From the Institute for Chemical Reaction Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan
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ABSTRACT |
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Geranylgeranyl diphosphate (GGPP) synthase
(GGPPSase) catalyzes the synthesis of GGPP, which is an important
molecule responsible for the C20-prenylated protein
biosynthesis and for the regulation of a nuclear hormone receptor
(LXR·RXR). The human GGPPSase cDNA encodes a protein of 300 amino
acids which shows 16% sequence identity with the known human farnesyl
diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed in
Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase
behaves as an oligomeric molecule with 280 kDa on a gel filtration
column and cross-reacts with an antibody directed against bovine brain
GGPPSase, which differs immunochemically from bovine brain FPPSase.
Northern blot analysis indicates the presence of two forms of the mRNA.
Since Schmidt et al. (1) first detected the
incorporation of a mevalonate-derived intermediate into protein in
Swiss 3T3 cells, a number of prenylated proteins have been found in
various organisms (2-6). The prenylation of these proteins is
essential for their function in the cells (7-9), and the direct
precursors of the prenyl moiety have been elucidated to be farnesyl
diphosphate (FPP)1 and
geranylgeranyl diphosphate (GGPP) by identifying
protein-prenyltransferases in yeast and mammalian brains (10). Further,
the number of geranylgeranylated proteins has been shown to be larger
than that of farnesylated proteins (11-13). Recently, these shorter
chain prenyl diphosphates also have been shown to be involved in the
function of nuclear hormone receptors. Forman et al. (14)
have isolated a mammalian orphan receptor, FXR, which forms a
heterodimeric complex with RXR and identified farnesol and the related
metabolites as effective activators of this complex (FXR·RXR). In a
study of LXR FPP and GGPP are mevalonate-derived intermediates. The biosynthesis of
mevalonate is regulated by a finely tuned mechanism, and the
rate-limiting enzyme is 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase in animal cells (16). FPP, whose carbon chain length is
C15, is generally accepted to be the common intermediate occupying the branch point in the biosynthetic pathways to cholesterol, dolichol, ubiquinone, heme a, and farnesylated proteins. This compound
is synthesized from dimethylallyl diphosphate by the action of FPP
synthase (FPPSase), the most abundant and widely occurring
prenyltransferase in mammalian tissues. FPPSase is composed of two
identical subunits (17), and several FPPSase cDNAs have already
been cloned in animals such as chicken, rat, and human (18). Genomic
Southern blot analyses suggested that rat and human have multiple genes
encoding divergent FPPSases (19, 20). On the other hand, GGPP, whose
carbon chain length is C20, was initially thought to be
supplied from FPP by the action of the FPPSase because FPPSase
crystallized from avian liver had a poor but significant ability to
synthesize GGPP from FPP and isopentenyl diphosphate (IPP) (21).
Thereafter, we succeeded in isolating GGPP synthase (GGPPSase) in the
different fraction from FPPSase by ion exchange chromatography of pig
liver (22), rat liver (23), and bovine brain (24) cytosols. The
GGPPSase has been purified from bovine brain in a homogeneous form (25)
using a one-step procedure by affinity chromatography which contained an FPP analog as a ligand. The purified GGPPSase catalyzed a single reaction C15 Considering that GGPP plays a role in the function of a nuclear hormone
receptor and of geranylgeranylated proteins, it would be very important
to characterize GGPPSase. Our original interest is in characterizing
GGPPSase and FPPSase that occur in the same mammal or organ because
GGPPSase could be regulated differently from FPPSase for their
functions, although both of them catalyze the
prenyl(C5)-transferring reaction. It is also interesting to learn the tissue-specific expression of GGPPSase because
geranylgeranylated proteins have been reported to be rich specifically
in brain (13). In the present study, we purified the GGPPSase of bovine
brain in large amounts, isolated cDNAs of bovine and human
GGPPSases using information from the bovine brain GGPPSase amino acid
sequence, expressed the human GGPPSase in Escherichia coli,
and characterized it. These analyses revealed that GGPPSase was a
considerably different protein from FPPSase. Northern blot analysis
indicated the presence of two mRNAs in various tissues.
