reported for Xenopus gastrulae (Goodman et al., 1998). In contrast, expression of mTLL-1 and mTLL-2 was limited to two mutually exclusive regions of the egg cylinder. Punctate signals for mTLL-1 were localized in the anterior portion of the egg cylinder, to either side of the neural groove (Figs. 5C and 5D). As previously reported (Clark et al., 1999), these punctate mTLL-1 signals lie within the expression domain of the cardiac-specific transcription factor Nkx-2.5 in precardiac mesoderm. Expression of mTLL-2 was limited to the posterior portion of the egg cylinder in the nascent mesoderm streaming off the primitive streak (Figs. 5E and 5F). This expression was found to extend up and down almost the entire length of the streak, but was not found in the streak itself. At the distal end of the embryo cylinder, mTLL-2 expression ended at the node. Proximally, extraembryonic mesoderm, including the allantois were negative (data not shown). In situ hybridization with a probe for mouse Chordin showed signal to be localized to the notochordal plate (Figs. 5G and 5H) and to be at high levels in the node (not shown). Thus, in 7.5 dpc mouse embryos, the expression domain for Chordin is overlapped by those of BMP-1 and mTLD, is flanked in some regions (notochord) but not others (node) by the expression domain of mTLL-1, and only nears the expression domain of mTLL-2 where the primitive streak approaches the posterior aspect of the node.

Expression Patterns of BMP-1, mTLD, mTLL-1, mTLL-2, and Chordin in Endochondral Bone Formation Peptides common to BMP-1 and mTLD copurify from osteogenic extracts of bone with peptides of TGFb-like BMPs that are capable of inducing endochondral bone formation (Wozney et al., 1988). This suggests roles for BMP-1 and/or mTLD in endochondral bone formation, beyond their roles as procollagen C-proteinases. The finding

FIG. 5. Expression of BMP-1, mTLD, mTLL-1, mTLL-2, and Chordin RNA in 7.5 dpc mouse embryos. Bright-field (A,C,E,G) and corresponding dark-field (B,D,F,H) photomicrographs are shown for in situ hybridization of BMP-1 (A,B), mTLL-1 (C,D), mTLL-2 (E,F), and Chordin (G,H) anti-sense riboprobes to transverse sections of 7.5 dpc embryos. (A–F) Serial sections taken about 135 mm from the proximal tip of a single 500-mm-long embryo. (G,H) Serial sections taken from the same region (135 mm from the proximal tip) of a different 500-mm embryo of the same stage. The region from which the serial sections were taken is shown in a drawing of a late allantoic bud stage (Downs and Davies, 1993) embryo (bottom). The anterior of each embryo section is facing upward, and that of the drawing is toward the left. Arrows point to specific signals from hybridization of mTLL-1 (D), mTLL-2 (F), and Chordin probes.

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that BMP-1 and mTLL-1 can cleave Chordin (above) suggested that such proteases may also act in endochondral bone formation through liberation of TGFb-like BMPs. Possible roles for Chordin in bone development have not previously been explored, although the importance of at least some antagonists of BMP signaling in skeletogenesis is evident in the phenotype of Noggin-null mutant mice, which have defects in cartilage and joint formation (Brunet et al., 1998). To ascertain whether Chordin is expressed in developing bone and, if so, how its distribution of expression in bone might compare with those of BMP-1, mTLD, mTLL-1, and mTLL-2, in situ hybridization analysis was performed on serial sections of a 15.5 dpc murine hindlimb (Fig. 6). As is shown, patterns of expression for Chordin are similar to those of BMP-1 and mTLD (Figs. 6B, 6D, and 6F). All three show distribution throughout limb mesenchyme, with particularly high levels of expression overlying perichondrium of the cartilage primordia of the tarsals, metatarsals, and phalanges and overlying perichondrium of the ossifying cartilage of the femur. High expression is also seen for all three overlying a tendon inserted into the femur. Expression of mTLL-1 (Fig. 6C), while more limited in distribution and at lower levels than expression of BMP-1, mTLD, or Chordin, is also found in perichondrium. In contrast to the four other gene products analyzed, mTLL-2 expression (Fig. 6E) was not detected in perichondrium, but seemed confined to the developing musculature of the limb. In fact, in situ hybridization of various sections of 10.5–17.5 dpc embryos has confirmed that mTLL-2, unlike any of the other known mammalian BMP-1/TLD-like proteases, is not expressed in developing bone or cartilage, with expression being detected only in certain developing skeletal muscles and within portions of the developing central nervous system (Scott et al., unpublished data). To investigate possible roles for Chordin and the proteases of this study in endochondral bone formation after birth, the distribution of each was analyzed by in situ hybridization in the area of the femoral growth plate of a 21-day-old mouse. As is shown (Fig. 7), the distribution of Chordin expression (Fig. 7D) is similar to that of mTLD (Fig. 7F) with high expression for both in the areas of ossification in both epiphysis and metaphysis. Interestingly, although the pattern of expression for BMP-1 is similar, with high levels of expression in centers of ossification, it also differs in that relatively high levels of BMP-1 expression are seen in cartilage of the epiphyseal plate (Fig. 7E). Although this may suggest a greater role for BMP-1 in the cartilaginous portion of the growth plate, signals for mTLD and Chordin, although markedly lower than for BMP-1, are also visible in growth plate cartilage (Figs. 7D and 7F). To emphasize the latter point, in situ hybridization is shown for a serial section of growth plate hybridized to a probe for the type I procollagen C-proteinase enhancer protein (PCPE) (Fig. 7G), a secreted glycoprotein that binds specifically to the C-propeptide of type I procollagen (Kessler and Adar, 1989; Takahara et al., 1994a). Signal for PCPE, like those for BMP-1, mTLD, and Chordin is high

