From the Departments of Biology and ¶ Human
Genetics, University of Utah, Salt Lake City, Utah 84112-0840
Received for publication, October 19, 2000
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ABSTRACT |
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The vitamin K-dependent
The functions of proteins are coordinated physiologically by
post-translational modification. For example,
phosphorylation-dephosphorylation cascades integrate the biochemistry
of individual proteins into cellular physiology. In addition to
post-translational modifications that occur primarily within cells,
post-translational modifications also occur on extracellular proteins.
The most familiar of these are N-glycosylation of asparagine
residues and O-glycosylation of serine and threonine residues.
One of the most distinctive of the extracellular post-translational
modifications is the vitamin K-dependent Long after its characterization in blood-clotting factors, vitamin
K-dependent In the conantokins, the significance of the post-translational
modification can readily be demonstrated: these peptides are inactive
in analogs without The enzymatic reaction in the invertebrate system has recently been
shown to have many striking similarities (e.g. a requirement for reduced vitamin K and the presence of a Materials--
Conus textile venom ducts were
obtained from Dr. L. J. Cruz (University of the Philippines).
Vitamin K (phytonadione) was from Abbott Laboratories, and
NaH14CO3 (55 mCi/mmol) was from PerkinElmer
Life Sciences. Enzymes were purchased from Life Technologies, Inc.
PCR1 reactions were performed
in an Air Thermo-Cycler (Idaho Techology). Oligonucleotides were
synthesized at the peptide sequencing facility at the University of Utah.
Preparation of mRNA--
Adult Drosophila
(Oregon) were frozen in liquid nitrogen and ground to a fine
powder, and total RNA was isolated (21). Poly(A)+ RNA was
isolated using a Qiagen Oligotex mRNA kit according to the
vendor's instructions. Molecular biology experiments were done
according to methods described by Sambrook et al. (22).
Sequence Analysis--
The cDNA sequence of
Drosophila
Analyses of amino acid homology between human (24), bovine (25), rat
(26), and Drosophila Northern Blot Analysis--
Total RNA was isolated from
Drosophila at various stages of development. Embryos at
0-2, 2-4, 4-8, 12-16, and 16-24 h of development, larval stages
1-3, pupae, and adult flies were used in the experiment. Northern blot
analysis was performed using reagents provided in the Northern Max kit
(Ambion). Nine µg of total RNA from each sample were electrophoresed
in a 1.5% denaturing agarose gel, transferred to a Gene Screen Plus
membrane (PerkinElmer Life Sciences), and hybridized to
[ In Situ Hybridization--
The spatial distribution of
Transient Expression of Enzyme Assays--
Characterization of a Putative Drosophila
We carried out further PCR analysis of the cDNA using the primers
indicated schematically in Table I and Fig.
2A. The 3'-end of the
transcript to the poly(A) addition site was determined using 3'-rapid
amplification of cDNA ends. Polyadenylation takes place 20 bases
downstream of a consensus Poly(A) signal, AATAAA. The remainder of the
cDNA was characterized by PCR amplification using primers shown in
Table I; PCR products were cloned, and overlapping sequences were
combined to yield the cDNA shown schematically in Fig.
2A.
The cDNA sequence encodes an open reading frame of 670 amino acids.
Examination of the genomic sequence revealed two notable differences
from the cDNA. First, there are two short introns. A schematic
comparison of the genomic and cDNA, shown in Fig. 2B,
illustrates the position of these introns. The nucleic acid sequences
at the splice junction are shown in Fig. 2C. Second, an as
yet uncharacterized processing event removes 6 nucleotides from the
cDNA (Fig. 2D).
