Eur J Biochem 269, 6162–6172 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03335.x Expression and characterization of recombinant vitamin K-dependent c-glutamyl carboxylase from an invertebrate, Conus textile Eva Czerwiec1, Gail S Begley1, Mila Bronstein2, Johan Stenflo1,3, Kevin Taylor1, Barbara C Furie1,2 and Bruce Furie1,2 Marine Biological Laboratory, Woods Hole, MA, USA; 2Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA; 3Department of Clinical Chemistry, Lund University, University Hospital, Malmo, Sweden The marine snail Conus is the sole invertebrate wherein both the vitamin K-dependent carboxylase and its product, c-carboxyglutamic acid, have been identified To examine its biosynthesis of c-carboxyglutamic acid, we studied the carboxylase from Conus venom ducts The carboxylase cDNA from Conus textile has an ORF that encodes a 811amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and  60% sequence similarity to the bovine carboxylase Expression of this cDNA in COS cells or insect cells yielded vitamin K-dependent carboxylase activity and vitamin Kdependent epoxidase activity The recombinant carboxylase has a molecular mass of  130 kDa The recombinant Conus carboxylase carboxylated Phe-Leu-Glu-Glu-Leu and the 28-residue peptides based on residues )18 to +10 of human proprothrombin and proFactor IX with Km values of 420 lM, 1.7 lM and lM, respectively; the Km for vitamin K is 52 lM The Km values for peptides based on the sequence of the conotoxin e-TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 lM, 75 lM and 74 lM, respectively The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides These results suggest two propeptide-binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K-dependent c-carboxylases argue for conservation of a vitamin K-dependent carboxylase across animal species and the importance of c-carboxyglutamic acid synthesis in diverse biological systems The vitamin K-dependent carboxylase catalyzes the posttranslational conversion of glutamic acid into c-carboxyglutamic acid in prothrombin, other blood coagulation proteins, and various vitamin K-dependent proteins [1,2] In this reaction, CO2 replaces the c-proton on specific glutamic acid residues of the peptide substrate to yield c-carboxyglutamic acid This enzymatic reaction is unique in that it involves a strong base catalysis mechanism that requires a labile oxidized form of vitamin K [3] The mammalian vitamin K-dependent carboxylase exhibits both carboxylase activity and vitamin K epoxidase activity [4] Precursor proteins bearing within their propeptides the c-carboxyla- tion-recognition site that binds directly to the carboxylase serve as substrates for this enzyme [5,6] The recognition site is sufficient to direct c-carboxylation [7] Cloning of the human and bovine carboxylases revealed a protein of 758 amino acids without obvious homology to other known proteins [8,9] Cloning of other mammalian vitamin Kdependent carboxylases revealed marked (> 90%) aminoacid sequence conservation, and the toadfish carboxylase showed 70% amino-acid sequence similarity to the mammalian carboxylases [8–11] The bovine c-carboxylase, composed of a single polypeptide chain rich in hydrophobic amino acids, is an integral membrane protein of molecular mass 94 kDa that resides in the endoplasmic reticulum [12– 14] The propeptide-binding site, the active site, and the vitamin K-binding site of the c-carboxylase have not been defined with certainty by affinity labeling and site-specific mutagenesis [15–20] Cysteine residues are important within the active site [21], and Cys99 and Cys450 have been proposed as critical residues [22] To understand the synthesis of c-carboxyglutamic acid in nonvertebrates and to define structure–function relationships in the vitamin K-dependent carboxylase, we have studied the carboxylase from a marine snail, Conus textile Cone snails of the genus Conus are the sole invertebrates wherein both the vitamin K-dependent carboxylase and its product, c-carboxyglutamic acid, have been identified Correspondence to B Furie, Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA E-mail: bfurie@caregroup.harvard.edu Abbreviations: proPT18, residues )18 to )1 of proprothrombin; proPT28, residues )18 to +10 of proprothrombin; proFIX18, residues )18 to )1 of proFactor IX; proFIX28, residues )18 to +10 of proFactor IX; pro-e-TxIX/12, residues )12 to )1 of e-TxIX precursor; e-TxIX12, residues 1–12 of e-TxIX; pro-e-TxIX/24, residues )12 to + 12 of e-TxIX precursor; pro-e-TxIX/41, residues )29 to +12 of e-TxIX precursor (Received 12 September 2002, accepted 24 October 2002) Keywords: blood coagulation; conotoxins; hemophilia; posttranslational processing; vitamin K Ó FEBS 2002 Conus vitamin K-dependent c-carboxylase (Eur J Biochem 269) 6163 [23,24] These marine gastropods use small biologically active peptides (conotoxins) to paralyze fish, marine worms and molluscs [25,26] Many c-carboxyglutamic acid-containing conotoxins have been identified [27–31] The metalbinding properties [32–35] and the 3D structures of some of these conopeptides suggest a specific structural role for c-carboxyglutamic acid [36–40] Experiments with crude preparations of Conus carboxylase have shown that this enzymatic reaction requires vitamin K [24,41] Efficient carboxylation requires a carboxylation-recognition site located on a precursor form of the conotoxin [42,43] However, the Conus carboxylation-recognition site is different from the carboxylation-recognition site in mammalian carboxylase substrates We have previously isolated a highly conserved region from the Conus carboxylase gene that exhibits marked sequence similarity to other c-carboxylases [10] This observation coupled to the finding of a carboxylase gene in the Drosophila genome [44,45] suggests a broad distribution for the vitamin K-dependent carboxylase in animal phyla We have cloned and expressed the Conus carboxylase in order to prove that this gene encodes a vitamin K-dependent carboxylase, and identified structural and functional similarities and differences between invertebrate and verterbrate