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J Biol Chem, Vol. 273, Issue 10, 5447-5450, March 6, 1998
-Carboxylation by a
Vitamin K-dependent Carboxylase from a Conus
Snail*
§,,,
, and
From the Departments of Biology and Pathology,
University of Utah, Salt Lake City, Utah 84112 and ¶ Cognetix
Inc., Salt Lake City, Utah 84108
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Conantokin-G isolated from the marine snail
Conus geographus is a 17-amino acid 
-carboxyglutamate
(Gla)-containing peptide that inhibits the
N-methyl-D-aspartate receptor. We describe the cloning and sequence of conantokin-G cDNA and the possible role of
the propeptide sequence. The cDNA encodes a 100amino acid
peptide. The N-terminal 80 amino acids constitute the prepro-sequence, and the mature peptide is derived from the remaining C-terminal residues after proteolysis, C-terminal amidation, and a unique post-translational modification, -carboxylation of glutamate residues to Gla. Mature conantokin-G peptide containing Glu residues (E.Con-G) in place of Gla is a poor substrate for the vitamin K-dependent -glutamyl carboxylase (apparent
Km = 3.4 mM). Using peptides
corresponding to different segments of the propeptide we investigated a
potential role for the propeptide sequences in -carboxylation.
Propeptide segment 
20 to 1 covalently linked to E.Con-G or the
synthetic pentapeptide FLEEL increased their apparent affinities 2 orders of magnitude. These substrates are not efficiently carboxylated
by the bovine microsomal -glutamyl carboxylase, suggesting
differences in specificities between the Conus and the
mammalian enzyme. However, the role of propeptide in enhancing the
efficiency of carboxylation is maintained.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The vitamin K-dependent -carboxylation of glutamate
residues was originally discovered as a novel post-translational
modification in the blood coagulation cascade (1); some of the key
clotting factors such as prothrombin must be -carboxylated in order
for proper blood clotting to occur. Somewhat later, this
post-translational modification was also found in certain bone proteins
(2). This modification was restricted to these rather specialized
mammalian systems until a very unusual peptide, conantokin-G, was
described from the venom of the predatory marine snail, Conus
geographus (3). Conantokin-G is a 17-amino acid peptide that
inhibits the N-methyl-D-aspartate receptor (4).
Unlike most Conus peptides, which are multiply
disulfide-bonded, conantokin-G has no disulfide cross-links but has 5 residues of -carboxyglutamate residues; this remains the highest
density of -carboxyglutamate found in any functional gene product
characterized to date.
Most of the biologically active components of the Conus
venom are multiply disulfide bonded peptides (the conotoxins). These have been shown to be initially translated as prepropeptide precursors, which are then post-translationally processed to yield the mature disulfide-cross-linked conotoxin. Conantokin-G differs strikingly from
most conotoxins not only in having -carboxyglutamate residues, but
also because it has no disulfide cross-links. We report below an
analysis of a cDNA clone encoding the conantokin-G precursor. Furthermore, we establish the probable function of one region of the
precursor that is excised during the maturation of the functional
conantokin-G peptide.
The presence of -carboxyglutamate in a non-mammalian system was
initially controversial because vitamin K-dependent
carboxylation of glutamate residues had primarily been thought to be a
highly specialized mammalian innovation. However, we have found that conantokin-G is only one member of a family of peptides; a variety of
other conantokins have been found including conantokin-T and conantokin-R from two other fish-hunting cone snails (5, 6). All three
peptides have a high content of -carboxyglutamate (4-5 residues).
-Glutamyl carboxylase has been purified from mammalian sources (7,
8) and has been expressed both in mammalian and insect cell lines (9,
10). Recently it was shown that, as is the case in the mammalian
system, the carboxylation reaction in Conus venom ducts
absolutely requires vitamin K, and the net carboxylation increases
greatly in the presence of high concentrations of ammonium sulfate. In
these respects, the mammalian and the Conus
-carboxylation venom systems are very similar (11).
The propeptides of vitamin K-dependent blood coagulation
proteins share extensive sequence similarity. This sequence is believed to interact with the carboxylase and constitutes the -carboxylation recognition sequence
(-CRS).1 In this report,
we analyze the conantokin-G precursor sequence for potential -CRS
sequences. The results described below identify a sequence present in
the 1 to 20 region of the conantokin-G prepropeptide, which when
covalently linked to the N-terminal of the substrate stimulates
carboxylation by the Conus enzyme.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Conus radiatus venom ducts were obtained from Dr. L. J. Cruz (University of the Philippines). Vitamin K (phytonadione) was from Abbot Laboratories, and NaH14CO3 55 mCi/mmol from NEN Life Science Products. Bovine microsomes were a gift from Dr. D. W. Stafford (University of North Carolina, Chapel Hill, NC).