The locations of the two introns are conserved between
Drosophila and mammals. As is generally found when comparing
Drosophila with mammalian introns (30), the
Drosophila introns are significantly shorter (for intron I,
58 versus 2204 nucleotides; for intron II, 72 versus 646 nucleotides). Comparison of the amino acid
sequences flanking Drosophila intron II and human intron VII
is shown in Fig. 2E. There are a total of 14 introns in both
the rat (26) and the human
The surprising finding from the sequencing described above is that an
uncharacterized mechanism of RNA processing results in the deletion of
6 nucleotides that would have been present if the DNA were faithfully
transcribed. The deletion does not change the amino acid homology to
the mammalian enzyme at this site. This region of cDNA was
sequenced at least three times as parts of PCR amplification products
synthesized by different primer pairs. To confirm the genomic sequence
of our strain, we directly sequenced the genomic DNA from the
Drosophila strain that was the source of our cDNA. It
was identical to that from BDGP, which confirmed that the deletion of 6 nucleotides was real and not an artifact of strain differences or
cloning or sequencing errors.
With regard to the protein length, the alignment in Fig. 3B
shows that although the Drosophila enzyme has 17 additional
amino acids at the N terminus compared with the mammalian enzymes, it is significantly shorter at the C-terminal end. All of the mammalian enzymes are longer (758 versus 670 amino acids). A recent
deletion analysis (32) of the bovine enzyme suggests that small
deletions at the C terminus may be tolerated by the wild-type mammalian enzyme. Interestingly, a deletion that resulted in a bovine enzyme that
was 676 amino acids in length had lower enzymatic activity (15-fold
lower with respect to Northern Blot Analysis--
Fig. 4
shows the results of Northern blot analysis. Drosophila
Spatial Expression of Assays for Substrate Specificity of In this work, we have characterized a cDNA clone derived from
adult D. melanogaster mRNA that encodes a protein with
Northern blot analysis indicates that the putative
Drosophila The discovery that a A recently elucidated hereditary disease further emphasizes the
functional significance of the total conservation of amino acid
sequence in this region: mutation of residue 395 (Leu Heterologous Because -carboxylation of glutamate to
-carboxyglutamate was originally
well characterized in the mammalian blood clotting cascade.
-Carboxyglutamate has also been found in a number of other mammalian
proteins and in neuropeptides from the venoms of marine snails
belonging to the genus Conus, suggesting wider prevalence
of
-carboxylation. We demonstrate that an open reading frame from a
Drosophila melanogaster cDNA clone encodes a protein
with vitamin K-dependent
-carboxylase activity. The open
reading frame, 670 amino acids in length, is truncated at the
C-terminal end compared with mammalian
-carboxylase, which is 758 amino acids. The mammalian gene has 14 introns; in
Drosophila there are two much shorter introns but in
positions precisely homologous to two of the mammalian introns. In
addition, a deletion of 6 nucleotides is observed when cDNA and
genomic sequences are compared. In situ hybridization to
fixed embryos indicated ubiquitous presence of carboxylase mRNA
throughout embryogenesis. Northern blot analysis revealed increased
mRNA levels in 12-24-h embryos. The continued presence of
carboxylase mRNA suggests that it plays an important role during
embryogenesis. Although the model substrate FLEEL is carboxylated by
the enzyme, a substrate containing the propeptide of a
Conus carboxylase substrate, conantokin G, is poorly
carboxylated. Its occurrence in vertebrates, molluscan systems
(i.e. Conus), and Drosophila and
the apparently strong homology between the three systems suggest that
this is a highly conserved and widely distributed post-translational
modification in biological systems.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxylation of glutamate residues to give
-carboxyglutamate (1). When it was
first characterized,
-carboxylation was thought to be a biochemical
specialization of the mammalian blood-clotting cascade. However,
several bone proteins (2, 3) as well as an extracellular ligand, gas6
(4), were subsequently identified as having the post-translational
modification, although in the latter cases the precise mechanistic role
of
-carboxylation for proper protein function has not been
established definitively. In addition, two novel proline-rich
-carboxyglutamic acid-containing proteins, PRGP1 and PRGP2,
of unknown function have been identified (5).
-carboxylation of glutamate residues was
discovered in a phylogenetically distant system: the neuropeptides made
in the venom duct of the predatory cone snails Conus (6, 7). The venoms of these snails have ~100 different peptides; ~5% of these are believed to be
-carboxylated (8). This post-translational modification has been found in a number of diverse Conus
peptides but has been studied most intensively in an unusual
Conus neuropeptide family, the conantokins, which are NMDA
receptor antagonists.
