A Complete Domain Structure of Drosophila Tolloid Is Required for Cleavage of Short Gastrulation
2006; Elsevier BV; Volume: 281; Issue: 19 Linguagem: Inglês
10.1074/jbc.m510483200
ISSN1083-351X
AutoresElizabeth G. Canty, Laure Garrigue‐Antar, Karl E. Kadler,
Tópico(s)TGF-β signaling in diseases
ResumoDrosophila tolloid (TLD) is a member of a family of proteinases that play important roles in development and includes mammalian tolloid (mTLD) and bone morphogenetic protein (BMP)-1. TLD accentuates the activity of decapentaplegic (DPP), a transforming growth factor β superfamily growth factor, by cleaving its antagonist Short gastrulation (Sog). Similarly, the activity of BMP-2/4 (vertebrate homologues of DPP) is augmented by cleavage of chordin. However, whereas TLD is an effective Sogase, mTLD is a poor chordinase and is functionally replaced by its smaller splice variant BMP-1, which lacks the most C-terminal epidermal growth factor (EGF)-like and CUB domains of mTLD. Moreover, the minimal chordinase activity resides in the N-terminal half of BMP-1. This study showed that the proteolytic activity of TLD is considerably enhanced by Ca2+ and tested the hypothesis that the Sogase activity of TLD resides in the N-terminal half of the proteinase. Unexpectedly, it was found that TLD lacking the CUB4 and CUB5 domains and/or the EGF-like domains was unable to cleave Sog. Loss of function mutations have been reported in the tld gene that result in amino acid substitutions at E835K (in CUB4), S915L (in CUB5), and N760I (in EGF2) in TLD. The CUB mutants were found to be ineffective Sogases, but the activity of the EGF2 mutant was unchanged. The results show that substrate recognition and cleavage by Drosophila tolloid and mTLD are different despite their identical domain structure and homologous functions in patterning. The result that the N760I mutant has full Sogase activity suggests that novel substrates for TLD exist. Drosophila tolloid (TLD) is a member of a family of proteinases that play important roles in development and includes mammalian tolloid (mTLD) and bone morphogenetic protein (BMP)-1. TLD accentuates the activity of decapentaplegic (DPP), a transforming growth factor β superfamily growth factor, by cleaving its antagonist Short gastrulation (Sog). Similarly, the activity of BMP-2/4 (vertebrate homologues of DPP) is augmented by cleavage of chordin. However, whereas TLD is an effective Sogase, mTLD is a poor chordinase and is functionally replaced by its smaller splice variant BMP-1, which lacks the most C-terminal epidermal growth factor (EGF)-like and CUB domains of mTLD. Moreover, the minimal chordinase activity resides in the N-terminal half of BMP-1. This study showed that the proteolytic activity of TLD is considerably enhanced by Ca2+ and tested the hypothesis that the Sogase activity of TLD resides in the N-terminal half of the proteinase. Unexpectedly, it was found that TLD lacking the CUB4 and CUB5 domains and/or the EGF-like domains was unable to cleave Sog. Loss of function mutations have been reported in the tld gene that result in amino acid substitutions at E835K (in CUB4), S915L (in CUB5), and N760I (in EGF2) in TLD. The CUB mutants were found to be ineffective Sogases, but the activity of the EGF2 mutant was unchanged. The results show that substrate recognition and cleavage by Drosophila tolloid and mTLD are different despite their identical domain structure and homologous functions in patterning. The result that the N760I mutant has full Sogase activity suggests that novel substrates for TLD exist. The tolloid/bone morphogenetic protein 1 (BMP-1) 2The abbreviations used