Materials--
[1-14C]IPP (55 mCi/mmol) and
[ Enzyme Preparation--
Bovine brain was homogenized with a
Polytron homogenizer in 50 mM Tris-HCl buffer (pH 7.5)
containing 0.1 mM leupeptin, 0.2 mM
phenylmethanesulfonyl fluoride, 1 mM EDTA, and 1 mM EGTA. The crude homogenate was centrifuged at
14,000 × g for 30 min. The supernatant was
fractionated with ammonium sulfate. For purification of GGPPSase, the
fraction precipitating between 40 and 60% saturation of ammonium
sulfate was subjected to Butyl-Toyopearl chromatography. The elution
from a Butyl-Toyopearl column was performed with a downward linear
gradient from 15 to 0% saturation of ammonium sulfate. The fractions
containing GGPPSase activities were pooled, concentrated, and dialyzed
against 10 mM BisTris buffer (pH 7.0) containing 10 mM 2-mercaptoethanol and 1 mM
MgCl2. The dialyzed fraction was then applied to an
affinity column with a farnesylmethyl phosphonophosphate ligand. The
GGPPSase was eluted with a linear gradient of FPP to 0.5 mM. The active fractions were pooled and then applied
directly to a Mono Q column and eluted with a linear gradient of NaCl
to 0.5 M. The GGPPSase was eluted at 0.15 M
NaCl. For purification of FPPSase, the fraction precipitated between 30 and 40% saturation of ammonium sulfate was dialyzed against 10 mM PIPES buffer (pH 7.0) containing 10 mM
2-mercaptoethanol and 1 mM MgCl2 and applied to
an affinity column with a geranylmethyl phosphonophosphate ligand. The
FPPSase was eluted with l mM inorganic diphosphate. The
active fractions were purified further by Mono Q chromatography.
Enzyme Assay--
The standard assay mixture contained, in a
final volume of 0.1 ml, 50 mM potassium phosphate buffer
(pH 7.0), 2 mM dithiothreitol, 5 mM
MgCl2, 20 µM [1-14C]IPP, 25 µM GPP or FPP, and an appropriate amount of enzyme
fraction. The mixture was incubated at 37 °C for 15 min and
terminated by the addition of 0.30 ml of a mixture of concentrated
HCl:methanol (4:1) followed by a 15-min incubation at 37 °C. The
hexane-soluble hydrolysates were analyzed in a liquid scintillation
fluid. For product analysis, the reaction products were extracted with
0.1 ml of 1-butanol saturated with water. The extracts were treated with potato acid phosphatase (2.4 units, 2.2 mg) according to the
method of Fujii et al. (28). After incubation at 37 °C
for 6 h, the hydrolysates were extracted with hexane and analyzed on a silica gel plate and a reverse phase C18 plate in a
solvent system of toluene:ethyl acetate (9:1) and in a solvent system of acetone:water (7:1), respectively. The hydrolysates were also analyzed by the two-plate thin layer chromatography (TLC) method (29).
The positions of authentic standards were visualized with iodine vapor.
The radioactivity of polyprenols developed on TLC was determined with a
Fuji Bioimage Analyzer BAS 1000.
SDS-PAGE and Immunoblotting--
Proteins were analyzed on 10 or
14% polyacrylamide gels (1.0 mm) containing 0.1% SDS and transferred
to a nitrocellulose membrane. FPPSase and GGPPSase were visualized
using the specific antibodies and the secondary antibody conjugated to
alkaline phosphatase.
Analysis of Amino Acid Sequence of Bovine Brain
GGPPSase--
The bovine brain GGPPSase preparation purified by
affinity chromatography as described above was subjected to 10%
SDS-PAGE and electrotransferred to a polyvinylidene difluoride
membrane. The membrane corresponding to a Ponsau Red-positive GGPPSase
band was cut off and analyzed by automated Edman degradation after treatment of lysyl endopeptidase. The sequences obtained were as
follows: KAYK (fragment 2), KHLSK (fragment 4), KMFK (fragment 9),
KTQETVQRILLEPY (fragment 14), and KQIDARGGNP (fragment 15).