-carboxylation of glutamate residues. Incomplete
-carboxylation of blood-clotting factors results in poor
coagulation. It has been postulated that
-carboxylation of both the
conantokins and of factors of the blood-clotting cascade induces a
helical conformation in the post-translationally modified regions. This
postulated role of
-carboxylation in determining conantokin
structure has been generally supported by a number of subsequent
structural studies on various conantokins (9-12).
-Carboxyglutamic
acid confers the property of Ca2+ binding to the modified
protein. In the case of the blood-clotting factors, the binding to
Ca2+ results in a conformational change exposing
hydrophobic residues for interaction with membranes (13-17).
-carboxylation
recognition site on the substrate) to that of the
-carboxylation of
factors involved in the mammalian blood-clotting cascade (18, 19). Despite the clear functional importance of
-carboxylation in these
two disparate phylogenetic systems,
-carboxylation of glutamate residues has been regarded as a highly specialized post-translational modification. In this report, we provide evidence that is strongly consistent with vitamin K-dependent
-carboxylation in
fact being a much more widely distributed biological phenomenon. We
demonstrate by molecular techniques the presence of a vitamin
K-dependent
-carboxylase-related protein that is
expressed in the fruit fly Drosophila melanogaster, which
has a high degree of sequence identity with the mammalian enzyme.
Similar observations have recently been reported by Li et
al. (20). Although the role of
-carboxylation in
Drosophila remains unknown, this post-translational
modification is present in arthropods, suggesting that it is generally
distributed in animal systems. The strong conservation in sequence of
the
-glutamyl carboxylase in Drosophila and in mammals
suggests an important functional role for the enzyme, resulting in
strong selection for sequence conservation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxylase was assembled from sequences of
PCR products obtained by amplification of oligo(dT)- or
QT-primed cDNA using primers shown in Table I. The
primer combinations used in the PCRs are shown in Table I. Primers 1 and 2 correspond to amino acid sequences conserved in human, bovine, and rat. Primers 5 and 6 were selected from the Drosophila
genomic sequence (Berkeley Drosophila Genome Project,
accession number AC005557). 3'-Sequences of the carboxylase mRNA
were determined by the technique of rapid amplification of cDNA
ends described by Frohman (23). The PCR product was cloned into the TA
cloning vector (Invitrogen), and the nucleic acid sequence was
determined. DNA sequencing was performed using ABI Prism BigDye
terminators and cycle sequencing with Taq FS DNA polymerase
(Life Technologies). The DNA sequence was collected and analyzed on an
ABI Prism 377 automated DNA sequencer (Applied Biosystems, Foster City,
CA). To obtain the 5' end of the coding sequences, we selected primer 6 from the genomic sequence. This allowed us to characterize a transcript
with an open reading frame from nucleotides 52 (ATG) to 2198 (TGA). The
sequences have been submitted to GenBank; the accession number is
AF170280.
-glutamyl carboxylases were carried
out using Gap and PileUp programs version 4.0, 1998 (Genetics Computer Group).
-32P]UTP-labeled antisense Drosophila
-carboxylase RNA. As a control for loading, a membrane containing
identical samples was hybridized to [
-32P]dCTP-labeled
Drosophila rp49. RNA molecular weight standards (RNA
Millennium) were purchased from Ambion. Membranes were exposed to
Molecular Dynamics (Sunnyvale, CA) Phosphor Screen and scanned. Images
were analyzed using the NIH Image processing program.
-glutamyl carboxylase RNA was probed by hybridization to whole-mount
embryos in situ (27). The cDNA was cloned into a
dual-promoter (T7 and Sp6) vector. Both sense and antisense RNAs were
synthesized using the appropriate RNA polymerase in the presence of
digoxigenin-labeled uridine triphosphate. The
digoxigenin-labeled RNA was used as probe in hybridizations to
fixed embryos. The hybridized digoxigenin-labeled RNA was detected by
incubating the embryos with alkaline phosphatase-conjugated anti-digoxigenin antibody and developed using nitroblue tetrzolium and
5-bromo-4-chloro-3-indolyl phosphate.
