(Received for publication, December 26, 1995; and in revised form, February 1, 1996)
From the
The importance of lowering serum cholesterol levels for the prevention of cardiovascular disease has been well documented. Because mevalonate pyrophosphate decarboxylase is a unique enzyme in the cholesterol biosynthetic pathway it is a potential therapeutic target for the treatment of hypercholesterolemia and other diseases. For this reason we cloned and expressed the cDNA for the human enzyme. We also cloned and expressed the yeast homolog using the human enzyme's similarity to a previously unidentified and incomplete genomic sequence. Northern blot analysis revealed a transcript of approximately 2 kilobases in a variety of human tissues. The recombinant human enzyme is a homodimer of 43-kDa subunits with a specific activity of 2.4 units/mg. Computer searches for similarity with known sequences showed that mevalonate pyrophosphate decarboxylase has little similarity to other proteins.
The mevalonate pyrophosphate decarboxylase (MPD) ()enzyme of the cholesterol/ergosterol biosynthetic pathway
converts the 6-carbon compound mevalonate pyrophosphate (MevPP) into
isopentenyl pyrophosphate, the 5-carbon isoprene building block of a
large family of biomolecules. Besides decarboxylation, this unusual
enzyme dehydrates its substrate while hydrolyzing ATP. MPD, which
performs the first committed step in the biosynthesis of isoprenes, has
been purified from several animal and plant sources, but a
comprehensive examination of the enzyme or its mechanism is
lacking(1, 2) .
The prodrug, 6-fluoromevalonate, has been shown to lower cholesterol biosynthesis(3, 4) , block protein prenylation(5, 6) , and inhibit the proliferation of various transformed cells(7, 8) . Upon pyrophosphorylation with endogenous cellular enzymes this compound becomes a potent competitive inhibitor of MPD(3, 4) . For these reasons MPD appears to be a potential pharmaceutical target for the treatment of hypercholesterolemia and unwanted cellular proliferation. In order to better study this enzyme we cloned and expressed the cDNA for MPD from human liver and, by sequence similarity, from yeast.
A DNA fragment from the incomplete rat liver cDNA clone
described above was then used to probe what was advertised as a human
liver cDNA library (Stratagene 937220). We isolated and sequenced
11 positive clones. The largest clone was designated 18-1, and it
encoded an estimated 90% of the ORF. This estimate was based on a
molecular mass of 45 kDa for the rat protein (9) and on the
absence of a methionine start codon.
Subsequent to our
demonstration of MPD activity with human liver cDNA derived from
another source (see below), we were informed by Stratagene that the
advertised human liver cDNA library was actually made from a non-human
tissue (possibly Chinese hamster ovary cells). This accident did not
alter our final results because we had used human liver RNA to isolate
the full-length cDNA sequence shown in Fig. 1(see below).
Figure 1: Nucleotide sequence of human liver mevalonate pyrophosphate decarboxylase cDNA and its deduced amino acid sequence. ORF region is shown in capital letters.
Primers for the 5`-RACE experiments were GSP1 (gene-specific primer 1, GGCCACATGCACCTTATAGC) and GSP2 (CTGTGAAGTCCTTGCTAATGG) and were purchased from Biosynthesis Inc. A 5`-RACE kit(18374-025) was purchased from Life Technologies, Inc. Total RNA was isolated from a 1-g sample of frozen human liver using a Life Technologies, Inc. total RNA isolation kit. A sample of human liver mRNA was also purchased from Clontech. Forty cycles of standard PCR conditions (94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2 min) were used to produce a 230-base pair (bp) band, which was subcloned into pCRII (Invitrogen) and transformed into Escherichia coli. Several independent subclones from the two different RNA sources were isolated and sequenced.
Primers for the 5`-RACE experiments were designed with
the DNA sequence of the 18-1 clone (see above). At that time we did not
know that 18-1 was derived from a non-human tissue. In spite of the
coding differences due to the nonhuman sequence of the primers, ()we obtained a 5`-end for the human liver MPD cDNA
(nucleotides 1-170 of Fig. 1).