vitamin K-dependent carboxylases We demonstrate amino-acid sequence similarities between the Conus and vertebrate c-glutamyl carboxylases The Conus carboxylase is also a vitamin K epoxidase, but several functional properties with regard to propeptide stimulation distinguish this enzyme from its mammalian counterpart Mammalian propeptides bind the enzyme, but are 10–80fold less potent in stimulating the Conus carboxylase Most importantly, Conus propeptides not stimulate either the Conus carboxylase or the mammalian carboxylase Yet, the presence of these propeptide sequences directs carboxylation and confers low Km on substrates in both the Conus and the mammalian system [43] This suggests that two sites may exist on the vitamin K-dependent carboxylase, one of which is a substrate-binding catalytic site and one a regulatory site In contrast with previous characterization of the Conus carboxylase activity in preparations derived from venom ducts, expression of the recombinant Conus carboxylase in a system free of endogenous carboxylase activity and substrates will considerably facilitate studies of the mechanistic properties of this unique enzyme EXPERIMENTAL METHODS Materials Live cone snails were obtained from Fiji, and frozen specimens of C textile were obtained from Vietnam FastTrack kits, TA cloning kits (with pCR2.1-TOPO and pCR4-TOPO vectors), the pcDNA 3.1/V5-His cloning kit, the pIB/V5-His TOPO TA cloning kit and anti-V5 horseradish peroxidase-conjugated antibody were from Invitrogen (Carlsbad, CA, USA) A kZAPII Custom cDNA library from C textile venom duct was prepared by Stratagene (La Jolla, CA, USA) TRIzol reagent, ThermoScript RT-PCR System, Platinum PCR Supermix, Platinum Pfx Polymerase, restriction enzymes, synthetic oligonucleotide primers, serum-free adapted Sf21 cells and Sf 900-II SFM medium were obtained from Gibco–BRL Life Tech- nologies (Grand Island, NY, USA) RACE kits and Advantage cDNA polymerase mix were from Clontech (Palo Alto, CA, USA) AmpliTaq Gold polymerase and buffer were from Perkin–Elmer (Branchburg, NJ, USA) Reagents for DNA purification were from Qiagen (Santa Clarita, CA, USA) Reagents for digoxygenin labeling of DNA and detection were obtained from Roche Biochemicals (Indianapolis, IN, USA) Superose 12 was from Pharmacia (Piscataway, NJ, USA) NaH14CO3 (55 mCiỈ mmol)1) and the ECL detection system were from Amersham Life Sciences (Arlington Heights, IL, USA) Atomlight scintillation fluid was from Packard (Meriden, CT, USA) Vitamin K1 was obtained from Abbott Laboratories (North Chicago, IL, USA) BSA (fraction V), FLEEL (PheLeu-Glu-Glu-Leu), L-phosphatidylcholine (type V-E) and Chaps were purchased from Sigma (St Louis, MO, USA) Kaleidoscope prestained standards were obtained from Bio-Rad (Hercules, CA, USA) Poly(vinylidene difluoride) membranes were from Millipore (Bedford, MA, USA) All other chemicals were of the highest grade commercially available Chemical synthesis of carboxylase peptide substrates proPT18 (residues )18 to )1 of proprothrombin), proPT28 (residues )18 to +10 of proprothrombin), proFIX18 (residues )18 to )1 of proFactor IX), proFIX28 (residues )18 to +10 of proFactor IX), pro-e-TxIX/12 (residues )12 to )1 of e-TxIX precursor), e-TxIX12 (residues 1–12 of e-TxIX), pro-e-TxIX/24 (residues )12 to +12 of e-TxIX precursor), pro-e-TxIX/41 (residues )29 to +12 of e-TxIX precursor) and FLEEL were synthesized using Fmoc/NMP chemistry on an Applied Biosystems model 430A peptide synthesizer [46] The amino-acid sequences of the synthetic substrates and propeptides are shown in Table Preparation of cone snail venom duct homogenates, microsomal preparations and cell homogenates Snails were extricated from their shell and laid flat on a cooled glass plate Venom ducts were removed and homogenized using a Tissue Tearor mixer for 10 s in : to 10 : (w/v) buffer A (250 mM sucrose, 500 mM KCl, 25 mM imidazole/HCl, pH 7.2) containing 0.1% (w/v) Chaps and · PIC (2 mM dithiothreitol, mM EDTA, 0.5 lgỈmL)1 leupeptin, lgỈmL)1 pepstatin A, lgỈmL)1 aprotinin) Homogenates were centrifuged at 12 000 g for min, and supernatants were subsequently centrifuged at 100 000 g for h at °C to separate the microsomal fraction The supernatant was discarded, and the pellet was resuspended in buffer B [25 mM Mops (pH 7.0), 500 mM NaCl, 0.1% (w/v) Chaps, 0.1% (w/v) phosphatidylcholine, 0.1 mM phenylmethanesulfonyl fluoride, 20% (v/v) glycerol] and sonicated using a model 220F sonicator (Heat SystemsUltrasonics) Sf21 cells were collected by centrifugation and washed in NaCl/Pi, pH 7.2 Cells were resuspended at a density of · 106 cellsỈmL)1 in lysis buffer [10 mM Mops (pH 7.0), 10 mM KCl, mM MgCl2, · PIC] containing 0.1% (w/v) Chaps Cells were homogenized in a glass homogenizer (10 strokes) and centrifuged at 500 g to separate cell debris The supernatant was centrifuged at 100 000 g for h at °C to separate the microsomal fraction The pellet was resuspended in NaCl/Pi (pH 7.2) Ó FEBS 2002 6164 E Czerwiec et al (Eur J Biochem 269) Table Amino-acid sequences of synthetic substrates and propeptides Bold type ¼ mature sequence Name Sequence proPT18 proPT28 proFIX18 proFIX28 pro-e-TxIX/12 e-TxIX12 pro-e-TxIX/24 pro-e-TxIX/41 HVFLAPQQARSLLQRVRR HVFLAPQQARSLLQRVRRANTFLEEVRK TVFLDHENANKILNRPKR TVFLDHENANKILNRPKRYNSGKLEEFV LKRTIRTRLNIR ECCEDGWCCTAA LKRTIRTRLNIRECCEDGWCCTAA ARTKTDDDVPLSSLRDNLKRTIRTRLNIRECCEDGWCCTAA containing 0.1% (w/v) Chaps, 0.1% (w/v) phosphatidylcholine, 0.1 mM phenylmethanesulfonyl fluoride and 20% (v/v) glycerol and sonicated COS7 cells (5 · 106 cells) were washed with NaCl/Pi, trypsinized and collected in NaCl/Pi (pH 7.2) containing 20% glycerol and · PIC Cells were homogenized in a glass homogenizer (3 · 10 strokes) and centrifuged at 500 g The pellet was rehomogenized and washed times with the same buffer Pooled supernatants were centrifuged at 100 000 g for h at °C to separate the microsomal fraction The pellet was resuspended in NaCl/Pi (pH 7.2) containing 0.5% (w/v) Chaps, 0.2% (w/v) phosphatidylcholine, · PIC and 20% (v/v) glycerol by sonication Enzyme assays The amount of 14CO2 incorporated into