-Glutamyl Carboxylase
Activity--
DNA containing the coding sequence of
Drosophila
-glutamyl carboxylase was obtained by PCR
amplification of the genomic sequences. The 3' PCR primer was designed
such that the
-carboxylase (GC) coding sequences would be in frame
with coding sequences of green fluorescent protein (GFP) in the
expression plasmid pRmHa-3.GFP (Fig. 1).
The
-glutamyl carboxylase encoding sequences include the two introns
in the genomic sequence. pRmHa-3.GFP was constructed by introducing the
coding sequence of GFP from the pEGFP vector (CLONTECH) into pRmHa-3 (28). Expression in this
plasmid is under the control of the inducible metallothionein promoter
and carries the alcohol dehydrogenase poly(A) addition signal.
Drosophila Schneider 2 (S2) cells were transfected with
pRmHa-3.GC.GFP DNA using CellFECTIN (Life Technologies). 24 h
after transfection cells were induced with 0.7 mM
CuSO4. 48 h after induction 50% of the cells
expressed GFP as judged by fluorescent microscopy. The results also
indicated that the introns were properly processed, and a continuous
reading frame was present in the cloned GC. pRmHa-3.GC.GFP was modified
to introduce a stop codon at the end of the GC coding sequences and to
delete the GFP coding sequences. The modified plasmid pRmHa-3.GC* was
transfected into S2 cells. The cells were induced with 0.7 mM CuSO4 and harvested 48 h after
induction. Cells were washed twice with phosphate-buffered saline and
resuspended in buffer containing 25 mM
4-morpholinepropanesulfonic acid, pH 7.0, 0.5 M NaCl, 0.2%
3-[(3-cholamidopropyl)dimenthylammonio]-1-propanesulfonic acid/phosphatidyl choline, 2 mM EDTA, 2 mM
dithiothreitol, 0.2 µg/ml leupeptin, 0.8 µg/ml pepstatin, and 0.04 mg/ml phenylmethylsulfonyl fluoride. The cell suspension was briefly
sonicated using a Branson 450 sonifier and incubated in ice for 20 min.
The lysate was assayed for carboxylase activity.
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Fig. 1.
Vectors used for the expression of GFP,
-carboxylase.GFP, and
-carboxylase in S2 cells.
-Glutamyl carboxylase assays were
performed as described by Stanley et al. (18). Reactions
were done in a total volume of 125 µl containing cell lysate and a
final concentration of reagents as follows: 25 mM
4-morpholinepropanesulfonic acid, pH 7.4, 0.5 M NaCl, 0.2%
3-[(3-cholamidopropyl)dimenthylammonio]-1-propanesulfonic acid, 0.2%
phosphatidyl choline, 0.8 M ammonium sulfate, 5 µCi of
NaH14CO3', 6 mM dithiothreitol, 222 µM reduced vitamin K, and 1.2 mM of a model
substrate, the pentapeptide FLEEL (29). Reaction mixtures were
incubated at 25 °C for 120 min and were quenched by addition of 75 µl of 1 N NaOH. The quenched reaction mixture (160 µl)
was transferred to 1 ml of 5% trichloroacetic acid and boiled to
remove unincorporated 14CO2. After cooling, 5 ml of Ecolite (PerkinElmer Life Sciences) was added, and the
14CO2 incorporated was determined in a Beckman
LS 9800 counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Carboxylase mRNA
Sequence--
Poly(A)+ RNA from adult
Drosophila was used as template for the reverse
transcription of cDNA using an oligo(dT) primer. An initial segment
of cDNA was amplified and sequenced using
-carboxylase primers
encoding amino acid sequences highly conserved between Drosophila sequences from the Berkeley Drosophila
Genome Project (BDGP) and all mammalian enzymes; the 5'-oligonucleotide
primer (primer 1; Table I) corresponded
to amino acids 395-402, YGYSWDMM, and the 3'-primer (primer 2; Table
I) corresponded to amino acids 465-471, IYFDIWC (the amino acid
positions correspond to human
-glutamyl carboxylase sequence). The
PCR product was cloned, and the nucleic acid sequence was determined;
the sequence obtained was identical to a DNA sequence in the
Drosophila genome (BDGP, accession number AC005557).
