Dolichyl Phosphate Biosynthesis Research Articles

Isoprenoid alcohols utilization by malaria parasites.

Plasmodium falciparum is the etiological agent of human malaria, one of the most widespread diseases in tropical and subtropical regions. Drug resistance is one of the biggest problems in controlling the disease, which leads to the need to discover new antimalarial compounds. One of the most promissory drugs purposed is fosmidomycin, an inhibitor of the biosynthesis of isoprene units by the methylerythritol 4-phosphate (MEP) pathway, which in some cases failed in clinical studies. Once formed, isoprene units are condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate, which are necessary for Heme O and A formation, ubiquinone, and dolichyl phosphate biosynthesis as well as for protein isoprenylation. Even though the natural substrates of polyprenyl transferases and synthases are polyprenyl pyrophosphates, it was already demonstrated that isoprenoid alcohols (polyprenols) such as farnesol (FOH) and geranylgeraniol (GGOH) can rescue parasites from fosmidomycin. This study better investigated how this rescue phenomenon occurs by performing drug-rescue assays. Similarly, to FOH and GGOH, it was observed that phytol (POH), a 20-carbon plant isoprenoid, as well as unsaponifiable lipid extracts from foods rescue parasites from the antimalarial effect of fosmidomycin. Contrarily, neither dolichols nor nonaprenol rescue parasites from fosmidomycin. Considering this, here we characterized the transport of FOH, GGOH, and POH. Once incorporated, it was observed that these substances are phosphorylated, condensed into longer isoprenoid alcohols, and incorporated into proteins and dolichyl phosphates. Through proteomic and radiolabelling approaches, it was found that prenylated proteins are naturally attached to several isoprenoids, derived from GGOH, dolichol, and POH if exogenously added. Furthermore, the results suggest the presence of at least two promiscuous protein prenyltransferases in the parasite: one enzyme which can use FPP among other unidentified substrates and another enzyme that can use GGPP, phytyl pyrophosphate (PPP), and dolichols, among other substrates not identified here. Thus, further evidence was obtained for dolichols and other isoprenoid products attached to proteins. This study helps to better understand the apicoplast-targeting antimalarial mechanism of action and a novel post-translational modification of proteins in P. falciparum.

Retinal Degeneration Caused by Rod-Specific Dhdds Ablation Occurs without Concomitant Inhibition of Protein N-Glycosylation.

SummaryDehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step in dolichol synthesis. Recessive mutations in DHDDS cause retinitis pigmentosa (RP59), resulting in blindness. We hypothesized that rod photoreceptor-specific ablation of Dhdds would cause retinal degeneration due to diminished dolichol-dependent protein N-glycosylation. Dhddsflx/flx mice were crossed with rod-specific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specific Dhdds knockout mice (Dhddsflx/flx iCre+). In vivo morphological and electrophysiological evaluation of Dhddsflx/flx iCre+ retinas revealed mild retinal dysfunction at postnatal (PN) 4 weeks, compared with age-matched controls; however, rapid photoreceptor degeneration ensued, resulting in almost complete loss of rods and cones by PN 6 weeks. Retina dolichol levels were markedly decreased by PN 4 weeks in Dhddsflx/flx iCre+ mice, relative to controls; despite this, N-glycosylation of retinal proteins, including opsin (the dominant rod-specific glycoprotein), persisted in Dhddsflx/flx iCre+ mice. These findings challenge the conventional mechanistic view of RP59 as a congenital disorder of glycosylation.

The ins(ide) and out(side) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum.

The precursor oligosaccharide donor for protein N-glycosylation in eukaryotes, Glc3Man9GlcNAc(2)-P-P-dolichol, is synthesized in two stages on both leaflets of the rough endoplasmic reticulum (ER). There is good evidence that the level of dolichyl monophosphate (Dol-P) is one rate-controlling factor in the first stage of the assembly process. In the current topological model it is proposed that ER proteins (flippases) then mediate the transbilayer movement of Man-P-Dol, Glc-P-Dol, and Man5GlcNAc(2)-P-P-Dol from the cytoplasmic leaflet to the lumenal leaflet. The rate of flipping of the three intermediates could plausibly influence the conversion of Man5GlcNAc(2)-P-P-Dol to Glc3Man(9)GlcNAc(2)-P-P-Dol in the second stage on the lumenal side of the rough ER. This article reviews the current understanding of the enzymes involved in the de novo biosynthesis of Dol-P and other polyisoprenoid glycosyl carrier lipids and speculates about the role of membrane proteins and enzymes that could be involved in the transbilayer movement of the lipid intermediates and the recycling of Dol-P and Dol-P-P discharged during glycosylphosphatidylinositol anchor biosynthesis, N-glycosylation, and O- and C-mannosylation reactions on the lumenal surface of the rough ER.