exogenous substrates was measured in reaction mixtures of 125 lL containing substrate at the indicated concentration, 222 lM reduced vitamin K1, 0.72 mM NaH14CO3 (5 mCi), 28 mM Mops (pH 7.0), 500 mM NaCl, 0.16% (w/v) phosphatidylcholine, 0.16% (w/v) Chaps and 0.8 M ammonium sulfate, unless stated otherwise All of the assay components except carboxylase were prepared as a master mixture The reaction was initiated by adding the master mixture to carboxylase-containing preparations 14CO2 incorporated into peptide substrates over 30 was assayed in a scintillation counter [6] Stimulation experiments with propeptide were performed at a constant concentration of enzyme and substrate (3.6 mM FLEEL or 1.6 mM e-TxIX12) and increasing concentrations of the propeptide proPT18, proFIX18 or pro-e-TxIX/12, as indicated Vitamin K epoxidase activity was assayed by HPLC as previously described [21] Molecular cloning of C textile vitamin K-dependent carboxylase All PCRs were performed in a PE Applied Biosystems 9700 thermocycler Degenerate primers were used at a final concentration of lM, and gene-specific primers at a final concentration of 0.2 lM Sequences of PCR products were obtained after cloning into the pCR2.1-TOPO or pCR4.0TOPO vector Ligation reactions were subsequently used to transform chemically competent Escherichia coli TOP10 cells Transformants were selected on Luria–Bertani agar plates containing 50 lgỈmL)1 kanamycin and 5-bromo-4chloro-3-indolyl-D-galactoside for blue/white screening Positive colonies were grown in Luria–Bertani broth containing 50 lgỈmL)1 ampicillin Plasmid DNA was extracted by alkaline lysis column mini preps (Qiagen) DNA was sequenced in an Applied Biosystems 373 DNA sequencer The full-length nucleotide sequence of the gene for the vitamin K-dependent carboxylase from C textile was obtained by assembling sequence information from screening a C textile venom duct kZAPII Custom cDNA library and from specific products amplified by PCR The kZAPII Custom library was screened with Probe (121 bp), based on the nucleotide sequence of the conserved motif identified in C textile cDNA [10], and two identical clones were identified in a pool of · 105 clones The insert contained a fragment encoding a polypeptide of 192 amino acids homologous to the region starting at residue 386 in the bovine carboxylase This fragment contains 18 residues of the 38-residue conserved motif previously identified [10] Sequence 3¢ of the clone from the kZAPII library was obtained with gene-specific primers using RACE-PCR (3¢ RACE primer and 2) Overlapping sequence from the PCR products obtained with both primers was identical and included the Ochre STOP codon (TAA) in the same ORF Sequence for the region located 5¢ of the conserved motif was obtained by PCR using a degenerate primer and a gene-specific primer The degenerate primer [F(L/I)(L/I/S)(P/S)YWY(V/I)F (L/F)LDK(T/P)(S/T/A)WNNHSYL] was designed based on the sequence of the region that was identified in vertebrate and invertebrate carboxylases (residues 142– 163) The degenerate primer in combination with a genespecific primer yielded a specific product that encodes 260 residues of a carboxylase homolog (homologous to region 164–401 of bovine carboxylase) This sequence information was used to design a new probe (Probe 2, 537 bp), complementary to the region ending 186 bp 5¢ (C textile sequence) of the codon for Gly386 This probe identified a clone with an insert of 1740 bp that contained the start codon of the Conus carboxylase at position 67 The insert encodes a protein of 557 amino acids that is homologous to the region 1–518 of bovine carboxylase The full length of the Conus carboxylase was obtained by assembling the sequences from the phage library clones and RACE-PCR reaction products Expression of vitamin K-dependent carboxylase cDNA Gene fusion constructs encoding a C-terminal V5-tagged and His-tagged enzyme were made in the pIB-V5/His TOPO vector and in the pcDNA3.1-V5/His vector The Ó FEBS 2002 Conus vitamin K-dependent c-carboxylase (Eur J Biochem 269) 6165 ORF for the cone snail carboxylase was amplified by Platinum Pfx Polymerase with the carboxylase 5¢ ORF and 3¢ ORF primers using cDNA as a template The fragment was ligated into the pIB-V5/His TOPO vector after addition of A overhangs by AmpliTaq Gold polymerase generating the pIB-CCbx-V5/His expression construct The pcDNA3.1-CCbx-V5/His construct was made by adding KpnI and XhoI restriction sites to the ORF using the carboxylase 5¢ ORF-KpnI and 3¢ ORF-XhoI primers during amplification, followed by restriction digest and ligation into the pcDNA3.1-V5/His vector digested with the same enzymes The plasmids were transformed into E coli, and transformants were screened for the insert by restriction digestion of plasmid DNA Recombinant plasmids were subjected to DNA sequencing Serum-free adapted Sf21 cells were transfected with pIBCCbx-V5/His plasmid DNA using the empty vector and the pIB-CAT-V5/His construct provided by the manufacturer as a control Transfectants were selected by blasticidin and expanded to suspension cultures COS7 cells were transfected with pcDNA3.1-CCbx-V5/ His using the empty vector as a control Cells were harvested after 48 h and monitored for transient expression Cell homogenates from COS7 cells or from stable Sf21 transfectants were assayed for carboxylase activity using FLEEL Western-blot analysis of the recombinant Conus vitamin K-dependent carboxylase The cell homogenate preparations were evaluated for recombinant carboxylase by Western-blot analysis after transfer to a poly(vinylidene difluoride) membrane after electrophoresis on a 10% SDS/polyacrylamide gel The expressed protein was detected using the horseradish peroxidase-conjugated anti-V5 Ig (1 lgỈmL)1) The CATV5/His protein was used as a positive control Positive bands were detected by chemiluminescence Quantitative Western-blot analysis was performed using Positope as a protein standard Carboxylase was quantitated in microsomal preparations from transfected Sf21 cells and COS7 cells Densitometric analysis was performed using the GELPRO ANALYZER program (Media Cybernetics, North Reading, MA, USA) RESULTS Cloning of the vitamin K-dependent carboxylase cDNA Vitamin K-dependent carboxylase activity was measured in venom duct homogenates from Conus bandanus, Conus geographus, Conus leopardus, Conus marmoreus, Conus striatus, C textile and Conus virgo In contrast with mammalian tissue preparations, crude cone snail venom duct homogenates contain large amounts of endogenous substrates which become labeled with 14CO2 during the carboxylase assay in the absence of added exogenous peptide substrate Carboxylase assay of crude venom duct homogenates from seven Conus species showed 14CO2 incorporation into endogenous substrates alone or into endogenous substrates plus exogenous peptide substrate The level of activity is species-dependent and varies up to 400-fold The highest specific activity was measured in venom duct homogenates from C marmoreus [3.9 · 106 c.p.m.