Using a unique DNA
sequence obtained from the 5`-RACE experiments and a sample of human
liver total RNA, we performed a 3`-RACE experiment and isolated the
3`-end of the cDNA (nucleotides 151-1812 of Fig. 1). The primers
used for the 3`-RACE experiment were GSP1 (TGGTTCTGCCCATCAACTC) and
GSP2 (ACTCTGCACCAGGACCAGTT). A 3`-RACE kit was purchased from Life
Technologies, Inc. Total RNA prepared in the 5`-RACE experiment was
utilized. PCR conditions were altered using the PCR Optimizer kit
(Invitrogen). Conditions were: 30 mM Tris-HCl, pH 9.0, 7.5
mM (NH)SO
, 1.3 mM MgCl
, 10% Me
SO; 30 cycles of 94 °C for
1 min, 42 °C for 1 min, and 72 °C for 2 min. A 1.7-kbp band was
isolated and subcloned, and several transformants were sequenced.
The PCR primers used to modify the 1.8-kbp full-length cDNA fragment for subcloning into the expression vectors were 5`-BamNde (ACGGGATCCATATGGCCTCGGAG) and the 3` primer of the full-length amplification. We used the 1.8-kbp DNA fragment from the full-length amplification and 20 standard PCR cycles. The resulting amplified DNA was first subcloned into pCRII and then into pET-14b (Novagen) using NdeI and XhoI or into pVL1392 (Invitrogen) using EcoRI.
The original subclone from the full-length PCR
reaction contained five mutations compared with the consensus sequence
of Fig. 1. Three of these mutations were coding changes
(AT gave Thr
Ser, G
C
gave Glu
Asp, and T
A gave
Cys
Ser) and two were noncoding (T
G
gave AGT to AGG at Ser
, and a
g in the
3`-noncoding region). We used the Sculptor in vitro mutagenesis kit (Amersham Corp.) and three mutagenic
oligonucleotides (at one time) to change the coding sequence of the
original full-length subclone to the consensus sequence shown in Fig. 1. The activity of human MPD shown in Table 1comes
from this corrected subclone.
Cell pellets from both expression systems were sonicated in
homogenization buffer (20 mM Tris/HCl, pH 7.5, at 20 °C,
15 mM EDTA, 15 mM EGTA, 100 µM leupeptin, 0.75 mg/liter aprotinin, 0.1 mM phenylmethanesulfonyl fluoride) and assayed for enzyme activity
and protein concentration. Activity was measured as the conversion of
labeled MevPP into isopentenyl pyrophosphate as adapted from Cardemil
and Jabalquinto(10) . The assay solution contained 50 mM BisTris/HCl, pH 7.0, at 20 °C, 1 mM dithiothreitol,
10 mM MgCl, 5 mM ATP, and 3.8 µM [3-
C]mevalonate pyrophosphate (DuPont NEN,
54 mCi/mmol). Assays were conducted at 37 °C for 1 h. To stop the
reaction and to convert the substrates/products, 2 mg of alkaline
phosphatase (Sigma P-3877) dissolved in 25 µl of 1 M Tris
was introduced, and the sample was incubated for 30 more min. Two ml of
Econofluor-2 (DuPont NEN) were then added, and the sample was capped,
shaken, and counted on a scintillation counter. Because of the organic
nature of the scintillation fluid, the dephosphorylated product
(isopentenyl) is solubilized and counted whereas the dephosphorylated
substrate (mevalonic acid) is not. A ``no enzyme'' sample was
used to determine the background. Protein concentrations were
determined using the dye-binding method (Bio-Rad).
Figure 3: Comparison between the deduced human liver and yeast MPD protein sequences. Identical regions are boxed.
The cDNA for human liver MPD was identified by using DNA
sequence information originally obtained from a partial cDNA
clone of rat liver MPD (see ``Experimental Procedures'') and
from RACE experiments. We used the sequence information obtained in 5`-
and 3`-RACE experiments to amplify a 1.8-kbp full-length cDNA directly
from a sample of human liver total RNA. We sequenced three independent
clones from two different full-length PCR reactions and obtained a
consensus. Fig. 1shows this consensus cDNA sequence with a
representative poly(A) tail attached at the 3`-end. Large stretches of
sequence identity with several expression sequence tag clones (EST)
further substantiated the MPD cDNA sequence (see below). Inspection of Fig. 1suggests that transcription starts at the methionine codon
at bp 8 within a Kozak box sequence and continues for 400 amino acids
producing a 43-kDa protein. A large 3`-untranslated region is also
evident.
Fig. 2shows a Northern blot of various human tissues probed with the human MPD cDNA sequence. A transcript of approximately 2 kilobases in size was observed for liver, skeletal muscle, heart, brain, placenta, lung, kidney, and pancreatic tissues.