Amplification of DNA by PCR--
The primers used for PCR were
commercially synthesized: primer-1 was TCCCATGGAGAAGACTCAA; primer-2,
ACTCA(AG)GA(AG)ACAGT(ACGT)CA; primer-3,
TT(CT)GT(AG)AA(CT)TC(AG)TT(CT)TACAA; and primer-4,
TTACTTATTACAATTCGAAAA. The PCR mixture (50 µl) consisted of 0.5 ng of
cDNA, 100 pmol of primers, 0.20 µM each
deoxynucleotide triphosphate, and 1.25 units of Ex-Taq DNA
polymerase. Thirty-five cycles of amplification (0.5 min at 94 °C
for denaturation, 1 min at 50 °C for annealing, and 1 min at
72 °C for extension) were carried out by using a Perkin-Elmer
thermalcycler. The amplified products were analyzed on 1.4% agarose
electrophoresis and recovered from the gels by use of a Geneclean II
kit (BIO 101). The products were ligated directly into a vector pT7
blue T, and the insert DNA sequence was analyzed using the
Thermosequenase Cycle Sequence kit (Amersham Pharmacia Biotech US78500).
Expression in E. coli--
The 919-base pair DNA containing
human GGPPSase cDNA was isolated from pT7 blue T vector after
double digestion with NcoI and BamHI, and the
fragment was ligated into the NcoI and BamHI sites of a bacterial expression vector pTrc 99A. E. coli
JM109 cells transformed with the vector alone or with the plasmid
containing human GGPPSase cDNA (pTrc-HGG) were grown in 50 ml of LB
medium to an A600 of 0.5. Isopropyl
1-thio- Purification of Bovine Brain GGPPSase and No Cross-reactivity of an
Anti-GGPPSase Antibody with FPPSase--
In a previous report (25) we
described a one-step purification of GGPPSase from bovine brain. The
GGPPSase fraction still contained several minor proteins. We tried to
improve the purification procedure with a combination of several
chromatographies including affinity chromatography. As a result,
Butyl-Toyopearl chromatography completely separated GGPPSase from
FPPSase and IPP isomerase. This enabled us to prepare the purified
GGPPSase with large amounts. Table I
shows the entire purification steps of GGPPSase and FPPSase. The former
enzyme (297 µg) and the latter enzyme (725 µg) were purified with
specific activities of 294 and 1,070 nmol/min/mg from 6.01 and 10.8 kg
of bovine brain, respectively. Fig.
1A shows SDS-PAGE of the
purified GGPPSase and FPPSase. The GGPPSase (35.0 kDa) migrated faster
than the FPPSase (37.5 kDa), suggesting that the entire polypeptide
chain length of GGPPSase is shorter than that of FPPSase unless a
post-translationary modification is present. A polyclonal antibody
directed against the GGPPSase did not recognize the FPPSase (Fig.
1B), and a polyclonal antibody directed against the FPPSase
did not recognize the GGPPSase (Fig. 1C). The Western blot
analysis of crude extracts did not show any positive protein bands with
the anti-GGPPSase antibody. Only affinity-purified GGPPSase was
recognized by the antibody, suggesting that the GGPPSase content in the
brain is low and that the current antibody is either of low affinity or
titer.
Cloning of GGPPSase cDNA--
Edman analysis of the purified
bovine brain GGPPSase suggested that the
NH2-terminal amino acid was blocked. Treatment
of the enzyme protein with lysyl endopeptidase gave partial amino acid
sequences of five fragments (fragments 2, 4, 9, 14, and 15). We
searched various cDNAs registered in GenBank and found 22 partial human cDNA fragments as sequence homologs to a Neurospora
crassa GGPPSase gene. Based on the sequence homology, we arranged
the human cDNA fragments as shown in Fig.
2. The amino acid sequences KTQETVQRILLEPY (fragment 14) and KHLSKMFK (fragments 4 and 9) identified in bovine brain GGPPSase were found in the deduced amino
acid sequence of these fragments. Based on the partial amino acid
sequences of the bovine brain GGPPSase described above, we synthesized
nucleotide primers (sense primer-2 and antisense primer-3).
PCR with primers-2 and -3 using human testis cDNA
(CLONTECH) as a template gave an 875-base product.
Sequence analysis revealed that this fragment contained the sequence of
the partial human cDNA fragments picked up in Fig. 2. We
synthesized sense primer-1 and antisense primer-4 containing a starting
codon (ATG) and a stop codon (TAA), respectively. As expected, PCR of
human testis cDNA with primers-1 and -4 gave a 919-base product
that covered an entire coding sequence of human GGPPSase.