overlying areas of ossification, but unlike BMP-1, mTLD, and Chordin, it is seen to be truly excluded from the cartilage of the epiphyseal plate. The data of Fig. 7 thus show BMP-1, mTLD, and Chordin to be correctly placed to play roles in postnatal growth plates. Of particular note, in light of the high Chordin-cleaving activity of BMP-1, is the colocalization of signal for Chordin and BMP-1 in Figs. 6 and 7, suggesting functional interaction between these two in pre- and postnatal endochondral bone formation. Signal for mTLL-1 is visible in the vicinity of the growth plate (Fig. 7B), however, in contrast to Chordin, BMP-1, and mTLD, signal for mTLL-1 is at relatively low levels in centers of ossification and is most notable in the perichondrium/periosteum. Signal for mTLL-2 did not appear to be above background in developing bone (Fig. 7C).

DISCUSSION In a screen for additional mammalian BMP-1/TLD-like proteases, we have isolated the novel BMP-1/TLD-like protease mammalian Tolloid-like 2 (mTLL-2). Thus, mammals possess at least three genes that encode four BMP-1/ TLD-like proteins, showing a previously unsuspected level of genetic complexity for this family of developmental proteases. Moreover, the synthesis and purification of recombinant versions of each mammalian BMP-1/TLD-like protease has enabled us to directly compare their enzymatic activities. Interestingly, while we have found some functional redundancy between family members, we have also found significant differences. Previously, BMP-1 and mTLD have been shown to provide the procollagen C-proteinase (PCP) activity that processes procollagen precursors into monomers capable of forming the major fibrous structures of vertebrate extracellular matrix (Kessler et al., 1996; Li et al., 1996). In contrast, Xenopus Xolloid (XLD) (Piccolo et al., 1997), zebrafish Tolloid (zTLD) (Blader et al., 1997), and Drosophila Tolloid (TLD) (Marque´s et al., 1997) have previously been shown to cleave Chordin or its orthologue SOG. Here we show that two mammalian BMP-1/TLD-like proteases, BMP-1 and mTLL-1, are each capable of both activities. Thus, an important concept emerges, that there is not a strict separation of those BMP-1/TLD proteases which have direct roles in the deposition of matrix and those with direct roles in the potentiation of signaling by TGFb-like molecules. Instead, the dual activities of enzymes such as BMP-1 and mTLL-1 suggest that such enzymes may serve to orchestrate the deposition of matrix and the activation of TGFblike BMPs in morphogenetic events. Cleavage of Chordin by both BMP-1 and mTLL-1 is shown to be highly specific, with both enzymes cleaving at the same two sites, each of which resembles the physiological cleavage sites at which BMP-1 has previously been shown to cleave the C-propeptides of the major fibrillar procollagens I–III (Kessler et al., 1996). As predicted (Piccolo et al., 1997), the N-terminal Chordin cleavage site occurs

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FIG. 6. Expression of BMP-1, mTLD, mTLL-1, mTLL-2, and Chordin RNA in 15.5 dpc mouse embryo hindlimb. Bright-field (A) or dark-field (B–F) photomicrographs are shown for serial sections of a 15.5 dpc mouse embryo hindlimb characterized by staining with hematoxylin– eosin and alcian blue (A) or by in situ hybridization with anti-sense Chordin (B), mTLL-1 (C), BMP-1 (D), mTLL-2 (E), or mTLD (F) riboprobes. T, tendon; CP, cartilage primordia; F, femur; M, muscle.

