The carboxylase cDNA from Conus textile has an ORF that encodes a 811-amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and 60% seq
Trang 1Expression and characterization of recombinant vitamin K-dependent
Eva Czerwiec1, Gail S Begley1, Mila Bronstein2, Johan Stenflo1,3, Kevin Taylor1, Barbara C Furie1,2 and Bruce Furie1,2
1
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
811-amino-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
vita-min dependent carboxylase activity and vitavita-min
K-dependent 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 6 lM, respectively; the Km for
vitamin K is 52 lM The Kmvalues for peptides based on the
sequence of the conotoxin e-TxIX and two precursor
analogs containing 12 or 29 amino acids of the pro-peptide region are 565 lM, 75 lM and 74 lM, respect-ively 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 carb-oxylase across animal species and the importance of c-carboxyglutamic acid synthesis in diverse biological systems
Keywords: blood coagulation; conotoxins; hemophilia; post-translational processing; vitamin K
The vitamin K-dependent carboxylase catalyzes the
post-translational conversion of glutamic acid into
c-carboxy-glutamic acid in prothrombin, other blood coagulation
proteins, and various vitamin K-dependent proteins [1,2] In
this reaction, CO2replaces the c-proton on specific glutamic
acid residues of the peptide substrate to yield
c-carboxy-glutamic 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 K-dependent carboxylases revealed marked (> 90%) amino-acid sequence conservation, and the toadfish carboxylase showed 70% amino-acid sequence similarity to the mam-malian 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 relation-ships 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,
resi-dues )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)
Trang 2[23,24] These marine gastropods use small biologically
active peptides (conotoxins) to paralyze fish, marine worms
and molluscs [25,26] Many c-carboxyglutamic
acid-con-taining conotoxins have been identified [27–31] The
metal-binding properties [32–35] and the 3Dstructures 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
dif-ferent from the carboxylation-recognition site in
mamma-lian 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
distribu-tion 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
Conusand 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
10–80-fold less potent in stimulating the Conus carboxylase Most
importantly, Conus propeptides do not stimulate either the
Conuscarboxylase or the mammalian carboxylase Yet, the
presence of these propeptide sequences directs
carboxyla-tion and confers low Kmon 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
sub-strates will considerably facilitate studies of the mechanistic
properties of this unique enzyme
E X P E R I M E N T A L M E T H O D S
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
horse-radish peroxidase-conjugated antibody were from
Invitro-gen (Carlsbad, CA, USA) A kZAPII Custom cDNA
library from C textile venom duct was prepared by
Stratagene (La Jolla, CA, USA) TRIzol reagent,
Thermo-Script RT-PCR System, Platinum PCR Supermix, Platinum
PfxPolymerase, restriction enzymes, synthetic
oligonucleo-tide 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 Biochem-icals (Indianapolis, IN, USA) Superose 12 was from Pharmacia (Piscataway, NJ, USA) NaH14CO3 (55 mCiÆ mmol)1) and the ECL detection system were from Amer-sham Life Sciences (Arlington Heights, IL, USA) Atom-light scintillation fluid was from Packard (Meriden, CT, USA) Vitamin K1was obtained from Abbott Laboratories (North Chicago, IL, USA) BSA (fraction V), FLEEL (Phe-Leu-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 1
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 homo-genized using a Tissue Tearor mixer for 10 s in 5 : 1 to
10 : 1 (w/v) buffer A (250 mM sucrose, 500 mM KCl,
25 mM imidazole/HCl, pH 7.2) containing 0.1% (w/v) Chaps and 1· PIC (2 mM dithiothreitol, 2 mM EDTA, 0.5 lgÆmL)1leupeptin, 1 lgÆmL)1pepstatin A, 2 lgÆmL)1 aprotinin) Homogenates were centrifuged at 12 000 g for
5 min, and supernatants were subsequently centrifuged at
100 000 g for 3 h at 4C 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 mMphenylmethanesulfonyl fluoride, 20% (v/v) glycerol] and sonicated using a model 220F sonicator (Heat Systems-Ultrasonics) Sf21 cells were collected by centrifugation and washed in NaCl/Pi, pH 7.2 Cells were resuspended at a density of 4· 106cellsÆmL)1in lysis buffer [10 mMMops (pH 7.0), 10 mM KCl, 1 mM MgCl2, 1· 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 3 h at 4C to separate the microsomal fraction The pellet was resuspended in NaCl/P (pH 7.2)