are: BMP-1, bone morphogenetic protein-1; TLD, Drosophila tolloid; mTLD, mammalian tolloid; 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; PSP, porcine sperm adhesion protein; HRP, horseradish peroxidase; Sog, Short gastrulation; DPP, decapentaplegic; Scw, Screw; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; HA, epitope from the influenza A virus hemagglutinin. metalloproteinases are fundamental to the normal development of animals because they cleave antagonists of the BMP subfamily of transforming growth factor β signaling molecules (1Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar). For example, Drosophila tolloid cleaves Short gastrulation (Sog), thereby augmenting the activities of decapentaplegic (DPP) and Screw (SCW) in dorsal-ventral patterning of the fly (2Marques G. Musacchio M. Shimell M.J. Wunnenberg-Stapleton K. Cho K.W. O'Connor M.B. Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 3Nguyen M. Park S. Marques G. Arora K. Cell. 1998; 95: 495-506Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 4Shimmi O. Umulis D. Othmer H. O'Connor M.B. Cell. 2005; 120: 873-886Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Similarly in vertebrates, chordin, which is a functional homologue of Sog and sequesters BMPs, is cleaved by tolloid family members (5Blader P. Rastegar S. Fischer N. Strahle U. Science. 1997; 278: 1937-1940Crossref PubMed Scopus (169) Google Scholar, 6Piccolo S. Agius E. Lu B. Goodman S. Dale L. De Robertis E.M. Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 7Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (232) Google Scholar). On the face of it, the mechanism of proteolytic activation of the BMP·antagonist complex by tolloid proteinases would appear to be identical in all animals, from flies to man. However, evidence from in vitro and in vivo studies suggests that there are fundamental differences in the way tolloids recognize their substrates in flies and vertebrates. An obvious difference is that Drosophila only has two tolloid genes, which encode two distinct proteins, whereas vertebrates have three tolloid genes and at least five tolloid and tolloid-like proteins (Fig. 1). The Drosophila genes code for tolloid (TLD) (8Shimell M.J. Ferguson E.L. Childs S.R. O'Connor M.B. Cell. 1991; 67: 469-481Abstract Full Text PDF PubMed Scopus (254) Google Scholar) and tolloid-related (TLR), also known as tolloid-related 1 and tolkin (9Nguyen T. Jamal J. Shimell M.J. Arora K. O'Connor M.B. Dev. Biol. 1994; 166: 569-586Crossref PubMed Scopus (70) Google Scholar, 10Finelli A.L. Xie T. Bossie C.A. Blackman R.K. Padgett R.W. Genetics. 1995; 141: 271-281Crossref PubMed Google Scholar, 11Serpe M. Ralston A. Blair S.S. O'Connor M.B. Development (Camb.). 2005; 132: 2645-2656Crossref PubMed Scopus (58) Google Scholar). These proteins contain a signal peptide (which directs the newly synthesized protein to the endoplasmic reticulum), a prodomain (which is cleaved by dibasic pro-protein convertases in the secretory pathway), a zinc-binding astacin-like metalloproteinase domain, and five CUB domains (numbered 1-5) that are interspersed with two EGF-like domains between CUB2 and CUB3 and between CUB3 and CUB4. The three tolloid genes in vertebrates code for proteins with identical domain structure, namely mammalian tolloid (mTLD), tolloid like-1 (mTLL-1), and tolloid like-2 (mTLL-2) (7Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (232) Google Scholar, 12Takahara K. Lyons G.E. Greenspan D.S. J. Biol. Chem. 1994; 269: 32572-32578Abstract Full Text PDF PubMed Google Scholar, 13Takahara K. Brevard R. Hoffman G.G. Suzuki N. Greenspan D.S. Genomics. 