FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1993573607> ?p ?o ?g. }

• W1993573607 endingPage "5894" @default.
• W1993573607 startingPage "5888" @default.
• W1993573607 abstract "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 inEscherichia 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. 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 inEscherichia 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. farnesyl diphosphate geranylgeranyl diphosphate 3-hydroxy-3-methylglutaryl coenzyme A FPP synthase isopentenyl diphosphate GGPP synthase geranyl diphosphate 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol 1,4-piperazinediethanesulfonic acid thin layer chromatography polyacrylamide gel electrophoresis polymerase chain reaction isopropyl 1-thio-β-d-galactopyranoside Since Schmidt et al. (1Schmidt R.A. Schneider C.J. Glomset J.A. J. Biol. Chem. 1984; 259: 10175-10180Abstract Full Text PDF PubMed Google Scholar) 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 (2Clarke S. Annu. Rev. Biochem. 1992; 61: 355-386Crossref PubMed Scopus (785) Google Scholar, 3Stimmel J.B. Deschenes R.J. Volker C. Stock J. Clarke S. Biochemistry. 1990; 29: 9651-9659Crossref PubMed Scopus (46) Google Scholar, 4Maltese W.A. FASEB J. 1990; 4: 3319-3328Crossref PubMed Scopus (429) Google Scholar, 5Rine J. Kim S.-H. New Biol. 1990; 2: 219-226PubMed Google Scholar, 6Takai Y. Kaibuchi K. Kikuchi A. Kawata M. Int. Rev. Cytol. 1992; 133: 187-230Crossref PubMed Scopus (254) Google Scholar). The prenylation of these proteins is essential for their function in the cells (7Itoh T. Kaibuchi K. Masuda T. Yamamoto T. Matsuura Y. Maeda A. Shimizu K. Takai Y. J. Biol. Chem. 1993; 268: 3025-3028Abstract Full Text PDF PubMed Google Scholar, 8Horiuchi H. Kaibuchi K. Kawamura M. Masuura Y. Suzuki N. Kuroda Y. Kataoka T. Takai Y. Mol. Cell. Biol. 1992; 12: 4515-4520Crossref PubMed Scopus (25) Google Scholar, 9Kuroda Y. Suzuki N. Kataoka T. Science. 1993; 259: 683-686Crossref PubMed Scopus (119) Google Scholar), 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 (10Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1751) Google Scholar). Further, the number of geranylgeranylated proteins has been shown to be larger than that of farnesylated proteins (11Rilling H.C. Bruenger E. Epstein W.W. Crain P.F. Science. 1990; 247: 318-320Crossref PubMed Scopus (112) Google Scholar, 12Farnsworth C.C. Gelb M.H. Glomset J.A. Science. 1990; 247: 320-322Crossref PubMed Scopus (162) Google Scholar, 13Epstein W.W. Lever D. Leining L.M. Bruenger E. Rilling H.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9668-9670Crossref PubMed Scopus (120) Google Scholar). Recently, these shorter chain prenyl diphosphates also have been shown to be involved in the function of nuclear hormone receptors. Forman et al. (14Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Noonan D.J. Burka L.T. McMorris T. Lamph W.W. Evans R.M. Weinberger C. Cell. 1995; 81: 687-693Abstract Full Text PDF PubMed Scopus (937) Google Scholar) 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 LXRα, another orphan nuclear receptor, they have also demonstrated that the DNA binding activity of LXRα·RXR complex is inhibited by GGPP (15Forman B.M. Ruan B. Chen J. Schroepfer Jr., G.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10588-10593Crossref PubMed Scopus (248) Google Scholar). These reports imply that not only FPP but also GGPP may contribute to signaling pathways in addition to protein prenylation. 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 (16Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4478) Google Scholar). 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 (17Ding V.D. Sheares B.T. Bergstrom J.D. Ponpipom M.M. Perez L.B. Poulter C.D. Biochem. J. 1991; 275: 61-65Crossref PubMed Scopus (30) Google Scholar), and several FPPSase cDNAs have already been cloned in animals such as chicken, rat, and human (18Chen A. Kroon P.A. Poulter C.D. Protein Sci. 1994; 3: 600-607Crossref PubMed Scopus (218) Google Scholar). Genomic Southern blot analyses suggested that rat and human have multiple genes encoding divergent FPPSases (19Clarke C.F. Tanaka R.D. Svenson K. Wamsley M. Fogelman A.M. Edwards P.A. Mol. Cell. Biol. 1987; 7: 3138-3146Crossref PubMed Scopus (92) Google Scholar, 20Sheares B.T. White S.S. Molowa D.T. Chan K. Ding V.D.