Trang 3containing 0.1% (w/v) Chaps, 0.1% (w/v)
phosphatidyl-choline, 0.1 mMphenylmethanesulfonyl fluoride and 20%
(v/v) glycerol and sonicated COS7 cells (5· 106cells) were
washed with NaCl/Pi, trypsinized and collected in NaCl/Pi
(pH 7.2) containing 20% glycerol and 1· PIC Cells were
homogenized in a glass homogenizer (3· 10 strokes) and
centrifuged at 500 g The pellet was rehomogenized and
washed 3 times with the same buffer Pooled supernatants
were centrifuged at 100 000 g for 3 h at 4C 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)
phos-phatidylcholine, 1· PIC and 20% (v/v) glycerol by
soni-cation
Enzyme assays
The amount of 14CO2 incorporated into exogenous
sub-strates was measured in reaction mixtures of 125 lL
containing substrate at the indicated concentration,
222 lMreduced vitamin K1, 0.72 mMNaH14CO3(5 mCi),
28 mMMops (pH 7.0), 500 mMNaCl, 0.16% (w/v)
phos-phatidylcholine, 0.16% (w/v) Chaps and 0.8Mammonium
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 min 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
Vita-min K epoxidase activity was assayed by HPLC as
previ-ously described [21]
Molecular cloning ofC 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 1 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
pCR4.0-TOPO vector Ligation reactions were subsequently used to
transform chemically competent Escherichia coli TOP10
cells Transformants were selected on Luria–Bertani agar
plates containing 50 lgÆmL)1kanamycin and
5-bromo-4-chloro-3-indolyl-D-galactoside for blue/white screening
Positive colonies were grown in Luria–Bertani broth
containing 50 lgÆmL)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 1 (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 4· 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 1 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 gene-specific primer yielded a gene-specific product that encodes 260 residues of a carboxylase homolog (homologous to region 164–401 of bovine carboxylase) This sequence informa-tion 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
Table 1 Amino-acid sequences of synthetic substrates and propeptides Bold type ¼ mature sequence.
proFIX28 TVFLDHENANKILNRPKRYNSGKLEEFV
pro-e-TxIX/24 LKRTIRTRLNIRECCEDGWCCTAA
pro-e-TxIX/41 ARTKTDDDVPLSSLRDNLKRTIRTRLNIRECCEDGWCCTAA
Trang 4ORF 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
pIB-CCbx-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
trans-fectants were assayed for carboxylase activity using FLEEL
Western-blot analysis of the recombinantConus
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 lgÆmL)1) The
CAT-V5/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
micro-somal preparations from transfected Sf21 cells and COS7
cells Densitometric analysis was performed using the
GELPRO ANALYZER program (Media Cybernetics, North
Reading, MA, USA)
R E S U L T S
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· 106c.p.m.Æ(mg protein))1Æ(30 min))1], C textile [1.1· 106c.p.m.Æ(mg protein))1Æ(30 min))1] and C band-anus [1.1· 106c.p.m.Æ(mg protein))1Æ(30 min))1], and the lowest in venom duct homogenates from C striatus [9· 103c.p.m.Æ(mg protein))1Æ(30 min))1] Relative amo-unts 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 Conuscarboxylase from C textile
The full-length cDNA encoding the vitamin K-depend-ent 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 theConus 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 membrane-spanning 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
carboxylas-es 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 Align-ment of the Conus carboxylase sequence with the primary structure of the vertebrate carboxylase homologs indicates the presence of one one-residue insertion, four two-resi-due insertions, one three-resitwo-resi-due insertion, one six-resitwo-resi-due insertion, and one 19-residue insertion; there are two one-residue 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
Trang 5yellow in Fig 1 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 ( method with the
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
Fig 1 Alignment of the amino-acid sequence of the vitamin K-dependent carboxylase from Conus (C textile) with the sequences from the vita-min 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 (fly) 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.