1996; 34: 157-165Crossref PubMed Scopus (84) Google Scholar). However, the gene encoding mTld also gives rise to two smaller splice variants that lack the EGF2-CUB4-CUB5 domains of the molecule. One molecule, BMP-1, contains a short unique sequence at its C terminus and the other, BMP-1His, contains a sequence rich in histidine residues. Comparison of the fly and vertebrate tolloids highlights further differences. For example, mTLD is a poor chordinase, whereas its smaller splice variant, BMP-1, is a highly effective chordinase (7Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (232) Google Scholar). Furthermore, the chordinase activity of mTLD can be greatly enhanced by genetically engineering a mTld protein that lacks the EGF-like domains (14Garrigue-Antar L. Francois V. Kadler K.E. J. Biol. Chem. 2004; 279: 49835-49841Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The differences in mechanism are further apparent because TLD will only cleave Sog in the presence of DPP or SCW (2Marques G. Musacchio M. Shimell M.J. Wunnenberg-Stapleton K. Cho K.W. O'Connor M.B. Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 3Nguyen M. Park S. Marques G. Arora K. Cell. 1998; 95: 495-506Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), whereas vertebrate tolloid proteinases do not require complexed BMPs to cleave chordin. Vertebrate tolloids also have additional developmental functions in that they are involved in the biosynthetic processing of extracellular matrix macromolecules (15Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google Scholar). It is intriguing that BMP-1, which is the shortest tolloid proteinase in vertebrates and lacks the most C-terminal CUB and EGF-like domains that are present in mTLD, is a highly effective chordinase. This implies that the Sogase activity of TLD could be increased by removal of the C-terminal CUB domains, i.e. a fly equivalent of BMP-1 could be a more effective Sogase. However, mutations E835K (in CUB4) and S915L (in CUB5) disrupt dorsal-ventral patterning during Drosophila embryogenesis (16Finelli A.L. Bossie C.A. Xie T. Padgett R.W. Development (Camb.). 1994; 120: 861-870Crossref PubMed Google Scholar, 17Childs S.R. O'Connor M.B. Dev. Biol. 1994; 162: 209-220Crossref PubMed Scopus (62) Google Scholar), implying that these domains have an important function. The effect of tolloid mutations on patterning is similar to, but less severe than, the effect of mutations in the dpp gene (18Ferguson E.L. Anderson K.V. Development (Camb.). 1992; 114: 583-597Crossref PubMed Google Scholar). In this study we have shown that the complete domain structure of TLD is required for Sogase activity. Mutant TLD proteins lacking the EGF-like domains or truncated to remove the C-terminal CUB and EGF domains were ineffective proteases. Furthermore, Sogase activity was significantly reduced for the E835K and S915L mutants. These results demonstrate the importance of the C-terminal CUB domains for TLD activity. Source of Materials—Plasmids encoding full-length Drosophila Sog (pBSsog) and tolloid (pNBtld) and the plasmid SKAsc2 (pBluescript II SK± modified to contain two AscI sites in the multiple cloning site) were a kind gift from Dr. Hilary Ashe (University of Manchester). cDNA Manipulations—The tolloid cDNA was subcloned from plasmid pNB40 (19Brown N.H. Kafatos F.C. J. Mol. Biol. 1988; 203: 425-437Crossref PubMed Scopus (510) Google Scholar) into pSKAsc2 using HindIII and NotI (to give pSKTld). Subsequent site-directed mutagenesis was then used to convert the stop codon to an XbaI restriction site (to give pSKTldXbaI). The tolloid cDNA cassette was transferred to