just downstream of the first cysteine-rich repeat. However, in contrast to previous predictions, the C-terminal cleavage site does not occur within cysteine-rich repeat 3, but instead occurs downstream of this domain. The cysteine-

rich repeats of Chordin show homology to similar domains found in the N-propeptides of some procollagen I–III chains (Sasai et al., 1994; Zhu et al., 1999). Interestingly, the single cysteine-rich domain within a splice variant of the

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FIG. 7. Expression of BMP-1, mTLD, mTLL-1, mTLL-2, Chordin, and the procollagen C-proteinase enhancer protein (PCPE) in femoral growth plate from a 21-day-old mouse. Bright-field (A) or dark-field (B–G) photomicrographs are shown for serial sections of the demineralized femoral growth plate from a 21-day-old mouse. Sections were characterized by staining with hematoxylin– eosin and alcian blue (A) or by in situ hybridization with anti-sense mTLL-1 (B), mTLL-2 (C), Chordin (D), BMP-1 (E), mTLD (F), or PCPE (G) riboprobes.

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N-propeptide of procollagen II has recently been shown to bind BMP-2 (Zhu et al., 1999). However, since data in the present report show that none of the cysteine-rich repeats is cleaved in the inactivation of Chordin by mammalian BMP-1/TLD-like proteases, no single Chordin cysteine-rich repeat is responsible for binding TGFb-like molecules in an inactive complex. Rather, it is the overall configuration of intact Chordin, or the covalent joining of the various Chordin cysteine-rich repeats into a single polypeptide that seems necessary for the latter function. Interestingly, mTLD, a product of alternatively spliced mRNAs of the BMP1 gene, while a capable PCP, is shown to be incapable of cleaving Chordin by two independent assays. This is the first indication that enzymatic differences have evolved in the two proteases encoded by the BMP1 gene. Moreover, the functional importance of this difference in enzymatic activities is underscored by its evolutionary persistence, since it is demonstrated to be conserved in humans and frogs. Thus, while BMP-1 may serve to coordinate the deposition of matrix and the activation of TGFb-like proteins, mTLD may function, in some tissues, solely as a PCP. However, mTLD is also expressed in developing and adult central nervous systems (Takahara et al., 1994b) where there is a paucity of fibrillar collagens, suggesting that mTLD has specificity for additional, yet to be identified substrate(s). It should be noted that BMP-1 and mTLD, as products of the same gene, have identical proregion, protease, CUB1, CUB2, EGF1, and CUB3 sequences and differ only at C-termini, where the final 27 amino acids of BMP-1 are replaced by additional C-terminal EGF2, CUB4, and CUB5 domains in mTLD (see Wozney et al., 1988 and Takahara et al., 1994b). Therefore, the C-terminal residues unique to BMP-1 or mTLD determine the ability or inability of these two proteases to cleave Chordin. Future studies involving BMP-1 and mTLD C-terminal residues should, thus, provide further insights into the nature of interactions between BMP-1/TLD-like proteases and Chordin/SOG. In contrast to BMP-1, mTLD, and mTLL-1, novel family member mTLL-2 does not process either procollagens or Chordin. In addition, mTLL-2 is also the first mammalian family member that is not expressed at high levels in developing skeletal elements. Instead, fetal mTLL-2 expression seems limited to developing skeletal muscle (this study) and central nervous system (Scott et al., unpublished data). Earlier in development, expression of mTLL-2 is uniquely localized to nascent mesoderm in 7.5 dpc mouse embryos. The substrate specificity and developmental roles of mTLL-2, which remain to be characterized, therefore appear to be quite distinct from those of BMP-1, mTLD, and mTLL-1. Thus, despite the high similarities in sequence and domain structure, which indicate a probable common origin from simple gene duplications, the mammalian BMP-1/TLD proteases have diverged markedly in their functions, which appear to be more far-ranging than previously suspected. Previously, in vitro assays have shown Drosophila TLD