Conus microsome preparation from frozen venom ducts of C. radiatus was performed as described by Stanley et al. (11). Carboxylase assay using 1 µg of Conus microsomal protein per assay was performed as follows: Conus microsomes were solubilized in 0.7% CHAPS/0.7% phosphatidyl choline/1.5 M NaCl for 20 min on ice. Final reactions were done in a total volume of 125 µl containing solubilized microsomes and a final concentration of reagents as follows: 25 mM MOPS, pH 7.4, 0.5 M NaCl, 0.2% CHAPS, 0.2% phosphatidyl choline, 0.8 M ammonium sulfate, 5 µCi of NaH14 CO3, 6 mM dithiothreitol, 222 µM reduced vitamin K (prepared as described by Ref. 12). Substrate and inhibitor concentrations are indicated in the legends to the figures and tables. For experiments with bovine microsomes, 380 µg of microsomal protein was present in each reaction. Reaction mixtures were incubated at 25 °C for 30 min and were quenched by the addition of 75 µl of 1 N NaOH. 160 µl of the quenched reaction mixture was transferred to 1 ml of 5% trichloroacetic acid and boiled to remove unincorporated 14CO2. After cooling, 5 ml of Ecolite (NEN Life Science Products) was added, and the 14CO2 incorporated was determined in a Beckman LS 9800 counter. The amount of microsomal proteins present in the various experiments are indicated under "Results." All reported values are averages of three independent determinations.
Peptides were synthesized on a 357ACT peptide synthesizer (Advanced ChemTech) using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry strategy. The peptides were cleaved from the solid support by treatment with trifluoroacetic acid/phenol/ethanedithiol/thioanisole (90/5/2.5/2.5 by volume) and purified by reverse-phase high pressure liquid chromatography. The integrity of the peptides were verified by electrospray mass spectroscopy. Experiments were done in triplicate, and the data were analyzed using Graph Pad Prism from GraphPad Software, Inc. (San Diego, CA).
Isolation of conantokin-G cDNA was as follows. A cDNA library from C. geographus, in a pUC plasmid derivative, was plated out in duplicate and probed with end-labeled degenerate oligonucleotides corresponding to the mature toxin sequences. Hybridization was done in 3 M tetramethyl ammonium chloride (Aldrich), 0.1 M Na2HPO4, 0.001 M EDTA, 5× Denhardt's solution, 0.6% SDS, 100 µg/ml sheared salmon sperm DNA for 24 h at 48 °C. Washes were done at room temperature in 3 M tetramethyl ammonium chloride, 0.05 M Tris-HCl, pH 8, 0.2% SDS for 15 min and 1 h at 50 °C in a solution of the same composition. The filters were washed twice in 2× SSC, 0.1% SDS at room temperature for 15 min each. The filters were then exposed to x-ray films. Double stranded DNA from purified clones were sequenced by dideoxynucleotide chain terminating method (13).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Characterization of the Conantokin-G Precursor-- A cDNA clone encoding conantokin-G was identified as described under "Materials and Methods." The clone was sequenced in both strands, and the nucleotide sequence obtained is shown in Fig. 1. The predicted amino acid sequence of the open reading frame encoded by this cDNA clone and the post-translational modifications that take place to yield the mature peptide are also indicated in the figure. The cDNA sequence predicts the presence of a valine residue at position 5 of the mature conantokin-G sequence. However, active peptide isolated from the venom contains leucine at this position. Using oligonucleotides corresponding to the 5' and 3' sequences of the cDNA as primers, we have also isolated the leucine containing cDNA by polymerase chain reaction amplification of total venom duct cDNA. We have used leucine containing conantokin-G peptides in our experiments.
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-carboxyglutamate residues are encoded by the codons for glutamate.
At the C-terminal end of the predicted open reading frame are codons
encoding -Asn-Gly-Lys-Arg-Stop, which signals processing to a
C-terminal Asn-NH2.
Immediately adjacent to Gly1 of the mature conantokin-G is
a typical proteolytic signal (Ala-Arg) for Conus
prepropeptides. Proteolysis after the Arg residue would excise the
N-terminal prepro-region of 80 amino acids containing a signal sequence
of 21 amino acids at the N terminus. (However, at this point, we do not
know the order of cleavage of the signal sequence and propeptide sequences.) In contrast to previously described signal sequences for
conotoxins, the signal sequence for the conantokin-G precursor lacks a
Cys residue (14).