pAc5.1/V5-HisA (Invitrogen) using KpnI and XbaI such that the 3′-end of the tolloid cDNA was in-frame with the downstream V5 tag provided by the expression vector (to give pAcTld). The Sog cDNA had been previously subcloned from pNB40 into Bluescript SK± to give pBSsog. The stop codon was similarly removed, and the Sog cDNA subcloned into pAc5.1/V5-HisA (Invitrogen). Site-directed Mutagenesis—A two-step strand overlap PCR strategy, or a single PCR amplification, using Expand High Fidelity polymerase (Roche Applied Science) was used to create cDNA fragments containing the desired nucleotide changes. The sequences of the primers used for amplification are listed in Table 1. The PCR products were cloned into pCR2.1 using the TOPO TA cloning kit (Invitrogen) and sequenced using M13 or internal primers. For truncated tolloid a single PCR reaction using the primers BstEIIF and EGF2toXbaIR was performed. Subcloning from the TA cloning vector to pAcTld was accomplished using BstEII and XbaI restriction enzymes, resulting in the truncated tolloid cDNA in pAc5.1/V5HisA. The EGF deletion mutants were created using primers comprising 10 bp of one, and 20 bp of the other, flanking CUB domain. For ΔEGF1, the initial PCR reactions were performed using BstEIIF with C2C3R and C3C2F with Bsu36IR. These PCR products were then gel purified and amplified separately using BstEIIF and Bsu36IR prior to TA cloning. Subcloning into pAc5.1/V5-HisA was accomplished using BstEII and Bsu36I restriction enzymes to produce the EGF deletion constructs in pAc5.1/V5HisA. For ΔEGF2, the initial PCR reactions were performed using Bsu36IF with C3C4R and C4C3F with BpuA1R. These PCR products were then gel purified and amplified separately using Bsu36IF and BpuA1R prior to TA cloning. There are two BpuA1 sites in pAc5.1/V5-His. To circumvent this problem, the fragment was first subcloned into pSKTldXbaI along with the ΔEGF1 fragment for the double deletion mutant, and the entire cDNA cassette was transferred to pAc5.1/V5-HisA using KpnI and XbaI. HA-tagged tolloid was produced using a single PCR reaction with the primers Bsu36IF and HA+STOP+XbaIR. After TA cloning and sequencing, Bsu36I and XbaI restriction enzymes were used to obtain the full-length HA-tagged tolloid cDNA in pAc5.1/V5HisA. For the HA-tagged tolloid mutants, the initial PCR reactions were performed using Bsu36IF with N760IR, E835KR, or S915LR and N760IF, E835KF, or S915LF with HA+STOP+XbaIR. The second round of PCR was performed using Bsu36IF with HA+STOP+XbaIR and the appropriate gel-purified first-round PCR products. Digestion with Bsu36I and XbaI was used to transfer the cDNA fragments containing the HA tags and mutations into pAcTld. For the EGF1 mutations, the initial PCR reactions were performed using BstEIIF with D581ER or D581AR and D581EF or D581AF with Bsu36IR. These PCR products were then gel purified and amplified separately using BstEIIF and Bsu36IR prior to TA cloning. The resulting cDNA fragments were subcloned into pAcTld using BstEII and Bsu36I restriction enzymes to produce the required constructs.TABLE 1Primer sequences used to perform site-directed mutagenesis The primers were used as described under “Experimental Procedures.”Primer namePrimer sequenceBpuA1R5′-TCTCTGAAGACCCGCATCCGT-3′BstEIIF5′-GCCAAAGGTTACCGTGGATCC-3′Bsu36IF5′-GAGTATCCTCAGGCTGGAGTT-3′Bsu36IR5′-AACTCCAGCCTGAGGATACTC-3′C2C3R5′-CTCCTCCACACAGCATCAAGGCGGCTGAGA-3′C3C2F5′-CTTGATGCTGTGTGGAGGAGTCGTGGACGC-3′C3C4R5′-CGAACTTGCATATTACAAACTTGGCCACAA-3′C4C3F5′-GTTTGTAATATGCAAGTTCGAGATCACCAC-3′D581AF5′-TTGATGCTGGCCGTGGATGAG-3′D581AR5′-CTCATCCACGGCCAGCATCAA-3′D581EF5′-TTGATGCTGGAGGTGGATGAG-3′D581ER5′-CTCATCCACCTCCAGCATCAA-3′E835KF5′-ACTTTGAGGTGAAGAGCCACCA-3′E835KR5′-TGGTGGCTCTTCACCTCAAAGT-3′EGF2toXbaIR5′-TCTAGATATTACAAACTTGGCCACAAATC-3′HA+STOP+XbaIR5′-TCTAGACTAAGCGTAGTCTGGGACGTCGTATGGGTAAATGAGTCTGCTGGGTGGC-3′N760IF5′ -CGATGCCGAATCACCTTTGGAT-3′N760IR5′-ATCCAAAGGTGATTCGGCATCG-3′S915LF5′-GACCTTCTACTTGCATCCACGA-3′S915LR5′-TCGTGGATGCAAGTAGAAGGTC-3′ Open table in a new tab Protein Expression—The Drosophila Expression System (Invitrogen) was used to express recombinant proteins in Drosophila melanogaster S2 cells according to the manufacturer's instructions. Briefly, S2 cells were maintained at 24 °C in Schneider's Drosophila medium supplemented with 10% fetal calf serum and 1% Pen-Strep. Calcium phosphate-based transient transfections were carried out using 5 or 19 μgof DNA (without any observable difference in expression levels) per 35-mm plate. Expression of recombinant tolloid contructs was analyzed by Western blotting at 24, 48, and 72 h post-transfection. The expression of V5-tagged Sog was investigated further to maximize the amount of substrate obtained from each transient transfection and was found to continue up to 7 days post-transfection. Depending on the volume to be processed and where required, the medium was dialysed into 50 mm Tris-HCl, pH 7.4, 100 mm NaCl or loaded onto a HiTrap or disposable PD10 desalting column (Amersham Biosciences) and eluted with 50 mm Tris-HCl buffer (pH 7.4) containing 100 mm NaCl, and 0.01% NaN3 (TBS). Preparation of Medium and Cell Lysates—S2 cells in suspension were separated from the cell culture medium by centrifugation at 1000 × g for 3 min. The medium was decanted and the cells washed by resuspension in phosphate-buffered saline. The cells were respun and the supernatant discarded. The cell pellet was lysed in ice-cold Nonidet P-40 lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40) and the cell lysates clarified by centrifugation at 13,000 × g for 1 min. Electrophoresis and Western Blotting—Samples were resolved by electrophoresis on 8 or 12% gels by SDS-Page or on NuPAGE Novex 4-12% Bis-Tris gels using NuPAGE MES or MOPS running buffer (Invitrogen) under reducing conditions and subjected to Western immunoblotting. The mouse monoclonal horseradish peroxidase (HRP)-conjugated anti-V5 antibody (Invitrogen) or the mouse monoclonal HRP-conjugated 3F10 antibody directed against the HA tag (Roche Applied Science) was used as appropriate. The signal was detected by enhanced chemiluminescence using ECL (Amersham Biosciences) or SuperSignal West Dura Extended Duration Substrate (Pierce). Where necessary, the intensity of the bands corresponding to the active forms of the enzymes, or of the Sog cleavage products, was quantified by densitometry using AIDA 2.0 software. Assay of Sogase Activity—Digestions were performed in TBS or in cell culture medium in the presence of protease inhibitors at 25 °C overnight or for the indicated times. A concentrated stock solution of protease inhibitors was made using EDTA-free Protease Inhibitor Mixture tablets (Roche Applied Science). Digests also contained recombinant DPP (0.5 ng/μl, 33 nm) (R&D Systems), CaCl2 (0.1-5 mm), and EDTA (5 mm) as required and where indicated. The V5-tagged Sog cleavage products were detected by electrophoresis and Western blotting. The use of V5-tagged enzymes did not affect the detection of V5-tagged Sog fragments because they were present at least an order of magnitude less than the substrate in the assay. Location of Exon/Intron Junctions within the Tolloid-related Domain Structure—The genomic organization and sequence for tolkin was obtained from Flybase (flybase.bio.indiana.edu). The