-H. Kroon P.A. Bostedor R.G. Karkas J.D. Biochemistry. 1989; 28: 8129-8135Crossref PubMed Scopus (48) Google Scholar). 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) (21Reed B.C. Rilling H.C. Biochemistry. 1975; 14: 50-54Crossref PubMed Scopus (102) Google Scholar). Thereafter, we succeeded in isolating GGPP synthase (GGPPSase) in the different fraction from FPPSase by ion exchange chromatography of pig liver (22Sagami H. Ishii K. Ogura K. Biochem. Int. 1981; 3: 669-675Google Scholar), rat liver (23Sagami H. Korenaga T. Ogura K. Steiger A. Pyun H.-J. Coates R.M. Arch. Biochem. Biophys. 1992; 297: 314-320Crossref PubMed Scopus (24) Google Scholar), and bovine brain (24Sagami H. Korenaga T. Ogura K. J. Biochem. (Tokyo). 1993; 114: 118-121Crossref PubMed Scopus (15) Google Scholar) cytosols. The GGPPSase has been purified from bovine brain in a homogeneous form (25Sagami H. Morita Y. Ogura K. J. Biol. Chem. 1994; 269: 20561-20566Abstract Full Text PDF PubMed Google Scholar) using a one-step procedure by affinity chromatography which contained an FPP analog as a ligand. The purified GGPPSase catalyzed a single reaction C15 → C20 and was composed of four or five subunits of identical size. 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 (13Epstein W.W. Lever D. Leining L.M. Bruenger E. Rilling H.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9668-9670Crossref PubMed Scopus (120) Google Scholar). 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. [1-14C]IPP (55 mCi/mmol) and [α-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-TaqDNA polymerase was obtained from Takara Shuzou.Z,E,E-Geranylgeraniol was given by the Kuraray Corp. IPP, geranyl diphosphate (GPP), E, E-FPP, andE,E,E-GGPP were prepared as described in a previous report (26Davisson V.J. Woodside A.B. Poulter C.D. Methods Enzymol. 1985; 110: 130-144Crossref PubMed Scopus (226) Google Scholar). 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. (27Bartlett D.L. King C.-H.R. Poulter C.D. Methods Enzymol. 1985; 110: 171-184Crossref PubMed Scopus (16) Google Scholar). 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. Bovine brain was homogenized with a Polytron homogenizer in 50 mm Tris-HCl buffer (pH 7.5) containing 0.1 mm leupeptin, 0.2 mmphenylmethanesulfonyl 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 mmMgCl2. 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 mNaCl. 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 mm2-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. 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 mmMgCl2, 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. (28Fujii H. Koyama T. Ogura K. Biochim. Biophys. Acta. 1982; 712: 716-718Crossref PubMed Scopus (156) Google Scholar). 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 (29Sagami H. Kurisaki A. Ogura K. Chojnacki T. J. Lipid Res. 1992; 33: 1857-1861Abstract Full Text PDF PubMed Google Scholar). 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. 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. 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). 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). 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. coliJM109 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 A 600 of 0.5. Isopropyl 1-thio-β-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. In a previous report (25Sagami H. Morita Y. Ogura K. J. Biol. Chem. 1994; 269: 20561-20566Abstract Full Text PDF PubMed Google Scholar) 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 Ishows 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.1 A 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.1 B), and a polyclonal antibody directed against the FPPSase did not recognize the GGPPSase (Fig. 1 C). 