Primers used in PCR to identify Drosophila -glutamyl carboxylase
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Fig. 2.
A, schematic of PCR products.
Coordinates refer to the genomic sequence. Positions of initiation and
termination codons, introns, processing site (DELETE), and
Poly(A) addition signals are shown. B, schematic comparison
of genomic and cDNA maps of Drosophila -carboxylase.
C, nucleic acid and amino acid sequences at the splice
junctions of Drosophila introns I and II. D,
cDNA sequences at the processing site (DELETE).
E, comparison of amino acid sequences flanking
Drosophila intron II and human intron VII. Amino acids at
the splice junction are shown in bold.
-carboxylase genes (31); therefore,
Drosophila has both fewer and shorter introns compared with
the mammalian gene. Fig. 3A
shows a schematic of amino acid homology between human and
Drosophila
-carboxylase.
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Fig. 3.
A, schematic of amino acid homology
between human and the Drosophila -glutamyl carboxylase.
The two proteins are strongly homologous over the entire length of the
predicted Drosophila open reading frame. B,
protein sequence homology between human and Drosophila
vitamin K-dependent carboxylases. Sequences in black
boxes indicate identical residues; those in gray boxes
are homologous residues. Dots indicate gaps.
-carboxylation and 400-fold lower than for
vitamin K epoxidation). It remains to be determined whether other
Drosophila subunits are necessary to compensate for the shorter length of the Drosophila open reading frame.
-glutamyl carboxylase mRNA is ~2.7 kb in size (Fig.
4A) and is predominantly expressed in 12-24-h embryos (Fig.
4B). (However, the more sensitive in situ
hybridization experiments presented below reveal the presence of
carboxylase mRNA throughout embryogenesis.) Ribosomal protein rp49
mRNA was also monitored in these experiments (Fig. 4C).
Although similar amounts of rp49 mRNA were present in the samples
from embryos at different developmental stages (0-24 h), there was
less RNA in larvae, pupae, and adult. The reduced level of rp49
mRNA observed at later stages is a reflection of reduced synthesis
at these developmental stages (33, 34). From densitometric measurements
using the NIH Image analysis program, we determined the amount of
-carboxylase RNA present relative to rp49 RNA. Normalized to rp49
RNA, we estimate that there is at least three times as much
-carboxylase RNA in late stage embryos (16-24 h) relative to that
in adults.
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Fig. 4.
Northern blot analysis. A, total RNA
from Drosophila embryos (0-24 h) probed with
32P-labeled antisense -carboxylase RNA. Molecular weight
standards represent ethidium bromide-stained RNA Millennium markers
(Ambion). B and C, analysis of developmental
expression of
-carboxylase (B) and rp49 (C)
RNAs. Lanes containing RNA from embryos at 0-2, 2-4, 4-8,
12-16, and 16-24 h of development, larval stages 1-3, pupae, and
adult flies are indicated (the lane between
4-8-h and 12-16-h embryo samples is
blank).
-Carboxylase--
When embryos were
examined to identify the sites of expression of
-glutamyl
carboxylase, ubiquitous expression of
-carboxylase RNA was observed
throughout embryogenesis. Only antisense transcript probes
cross-hybridized to embryonic mRNAs in situ (Fig.
5, B and D). No
hybridization-positive embryos were observed when sense transcripts
were used as probes (Fig. 5, A and C).
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Fig. 5.
Ubiquitous transcription of
-carboxylase in Drosophila
embryos. In situ hybridization to stage 5 (A
and B) and 16 (C and D) whole-mount
embryos with digoxigenin labeled strand-specific probes is shown.