entire genomic sequence was translated in all reading frames using the Expasy proteomics server translate tool (us.expasy.org/tools/dna.html). cDNA sequences are also available on GenBank™ (accession numbers U34777 and U12634). Comparison of the conceptual translation products, the cDNA and genomic sequences, the amino acid sequence of tolkin, and the location of the domains allowed the identification of the location of the introns on the tolkin domain structure. V5-His-tagged Sog and Active V5-His-tagged Tolloid Are Expressed and Secreted by D. melanogaster Schneider S2 Cells—Full-length tolloid and Sog cDNAs were subcloned in-frame into the vector pAc5.1V5HisA (Invitrogen) and used to transfect Drosophila S2 cells. Western blot analysis of the cell culture medium using the anti-V5 antibody showed that TLD-V5-His and Sog-V5-His (Fig. 2A) were readily secreted from the S2 cells (Fig. 2B) and that cleavage of Sog-V5-His by TLD-V5-His required the addition of exogenous DPP (not shown) (2Marques G. Musacchio M. Shimell M.J. Wunnenberg-Stapleton K. Cho K.W. O'Connor M.B. Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Hence, the addition of two tandem epitope tags at the C termini of the molecules does not abolish the secretion of the proteins from S2 cells or the ability of TLD to cleave Sog. The Sogase Activity of Drosophila Tolloid Is Substantially Promoted by Exogenous Calcium Ions—It was previously shown that procollagen C proteinase, purified from chick embryo tendons, required 10 mm calcium chloride for maximal cleavage of the C-propeptides from type I procollagen and that the enzyme was inactive in the absence of exogenous calcium ions (20Hojima Y. van der Rest M. Prockop D.J. J. Biol. Chem. 1985; 260: 15996-16003Abstract Full Text PDF PubMed Google Scholar). Subsequent studies showed that procollagen C proteinase activity is exhibited by BMP-1 and mTLD (21Li S.W. Sieron A.L. Fertala A. Hojima Y. Arnold W.V. Prockop D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5127-5130Crossref PubMed Scopus (201) Google Scholar, 22Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (457) Google Scholar) as well as mammalian tolloid-like 1 (7Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (232) Google Scholar). These results have been used to imply that all members of the tolloid family require relatively high concentrations of Ca2+ for proteolytic activity. TLD is able to cleave Sog in the presence of exogenous DPP in Schneider's cell culture medium where the concentration of CaCl2 is 5.41 mm and in M3 insect medium where the concentration is 7 mm (2Marques G. Musacchio M. Shimell M.J. Wunnenberg-Stapleton K. Cho K.W. O'Connor M.B. Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 11Serpe M. Ralston A. Blair S.S. O'Connor M.B. Development (Camb.). 2005; 132: 2645-2656Crossref PubMed Scopus (58) Google Scholar, 23Yu K. Srinivasan S. Shimmi O. Biehs B. Rashka K.E. Kimelman D. O'Connor M.B. Bier E. Development (Camb.). 2000; 127: 2143-2154Crossref PubMed Google Scholar, 24Shimmi O. O'Connor M.B. Development (Camb.). 2003; 130: 4673-4682Crossref PubMed Scopus (93) Google Scholar). To determine whether Drosophila tolloid requires Ca2+, the cell culture medium was subjected to dialysis or gel filtration prior to the digestion assay. TLD exhibited negligible Sogase activity in the absence of exogenous Ca2+ ions but could be recovered by the addition of 0.1-5 mm CaCl2 (Fig. 2C). A Drosophila-equivalent BMP-1 Does Not Cleave Sog—The Drosophila tolloid gene contains six short introns that are thought to facilitate rapid transcription and splicing during the short cell division cycles of the developing Drosophila embryo (10Finelli A.L. Xie T. Bossie C.A. Blackman R.K. Padgett R.W. Genetics. 