Characterization and Localization of a Long-Chain Isoprenyltransferase Activity in Porcine Brain: Proposed Role in the Biosynthesis of Dolichyl Phosphate

Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from [1-14C]isopentenyl pyrophosphate [( 1-14C]I-P-P) into long-chain polyisoprenyl pyrophosphates (Poly-P-P) and unidentified neutral lipids. The brain isoprenyltransferase activity synthesizing the Poly-P-P (1) required 5 mM Mg2+ and 10 mM vanadate ions for maximal activity; (2) exhibited an apparent Km of 8 microM for I-P-P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (5) was stimulated by dithiothreitol. The major products were identified as C90 and C95 allylic Poly-P-P on the basis of the following chemical and chromatographic properties: (1) the intact product co-chromatographed with authentic Poly-P-P on silica-gel-impregnated paper; (2) the major product was converted to a compound chromatographically identical to polyisoprenyl monophosphate (Poly-P) by alkaline hydrolysis; (3) treatment of the labeled Poly-P with wheat germ acid phosphatase or mild acid yielded neutral labeled products; (4) the KOH hydrolyzed product coeluted with authentic Poly-P from lipophilic Sephadex LH-20; and (5) the labeled lipids produced by enzymatic dephosphorylation had mobilities identical to fully unsaturated polyisoprenols containing 18 (C90) and 19 (C95) isoprene units when analyzed by reverse-phase chromatography. When subcellular fractions from rat brain gray matter were compared, the highest specific activity was found in the heavy microsomes. These results demonstrate that brain contains an isoprenyltransferase activity, associated with the rough endoplasmic reticulum, capable of synthesizing long-chain Poly-P-P. The enzymatic reactions by which the Poly-P-P intermediate is converted to dolichyl phosphate remain to be elucidated.

Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis

The prenyltransferase involved in the biosynthesis of dolichyl phosphate has been characterized in Saccharomyces cerevisiae. Although the enzyme is predominantly membrane-bound, a significant percentage was found in the soluble fraction. The prenyltransferase preferentially utilizes farnesyl pyrophosphate as the allylic substrate and isopentenyl pyrophosphate as cosubstrate with half-maximal velocities obtained at 25 and 6.7 μ m, respectively. The enzymatic activity is sensitive to sulfhydryl reagents and is inhibited by all detergents tested, except 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at concentrations less than 5 m m. The product of the reaction has been characterized as an α-unsaturated polyprenyl pyrophosphate, containing 12–15 isoprene units, approximately two isoprene units shorter than the endogenous yeast dolichyl phosphate. The stereochemistry of addition of isoprene units by the prenyltransferase was shown to be cis by a comparison of the HPLC retention time for a pentadecaprenyl phosphate derived from the in vitro reaction product with that for an authentic mixture of α- cis- and α- trans-pentadecaprenyl phosphates.

Cell-cycle dependence of dolichyl phosphate biosynthesis

The cell-cycle dependence of dolichyl phosphate biosynthesis has been investigated in mouse L-1210 cells fractionated by centrifugal elutriation. Dolichyl phosphate levels increased linearly through the cell cycle, reaching a value in late S phase twice that of early G1. The cell-cycle dependences of four dolichyl phosphate metabolizing enzymes have been measured: cis-prenyltransferase, CTP-dependent dolichol kinase, dolichyl phosphatase, and dolichyl pyrophosphatase. The kinase, the cis-prenyltransferase, and the pyrophosphatase showed cell-cycle variations, increasing through G1 to a maximum in S phase while the monophosphatase activity was cell-cycle independent. The rate of accumulation of dolichyl phosphate was not affected by growing the cells in mevalonolactone-supplemented media. The evidence presented is consistent with models in which either the cis-prenyltransferase or the kinase/phosphatase couple (or both) regulates the levels of dolichyl phosphate in the cell.

The mechanism and regulation of dolichyl phosphate biosynthesis in rat liver.

