Báo cáo Y học: Expression and characterization of recombinant vitamin K-dependent c-glutamyl carboxylase from an invertebrate, Conus textile doc

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
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 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 6 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:
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.

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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 do 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 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 homogenized 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 lgỈmL)1 leupeptin, 1 lgỈmL)1 pepstatin A, 2 lgỈmL)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 4 °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 4 · 106 cellsỈmL)1 in lysis buffer [10 mM Mops
(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 4 °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 1. 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 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 4 °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, 1 · 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 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. 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 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 pCR4.0TOPO 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 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 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. 1. 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. 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 (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 do 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. 2. 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 do 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 2 ± 0.3 lgỈmL)1
in Sf21 microsomes and 9 ± 1 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 3. 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. 3. 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, 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.

(10 ± 1 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 2. 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 ± 7

Sf21 cells
–proPT18
+ proPT18

48 ± 7
120 ± 20

2.1 ± 0.6
29 ± 6

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
6
1.7
565
75
74
b

±
±
±
±
±
±
±

Ulrich et al. [6].

10
100
2

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 6 ± 2 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 do 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. 4 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. 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 recombinant Conus carboxylase (open symbols) was determined with

increasing concentrations of propeptide. The results were analyzed
with the GRAPHPAD PRISM 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-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 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
[pmolỈ(30 min))1]

Carboxylase activity
[pmolỈ(30 min))1]

–Carboxylase
–Vitamin KH2
–Substrate
–Propeptide
Complete assay

ND
ND
10

175
440

0
0
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 do 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 do 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 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.

REFERENCES
1. Furie, B., Bouchard, B.A. & Furie, B.C. (1999) Vitamin Kdependent 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., Shoemaker, 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 carboxylase in vitro. J. Biol. Chem. 263, 9697–9702.
7. Furie, B.C., Ratcliffe, J.V., Tward, J., Jorgensen, M.J., Blaszkowsky, L.S., DiMichele, D. & Furie, B. (1997) The gamma-carboxylation 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 distributed 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 carboxylase 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 carboxylase: 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 Kdependent 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 vitamin K-dependent carboxylase identified by site-directed mutagenesis. 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-dependent 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 substrate-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) Gammacarboxyglutamate 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 Advances 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 cysteinerich 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 ionization mass spectrometry and biochemical methods. J. Mass Spectrom. 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


6172 E. Czerwiec et al. (Eur. J. Biochem. 269)

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

neuronal pacemaker cation currents. Biochemistry 37,
1470–1477.
Prorok, M., Warder, S.E., Blandl, T. & Castellino, F.J. (1996)
Calcium binding properties of synthetic gamma-carboxyglutamic
acid-containing marine cone snail ÔsleeperÕ peptides, conantokin-G
and conantokin-T. Biochemistry 35, 16528–16534.
Blandl, T., Zajicek, J., Prorok, M. & Castellino, F.J. (1997) Metalion-binding properties of synthetic conantokin-G. Biochem. J.
328, 777–783.
Prorok, M. & Castellino, F.J. (1998) Thermodynamics of binding
of calcium, magnesium, and zinc to the N-methyl-D-aspartate
receptor ion channel peptidic inhibitors, conantokin-G and conantokin-T. J. Biol. Chem. 273, 19573–19578.
Blandl, T., Warder, S.E., Prorok, M. & Castellino, F.J. (2000)
Structure-function relationships of the NMDA receptor antagonist peptide, conantokin-R. FEBS Lett. 470, 139–146.
Skjaerbaek, N., Nielsen, K.J., Lewis, R.J., Alewood, P. & Craik,
D.J. (1997) Determination of the solution structures of conantokin-G and conantokin-T by CD and NMR spectroscopy.
J. Biol. Chem. 272, 2291–2299.

Rigby, A.C., Baleja, J.D., Furie, B.C. & Furie, B. (1997) Threedimensional structure of a gamma-carboxyglutamic acid-containing conotoxin, conantokin G, from the marine snail Conus
geographus: the metal-free conformer. Biochemistry 36, 6906–
6914.
Chen, Z., Blandl, T., Prorok, M., Warder, S.E., Li, L., Zhu, Y.,
Pedersen, L.G., Ni, F. & Castellino, F.J. (1998) Conformational
changes in conantokin-G induced upon binding of calcium and
magnesium as revealed by NMR structural analysis. J. Biol. Chem.
273, 16248–16258.
Rigby, A.C., Baleja, J.D., Li, L., Pedersen, L.G., Furie, B.C. &
Furie, B. (1997) Role of gamma-carboxyglutamic acid in the calcium-induced structural transition of conantokin G, a conotoxin
from the marine snail Conus geographus. Biochemistry 36, 15677–
15684.
Rigby, A.C., Lucas-Meunier, E., Kalume, D.E., Czerwiec, E.,
Hambe, B., Dahlqvist, I., Fossier, P., Baux, G., Roepstorff, P.,
Baleja, J.D., Furie, B.C., Furie, B. & Stenflo, J. (1999) A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx. Proc. Natl Acad. Sci.
USA 96, 5758–5763.
Stanley, T.B., Stafford, D.W., Olivera, B.M. & Bandyopadhyay,
P.K. (1997) Identification of a vitamin K-dependent carboxylase
in the venom duct of a Conus snail. FEBS Lett. 407, 85–88.
Bandyopadhyay, P.K., Colledge, C.J., Walker, C.S., Zhou, L.M.,
Hillyard, D.R. & Olivera, B.M. (1998) Conantokin-G precursor
and its role in gamma-carboxylation by a vitamin K-dependent
carboxylase from a Conus snail. J. Biol. Chem. 273, 5447–5450.

Ó FEBS 2002
43. Bush, K.A., Stenflo, J., Roth, D.A., Czerwiec, E., Harrist, A.,
Begley, G.S., Furie, B.C. & Furie, B. (1999) Hydrophobic amino
acids define the carboxylation recognition site in the precursor of
the gamma-carboxyglutamic-acid-containing conotoxin epsilonTxIX from the marine cone snail Conus textile. Biochemistry 38,
14660–14666.

44. Li, T., Yang, C. & Stafford, D. (2000) Identification of a Drosophila vitamin K-dependent gamma-glutamyl carboxylase. J. Biol.
Chem. 275, 18291–18296.
45. Walker, C.S., Shetty, R.P., Clark, K., Kazuko, S.G., Letsou, A.,
Olivera, B.M. & Bandyopadhyay, P.K. (2001) On a potential
global role for vitamin K-dependent gamma-carboxylation in
animal systems. Evidence for a gamma-glutamyl carboxylase in
Drosophila. J. Biol. Chem. 276, 7769–7774.
46. Jacobs, M., Freedman, S.J., Furie, B.C. & Furie, B. (1994)
Membrane binding properties of the Factor IX c-carboxyglutamic
acid-rich domain prepared by chemical synthesis. J. Biol. Chem.
269, 25494–25501.
47. Tie, J., Wu, S.M., Jin, D., Nicchitta, C.V. & Stafford, D.W. (2000)
A topological study of the human gamma-glutamyl carboxylase.
Blood 96, 973–978.
48. Hubbard, B.R., Jacobs, M., Ulrich, M.M., Walsh, C., Furie, B. &
Furie, B.C. (1989) Vitamin K-dependent carboxylation. In vitro
modification of synthetic peptides containing the gamma-carboxylation recognition site. J. Biol. Chem. 264, 14145–14150.
49. Knobloch, J.E. & Suttie, J.W. (1987) Control of 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.