Ubiquitous gene expression is visible when the antisense strand is used
as probe (B and D) but not when the sense strand
is used (A and C).
-Carboxylase Activity--
Lysates of S2 cells
transfected with DNA encoding the putative
-glutamyl carboxylase
were assayed for carboxylase activity. Table
II demonstrates that S2 cells transfected
with pRmHa-3.GC* expresses vitamin K-dependent
-carboxylase activity that is not present in mock-transfected cells
or cells transfected with pRmHa-3.GFP (data not shown).
Carboxylase activity in transfected Drosophila S2 cells
-Carboxylation--
Endogenous
substrates of both the mammalian (35) and Conus
-glutamyl
carboxylases contain a
-carboxylation recognition signal (
-CRS)
in the propeptide region (19, 36). The presence of a
-CRS at the
amino terminus of a substrate greatly enhances the efficiency of
carboxylation. We have previously identified the
-CRS of a
-carboxylated Conus peptide, conantokin-G (19). A peptide
containing proconantokin-G sequences (
20 to +5,
20GKDRLTQMKRILKQRGNKAR
1GEEEL+5Y) referred to as
20Y in the text below,
is efficiently carboxylated by Conus carboxylase (19). We
determined the carboxylation of both FLEEL and
20Y by
Conus and Drosophila
-glutamyl carboxylases. The ratio of 14CO2 incorporated in the two
substrates (
20Y/FLEEL) by the Conus enzyme was 4, whereas
that for the Drosophila enzyme was 0.26.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamyl carboxylase activity. The open reading frame is homologous
to the mammalian
-glutamyl carboxylase throughout its length of 670 amino acids; the degree of sequence identity is strikingly high (39%
identity and 55% homology) and comparable with the subset of enzymes
highly conserved between Drosophila and mammals. One unexpected result when comparing the putative genomic DNA sequence with
the cDNA is that there is an apparent small deletion in the cDNA sequence, suggesting that RNA processing of the
Drosophila
-glutamyl carboxylase mRNA occurs.
-glutamyl carboxylase mRNA is ~2.7 kb in
size. This is ~350 nucleotides longer than the open reading frame and
3'-untranslated region previously characterized from cDNA. Because
a single RNA isoform is observed, and the 3'-end of the RNA determined
by cDNA analysis is unique, the additional 350 nucleotides
represent the length of the poly(A) tail together with the
5'-untranslated region. In situ hybridization reveals the
ubiquitous presence of carboxylase RNA throughout embryogenesis. By
reverse transcription-PCR analysis, Li et al. (20) have also
reported similar observations. In early embryos (0-2 h) it is probably
of maternal origin, whereas in later embryos (2-12 h), there is
considerable contribution from embryonic synthesis as suggested by the
kinetics of RNA synthesis. In this regard, carboxylase mRNA
expression has been demonstrated in neuronal and skeletal tissue of
postimplantation rat embryos during early organogenesis (26).
-carboxylase is expressed in
Drosophila opens the door to genetic analysis of vitamin
K-dependent
-carboxylation in this model system. The
availability of the Drosophila
-glutamyl carboxylase
sequence already provides significant structure-function information.
Some segments are clearly much more highly conserved when the
Drosophila sequence is compared with that of mammalian enzymes; presumably, these are domains of the enzyme most critical for
function. Among the most noteworthy, mammalian and
Drosophila sequences corresponding to amino acids 385-404
in the Drosophila sequence (see Fig. 3) are identical. An
extended conserved motif in this region has been suggested by Begley
et al. (37). However, nucleic acid sequences reported here,
by Li et al. (20) and by the BDGP (AC005557), are not
consistent with the suggestion. The sequences reported here, by Li
et al. (20) and by the BDGP (AC005557), are
385GYNNWTNGLYGYSWDMMVH404SYDTLQTSIQVVD ... ,
whereas the sequences at this site reported by Begley et al.
(37) are
385GYNNWTNGLYGYSWDMMVH404SRSHQHVKITYRD.