A noteworthy feature of the prepropeptide is the relatively long
intervening pro-region (59 amino acids). Among precursors of small
Conus peptides (under 30 amino acids in length) sequenced to
date, this is the longest pro-region that has been characterized so
far. As we establish below, this 59-amino acid pro-region contains a
-carboxylation recognition sequence that presumably plays a critical
role in the conversion of Glu to Gla.
Identification of -CRS of Conantokin-G--
In the case of
mammalian proteins that undergo -carboxylation, the -CRS is
contained in a 18-amino acid sequence (16-amino acid sequence in the
case of bone peptides) immediately N-terminal to the mature peptide
sequence (15-17). We investigated the ability of peptides shown in
Table I to serve as substrates or affect the activity of the Conus carboxylase.
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20.Pro-E.Con-G is 28 ± 3 µM. The results clearly indicate that covalent linkage of
the propeptide sequence 20 to 1 makes E.Con-G into an efficient substrate (note that the 20 to 1 sequence has no Glu residues that
would be substrates for carboxylation). Similar observations were made
when the 20 to 1 peptide was covalently linked to FLEEL (Table I).
The apparent Km for peptide 3 is less than that of
peptide 2, suggesting that propeptide sequences between 20 and 11
also interact with the carboxylase (Table I).
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20 to 1) in
trans, on the carboxylation of FLEEL, E.Con-G, 10.Pro-E.Con-G, and
20.Pro-E.Con-G. We also determined the effect of addition of Pro(20
to 11) and Pro(11 to 1) on the carboxylation of 10.Pro-E.Con-G.
Inspection of the Km values in Table I clearly
indicates that substrates in which the propeptide sequences 20 to 1
are covalently linked to the N terminus are efficient substrates for
carboxylation. In the case of both E.Con-G and FLEEL, the Km decreases by 2 orders of magnitude. Pro(20 to
1) stimulates the carboxylation of both FLEEL and E.Con-G when added in trans, but the effects are quite small: ~25% enhancement in the
case of E.Con-G and 40% for FLEEL. Pro(40 to 21) and Pro(60 to
41) have no effect on the carboxylation of FLEEL or E.Con-G (data not
shown).
Propeptide sequence 20 to 1 is an inhibitor for the carboxylation
of 10.Pro-E.Con-G (Fig. 3 and Table I)
and 20.Pro-E.Con-G. The IC50 of Pro(20 to 1) is very
similar to Pro(30 to 1), suggesting that the interaction of the
propeptide with the carboxylase in the region between 30 and 1 is
probably limited to 20 to 1. Pro(40 to 21) and Pro(60 to
41) did not inhibit carboxylation of 10.Pro-E.Con-G even at
concentrations 100-fold greater.
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20GKDRLTQMKRILKQRGNKAR1GEEELY-NH2,
a 26-amino acid peptide containing propeptide sequences 20 to 1 of
conantokin-G was used as the wild type substrate. Individual peptides
in which KR at positions 12 and 11, KQR at positions 8, 7, and
6, NK at positions 4 and 3, and NK-R at positions 4, 3, and
1 were substituted by alanine were used as substrates in the
carboxylation reaction. Carboxylation reactions were done at a
substrate concentration of 40 µM, the apparent Km of carboxylation for the wild type substrate.
Alanine substitutions in the context of the remaining propeptide
sequences had little effect (<10%) on carboxylation.
We examined the ability of bovine microsomes to use 20.Pro-E.Con-G as
substrate for -carboxylation. As shown in Table
II, although this peptide is an excellent
substrate for the Conus enzyme, it is extremely poor for the
mammalian enzyme.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have demonstrated that the -carboxylated 17-amino acid
Conus peptide conantokin-G is initially translated as a
prepropeptide of 100 amino acids. In general, the organization of the
conantokin-G precursor is similar to that previously reported for
disulfide-rich conotoxins from Conus venoms. The mature
peptide is found in a single copy at the C-terminal end of the
precursor. Before maturation of the peptide, a number of
post-translational processing events have to take place (Fig. 1). These
events include the -carboxylation of five glutamate residues,
C-terminal amidation of asparagine-17 following excision of the
C-terminal tripeptide, and a proteolytic event between
Arg1 and Gly1.
One notable feature of the conantokin-G prepropeptide is the length of
the intervening region between the signal sequence and the mature
peptide. The 59 amino acids in the intervening pro-region is the
longest so far reported for any Conus venom peptide. We have
demonstrated one potential function of this extended region: the
presence of a -carboxylation recognition sequence for the
Conus venom duct -glutamyl carboxylase in the 1 to 20 region.