to efficiently cleave SOG in the presence, but not in the absence, of TGFb-like ligands DPP, SCW, or 60A (Marque´s et al., 1997), whereas this ligand dependence is not observed for XLD, which cleaves Chordin with similar efficiency whether or not ligand is present (Piccolo et al., 1997). The present study shows BMP-1 and mTLL-1 to be similar to XLD, in that they cleave Chordin in vitro in the absence of ligand. Interestingly, however, mTLD and mTLL-2 differ from both XLD and Drosophila TLD in that they do not counteract the dorsalizing effects of Chordin upon coinjection of cognate RNAs into Xenopus embryos. In contrast, levels of endogenous ligand appear sufficient to stimulate the cleavage of SOG by Drosophila TLD in similar types of coinjection experiments (Marque´s et al., 1997). Thus, present results suggest that mTLD and mTLL-2 do not cleave Chordin even in the presence of ligand. Nevertheless, the possibility remains that cleavage specificities and/or activity levels of the various enzymes described here may be changed in the presence of sufficient local concentrations of BMP ligands or other cofactors, and the possibility of such effects merits further study. Bmp1 homozygous null mice are perinatal lethal, with defects in formation of the ventral body wall, in procollagen processing, and in fibrillogenesis (Suzuki et al., 1996). Nevertheless, these BMP-1/mTLD-null embryos lack obvious patterning defects or gross defects in formation of the axial or appendicular skeleton and have residual procollagen C-protease activity (Suzuki et al., 1996). The latter observations suggested genetic redundancy and led to the isolation of sequences for mTLL-1 (Takahara et al., 1996). Similarly, mTLL-1-null mice, although embryonic lethal with a spectrum of defects apparently confined to the heart, lack obvious patterning defects or defects in procollagen processing or fibrillogenesis (Clark et al., 1999). The findings of the present report appear to provide the molecular bases for the phenotypes of the BMP-1/mTLD-null and mTLL-1-null mice. Thus, mTLL-1 is shown to be a PCP capable of providing the residual PCP activity found in BMP-1/mTLD-null mice and providing what appears to be sufficient Chordin-cleaving activity to prevent gross patterning defects in these animals. In mTLL-1-null mice, the more robust PCP activity of BMP-1 combined with that of mTLD, seems sufficient to prevent defects in procollagen processing and fibrillogenesis, while the Chordin-cleaving activity of BMP-1 seems sufficient to prevent derangement of BMP signaling except in certain structures of the heart in which the expression domain of BMP-1 does not appear to overlap that of mTLL-1 (Clark et al., 1999). Peptides leading to the isolation of BMP-1 were originally copurified with TGFb-like BMPs from osteogenic extracts of bone (Wozney et al., 1988). Here, in addition to demonstrating that BMP-1 has high Chordin-cleaving activity, data also show that expression patterns for BMP-1 and Chordin are highly similar in both pre- and postnatal sites of endochondral bone formation. Thus, although it is clear from the skeletal defects of Noggin-null mice that Noggin plays an important role in regulating BMP activity in the

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formation of bone (Brunet et al., 1998), the present data indicate that the interplay of BMP-1 and Chordin may normally play an important role in skeletogenesis as well. Such an interplay in skeletogenesis may well recapitulate earlier interactions of BMP-1, Chordin, and TGFb-like BMPs involved in the dorsoventral patterning of vertebrates. In early development, BMP-1, with the highest levels of Chordin-cleaving activity of any of the mammalian BMP1/TLD-related proteases, is also shown to be broadly expressed throughout 7.5 dpc murine embryonic and extraembryonic tissues. Thus, BMP-1 has the enzymatic activity and seems properly distributed in the gastrulating embryo to provide the postulated function of acting as a “clearing system” (Piccolo et al., 1997) that prevents excess accumulation of extracellular Chordin, thereby preserving a Chordin gradient. In contrast, the Chordin-cleaving activity of mTLL-1 is less than that of BMP-1 and its distribution of expression in 7.5 dpc murine embryos is much more limited, suggesting a less-prominent role in the clearing of Chordin in the normal course of dorsoventral patterning. This suggests that the lack of patterning defects in BMP-1null embryos may be due to compensatory upregulation of mTLL-1 expression. Alternatively, mTLL-1 may normally be more efficiently translated or have greater stability than BMP-1, giving it greater relative abundance in tissues, at the protein level, than is suggested by levels or distribution of mTLL-1 RNA. Since the genes for BMP-1 and mTLL-1 are located on different murine chromosomes (Ceci et al., 1990; Takahara et al., 1996), “double knockout” embryos, homozygous null for both genes, can be generated by simple matings of Bmp1 1/2 and Tll1 1/2 heterozygous animals. The presence of early patterning defects in such doubly homozygous null embryos would indicate that the absence of such defects in BMP-1-null mice is due to functional substitution by mTLL-1. The absence of patterning defects in doubly homozygous null embryos would indicate that additional BMP-1/TLD proteases with Chordin-cleaving activity remain to be discovered. Such studies will also help address the question of whether, despite differences in distributions of expression, mTLL-1 is likely to be the mammalian orthologue of XLD and/or of zebrafish TLD or whether such orthologues remain to be discovered.

ACKNOWLEDGMENTS We thank Guy G. Hoffman and Satoshi S. Kinoshita for excellent technical assistance and Brian Schmidt for help in constructing some of the expression vectors. This work was supported by National Institutes of Health Grants AR43621, GM46846 (to D.S.G.), and GM54704 (to K.W.Y.C) and by a grant from FibroGen, Inc., South San Francisco, CA (to D.S.G.). B.M.S. was supported by National Institutes of Health Predoctoral Training Grant T32 GM07215.

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