Arg) in the
human enzyme (38) results in a clinical syndrome characterized by a
general deficiency of blood clotting. Interestingly, this clinical
condition can be treated satisfactorily by an infusion of high doses of
vitamin K, consistent with an enzymatic defect in the affinity of the
enzyme for its substrate, reduced vitamin K. Thus, the comparison
between Drosophila and mammalian enzymes may have helped
define a conserved site involved in the binding of reduced vitamin K,
consistent with more conventional biochemical studies.
-CRS sequences are not or are poorly recognized
by the
-glutamyl carboxylases.
-CRS containing Conus
substrate, proconantokin G, is poorly carboxylated by the bovine
enzyme, whereas a peptide, factor IX-18-41, which consists of the
propeptide and all normally carboxylated residues of the vitamin
K-dependent clotting protein factor IX, is not carboxylated
by the Conus enzyme (18). The poor carboxylation of
20Y by
the Drosophila enzyme further strengthens the suggestion
that the enzymes have evolved to recognize their cognate
-CRSs. This
is also supported by the observation of Li et al. (20), who
found that the propeptide of human blood coagulation factor IX did not
stimulate carboxylation by the Drosophila enzyme. Because
the Drosophila and human
-glutamyl carboxylases share
considerable sequence homology, it should be possible to identify
substrate binding domains by studying carboxylation using chimeric enzymes.
-carboxylated molecules may serve as signals for growth and
differentiation, differential regulation of
-carboxylation may
operate at multiple levels during development. Control may be at the
level of synthesis of
-carboxylase mRNA or its translation, or
both. Although mRNA may be present, enzyme activity may not be
obvious. Future experiments will be aimed at determining possible differences among levels of mRNA, expressed protein, and activity by immunological methods (for protein) and
-carboxylase assay (for activity).
-Carboxyglutamate-containing proteins isolated to date are
extracellular proteins.
-Carboxyglutamate interacts with
Ca2+, induces a conformational change in the protein, and
facilitates binding to membrane phospholipids. A number of
-carboxyglutamate-containing vitamin K-dependent proteins
(thrombin, factor Xa, protein S, and Gas6) are ligands for cell surface
receptors. Interaction with the receptors induces cellular
proliferative responses (39, 40). In Drosophila, high levels
of
-carboxylase RNA are detected in late stage embryos. During this
period, a variety of developmental and morphogenetic events occur,
among them cuticle deposition and central nervous system, peripheral
nervous system, and gut differentiation. It is conceivable that some of
the gene products signaling these events are
-carboxylated and serve
as ligands for corresponding receptors. The effects of
-carboxylase
knockout in flies will enable a systematic study of probable targets
for this post-translational modification.
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ACKNOWLEDGEMENT |
---|
We thank the DNA Sequencing Facility at the University of Utah. The Peptide Sequencing facility at the University of Utah is supported by NCI Grant 5p30CA42014.
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FOOTNOTES |
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* This work was supported in part by a grant from the University of Utah Research Foundation (to P. K. B.), the Funding Initiative Seed Grant Program (1999), National Institutes of Health Grant GM48677, and Cognetix, Inc. (Salt Lake City, UT).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF170280.
§ Supported by a Pfizer summer undergraduate research fellowship and a Beckman undergraduate research fellowship.
To whom correspondence should be addressed: Dept. of Biology,
University of Utah, 257 S. 1400 E., Rm. 201, Salt Lake City, UT
84112-0840. Tel.: 801-581-5907; Fax: 801-585-5010; E-mail: bandyop@biology.utah.edu.
Published, JBC Papers in Press, December 7, 2000, DOI 10.1074/jbc.M009576200
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ABBREVIATIONS |
---|
The abbreviations used are:
PCR, polymerase
chain reaction;
GC, -carboxylase;
BDGP, Berkeley
Drosophila Genome Project;
-CRS,
-carboxylation
recognition signal sequence;
GFP, green fluorescent protein;
S2, Schneider 2.
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REFERENCES |
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