On the basis of the similarity of the peptide sequence between
NH2-terminal and COOH-terminal human and bovine
GGPPSases, we performed a PCR with a combination of primers-1 and -4 using bovine liver cDNA as a template. Multiple PCR products
were obtained in this case. However, further amplification of the
multiple PCR products with a combination of the inner primers
(primers-2 and -3) gave an 875-base product on an agarose
electrophoresis. The deduced amino acid sequence of the 875-base
product contained all of the partial sequence (fragments 2, 4, 9, 14, and 15) determined by Edman degradation of purified bovine brain
GGPPSase, indicating that the cDNA (875 base pairs) encodes a
partial bovine brain GGPPSase.
Expression of human GGPPSase in E. coli--
To confirm that the
human 919-base PCR product codes GGPPSase, we tried to express the
protein and assay the enzyme activity. E. coli JM109 cells
were transformed with the expression plasmid pTrc 99A containing the
GGPPSase cDNA, and the cell extracts were subjected to SDS-PAGE. As
shown in Fig. 3A, a
polypeptide corresponding to 33 kDa was induced by the addition of
IPTG. Western blot analysis showed that this polypeptide was
specifically recognized by anti-bovine brain GGPPSase antibody (Fig.
3B). To determine whether the induced protein is active, the
bacterial cell extracts were used as the enzyme source in GGPPSase
assays. The enzymatic activity with a combination of FPP and
[1-14C]IPP was enhanced about 40-fold in the expressed
cells compared with the control cells as shown in Table
II. The reaction products comigrated not
only with the spot of geranylgeranyl monophosphate but also with that
of GGPP on normal phase silica gel TLC (not shown). However, the
hydrolysates by acid phosphatase treatment of the products moved with
the spot of all-trans-geranylgeraniol on reverse phase
C18 TLC (Fig. 3C) and normal phase TLC (not
shown). These results indicate that the isolated cDNA codes an
active GGPPSase protein. The expressed human GGPPSase protein was
purified by Butyl-Toyopearl and Mono Q column chromatographies and by
Superdex 200 gel filtration. The enzyme was purified as a single peak
with a molecular mass of 280 kDa (Fig. 3D) on the gel
filtration. The purified GGPPSase catalyzed the formation of GGPP, and
the allylic substrate specificity for the enzyme is shown in Fig.
3E. Table III shows
quantitative analysis of the enzymatic products. These results
confirmed the identify of the expected 900-base cDNA as encoding
human GGPPSase and the similar properties of human GGPPSase to bovine
GGPPSase in the substrate specificity as characterized previously (25).
It should be emphasized that the human GGPPSase also behaves as an
oligomer composed of eight subunits of identical size (33 kDa) similar
to bovine brain GGPPSase. Fig.
4A shows the cDNA sequence
and the deduced amino acid sequence of human testis GGPPSase. The amino
acid sequence predicted from the GGPPSase gene corresponds to a
polypeptide chain with molecular mass of 34,870 Da. The human GGPPSase
only showed 16% sequence identity with the known human FPPSase (Fig.
4B) but 49.2% identity in the consensus sequences (I, II,
III, IV, and V) proposed for trans-prenyltransferase. The
total homology between the nucleotide sequences was 27.8%. The
GGPPSase had three potential N-glycosylation sites.
Multiple Transcripts Hybridized to a GGPPSase cDNA
Probe--
Northern blotting analyses with 32P-labeled
cDNA probes encoding the human testis GGPPSase revealed two sizes
of mRNAs (about 1.5 and 3.1 kilobases) in various tissues (Fig.
5). These mRNAs were abundant in
heart, skeletal muscle, and testis, with the shorter mRNA being the
major species in these tissues. The amount of GGPPSase mRNA in the
brain was lower than expected given the degree of enrichment of
geranylgeranylated proteins in this organ (11).
The original aim of our study was to learn how FPPSase and
GGPPSase are different from each other because these enzymes catalyze a
common isoprenyl-transferring reaction. Polyclonal antibodies directed
against the two enzymes of bovine brain did not show any
cross-reactivity with each other (Fig. 1). The immunochemical difference between these two proteins was confirmed with amino acid
sequence comparison between the GGPPSase determined in this study and
the known human FPPSase (30) (Fig. 4B). Five conserved amino
acid motifs (I, II, III, IV, and, V) common to
trans-prenyltransferases reported (18) were observed between
the two enzymes, but the entire identity was only 16%. The human
GGPPSase has three potential N-glycosylation sites, whereas
human FPPSase has no such sites (30). Any potential
N-glycosylation site is also not observed in the amino acid
sequences of FPPSase of rat (19). It is not clear at present whether
the GGPPSase is N-glycosylated. Human GGPPSase shows
extremely high homology to other GGPPSases of bovine, Drosophila, and N. crassa (Fig.