Trang 6Expression of theConus vitamin K-dependent
carboxylase
The Conus carboxylase was expressed in COS7 cells, a
mammalian cell line.14CO2incorporation into FLEEL was
40 292 c.p.m.Æ(30 min))1in the presence of vitamin K and
proPT18 when microsomes from cells transfected with the
plasmid vector containing Conus carboxylase cDNA were
assayed.14CO2incorporation into FLEEL in the absence of
vitamin K was 483 c.p.m.Æ(30 min))1with the same
micro-somal 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,
14CO2 incorporation into FLEEL was 6274 c.p.m.Æ(30
min))1 These experiments indicate increased expression of
carboxylase in COS cells of about sevenfold over
endo-genous 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
car-boxylase in Sf21 insect cells These cells do not express
endogenous carboxylase activity Cells transfected with the
construct containing the Conus carboxylase cDNA showed
significant carboxylase activity This activity had an
abso-lute requirement for vitamin K in that the carboxylase
activity was 1237 c.p.m.Æ(30 min))1 in the presence of
vitamin K and 181 c.p.m.Æ(30 min))1in its absence In the
presence of proPT18 and vitamin K, this activity increases
to 10 239 c.p.m.Æ(30 min))1 Carboxylase activity was not
detected in homogenates from nontransfected cells,
with carboxylase activity of 199 c.p.m.Æ(30 min))1 and
102 c.p.m.Æ(30 min))1with 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
Conuscarboxylase 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 recombinantConus
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-trans-fected cells, nontranscDNA-trans-fected cells and cells transcDNA-trans-fected with
chloramphenicol acetyltransferase (CAT) cDNA were
an-alyzed 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
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) Micro-somal preparations from nontransfected Sf21 cells or mock-transfected COS7 cells do not contain protein that can be detected by antibody to V5 (data not shown) Using densitometric analysis, the concentration of recombinant Conuscarboxylase was determined to be 2 ± 0.3 lgÆmL)1
in Sf21 microsomes and 9 ± 1 lgÆmL)1in COS7 micro-somes Quantitation of recombinant Conus carboxylase in COS7 microsomes using an anti-His Ig gave a similar result
Fig 2 Molecular mass analysis of Conus vitamin K-dependent car-boxylase 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 lgÆmL)1) Lane A, Homogenate from nontransfected cells; lane B, homogenates from Sf21 cells transfected with an expression plasmid containing the full-length Conus carboxylase cDNA; lane C, homogenates from Sf21 cells transfected with an expression plasmid containing the CAT cD NA; lane D, purified recombinant bovine carboxylase Bands were detected
by chemiluminescence.
Trang 7(10 ± 1 lgÆmL)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 recombinantConus
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
enzy-matic properties [41,43], our assays were performed in the
absence of endogenous carboxylase substrates, thus
elimin-ating interference of carboxylation of exogenous substrates
by endogenous substrates The apparent Kmof the
recom-binant 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 Kmvalues for peptides
based on mammalian vitamin K-dependent precursors,
including FLEEL, proPT28 and proFIX28, were
430 ± 100 lM, 1.7 ± 0.02 lM and 6 ± 2 lM, respect-ively; 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 precursor-based 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 Kmvalues 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 homogen-ate or bovine carboxylase (Table 3)
Effect of the propeptides of vitamin K-dependent protein precursors onConus 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 Kmvalues for pro-peptide-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-con-taining 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 coagu-lation 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 stimula-tion of the Conus carboxylase activity on FLEEL, we used recombinant Conus carboxylase expressed in Sf21 cells as these cells do not contain endogenous carboxylase activity
or carboxylase substrates Carboxylation of FLEEL by recombinant Conus carboxylase is increased about fivefold
Fig 3 Quantitative Western-blot analysis (A) Microsomal
prepar-ation from Sf21 cells expressing recombinant Conus carboxylase.
Known amounts of Positope protein (lane 1, 2.5 ng; lane 2, 5 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, 5 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, 2 ng;
lane 6, 4 ng; lane 7, 8 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.
Table 2 Specific carboxylase and epoxidase activity of recombinant
Conus carboxylase in the absence or presence of 400 l M proPT18 ND,
not determined.