Ỉ(mg protein))1Ỉ(30 min))1], C textile [1.1 · 106 c.p.m.Ỉ(mg protein))1Ỉ(30 min))1] and C bandanus [1.1 · 106 c.p.m.Ỉ(mg protein))1Ỉ(30 min))1], and the lowest in venom duct homogenates from C striatus [9 · 103 c.p.m.Ỉ(mg protein))1Ỉ(30 min))1] Relative amounts of carboxylation occurring on endogenous substrates varied from as high as 32% of total activity in C textile venom duct homogenate to as low as 0.5% in C geographus homogenate Because of the high specific activity and the availability of the species, we chose to clone and express the Conus carboxylase from C textile The full-length cDNA encoding the vitamin K-dependent carboxylase from C textile was assembled, as described in Experimental methods The entire cDNA sequence includes 3795 bp, with a 5¢ UTR of 66 nucleotides and a 3¢ UTR of 1296 nucleotides The translational start site begins at nucleotide 67 and the stop site (TAA) is at nucleotide 2499 The 5¢ untranslated region, which was not mapped, is presumably incomplete The 3¢ untranslated sequence includes a polyadenylation consensus sequence (AATAAA) located 17 nucleotides upstream of the polyA tail An ORF of 2433 bp encoding an 811-amino-acid protein is predicted (Supplementary material; GenBank accession number AF382823) Comparison of the primary structure of the Conus and vertebrate vitamin K-dependent carboxylases The N-terminal amino acids of the Conus carboxylase are dominated by acidic residues including three aspartate residues and a glutamate-rich region that includes stretches of three and five glutamate residues in the region 19–30 (bovine carboxylase numbering system) (Fig 1) This is in contrast with vertebrate vitamin K-dependent carboxylases, in which the charged residues are predominantly basic The ORF encodes a protein rich in hydrophobic residues, consistent with the prediction of multiple membranespanning regions for the human and bovine carboxylases [9,47] and with the functional properties of this enzyme as an integral membrane protein We aligned the amino-acid sequences of all of the cloned vertebrate vitamin K-dependent carboxylases with the invertebrate carboxylases from Drosophila and Conus (Fig 1) The Conus carboxylase has a 15-amino-acid N-terminal extension and a three-residue C-terminal extension relative to the mammalian carboxylases The Conus carboxylase has 811 amino acids compared with 758 amino acids for most of the mammalian carboxylases The large central portion of this enzyme shows sequence similarity to the vertebrate and invertebrate vitamin K-dependent carboxylases, and is flanked by divergent N-terminal and C-terminal sequences Alignment of the Conus carboxylase sequence with the primary structure of the vertebrate carboxylase homologs indicates the presence of one one-residue insertion, four two-residue insertions, one three-residue insertion, one six-residue insertion, and one 19-residue insertion; there are two oneresidue deletions From this alignment, seven conserved regions (CR) were identified These include CR1 (33–317), CR2 (356–415), CR3 (420–451), CR4 (465– 519), CR5 (528–544), CR6 (555–567), and CR7 (581–609) Residues that are identical among the Conus carboxylase and the vertebrate carboxylases are highlighted in deep 6166 E Czerwiec et al (Eur J Biochem 269) Ó FEBS 2002 Fig Alignment of the amino-acid sequence of the vitamin K-dependent carboxylase from Conus (C textile) with the sequences from the vitamin K-dependent carboxylase from bovine (Bos taurus), human (Homo sapiens), rat (Rattus norvegicus), mouse (Mus musculus), sheep (Ovis aries), Beluga whale (Delphinapterus leucas), toadfish (Opsanus tau), and fruitfly (Drosophila melanogaster) The bovine carboxylase numbering system is used Conserved regions (CR) are shown in light yellow and variable regions (VR) are shown in white Residues that are identical in the Conus sequence and all of the vertebrate carboxylase sequences are highlighted in deep yellow; valine, leucine and isoleucine are sufficiently similar that we have considered them as identical for this analysis The Drosophila sequence (ÔflyÕ) is shown for comparison Each residue of the Conus carboxylase is compared with the vertebrate carboxylase sequence Amino-acid similarity is depicted in red and nonconserved residues are shown in black yellow in Fig These identical residues are widely distributed within the conserved sequences of the Conus carboxylase The most extensive regions of high sequence identity among all of the carboxylases include residues 118–126 and 157–167 in CR1, residues 195–241 in CR1, residues 390–407 in CR2, and residues 528–544 in CR5 The bovine carboxylase and Conus carboxylase sequence share 42% identity (CLUSTAL method with the MEGALIGN program) Of the amino acids between residues 33 and 610, 52% are identical comparing the bovine and Conus carboxylase sequences (CLUSTAL method with MEGALIGN program), and 65% are conserved using BLAST analysis comparing all of the vitamin K-dependent carboxylases In contrast, the C-terminal 25% of the protein shows no homology to any of the other vitamin K-dependent carboxylases Ó FEBS 2002 Conus vitamin K-dependent c-carboxylase (Eur J Biochem 269) 6167 Expression of the Conus vitamin K-dependent carboxylase The Conus carboxylase was expressed in COS7 cells, a mammalian cell line 14CO2 incorporation into FLEEL was 40 292 c.p.m.