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.Table IPurification of GGPPSase and FPPSase of bovine brainStepsUnitsProteinSpecific activityYieldPurificationnmol/minmgnmol/min/mg%foldGGPPase40–60% (NH4)2SO43,63024,2000.151001Butyl Toyopearl4122701.5311.410F-affinityaF-affinity, farnesylmethyl phosphonophosphate affinity chromatography.1591.381154.4767Mono Q87.30.2972942.41,960FPPSase30–40% (NH4)2SO42,43015,2000.161001G-affinitybG-affinity, geranylmethyl phosphonophosphate affinity chromatography.6241.8334125.72,130Mono Q7750.7251,07031.96,690GGPPSase and FPPSase were purified starting from 6.01 kg and 10.8 kg of bovine brain, respectively. One unit was defined as the activity required to synthesize 1 nmol of product min−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.a F-affinity, farnesylmethyl phosphonophosphate affinity chromatography.b G-affinity, geranylmethyl phosphonophosphate affinity chromatography. Open table in a new tab GGPPSase and FPPSase were purified starting from 6.01 kg and 10.8 kg of bovine brain, respectively. One unit was defined as the activity required to synthesize 1 nmol of product min−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. 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. 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. 3 A, 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.3 B). 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 TableII. 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. 3 C) 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. 3 D) on the gel filtration. The purified GGPPSase catalyzed the formation of GGPP, and the allylic substrate specificity for the enzyme is shown in Fig.3 E. 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 (25Sagami H. Morita Y. Ogura K. J. Biol. Chem. 1994; 269: 20561-20566Abstract Full Text PDF PubMed Google Scholar). 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.4 A 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.4 B) 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.Table IIGGPPSase activity in E. coli extractsPlasmidIPTGFPPSpecific activityRelative specific activitynmol/min/mgpTrc 99A++0.1891.00pTrc-HGG++8.34544.15pTrc-HGG−+0.3401.80pTrc-HGG+−0.0360.19GGPPSase activities were measured in extracts from cells (JM109) transformed with plasmids as indicated. pTrc-HGG shows a plasmid containing human GGPPSase cDNA in the pTrc 99A vector. Assays were carried out in the presence (+) and absence (−) of FPP. Open table in a new tab Table IIIDistribution of enzymatic productsAllylic substrateGPPProducts FPPaβ-Ray intensity (PSL*) of the radioactivity corresponding to GPP (C10), FPP (C15), and GGPP (C20) on the normal-phase plate of Fig. 3E, was measured with a Fuji BAS 1000 bioimage analyzer.GGPPDMAPPbDMAPP, dimethylallyl diphosphate. + [14C]IPP0* (0.00)cThe figures in parentheses indicate the relative velocities for the C5→C20, C10→C20, and C15→20 steps of the reaction calculated on the assumption that the specific activities of GGPP formed from [1–14C]IPP (C5) with dimethylallyl diphosphate (C5) and with GPP relative to that of GGPP formed from [1–14C]IPP with FPP are 3 and 2, respectively.0 (0.00)380 (0.041)GPP + [14C]IPP0 (0.00)561 (0.25)2616 (0.58)FPP + [14C]IPP0 (0.00)0 (0.00)2244 (1.00)a β-Ray intensity (PSL*) of the radioactivity corresponding to GPP (C10), FPP (C15), and GGPP (C20) on the normal-phase plate of Fig. 3E, was measured with a Fuji BAS 1000 bioimage analyzer.b DMAPP, dimethylallyl diphosphate.c The figures in parentheses indicate the relative velocities for the C5→C20, C10→C20, and C15→20 steps of the reaction calculated on the assumption that the specific activities of GGPP formed from [1–14C]IPP (C5) with dimethylallyl diphosphate (C5) and with GPP relative to that of GGPP formed from [1–14C]IPP with FPP are 3 and 2, respectively. Open table in a new tab Figure 4Primary structure of human GGPPSase. Panel A, cDNA and deduced amino acid sequences of human GGPPSase. The stop codon is TAA (*). The potentialN-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.View Large Image Figure ViewerDownload (PPT) GGPPSase activities were measured in extracts from cells (JM109) transformed with plasmids as indicated. pTrc-HGG shows a plasmid containing human GGPPSase cDNA in the pTrc 99A vector. Assays were carried out in the presence (+) and absence (−) of FPP. 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 (11Rilling H.C. Bruenger E. Epstein W.W. Crain P.F. Science. 1990; 247: 318-320Crossref PubMed Scopus (112) Google Scholar). 