In mammalian blood coagulation and bone Gla proteins, -carboxylation
of glutamate residues is carried out by a vitamin
K-dependent carboxylase. A conserved motif (16)
-carboxylation recognition sequence in the propeptide sequence binds
the -carboxylase and is required for a polypeptide substrate to be a
high affinity target for the -carboxylase. In the experiments
described above, we carried out an analysis using segments of the
conantokin-G prepropeptide to identify potential sites that might serve
as -carboxylase recognition signals for the Conus enzyme.
The results reveal that a -carboxylation recognition sequence is
included in the 1 to 20 region of the conantokin-G prepropeptide.
This appears to increase the affinity of the Conus
carboxylase by approximately 2 orders of magnitude under the assay
conditions used.
Knobloch and Suttie (18) and others (19) found that the propeptide
sequences of Factors IX and X at micromolar concentrations stimulated
the carboxylation of oligopeptide substrates, suggesting a probable
positive allosteric effector role. In addition, the propeptide at
micromolar concentrations acted as a competitive inhibitor of
carboxylation of a substrate whose sequences were based on residues
18 to +10 of prothrombin (20). Similarly, the Conus
propeptide (20 to 1) inhibits the carboxylation of propeptide-containing substrates, (i.e. 10.Pro-E.Con-G and
20.Pro.E.Con-G). However, we were unable to observe equivalent,
strong stimulation of carboxylation of FLEEL and E.Con-G by the
Conus propeptide.
The relevant propeptide sequence (20 to 1) for conantokin-G, human
Factors IX (21) and X (22), and prothrombin (23) are shown in Table
III. Except for the presence of a
hydrophobic residue at position 16, the conantokin-G sequence does
not appear to share the conserved features of the mammalian propeptides
(16). Phenylalanine is present at position 16 in all the propeptide sequences except in gas6 and protein S, in which it is leucine as in
the case of Con-G. In addition, isoleucine is present at position 10
of Con-G in place of a conserved alanine residue. Besides, positions
6 and 7, which are always hydrophobic for the mammalian propeptide
sequences, are basic and polar residues, respectively, for Con-G. This
suggests differences between the recognition specificities of the
mammalian and Conus enzymes. A preliminary characterization
of the amino acids important for recognition by the Conus
enzyme has been carried out. The individual basic amino acids in the
propeptide region 20 to 1 can be replaced by alanine without
significantly affecting the apparent Km for
carboxylation. The importance of hydrophobic residues in the mammalian -CRS suggests that they may also be important for the Conus carboxylase, and this role needs to be
investigated.
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What is the role of the -CRS ? Does it merely tether the substrate
to the carboxylase, or does it play additional roles in the
-carboxylation reaction? How do multiple carboxylations occur on a
single substrate molecule? Does the -CRS set a limit to the distance
at which -carboxylation will take place? Is the -CRS sufficient
for the fidelity of carboxylation, or are additional membrane
components necessary? These are some of the questions that need to be
addressed to understand the mechanism of action of -glutamyl
carboxylase.
The orientation in which a Glu presents itself to the active site
of the carboxylase may determine whether it will be carboxylated. In
the case of Con-G not all the Glu residues are -carboxylated (Glu2 is not carboxylated, whereas Glu3 and
Glu4 are carboxylated). The solution structures of Con-G
and Con-T as determined by CD and NMR spectroscopy (24, 25) are a
mixture of 
and 310 helices. Rigby et al.
(26) also determined the structure of the metal-free conformer of
conantokin-G by NMR spectroscopy. In all these structures, the Gla
residues are on the same side of the conantokin structure; this would
allow a membrane-bound enzyme to carry out efficient carboxylation of
Glu residues oriented in the same direction with optimum
stereochemistry.
In the discussion above, we have emphasized the differences in the
-carboxylation recognition signal sequences in the mammalian and
Conus systems. These differences may be due to the large
evolutionary distance between the two species. However, there is an
underlying general similarity between the two enzymes: the catalytic
reaction they carry out, their cofactor requirements, and a recognition signal (albeit differing in sequence) in the 1 to 20 propeptide region. It will be important to purify the Conus enzyme and
characterize it to determine the relationship between the two
enzymes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Bob Schackmann of the
DNA/PEPTIDE facility, Huntsman Cancer Center (supported by Grant NCI
42014) for synthesis of 20.Pro-E.Con-G and Dr. J. Rivier of Salk
Institute for synthesis of Pro(30 to 1). We thank Tom Stanley and
D. Yoshikami for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 48677 (to B. M. O.) and by Cognetix Inc. (to P. K. B. and C. S. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 801-581-7312; Fax: 801-585-5010; E-mail bandyop{at}biology.utah.edu.
1
The abbreviations used are: -CRS,
-carboxylation recognition sequence; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
MOPS, 4-morpholinepropanesulfonic acid; Gla,
-carboxyglutamate.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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