6). Three potential
N-glycosylation sites observed in human GGPPSase are also
found in the bovine enzyme, and two of them are conserved in the insect
enzyme, although two Asn-X-Ser/Thr motifs are found at
different sites in the N. crassa enzyme.
INTRODUCTION
Top
Abstract
Introduction
References
, another orphan nuclear receptor, they have also
demonstrated that the DNA binding activity of LXR
·RXR complex is
inhibited by GGPP (15). These reports imply that not only FPP but also
GGPP may contribute to signaling pathways in addition to protein prenylation.
C20 and was composed of four
or five subunits of identical size.
EXPERIMENTAL PROCEDURES
-32P]dCTP (6,000 Ci/mmol) were purchased from
Amersham Pharmacia Biotech. [1-3H] GGPP (20 Ci/mmol),
and [1-3H]geranylgeranyl monophosphate (15 Ci/mmol) were
purchased from American Radiolabeled Chemicals Inc. Ex-Taq
DNA polymerase was obtained from Takara Shuzou.
Z,E,E-Geranylgeraniol was given by the Kuraray Corp. IPP,
geranyl diphosphate (GPP), E, E-FPP, and E,E,E-GGPP were prepared as described in a previous report
(26). Potato germ acid phosphatase (type I) was obtained from Sigma. All other chemicals were of reagent grade. Butyl-Toyopearl was obtained
from Toyo Soda Co. Mono Q and Superdex 200 HR 10/30 were obtained from
Amersham Pharmacia Biotech. Affinity gels with a geranylmethyl
phosphonophosphate ligand and with a farnesylmethyl phosphonophosphate
ligand were prepared by the method of Bartlett et al. (27).
Bovine brain was obtained from a local slaughterhouse. Goat anti-guinea
pig IgG heavy and light chain (alkaline phosphatase conjugate) were
obtained from Bethyl Laboratories Inc. Guinea pig polyclonal antibodies
were obtained by directing against purified bovine brain GGPPSase and
FPPSase.
-D-galactopyranoside (IPTG) was added to a final
concentration of 0.4 mM, and after 2.5 h culture the
cells were collected and suspended in 2.0 ml of the TE buffer (pH 8.0).
The cell suspensions were sonicated with a Branson sonifier before
centrifugation at 14,000 × g to remove unbroken cells.
The supernatants were frozen in aliquots for protein determination,
SDS-PAGE, and GGPPSase assays.
RESULTS
Purification of GGPPSase and FPPSase of bovine brain
1 at 37 °C.
FPP and [1-14C]IPP were used as substrates for GGPPSase. GPP
and [1-14C]IPP were used as substrates for FPPSase.
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Fig. 1.
SDS-PAGE of FPPSase and GGPPSase from bovine
brain. Panel A, after electrophoresis, the gel was
stained with Coomassie Brilliant Blue. Lane 1, FPPSase;
lane 2, GGPPSase; lane 3, mixture of FPPSase and
GGPPSase. Panels B and C, after electrophoresis,
FPPSase (lanes 1 and 3) and GGPPSase (lanes
2 and 4) were transblotted to a nitrocellulose membrane
and detected with anti-GGPPSase antibody (panel B) and
anti-FPPSase antibody (panel C).
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Fig. 2.
Strategy for cloning human and bovine
GGPPSase cDNA. Partial cDNA sequences similar to N. crassa GGPPSase registered in GenBank were arranged from the
5'-end in each tissue. A single line was arranged in the middle,
considering the high frequency of the common sequence (closed
bar). The left and right continuous DNA
sequences contain stop codons TGA and TAA, respectively. Immediately
below the two nucleotide sequences are the deduced amino acid
sequences. The actual amino acid sequences (fragments 14, 4, and 9)
determined by Edman degradation of purified bovine brain GGPPSase are
underlined. Combinations of primers-1 (sense) and -4 (antisense) and of primers-2 (sense) and -3 (antisense) were used for
PCRs.