Figure 2: Northern blot of various human tissues probed with human MPD cDNA. Size standards are along the left margin. See text for details.
Because the clones from the full-length amplification had coding mutations compared with Fig. 1, one clone was modified and mutated so as to encode the consensus ORF (see ``Experimental Procedures''). The resulting DNA fragment was subcloned into E. coli and baculovirus expression vectors. Shown in Table 1are the MPD activity levels of extracts made from cells with these expression systems. In the E. coli system the cells containing human liver MPD cDNA showed substantial enzyme activity whereas the control cells had no detectable activity. Because E. coli does not inherently possess MPD activity (12) its presence indicates that we have cloned and expressed the human enzyme. The baculovirus/insect cell system showed a >50-fold increase in activity compared with the background level of uninfected cells.
To
find if there was any similarity between MPD and any reported sequence
we searched the publicly available data banks with the BLASTN, BLASTP,
BLASTX, and TBLASTN programs(13) . Only two classes of
similarity emerged. One class included over 20 EST clones while the
other class was an unidentified and incomplete ORF adjacent to the COQ2 gene of Saccharomyces cerevisiae(11) .
From the level of similarity it appeared that the unidentified ORF was
the yeast MPD gene. Based on the desirability of obtaining the yeast
homolog of MPD, we decided to clone and to express this sequence.
Consequently, we screened a yeast cDNA library with the unidentified
ORF and obtained three overlapping clones. We subcloned the yeast cDNA
sequence into the same E. coli expression system that was used
for the human enzyme and found MPD activity as shown in Table 1.
In this expression system the yeast MPD clone yielded more enzyme
activity in the crude extract than the human clone. The reason for this
difference may be related to the presence of insoluble MPD protein only
with the human clone.
Fig. 3compares the predicted peptide sequences for both MPD enzymes, and it shows a 45% level of identity between the two homologs. Because the neighboring COQ2 gene encodes para-hydroxybenzoate:polyprenyltransferase, it appears that this region of the yeast genome is involved with isoprene metabolism.
Significant DNA sequence identity to human liver MPD was also found
with 20 EST clones derived from human tissues such as brain, spleen,
and liver. We obtained and sequenced one of the EST clones
(ATCC 85596) that was derived from human fetal brain tissue and found
it to be nearly identical to Fig. 1. Besides liver, brain tissue
is another rich source of cholesterol biosynthesis. Apparently the
human MPD cDNAs from infant brain and adult liver are identical.
We also found similarities with EST clones derived from Arabidopsis thaliana, Oryza sativa, and Caenorhabditis elegans. Though only partial sequence information is available, these EST clones would appear to be partial MPD cDNA sequences from those organisms.
We performed a scan for pattern recognition elements in the human MPD protein sequence using the PROSITE program from the EMBL-Heidelberg (14) . No patterns were observed out of the 1011 tested. Combining the results of the BLAST and PROSITE searches, it appears that MPD is unique with little similarity to other protein sequences or to other non-MPD cDNA sequences.
We purified
human MPD from the baculovirus/insect cell expression system to 50%
purity as judged by SDS-polyacrylamide gel electrophoresis. We measured
a specific activity of 2.4 units/mg and determined the apparent K values for MEVPP and ATP to be 2 and 600
µM, respectively. Further analysis using gel filtration
chromatography revealed that the inclusion of 1 mM dithiothreitol was necessary to retain enzyme activity during
elution. Omission of this reducing agent caused a loss of activity and
a reduction in apparent molecular mass of the protein from 100 to 50
kDa. We interpret this to mean that active recombinant human MPD is a
homodimer of 43-kDa subunits. These parameters are similar to those
reported for MPD isolated from chicken liver(10) , pig liver (15) , and rat liver(9) .
In summary, we have cloned and expressed the cDNAs for human liver and yeast MPD. Because of the relevance of MPD to cholesterol metabolism, studies are under way to observe if inhibitors of this enzyme will alter serum lipid levels. Because the prodrug 6-fluoromevalonate inhibits MPD activity after it becomes pyrophosphorylated and subsequently blocks the proliferation of Ras-transformed cells(7, 8) , we also consider this enzyme to be a target for diseases such as cancer and restenosis. The production of recombinant MPD may allow a more complete picture of the mechanism and the structure of this remarkable enzyme. Furthermore, the cloning of yeast MPD should extend our understanding of sterol biosynthesis in this well studied eukaryote.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U49260 [GenBank]and U49261[GenBank].