Expression
system
Specific epoxidase activity
(nmolÆmg)1Æmin)1)
Specific carboxylase activity
(nmolÆmg)1Æmin)1) COS7 cells
–proPT18 ND1.9 ± 0.6
+ proPT18 ND56 ± 7
Sf21 cells
–proPT18 48 ± 7 2.1 ± 0.6
+ proPT18 120 ± 20 29 ± 6
Table 3 Comparison of kinetic properties of recombinant Conus and recombinant bovine c-carboxylases Recombinant Conus carboxylase was expressed in Sf21 cells.
K m (l M ) Conus Bovine Vitamin KH 2 52 ± 10 23a FLEEL 430 ± 100 2200 b
proPT28 1.7 ± 0.02 3.6c Pro-e-TxIX/12 565 ± 60 1500 d Pro-e-TxIX/24 75 ± 20 69 d Pro-e-TxIX/41 74 ± 18 117d
a Roth et al [19] b Ulrich et al [6] c Hubbard et al [48] d Bush
et al [43].
Trang 8by 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
carboxy-lation 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
Conuscarboxylase, 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
stimu-lation 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 4 inset) Masking of stimulation by the Conus
propeptides resulting from the presence of high
concentra-tions 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
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
D I S C U S S I O N
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-carboxyglut-amic acid by the vitamin K-dependent c-glutamyl carboxylase Previous studies of the mammalian vita-min 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 carboxy-lases, 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 Conusand vertebrate vitamin K-dependent carboxylases in the central region of the protein, confirming the results of a recent independent report [51] Furthermore, our expres-sion 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 represen-ted 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
Fig 4 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
recom-binant Conus carboxylase (open symbols) was determined with
increasing concentrations of propeptide The results were analyzed
with the GRAPHPADPRISM 3 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-e-TxIX/12 (s, d) Inset: Effect of pro-e-pro-e-TxIX/12 on carboxylation of
e-TxIX12 (1.6 m M ) by the recombinant bovine carboxylase (closed
symbols) and the recombinant Conus carboxylase (open symbols).
Incorporation of 14 CO 2 into e-TxIX12 was measured in the presence of
increasing concentrations of pro-e-TxIX/12.
Table 4 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 [pmolÆ(30 min))1]
Carboxylase activity [pmolÆ(30 min))1] –Carboxylase ND0
–Vitamin KH 2 ND0 –Substrate 10 0 –Propeptide 175 18 Complete assay 440 111
Trang 9The 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
Drosophilacarboxylase 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
equival-ently 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
car-boxylase 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 do not know whether
these are polymorphisms or sequencing artefacts in either of
the clones
To prove that the cloned cDNA encoded a vitamin
K-dependent 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
interfer-ence 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 Kmfor vitamin K was
found to be 52 lM, which is comparable to the value
measured for both bovine and human carboxylase FLEEL,
a high-Kmsubstrate for the bovine carboxylase because it
lacks the propeptide [6], is a lower-Km substrate for the
Conuscarboxylase, with a Kmof 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
propro-thrombin and proFactor IX direct carboxylation by
redu-cing the Km 1000-fold for substrates bearing the
propeptide However, the Conus propeptides do not
stimu-late 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
enco-ded in the amino-acid sequence of the substrate In addition,
we demonstrate in this study that the recombinant Conus carboxylase has epoxidase activity The carboxylation-recognition site is most often found in a precursor form of the c-carboxyglutamic acid-containing substrates in both the Conus and mammalian systems, although uncarboxyl-ated osteocalcin is a low-Kmsubstrate 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 carboxy-lases are noteworthy First, the propeptide sequence that directs c-carboxylation in conotoxins is different from the propeptide sequences of blood coagulation proteins Sec-ond, 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 propeptide-binding 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 carboxy-lation 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 con-served 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-carbo-xyglutamic 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 epox-ide, 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 implica-tions However, it would seem that this system was not conserved in invertebrates and vertebrates to post-trans-lationally 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
Trang 10acid synthesis Indeed, synthesis of blood coagulation
proteins in vertebrates and toxins in the cone snail may be
secondary functions of this enzyme
A C K N O W L E D G E M E N T S
We especially appreciate the efforts of Tony Nahacky in providing us
with cone snails, and Drs David Roth and Takako Hirata for helpful
discussions This work was supported by grants (HL38216 and
HL42443) from the National Institutes of Health.