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 (30Wilkin D.J. Kutsunai S.Y. Edwards P.A. J. Biol. Chem. 1990; 265: 4607-4614Abstract Full Text PDF PubMed Google Scholar) (Fig. 4 B). Five conserved amino acid motifs (I, II, III, IV, and, V) common totrans-prenyltransferases reported (18Chen A. Kroon P.A. Poulter C.D. Protein Sci. 1994; 3: 600-607Crossref PubMed Scopus (218) Google Scholar) 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 (30Wilkin D.J. Kutsunai S.Y. Edwards P.A. J. Biol. Chem. 1990; 265: 4607-4614Abstract Full Text PDF PubMed Google Scholar). Any potentialN-glycosylation site is also not observed in the amino acid sequences of FPPSase of rat (19Clarke C.F. Tanaka R.D. Svenson K. Wamsley M. Fogelman A.M. Edwards P.A. Mol. Cell. Biol. 1987; 7: 3138-3146Crossref PubMed Scopus (92) Google Scholar). 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 potentialN-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. Sheares et al. (20Sheares B.T. White S.S. Molowa D.T. Chan K. Ding V.D.-H. Kroon P.A. Bostedor R.G. Karkas J.D. Biochemistry. 1989; 28: 8129-8135Crossref PubMed Scopus (48) Google Scholar) 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 (16Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4478) Google Scholar), suggesting a common control mechanism for these enzymes. Recently, Guan et al. (31Guan G. Dai P. Shechter I. J. Biol. Chem. 1998; 273: 12526-12535Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) 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.(32Lutz R.J. McLain T.M. Sinensky M. J. Biol. Chem. 1992; 267: 7983-7986Abstract Full Text PDF PubMed Google Scholar) 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 (25Sagami H. Morita Y. Ogura K. J. Biol. Chem. 1994; 269: 20561-20566Abstract Full Text PDF PubMed Google Scholar). GGPP acts as a precursor for the C20 lipid moiety in prenylated proteins and also as a modulator for nuclear hormone receptor (LXRα·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. (11Rilling H.C. Bruenger E. Epstein W.W. Crain P.F. Science. 1990; 247: 318-320Crossref PubMed Scopus (112) Google Scholar) 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 α and β subunits (component B) and an escort protein (component A) (33Armstrong S.A. Seabra M.C. Südhof T.C. Goldstein J.L. Brown M.S. J. Biol. Chem. 1993; 268: 12221-12229Abstract Full Text PDF PubMed Google Scholar, 34Andres D.A. Seabra M.C. Brown M.S. Armstrong S.A. Smeland T.E. Cremers F.P.M. Goldstein J.L. Cell. 1993; 73: 1091-1099Abstract Full Text PDF PubMed Scopus (283) Google Scholar), the α and β 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 α subunit of rat protein geranylgeranyl transferase I (35Chen W.-J. Andres D.A. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11368-11372Crossref PubMed Scopus (88) Google Scholar), 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 (19Clarke C.F. Tanaka R.D. Svenson K. Wamsley M. Fogelman A.M. Edwards P.A. Mol. Cell. Biol. 1987; 7: 3138-3146Crossref PubMed Scopus (92) Google Scholar, 20Sheares B.T. White S.S. Molowa D.T. Chan K. Ding V.D.-H. Kroon P.A. Bostedor R.G. Karkas J.D. Biochemistry. 1989; 28: 8129-8135Crossref PubMed Scopus (48) Google Scholar). Further analysis of mammalian GGPPSase will contribute to the understanding of a finely tuned mechanism of mevalonate pathway. Sepp-Lorenzino et al. (36Sepp-Lorenzino L. Rao S. Coleman P.S. Eur. J. Biochem. 1991; 200: 579-590Crossref PubMed Scopus (28) Google Scholar) reported cell cycle-dependent differential prenylation of proteins. LXRα has been reported to display constitutive transcriptional activity that is negatively regulated with GGPP (15Forman B.M. Ruan B. Chen J. Schroepfer Jr., G.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10588-10593Crossref PubMed Scopus (248) Google Scholar). It remains unclear how GGPP inhibits LXRα 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." @default.
• W1993573607 created "2016-06-24" @default.
• W1993573607 creator A5018232832 @default.
• W1993573607 creator A5028554460 @default.
• W1993573607 creator A5044659450 @default.
• W1993573607 creator A5055140173 @default.
• W1993573607 creator A5061816559 @default.
• W1993573607 date "1999-02-01" @default.
• W1993573607 modified "2023-09-30" @default.
• W1993573607 title "Human Geranylgeranyl Diphosphate Synthase" @default.
• W1993573607 cites W1482012198 @default.
• W1993573607 cites W1498067249 @default.
• W1993573607 cites W1517640879 @default.
• W1993573607 cites W1519293659 @default.