Ỉ(30 min))1 in the presence of vitamin K and proPT18 when microsomes from cells transfected with the plasmid vector containing Conus carboxylase cDNA were assayed 14CO2 incorporation into FLEEL in the absence of vitamin K was 483 c.p.m.Ỉ(30 min))1 with the same microsomal fraction As COS7 cells have endogenous carboxylase activity, COS7 cells transfected with a plasmid vector lacking the Conus carboxylase cDNA were tested for carboxylase activity for comparison In these experiments, 14 CO2 incorporation into FLEEL was 6274 c.p.m.Ỉ(30 min))1 These experiments indicate increased expression of carboxylase in COS cells of about sevenfold over endogenous carboxylase levels To ensure that the increased caboxylase activity observed in COS cells transfected with a plasmid vector containing carboxylase cDNA arose from expression of carboxylase from this cDNA, we expressed recombinant Conus carboxylase in Sf21 insect cells These cells not express endogenous carboxylase activity Cells transfected with the construct containing the Conus carboxylase cDNA showed significant carboxylase activity This activity had an absolute requirement for vitamin K in that the carboxylase activity was 1237 c.p.m.Ỉ(30 min))1 in the presence of vitamin K and 181 c.p.m.Ỉ(30 min))1 in its absence In the presence of proPT18 and vitamin K, this activity increases to 10 239 c.p.m.Ỉ(30 min))1 Carboxylase activity was not detected in homogenates from nontransfected cells, with carboxylase activity of 199 c.p.m.Ỉ(30 min))1 and 102 c.p.m.Ỉ(30 min))1 with and without added vitamin K, respectively Similarly, cells expressing a C-terminally tagged chloramphenicol acetyltransferase expressed from the same vector as the Conus carboxylase cDNA had no significant carboxylase activity in the presence or absence of vitamin K Together, these results indicate that the recombinant Conus carboxylase activity can be functionally expressed in two different eukaryotic systems These results prove that the identified coding sequence encodes a protein with vitamin K-dependent carboxylase activity Further, using insect cells, this expression system provides a source of Conus carboxylase free of endogenous carboxylase and carboxylase substrates Molecular mass analysis of the recombinant Conus vitamin K-dependent carboxylase The molecular mass of the expressed Conus vitamin K-dependent carboxylase containing a C-terminal V5 and His tag was determined using antibodies to the V5 epitope Cell homogenates from Conus carboxylase cDNA-transfected cells, nontransfected cells and cells transfected with chloramphenicol acetyltransferase (CAT) cDNA were analyzed by Western blot (Fig 2) Sf21 cells transfected with the carboxylase cDNA-containing plasmid show a major band at  130 kDa (Fig 2, lane B) Cells transfected with the CAT-V5/His plasmid show the expected band of  33 kDa (Fig 2, lane C) In contrast, no bands from homogenates from nontransfected cells and a preparation Fig Molecular mass analysis of Conus vitamin K-dependent carboxylase expressed in Sf21 insect cells The homogenates from Sf21 cells transfected with an expression plasmid containing the full length Conus carboxylase cDNA (lane B) or the CAT cDNA (lane C) were evaluated for expressed protein by Western-blot analysis after SDS/PAGE (7.5% gels) The expressed protein was detected using the horseradish peroxidase-conjugated anti-V5 Ig (1 lgỈmL)1) Lane A, Homogenate from nontransfected cells; lane B, homogenates from Sf21 cells transfected with an expression plasmid containing the fulllength Conus carboxylase cDNA; lane C, homogenates from Sf21 cells transfected with an expression plasmid containing the CAT cDNA; lane D, purified recombinant bovine carboxylase Bands were detected by chemiluminescence of purified flag-tagged bovine carboxylase are detected using the anti-V5 antibody (Fig 2, lanes A and D) When expressed in COS7 cells, the Conus carboxylase migrates with an apparent molecular mass of 130 kDa (data not shown) Specific carboxylase activity of the recombinant Conus carboxylase The concentration of expressed Conus carboxylase in microsomes from transfected Sf21 cells and COS7 cells was determined by quantitative Western-blot analysis using the 53-kDa Positope protein as a standard (Fig 3A,B) Microsomal preparations from transfected Sf21 or COS7 cells show the carboxylase at 130 kDa (Fig 3A,B) Microsomal preparations from nontransfected Sf21 cells or mocktransfected COS7 cells not contain protein that can be detected by antibody to V5 (data not shown) Using densitometric analysis, the concentration of recombinant Conus carboxylase was determined to be ± 0.3 lgỈmL)1 in Sf21 microsomes and ± lgỈmL)1 in COS7 microsomes Quantitation of recombinant Conus carboxylase in COS7 microsomes using an anti-His Ig gave a similar result Ó FEBS 2002 6168 E Czerwiec et al (Eur J Biochem 269) Table Comparison of kinetic properties of recombinant Conus and recombinant bovine c-carboxylases Recombinant Conus carboxylase was expressed in Sf21 cells Km (lM) Bovine Conus Fig Quantitative Western-blot analysis (A) Microsomal preparation from Sf21 cells expressing recombinant Conus carboxylase Known amounts of Positope protein (lane 1, 2.5 ng; lane 2, ng; lane 3, 10 ng; lane 4, 20 ng) were applied and used as a standard (B) Microsomal preparation from COS7 cells expressing recombinant Conus carboxylase Known amounts of Positope protein (lane 1, 2.5 ng; lane 2, ng; lane 3, 10 ng; lane 4, 20 ng) were applied and used as a standard The amount of protein in lanes 5–8 in (A) (lane 5, ng; lane 6, ng; lane 7, ng; lane 8, 16 ng) and (B) (lane 5, 10 ng; lane 6, 20 ng; lane 7, 40 ng; lane 8, 80 ng) was determined by densitometry (10 ± lgỈmL)1, data not shown) The difference in the expression level in the two systems is related to the efficiency of carboxylase expression in these systems The specific carboxylase activity of the Conus carboxylase is very similar when expressed in either Sf21 or COS7 cells and is stimulated by