R E F E R E N C E S
1 Furie, B., Bouchard, B.A & Furie, B.C (1999) Vitamin
K-dependent biosynthesis of gamma-carboxyglutamic acid Blood
93, 1798–1808.
2 Stenflo, J (1999) Contributions of Gla and EFG-like domains to
the function of vitamin K-dependent coagulation factors Crit.
Rev Eukaryotic Gene Expression 9, 59–88.
3 Dowd, P., Hershline, R., Ham, S.W & Naganathan, S (1995)
Vitamin K and energy transduction: a base strength amplification
mechanism Science 269, 1684.
4 Morris, D P., Soute, B.A.M., Vermeer, C & Stafford, D W.
(1993) Characterization of the purified vitamin K-dependent
c-glutamyl carboxylase J Biol Chem 268, 8735–8742.
5 Jorgensen, M.J., Cantor, A.B., Furie, B.C., Brown, C.L.,
Shoe-maker, C.B & Furie, B (1987) Recognition site directing vitamin
K-dependent c-carboxylation resides on the propeptide of factor
IX Cell 48, 185.
6 Ulrich, M.M., Furie, B., Jacobs, M.R., Vermeer, C & Furie, B.C.
(1988) Vitamin K-dependent carboxylation A synthetic peptide
based upon the gamma-carboxylation recognition site sequence of
the prothrombin propeptide is an active substrate for the
car-boxylase in vitro J Biol Chem 263, 9697–9702.
7 Furie, B.C., Ratcliffe, J.V., Tward, J., Jorgensen, M.J.,
Blasz-kowsky, L.S., DiMichele, D & Furie, B (1997) The
gamma-car-boxylation recognition site is sufficient to direct vitamin
K-dependent carboxylation on an adjacent glutamate-rich region
of thrombin in a propeptide-thrombin chimera J Biol Chem 272,
28258–28262.
8 Wu, S.-M., Cheung, W.-F., Frazier, D & Stafford, D.W (1991)
Cloning and expression of the cDNA for human c-glutamyl
carboxylase Science 254, 1634–1636.
9 Rehemtulla, A., Roth, D.A., Wasley, L.C., Kuliopulos, A., Walsh,
C.T., Furie, B., Furie, B.C & Kaufman, R.J (1993) In vitro and
in vivo functional characterization of bovine vitamin K-dependent
gamma-carboxylase expressed in Chinese hamster ovary cells.
Proc Natl Acad Sci USA 90, 4611–4615.
10 Begley, G.S., Furie, B.C., Czerwiec, E., Taylor, K.L., Furie, G.L.,
Bronstein, L., Stenflo, J & Furie, B (2000) A conserved motif
within the vitamin K-dependent carboxylase gene is widely
dis-tributed across animal phyla J Biol Chem 275, 36245–36249.
11 Romero, E.E., Deo, R., Velazquez-Estades, L.J & Roth, D.A.
(1998) Cloning, structural organization, and transcriptional
activity of the rat vitamin K-dependent gamma-glutamyl
car-boxylase gene Biochem Biophys Res Commun 248, 783–788.
12 Wu, S.-M., Morris, D.P & Stafford, D.W (1991) Identification
and purification to near homogeneity of the vitamin K-dependent
carboxylase Proc Natl Acad Sci USA 88, 2236–2240.
13 Carlisle, T.L & Suttie, J.W (1980) Vitamin K dependent
car-boxylase: subcellular localization of the carboxylase and enzymes
involved in vitamin K metabloism in rat liver Biochemistry 19,
1161–1167.
14 Bristol, J.A., Ratcliffe, J.V., Roth, D.A., Jacobs, M.A., Furie, B.C.
& Furie, B (1996) Biosynthesis of prothrombin: intracellular
localization of the vitamin K-dependent carboxylase and the sites
of gamma-carboxylation Blood 88, 2585–2593.
15 Kuliopulos, A., Nelson, N.P., Yamada, M., Walsh, C.T., Furie, B., Furie, B.C & Roth, D.A (1994) Localization of the affinity peptide-substrate inactivator site on recombinant vitamin K-dependent carboxylase J Biol Chem 269, 21364–21370.