• W1993573607 cites W1538564037 @default.
• W1993573607 cites W1582794275 @default.
• W1993573607 cites W1594736139 @default.
• W1993573607 cites W1851912372 @default.
• W1993573607 cites W1976621716 @default.
• W1993573607 cites W1979027806 @default.
• W1993573607 cites W1985299974 @default.
• W1993573607 cites W1992972153 @default.
• W1993573607 cites W1994105720 @default.
• W1993573607 cites W2000002322 @default.
• W1993573607 cites W2029392258 @default.
• W1993573607 cites W2037485198 @default.
• W1993573607 cites W2039837364 @default.
• W1993573607 cites W2056385716 @default.
• W1993573607 cites W2061302578 @default.
• W1993573607 cites W2066768225 @default.
• W1993573607 cites W2071124826 @default.
• W1993573607 cites W2075982708 @default.
• W1993573607 cites W2083308923 @default.
• W1993573607 cites W2092580882 @default.
• W1993573607 cites W2101222467 @default.
• W1993573607 cites W2156398385 @default.
• W1993573607 cites W2178557511 @default.
• W1993573607 cites W2337872512 @default.
• W1993573607 cites W2394552554 @default.
• W1993573607 cites W2804275696 @default.
• W1993573607 cites W4301626766 @default.
• W1993573607 cites W979882159 @default.
• W1993573607 doi "https://doi.org/10.1074/jbc.274.9.5888" @default.
• W1993573607 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10026212" @default.
• W1993573607 hasPublicationYear "1999" @default.
• W1993573607 type Work @default.
• W1993573607 sameAs 1993573607 @default.
• W1993573607 citedByCount "44" @default.
• W1993573607 countsByYear W19935736072012 @default.
• W1993573607 countsByYear W19935736072013 @default.
• W1993573607 countsByYear W19935736072014 @default.
• W1993573607 countsByYear W19935736072015 @default.
• W1993573607 countsByYear W19935736072016 @default.
• W1993573607 countsByYear W19935736072017 @default.
• W1993573607 countsByYear W19935736072018 @default.
• W1993573607 countsByYear W19935736072019 @default.
• W1993573607 countsByYear W19935736072021 @default.
• W1993573607 countsByYear W19935736072022 @default.
• W1993573607 countsByYear W19935736072023 @default.
• W1993573607 crossrefType "journal-article" @default.
• W1993573607 hasAuthorship W1993573607A5018232832 @default.
• W1993573607 hasAuthorship W1993573607A5028554460 @default.
• W1993573607 hasAuthorship W1993573607A5044659450 @default.
• W1993573607 hasAuthorship W1993573607A5055140173 @default.
• W1993573607 hasAuthorship W1993573607A5061816559 @default.
• W1993573607 hasBestOaLocation W19935736071 @default.
• W1993573607 hasConcept C112243037 @default.
• W1993573607 hasConcept C181199279 @default.
• W1993573607 hasConcept C185592680 @default.
• W1993573607 hasConcept C55493867 @default.
• W1993573607 hasConceptScore W1993573607C112243037 @default.
• W1993573607 hasConceptScore W1993573607C181199279 @default.
• W1993573607 hasConceptScore W1993573607C185592680 @default.
• W1993573607 hasConceptScore W1993573607C55493867 @default.
• W1993573607 hasIssue "9" @default.
• W1993573607 hasLocation W19935736071 @default.
• W1993573607 hasOpenAccess W1993573607 @default.
• W1993573607 hasPrimaryLocation W19935736071 @default.
• W1993573607 hasRelatedWork W1523820041 @default.
• W1993573607 hasRelatedWork W1569138873 @default.
• W1993573607 hasRelatedWork W2008886368 @default.
• W1993573607 hasRelatedWork W2023402920 @default.
• W1993573607 hasRelatedWork W2046313741 @default.
• W1993573607 hasRelatedWork W2052443581 @default.
• W1993573607 hasRelatedWork W2095027488 @default.
• W1993573607 hasRelatedWork W2095865899 @default.
• W1993573607 hasRelatedWork W284590271 @default.
• W1993573607 hasRelatedWork W3176632500 @default.
• W1993573607 hasVolume "274" @default.
• W1993573607 isParatext "false" @default.
• W1993573607 isRetracted "false" @default.
• W1993573607 magId "1993573607" @default.
• W1993573607 workType "article" @default.