the addition of proPT18 (Table 2) Compared with the recombinant bovine carboxylase, the recombinant Conus carboxylase activity has a 10-fold lower specific activity at maximum stimulation of both carboxylases by proPT18 Enzymatic properties of the recombinant Conus carboxylase The recombinant carboxylase has functional properties that are similar to those of bovine carboxylase, with the exception of propeptide binding and stimulation In contrast with previous reports of Conus carboxylase enzymatic properties [41,43], our assays were performed in the absence of endogenous carboxylase substrates, thus eliminating interference of carboxylation of exogenous substrates by endogenous substrates The apparent Km of the recombinant Conus carboxylase for reduced vitamin K was 52 ± 10 lM; this can be compared with a value of 23 lM for the bovine carboxylase [19] The Km values for peptides based on mammalian vitamin K-dependent precursors, including FLEEL, proPT28 and proFIX28, were Table Specific carboxylase and epoxidase activity of recombinant Conus carboxylase in the absence or presence of 400 lM proPT18 ND, not determined Expression system Specific epoxidase activity (nmolỈmg)1Ỉmin)1) Specific carboxylase activity (nmolỈmg)1Ỉmin)1) COS7 cells –proPT18 + proPT18 ND ND 1.9 ± 0.6 56 ± Sf21 cells –proPT18 + proPT18 48 ± 120 ± 20 2.1 ± 0.6 29 ± Vitamin KH2 FLEEL proFIX28 proPT28 Pro-e-TxIX/12 Pro-e-TxIX/24 Pro-e-TxIX/41 a Roth et al [19] et al [43] 52 430 1.7 565 75 74 b ± ± ± ± ± ± ± Ulrich et al [6] 10 100 0.02 60 20 18 c 23a 2200b 3.1a 3.6c 1500d 69d 117d Hubbard et al [48] dBush 430 ± 100 lM, 1.7 ± 0.02 lM and ± lM, respectively; these values are similar to those observed for carboxylation of these substrates by the bovine carboxylase [6,48] (Table 3) This value for the Factor IX precursorbased substrate is in contrast with a previous report that indicated that a Factor IX precursor-based substrate was not a substrate for the Conus radiatus carboxylase when measured in a microsomal preparation of crude venom duct [41] The Km values for peptides based on a conotoxin, e-TxIX [43] and its precursors, including e-TxIX12, pro-e-TxIX/24 and pro-e-TxIX/41, are 565 ± 90 lM, 75 ± 20 lM and 74 ± 18 lM, respectively (Table 3) These values are also similar to those obtained with crude venom duct homogenate or bovine carboxylase (Table 3) Effect of the propeptides of vitamin K-dependent protein precursors on Conus carboxylase activity Our work [43] and that of Bandyopadhyay et al [42] demonstrated the importance of the propeptide in directing c-carboxylation of Conus precursor substrates by the Conus carboxylase The propeptide of these substrates binds tightly to the carboxylase and, as with the bovine carboxylase, represents all or almost all of the binding energy for the enzyme–substrate interaction This is also the case for the recombinant Conus carboxylase The Km values for propeptide-containing substrates is decreased in parallel with both recombinant Conus carboxylase and recombinant bovine carboxylase (Table 3) In addition, the activity of mammalian carboxylases operating on nonpropeptide-containing substrates such as FLEEL is stimulated by the addition of synthetic peptides based on the sequences of residues )18 to )1 of the propeptides from blood coagulation precursors, including bovine Factor X, human proFactor IX and human proprothrombin [49] Whether the propeptide-binding site that directs carboxylation and the site that stimulates carboxylase activity are identical or separate remains unresolved To study propeptide stimulation of the Conus carboxylase activity on FLEEL, we used recombinant Conus carboxylase expressed in Sf21 cells as these cells not contain endogenous carboxylase activity or carboxylase substrates Carboxylation of FLEEL by recombinant Conus carboxylase is increased about fivefold Ó FEBS 2002 Conus vitamin K-dependent c-carboxylase (Eur J Biochem 269) 6169 by the addition of proFIX18 and proPT18, indicating that the Conus carboxylase, like the bovine carboxylase, is activated by the human propeptides To compare the potency of the effect of these propeptides on the recombinant Conus carboxylase and recombinant bovine carboxylase, we added increasing concentrations of these propeptides to a reaction mixture containing a fixed amount of either the recombinant Conus carboxylase or the recombinant bovine carboxylase, and monitored carboxylation of FLEEL or e-TxIX12 as a function of propeptide concentration The effect of proFIX18 on the recombinant bovine carboxylase is about 80-fold more potent than on the Conus carboxylase, with half-maximal stimulation at 0.2 lM and 16 lM, respectively The effect of proPT18 on the recombinant bovine carboxylase is  10-fold more potent than on the Conus carboxylase, with half-maximal stimulation at 0.54 lM and 5.5 lM, respectively These results show that propeptides based on human proprothrombin and human proFactor IX bind the Conus carboxylase The propeptide that directs c-carboxylation of the conotoxin precursor and lowers the apparent Km [43] , pro-e-TxIX/12, does not stimulate the carboxylation of FLEEL by either the bovine carboxylase or the Conus carboxylase (Fig 4) Identical results were obtained when e-TxIX12 was used as the substrate instead of FLEEL (Fig inset) Masking of stimulation by the Conus propeptides resulting from the presence of high concentrations of the stimulator ammonium sulfate [50] was ruled out by performing experiments in the absence of ammonium sulfate (data not shown) The bovine vitamin K-dependent carboxylase expresses vitamin K epoxidase activity The recombinant Conus Fig Effect of propeptides on carboxylase activity The effect of proFIX18, proPT18 and pro-e-TxIX/12 on FLEEL carboxylation by the recombinant bovine carboxylase (closed symbols) and the recombinant Conus carboxylase (open symbols) was determined with increasing concentrations of propeptide The results were analyzed with the GRAPHPAD PRISM program using nonlinear curve fitting The data are the mean of three experiments and the error bars represent standard deviation ProFIX18 (h, j), proPT18 (n, m) and pro-eTxIX/12 (s, d) Inset: Effect of pro-e-TxIX/12 on carboxylation of e-TxIX12 (1.6 mM) by the recombinant bovine carboxylase (closed symbols) and the recombinant Conus carboxylase (open symbols) Incorporation of 14CO2 into e-TxIX12 was measured in the presence of increasing concentrations of pro-e-TxIX/12 Table Epoxidase activity from recombinant Conus carboxylase Assays were performed as described in Experimental methods with the omissions as indicated ND, Not detectable Assay conditions Epoxidase activity [pmolỈ(30 min))1] Carboxylase activity [pmolỈ(30 min))1] –Carboxylase –Vitamin KH2 –Substrate –Propeptide Complete assay ND ND 10 175 440 0 18 111 carboxylase also functions as an epoxidase Formation of vitamin K epoxide associated with the formation of c-carboxyglutamic acid was measured by detection of the epoxide by HPLC In the absence of carboxylase, substrate or reduced vitamin K, no vitamin K epoxide was measured (Table 4) The addition of proPT18, the propeptide from human proprothrombin, stimulated epoxidation to about the same extent as it stimulated carboxylation DISCUSSION The sole known function of vitamin K, an essential vitamin in mammals, is to serve as a cofactor in the enzymatic conversion of glutamic acid to c-carboxyglutamic acid by the vitamin K-dependent c-glutamyl carboxylase Previous studies of the mammalian vitamin K-dependent carboxylases have shown that this enzyme has a unique primary structure and is not significantly homologous to any other known gene products Furthermore, the amino-acid sequences of the vitamin K-dependent carboxylase from human, bovine, ovine, rat, mouse and whale are more than 90% identical, and the sequence of the toadfish carboxylase is about 70% identical with bovine carboxylase [8–11] Our reason for cloning and expressing the Conus carboxylase was to compare the sequence and function of this invertebrate enzyme with the vertebrate vitamin K-dependent carboxylases, and to compare the structural and functional homologies of this enzyme between invertebrates and vertebrates Whereas the sequences of the N-terminus and C-terminus of the Conus carboxylase are quite divergent, we found significant amino-acid conservation between the Conus and vertebrate vitamin K-dependent carboxylases in the central region of the protein, confirming the results of a recent independent report [51] Furthermore, our expression of the recombinant Conus carboxylase proves that this cDNA indeed encodes a vitamin K-dependent carboxylase and reveals an enzyme with marked functional similarities to bovine carboxylase, including vitamin K epoxidase activity, but with several differences that yield additional insight into the enzymology of this protein We previously identified a conserved motif from the vitamin K-dependent carboxylase that is broadly represented in animal phyla [10] This 38-residue motif was identified in the human, bovine, rat, mouse, whale, toadfish, hagfish, horseshoe crab and cone snail carboxylase gene The cone snail motif was obtained by RT-PCR using primers based on conserved vertebrate sequences The C-terminal region of this motif differs from the Conus carboxylase cDNA obtained by library screening 6170 E Czerwiec et al (Eur J Biochem 269) The Conus carboxylase sequence is distinguished by a unique 47-residue N-terminal region (VR1) that bears no similarity to either the vertebrate carboxylases or the Drosophila carboxylase Furthermore, there is no homology in VR8 of the Conus carboxylase sequence It would appear that this region has no specific function related to either the carboxylase activity or the epoxidase activity Truncations of bovine carboxylase from the C-terminus at amino acids 712 or 676 result in carboxylase species that bind to substrates containing propeptide and glutamate equivalently to the wild-type enzyme, suggesting that the extreme C-terminal region is not involved in propeptide binding [19] Comparison of our C textile vitamin K-dependent carboxylase cDNA and that of Bandyopadhyay et al [51] reveals near sequence identity, but with several differences We report the entire 3¢ untranslated region, including  1.2 kb, and partial 5¢ untranslated sequence In the ORF, there are six single-base nucleotide differences in the two cDNA clones, five of which encode a different amino acid and one that is a silent substitution Using the bovine numbering system (Fig 1), we observe Arg179 rather than histidine, Thr430 rather than alanine, Pro654 rather than serine, Met726 rather than threonine, and Gly743 rather than valine At the present time, we not know whether these are polymorphisms or sequencing artefacts in either of the clones To prove that the cloned cDNA encoded a vitamin Kdependent carboxylase, we expressed this coding sequence in vertebrate and invertebrate cells In the absence of a molluscan heterologous expression system, we transfected Sf21 insect cells with an expression plasmid containing the Conus carboxylase coding sequence Conus carboxylase expressed by the transfected cells has a molecular mass of  130 kDa The Conus carboxylase contains 53 more amino acids than bovine carboxylase; the glycosylation state of this enzyme is not known nor can we comment on whether the recombinant carboxylase expressed in insects reflects the glycosylation state of the native protein As the Sf21 cells have no endogenous carboxylase activity, epoxidase activity or endogenous carboxylase substrates, recombinant Conus carboxylase can be analyzed without ambiguity or interference Like the bovine enzyme, the Conus carboxylase is also a vitamin K epoxidase The activity observed is similar to that of the bovine carboxylase The Km for vitamin K was found to be 52 lM, which is comparable to the value measured for both bovine and human carboxylase FLEEL, a high-Km substrate for the bovine carboxylase because it lacks the propeptide [6], is a lower-Km substrate for the Conus carboxylase, with a Km of  400 lM,  threefold to sixfold lower than the bovine carboxylase The structural basis for this difference is not known but may provide an approach to identifying the glutamate-binding