1995; 141: 271-281Crossref PubMed Google Scholar, 16Finelli A.L. Bossie C.A. Xie T. Padgett R.W. Development (Camb.). 1994; 120: 861-870Crossref PubMed Google Scholar, 17Childs S.R. O'Connor M.B. Dev. Biol. 1994; 162: 209-220Crossref PubMed Scopus (62) Google Scholar, 25Rothe M. Pehl M. Taubert H. Jackle H. Nature. 1992; 359: 156-159Crossref PubMed Scopus (119) Google Scholar). Tolloid-related plays a crucial role later in development, and its gene contains larger and more numerous introns (10Finelli A.L. Xie T. Bossie C.A. Blackman R.K. Padgett R.W. Genetics. 1995; 141: 271-281Crossref PubMed Google Scholar). No shorter tolloid or tolloid-related splice variants have been reported in D. melanogaster (10Finelli A.L. Xie T. Bossie C.A. Blackman R.K. Padgett R.W. Genetics. 1995; 141: 271-281Crossref PubMed Google Scholar, 26Takahara K. Lee S. Wood S. Greenspan D.S. Genomics. 1995; 29: 9-15Crossref PubMed Scopus (23) Google Scholar). Alternative splicing events between the exons coding for the CUB3 and EGF2 domains in the mammalian tolloid gene are responsible for the production of the shorter BMP-1 and BMP-1His isoforms (Fig. 1). BMP-1 lacks the C-terminal EGF2, CUB4, and CUB5 domains and is a more effective C-proteinase and chordinase. To test the hypothesis that a shorter splice variant of tolloid would be a more effective Sogase, we produced a BMP-1-like tolloid molecule lacking the EGF2, CUB4, and CUB5 domains (Fig. 3A). This molecule was constructed to contain a C-terminal V5-His tag and expressed in S2 cells. The Drosophila equivalent BMP-1 (truncated tolloid) was found in both the latent (i.e. retaining the prodomain) and mature (prodomain deficient) forms in the cell culture medium (Fig. 3B). Presumably, the proprotein was cleaved by a furinlike proprotein convertase to produce the mature protein. On assaying the recombinant tolloids it was found that the short form of tolloid did not cleave Sog (Fig. 3C). Removal of the EGF-like Domains from Drosophila Tolloid Affects the Secretion and Activity of the Enzyme Produced by S2 Cells—It has been shown that removal of the EGF-like domains from mTLD converts it to a chordinase and more effective C-proteinase (14Garrigue-Antar L. Francois V. Kadler K.E. J. Biol. Chem. 2004; 279: 49835-49841Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To investigate the role of the EGF-like domains in Drosophila TLD, a series of deletion mutants were generated that lacked either, or both, of the EGF-like domains (Fig. 4A). cDNA constructs encoding these proteins were transfected into S2 cells and the cell lysates and cell culture medium analyzed by Western blotting from 24 to 72 h post-transfection (Fig. 4B). Removal of the EGF-like domains markedly reduced the amount of enzyme secreted from S2 cells. To normalize the amounts of each enzyme and compare the relative Sogase activity for equivalent amounts of enzyme, a range of volumes of cell culture medium obtained 48 h post-transfection were analyzed by Western blotting using the anti-V5 antibody (Fig. 5A). The relative position of the immunoreactive bands indicates that the enzymes were present in both the latent and the mature (prodomain deficient) forms in the cell culture medium. By using equivalent amounts of the active forms of each enzyme in the Sogase assay we were able to show that only the ΔEGF1 TLD enzyme retained any activity, whereas proteins lacking the EGF2 domain were unable to cleave Sog (Fig. 5B). Sogase Activity Is Reduced by Point Mutations at Conserved Residues in CUB4 and CUB5, but Not by Mutations in EGF1 or EGF2—To investigate further the role of the CUB and EGF domains in TLD, the range of chemically induced mutations in the tolloid gene that affect dorsal-ventral patterning in the Drosophila embryo were collated from published data (16Finelli A.L. Bossie C.A. Xie T. Padgett R.W. Development (Camb.). 