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Fig. 3.
Characterization of human GGPPSase expressed
in E. coli. Panel A, SDS-PAGE analysis
of human GGPPSase in E. coli. The cell extracts were
electrophoresed on SDS-polyacrylamide gels. Lane 1, E. coli pTrc 99A; lane 2, E. coli pTrc-HGG
( IPTG); and lane 3, E. coli pTrc-HGG (+IPTG).
Molecular mass standards are shown on the left.
Panel B, Western blot analysis of human GGPPSase
production in E. coli. Proteins on electrophoresis similar
to panel A were transferred to a nitrocellulose
membrane and probed with an antibody directed against purified bovine
brain GGPPSase. Molecular mass standards are shown on the
left. Lanes 1, 2, and 3 are the same
as indicated in the legend to panel A. Panel
C, reverse phase C18 TLC radioautograms of the
hydrolysates obtained by phosphatase treatment of the products derived
from FPP and [1-14C]IPP with crude extracts of E. coli pTrc 99A (lane 1), E. coli pTrc-HGG
(
IPTG) (lane 2), or E. coli pTrc-HGG (+IPTG)
(lane 3). The samples were developed in acetone:water (7:1).
GGOH, geranylgeraniol (C20); FOH,
farnesol (C15); Ori., origin; and
S.F., solvent front. Panel D, gel
filtration of human GGPPSase. The closed and open
circles indicate the absorption at 280 nm and the enzyme activity
with FPP and [1-14C]IPP, respectively. The
inset shows SDS-PAGE stained with Coomassie Brilliant Blue
of the active fractions. Molecular mass standards: a,
ferritin 440,000; b, catalase 232,000; c, bovine
serum albumin 67,000. Panel E, thin layer radiochromatograms
of prenyl products with purified human GGPPSase. Enzymatic products
were developed in isopropyl alcohol:ammonia:water (6:3:1). Lane
1, [1-14C]IPP alone; lane 2,
dimethylallyl diphosphate + [1-14C]IPP; lane
3, GPP + [1-14C]IPP; lane 4, FPP + [1-14C]IPP.
GGPPSase activity in E. coli extracts
) of FPP.
Distribution of enzymatic products
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Fig. 4.
Primary structure of human GGPPSase.
Panel A, cDNA and deduced amino acid
sequences of human GGPPSase. The stop codon is TAA (*). The potential
N-glycosylation sites are underlined.
Panel B, comparison between human GGPPSase and
FPPSase (P14324). Five consensus sequences (I, II, III, IV, and V)
common to trans-prenyltransferase are underlined.
The common amino acids between the two enzymes are boxed.
Asterisks indicate the potential N-glycosylation
sites in the GGPPSase sequence.
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Fig. 5.
Tissue distribution of mRNA for human
GGPPSase. Northern blots (CLONTECH) containing
approximately 2 µg of poly(A)+ RNA/lane from 16 different
human tissues were used. Hybridization was done at 68 °C for 1 h with a 32P-labeled cDNA probe (2 × 106 cpm/ml) for human GGPPSase. Filters were washed three
times in 2 × SSC containing 0.05% SDS at room temperature for 10 min and twice in 0.1 × SSC containing 0.1% SDS at 50 °C for
20 min and exposed to an imaging plate overnight at room temperature.
As a loading control, the same filters were then reprobed with random
primed 32P-labeled oligonucleotides of human -actin
cDNA (2 × 106 cpm/ml). The reprobed filters were
exposed for 4 h at room temperature (lower panel). Size
markers are indicated by the arrows. Muscle,
skeletal muscle; Intestine, small intestine;
Leukocyte, peripheral blood leukocyte. kb,
kilobases.
DISCUSSION
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Fig. 6.
Comparison of the deduced amino acid sequence
of the human GGPPSase with several other GGPPSases.
Human, human GGPPSase; Bovine, a partial sequence
of bovine brain GGPPSase (T. Kuzuguchi, and H. Sagami,
unpublished results); Drosophila, Drosophila
melanogaster GGPPSase (AF049659);
Neurospora, N. crassa GGPPSase
(P24322). Consensus sequences shown below the five highly
conserved sequence domains (I-V) proposed for
trans-prenyltransferase by Chen et al. (18) are
underlined. The numbers of amino acids are indicated on the
left. Boxes enclose identical amino acids in at
least three of the four sequences compared. Asterisks
indicate the potential N-glycosylation sites in the human
GGPPSase sequence.