site on the Conus enzyme The propeptides of both human proprothrombin and proFactor IX direct carboxylation by reducing the Km  1000-fold for substrates bearing the propeptide However, the Conus propeptides not stimulate Conus carboxylase, in contrast with the mammalian propeptides From the current data as well as from previous studies [24,41–43], the Conus and mammalian enzymes (a) all require vitamin K for c-carboxylation and (b) recognize their substrate via the carboxylation-recognition site encoded in the amino-acid sequence of the substrate In addition, Ó FEBS 2002 we demonstrate in this study that the recombinant Conus carboxylase has epoxidase activity The carboxylationrecognition site is most often found in a precursor form of the c-carboxyglutamic acid-containing substrates in both the Conus and mammalian systems, although uncarboxylated osteocalcin is a low-Km substrate that lacks such an external site but instead contains a unique internal site [52] Despite these major similarities, several differences between the Conus and mammalian vitamin K-dependent carboxylases are noteworthy First, the propeptide sequence that directs c-carboxylation in conotoxins is different from the propeptide sequences of blood coagulation proteins Second, the stimulatory effect of the mammalian propeptides is less potent on the recombinant Conus carboxylase than for the mammalian carboxylases Most importantly, the Conus propeptide does not stimulate the carboxylation of FLEEL by the Conus or bovine carboxylase Since the discovery that the propeptide of the precursor forms of vitamin K-dependent proteins contain a c-carboxylation-recognition site that directs carboxylation [5] and that the free propeptide greatly stimulates the carboxylation of FLEEL by the carboxylase [49], it has remained unresolved whether the propeptide-binding site that directs carboxylation of low-Km substrates and the propeptide-binding site that enhances carboxylase activity are the same or distinct Analysis of enzymatic properties of the Conus carboxylation system, which has many functional similarities to the mammalian carboxylation system, suggests that there are two distinct propeptidebinding sites on the carboxylase The propeptide on the precursor forms of the Conus substrate e-TxIX greatly reduces the Km for the Conus carboxylase substrates; however, the free propeptide does not stimulate carboxylation of FLEEL The discovery of c-carboxyglutamic acid in 1974 identified a post-translational modification in prothrombin that was dependent on the action of vitamin K [53,54] In mammalian systems, it is now becoming clear that vitamin K-dependent proteins are important in processes other than blood coagulation This study shows that the vitamin K-dependent carboxylase has been strongly conserved in vertebrates and invertebrates, suggesting a fundamental function for this enzyme The vitamin K-dependent carboxylase and c-carboxyglutamic acid are phylogenetically older than blood coagulation, although carboxylation was initially discovered during the study of mammalian blood coagulation The synthesis of c-carboxyglutamic acid is complex in that the system requires a reduced form of vitamin K, molecular oxygen, carbon dioxide, a vitamin K-dependent carboxylase that also co-ordinately oxidizes vitamin K to the vitamin K epoxide, and a salvage enzyme, the vitamin K epoxide reductase, to cycle vitamin K epoxide to vitamin K The presence of a Conus vitamin K-dependent carboxylase with functional and structural similarity to the mammalian carboxylase has broad and important biological implications However, it would seem that this system was not conserved in invertebrates and vertebrates to post-translationally modify glutamic acids on blood coagulation proteins and conotoxins during c-carboxyglutamic acid synthesis Rather, it seems that this system, which developed early in evolution, has a more fundamental purpose that may or may not involve c-carboxyglutamic Ó FEBS 2002 Conus vitamin K-dependent c-carboxylase (Eur J Biochem 269) 6171 acid synthesis Indeed, synthesis of blood coagulation proteins in vertebrates and toxins in the cone snail may be secondary functions of this enzyme ACKNOWLEDGEMENTS We especially appreciate 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enzyme activity by the ÔpropeptideÕ region of factor X J Biol Chem 262, 15334 50 Soute, B.A.M., Acher, F., Azerad, R & Vermeer, C (1990) Vitamin K-dependent carboxylase: effect of ammonium sulfate on substrate carboxylation and on inhibition by stereospific substrate analogs Biochim Biophys Acta 1034, 11–16 51 Bandyopadhyay, P.K., Garrett, J.E., Shetty, R.P., Keate, T., Walker, C.S & Olivera, B.M (2002) Gamma-glutamyl carboxylation: an extracellular posttranslational modification that antedates the divergence of molluscs, arthropods, and chordates Proc Natl Acad Sci USA 99, 1264–1269 52 Houben, R.J., Rijkers, D.T., Stanley, T.B., Acher, F., Azerad, R., Kakonen, S.M., Vermeer, C & Soute, B.A (2002) Characteristics and composition of the vitamin K-dependent gamma-glutamyl carboxylase-binding domain on osteocalcin Biochem J 364, 323– 328 53 Stenflo, J., Ferlund, P., Egan, W & Roepstorff, P (1974) Vitamin K dependent modifications of glutamic acid residues in prothrombin Proc Natl Acad Sci USA 71, 2730–2733 54 Nelsestuen, G.L., Zytkovicz, T.H & Howard, J.B (1974) The mode of action of vitamin K Identification of gamma- carboxyglutamic acid as a component of prothrombin J Biol Chem 249, 6347–6350  ... expected band of  33 kDa (Fig 2, lane C) In contrast, no bands from homogenates from nontransfected cells and a preparation Fig Molecular mass analysis of Conus vitamin K-dependent carboxylase expressed... properties of recombinant Conus and recombinant bovine c-carboxylases Recombinant Conus carboxylase was expressed in Sf21 cells Km (lM) Bovine Conus Fig Quantitative Western-blot analysis (A) Microsomal... protein with vitamin K-dependent carboxylase activity Further, using insect cells, this expression system provides a source of Conus carboxylase free of endogenous carboxylase and carboxylase substrates