1994; 120: 861-870Crossref PubMed Google Scholar, 17Childs S.R. O'Connor M.B. Dev. Biol. 1994; 162: 209-220Crossref PubMed Scopus (62) Google Scholar, 18Ferguson E.L. Anderson K.V. Development (Camb.). 1992; 114: 583-597Crossref PubMed Google Scholar) and from Flybase (flybase.bio.indiana.edu) (Table 2). These mutations affect residues throughout the protease and regulatory domains, and many of the mutations introduce premature stop codons into the N-terminal half of tolloid (see “Discussion”). It was of particular interest to determine the effects of the single amino acid substitutions located in the C-terminal EGF2, CUB4, and CUB5 domains in light of the observation that these domains are crucial for the Sogase activity of TLD. There are eight substitutions in the CUB domains and one in EGF2. The positions of the substitutions within the CUB domain structure were identified (Fig. 6A) and superimposed onto the crystal structure of a single CUB domain from the spermadhesin porcine seminal plasma PSP-1/PSP-II heterodimer (Fig. 6B). This indicated that the CUB domain mutations cluster in a region that appears to be involved in calcium ion binding. The single mutation in EGF2 affects an asparagine (N) residue that is part of the consensus sequence for Ca2+ binding (Fig. 8A).TABLE 2Tolloid alleles resulting in disruptions to dorsal ventral patterning in the Drosophila embryo The allele strength given is as described in the published work. @, mutation that resulted in a stop codon.StrengthAllele namesAmino acid changeDomainReferencesNotesVery weaktld1, tld5H, tld5H56M487KCUB2(17Childs S.R. O'Connor M.B. Dev. Biol. 1994; 162: 209-220Crossref PubMed Scopus (62) Google Scholar, 18Ferguson E.L. Anderson K.V. Development (Camb.). 1992; 114: 583-597Crossref PubMed Google Scholar)Antimorphtld6, tld7 M, tld7M89R235HProtease(17Childs S.R. O'Connor M.B. Dev. Biol. 1994; 162: 209-220Crossref PubMed Scopus (62) Google Scholar, 18Ferguson E.L. Anderson K.V. Development (Camb.). 1992; 114: 583-597Crossref PubMed Google Scholar)AntimorphWeak, incompletely penetranttld2, tld6B, tld6B9, tld6B69E839KCUB4(16Finelli A.L. Bossie C.A. Xie T. Padgett R.W. Development (Camb.). 1994; 120: 861-870Crossref PubMed Google Scholar, 18Ferguson E.L. Anderson K.V. Development (Camb.). 1992; 114: 583-597Crossref PubMed Google Scholar)Weak, completely penetranttld7, tld7O, tld70D884VCUB4(16Finelli A.L. Bossie C.A. Xie T. Padgett R.W. Development (Camb.). 1994; 120: 861-870Crossref PubMed Google Scholar, 18Ferguson E.L. Anderson K.V. Development (Camb.). 1992; 114: 583-597Crossref PubMed Google Scholar)tld10, tld9D, tld9D36D728YCUB3(17Childs S.R. O'Connor M.B. Dev. Biol. 1994; 162: 209-220Crossref PubMed Scopus (62) Google Scholar, 18Ferguson E.L. Anderson K.V. Development (Camb.). 1992; 114: 583-597Crossref PubMed Google Scholar)Temperature sensitivetld12, tld9Q1, tld9Q, tld9Q19E517KCUB2(17Childs S.R. O'Connor M.B. Dev. Biol. 1994; 162: 209-220Crossref PubMed Scopus (62) Google Scholar, 18Ferguson E.L. Anderson K.V. Development (Camb.). 1992; 114: 583-597Crossref PubMed Google Scholar)AntimorphWeaktldE4N760IEGF2(16Finelli A.L. Bossie C.A. Xie T. Padgett R.W. Development (Camb.). 1994; 120: 861-870Crossref PubMed Google Scholar)tldE6V21 M & D728NSS & CUB3(16Fin
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