16 Sugiura, I., Furie, B., Walsh, C.T & Furie, B.C (1996) Profactor
IX propeptide and glutamate substrate binding sites on the vita-min K-dependent carboxylase identified by site-directed muta-genesis J Biol Chem 271, 17837–17844.
17 Yamada, M., Kuliopulos, A., Nelson, N.P., Roth, D.A., Furie, B., Furie, B.C & Walsh, C.T (1995) Localization of the Factor IX propeptide binding site on recombinant vitamin K dependent carboxylase using benzoylphenylalanine photoaffinity peptide inactivators Biochemistry 34, 481–489.
18 Wu, S.M., Mutucumarana, V.P., Geromanos, S & Stafford, D.W (1997) The propeptide binding site of the bovine gamma-glutamyl carboxylase J Biol Chem 272, 11718–11722.
19 Roth, D.A., Whirl, M.L., Velazquez-Estades, L.J., Walsh, C.T., Furie, B & Furie, B.C (1995) Mutagenesis of vitamin K-depen-dent carboxylase demonstrates a carboxyl terminus-mediated interaction with vitamin K hydroquinone J Biol Chem 270, 5305–5311.
20 Maillet, M., Morris, D., Gaudry, M & Marquet, M (1997) The active site region of the vitaminK-dependent carboxylase includes both the amino-terminal hydrophobic and carboxy-terminal hydrophilic domains of the protein FEBS Lett 413, 1.
21 Bouchard, B.A., Furie, B & Furie, B.C (1999) Glutamyl sub-strate-induced exposure of a free cysteine residue in the vitamin K-dependent gamma-glutamyl carboxylase is critical for vitamin
K epoxidation Biochemistry 38, 9517–9523.
22 Pudota, B.N., Miyagi, M., Hallgren, K.W., West, K.A., Crabb, J.W., Misono, K.S & Berkner, K.L (2000) Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation Proc Natl Acad Sci USA 97, 13033–13038.
23 McIntosh, J., Olivera, B., Cruz, L & Gray, W (1984) Gamma-carboxyglutamate in a neuroactive toxin J Biol Chem 259, 14343–14346.
24 Hauschka, P.V., Mullen, E.A., Hintsch, G & Jazwinski, S (1988) Abundant occurrence of c-carboxyglutamic acid (Gla)-containing peptides in the marine gastropod family conidae Current Advan-ces in Vitamin K Research (Suttie, J.W., ed.), pp 237–243 Elsevier, New York.
25 Olivera, B.M., Gray, W.R., Zeikus, R., McIntosh, J.M., Varga, J., Rivier, J., de Santos, V & Cruz, L.J (1985) Peptide neurotoxins from fish-hunting cone snails Science 230, 1338–1343.
26 Olivera, B.M., Rivier, J., Scott, J.K., Hillyard, D.R & Cruz, L.J (1991) Conotoxins J Biol Chem 266, 22067–22070.
27 Haack, J.A., Rivier, J., Parks, T.N., Mena, E.E., Cruz, L.J & Olivera, B.M (1990) Conantokin-T A gamma-carboxyglutamate containing peptide with N-methyl- D -aspartate antagonist activity.
J Biol Chem 265, 6025–6029.
28 Fainzilber, M., Gordon, D., Hasson, A., Spira, M.E & Zlotkin, E (1991) Mollusc-specific toxins from the venom of Conus textile neovicarius Eur J Biochem 202, 589–595.
29 Nakamura, T., Yu Z., Fainzilber, M & Burlingame, A.L (1996) Mass spectrometric-based revision of the structure of a cysteine-rich peptide toxin with gamma-carboxyglutamic acid, TxVIIA, from the sea snail, Conus textile Protein Sci 5, 524–530.
30 Kalume, D.E., Stenflo, J., Czerwiec, E., Hambe, B., Furie, B.C., Furie, B & Roepstorff, P (2000) Structure determination of two conotoxins from Conus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ioniza-tion mass spectrometry and biochemical methods J Mass Spec-trom 35, 145–156.
31 Fainzilber, M., Nakamura, T., Lodder, J.C., Zlotkin, E., Kits, K.S & Burlingame, A.L (1998) gamma-Conotoxin-PnVIIA,
a gamma-carboxyglutamate-containing peptide agonist of