Sheares et al. (20) reported that the expression of human FPPSase in Hep G2 cells is transcriptionally regulated: lovastatin, a potent inhibitor of HMG-CoA reductase, increased the level of the mRNA, whereas cholesterol in its 25-hydroxylated form or in low density lipoprotein particles reduced the amount of the mRNA, and mevalonate also decreased the mRNA levels. Similar regulation of HMG-CoA synthase, HMG-CoA reductase, and low density lipoprotein receptor had been already reported (16), suggesting a common control mechanism for these enzymes. Recently, Guan et al. (31) described differential, transcriptional regulation of the human gene encoding squalene synthase, which accepts FPP as a substrate, by variation in the level of cellular cholesterol. Concerning GGPPSase, which also accepts FPP as a substrate, the study on regulation is far behind those of the enzymes described above. Lutz et al. (32) have demonstrated that GGPP synthesis is specifically inhibited by GGPP, suggesting that the cellular GGPP pool may be regulated by product inhibition of GGPPSase. Our in vitro experiments using a purified bovine brain GGPPSase supported this regulation (25).
GGPP acts as a precursor for the C20 lipid moiety in
prenylated proteins and also as a modulator for nuclear hormone
receptor (LXR·RXR) complex formation. In the present study two
mRNAs of GGPPSase were identified. Arrangement of several partial
cDNA sequences registered in GenBank (Fig. 2) further implies the
presence of more than two GGPPSase mRNAs. Therefore, the GGPP
synthesis might be regulated at the transcriptional level of GGPPSase.
Rilling et al. (11) have reported the content of
geranylgeranylated proteins in mouse tissues; these proteins are
severalfold rich in brain compared with those in liver, kidney, and
lung. In human tissues, two GGPPSase mRNAs were also observed in
those tissues. Although they did not describe the content of
geranylgeranylated proteins in mouse heart, skeletal muscle, and
testis, much higher contents of human GGPPSase mRNAs were detected
in these tissues. Two kinds of protein geranylgeranyl transferase
responsible for the synthesis geranylgeranylated proteins have been
found and characterized in mammals. On Northern blot analysis for rat
protein geranylgeranyl transferase II consisting of
and
subunits (component B) and an escort protein (component A) (33, 34),
the
and
subunits mRNAs are abundant in heart and much lower
muscle and testis. The escort protein mRNA is also abundant in
heart, much lower in muscle, and not detectable in testis. In the case
of the
subunit of rat protein geranylgeranyl transferase I (35), which is the same protein as that of protein farnesyl transferase, the
amount of the mRNA is rather low in heart and muscle, whereas the
amount in testis is severalfold higher than in any other tissue. It
would be difficult at present to explain the reason why the mRNA
levels of these enzymes directly related to GGPP metabolism are
regulated differently especially in these tissues.
It is very important to establish how many genes encode GGPPSase, which
gene is transcripted in each tissue, and how each gene is regulated.
FPPSase has been reported to be encoded by multiple genes (19, 20).
Further analysis of mammalian GGPPSase will contribute to the
understanding of a finely tuned mechanism of mevalonate pathway.
Sepp-Lorenzino et al. (36) reported cell cycle-dependent differential prenylation of proteins.
LXR has been reported to display constitutive transcriptional
activity that is negatively regulated with GGPP (15). It remains
unclear how GGPP inhibits LXR
and contributes to signaling pathways. It would be also very interesting to know the regulation of GGPP formation in the process of embryonic development and cell differentiation.
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FOOTNOTES |
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* This research was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.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) AB017971.
To whom correspondence should be addressed. Tel.: 81-22-217-5622;
Fax: 81-22-217-5620; E-mail: yasagami{at}icrs.tohoku.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are:
FPP, farnesyl
diphosphate;
GGPP, geranylgeranyl diphosphate;
HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A;
FPPSase, FPP synthase;
IPP, isopentenyl diphosphate;
GGPPSase, GGPP synthase;
GPP, geranyl
diphosphate;
BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol;
PIPES, 1,4-piperazinediethanesulfonic acid;
TLC, thin layer
chromatography;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
IPTG, isopropyl
1-thio--D-galactopyranoside.
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REFERENCES |
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