Rat liver slices were pulse labeled for 6 min with [3H]mevalonolactone and then chased for 90 min with unlabeled mevalonolactone in order to study the mechanism of dolichyl phosphate biosynthesis. The cholesterol pathway was also monitored and served to verify the pulse-chase. Under conditions in which radioactivity in the methyl sterol fraction chased to cholesterol, radioactivity in alpha-unsaturated polyprenyl (pyro)-phosphate chased almost exclusively into dolichyl (pyro)phosphate. Lesser amounts of radioactivity appeared in alpha-unsaturated polyprenol and dolichol, and neither exhibited significant decline after 90 min of incubation. The relative rates of cholesterol versus dolichyl phosphate biosynthesis were studied in rat liver under four different nutritional conditions using labeled acetate, while the absolute rates of cholesterol synthesis were determined using 3H2O. From these determinations, the absolute rates of dolichyl phosphate synthesis were calculated. The absolute rates of cholesterol synthesis were found to vary 42-fold while the absolute rates of dolichyl phosphate synthesis were unchanged. To determine the basis for this effect, the rates of synthesis of cholesterol and dolichyl phosphate were quantitated as a function of [3H]mevalonolactone concentration. Plots of nanomoles incorporated into the two lipids were nearly parallel, yielding Km values on the order of 1 mM. In addition, increasing concentrations of mevinolin yielded parallel inhibition of incorporation of [3H]acetate into cholesterol and dolichyl phosphate. The specific activity of squalene synthase in liver microsomes from rats having the highest rate of cholesterol synthesis was only 2-fold greater than in microsomes from rats having the lowest rate. Taken together, the results suggest that the maintenance of constant dolichyl phosphate synthesis under conditions of enhanced cholesterogenesis is not due to saturation of the dolichyl phosphate pathway by either farnesyl pyrophosphate or isopentenyl pyrophosphate but coordinate regulation of hydroxymethylglutaryl-CoA reductase and a reaction on the pathway from farnesyl pyrophosphate to cholesterol.

Inhibition of dolichyl phosphate biosynthesis by compactin in cultured rat hepatocytes.

Isolated rat hepatocytes were cultured in monolayer for about 24 h. During this period the cells exhibited constant protein and lipid synthesis. When the culture medium contained compactin, a competitive inhibitor of the 3-hydroxyl-3-methylglutary-coenzyme-A reductase, dolichyl-P synthesis was inhibited by 91% at the end of the incubation, as estimated by the incorporation of [3H]acetate and by 77% as estimated by the incorporation of 32Pi. These results indicate that in primary cultures of rat hepatocytes dolichyl monophosphate is mainly synthesized through a de novo process, while phosphorylation through the CTP-mediated kinase is of limited functional importance.

Solubilization and characterization of the long chain prenyltransferase involved in dolichyl phosphate biosynthesis.

Membranes from Ehrlich ascites tumor cells possess an activity which, in the presence of farnesyl pyrophosphate, incorporates [14C]isopentenyl pyrophosphate into a product which is soluble in chloroform/ methanol and retained by DEAE-cellulose. The product co-migrated with authentic dolichyl phosphate on thin layer chromatography but was degraded to neutral labeled compounds when subjected to mild acid hydrolysis, suggesting the presence of an unsaturated alpha-isoprene unit. Triton X-100 (2%) liberated the activity from the membranes and the resulting solubilized preparation was further characterized. The enzyme was found to be sensitive to sulfhydryl reagents and stimulated by ionic strength. A strong dependence of activity on the addition of farnesyl pyrophosphate was observed, allowing several compounds to be tested for their ability to serve as potential primers. Geranyl pyrophosphate, neryl pyrophosphate, all-trans farnesyl pyrophosphate and all-trans geranylgeranyl pyrophospate were found to be effective substrates, although to different extents. Citronellyl pyrophosphate (which has a saturated alpha-isoprene) was inactive as a substrate. The chain length of the products generated was investigated by using a double label isotope procedure and by reverse-phase high performance liquid chromatography. All active primers yielded a product containing 16-19 isoprene units; the distribution of the individual isoprene species was essentially identical regardless of the primer used as substrate. These findings indicate that the specificity lies in the absolute chain length of the product released and not the number of isoprenes added. High performance liquid chromatography of the [14C]polyprenol resulting from enzymatic dephosphorylation of the reaction product indicated the presence of an unsaturated alpha-isoprene unit in a cis-configuration. It is proposed that the enzyme is the long chain cis-prenyltransferase involved in the biosynthesis of dolichyl phosphate.

Topography of dolichyl phosphate synthesis in rat liver microsomes transbilayer arrangement of dolichol kinase and long-chain prenyltransferase

The topography of the dolichyl phosphate biosynthetic enzymes within the plane of rat liver microsomes was investigated by the use of two impermeant inhibitors of enzyme activity: trypsin and mercury-dextran. Mercury-dextran was found to inactivate over 50% of the activities of the CTP-dependent dolichol kinase and the long-chain prenyltransferase. Trypsin caused over 90% inactivation of the long-chain prenyltransferase and 60% inactivation of the dolichol kinase. In addition, the CTP-dependent dolichol kinase was inhibited over 90% by CDP applied externally to sealed microsomes. Inactivation of the dolichyl phosphate biosynthetic enzymes by the impermeant probes occurred under conditions where the mannose-6-phosphatase activity was highly latent. It was concluded that the active sites of these two enzymes are located on the external surface of the microsomal membranes and that dolichyl phosphate biosynthesis occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.

Dolichol metabolism in rat liver. Determination of the subcellular distribution of dolichyl phosphate and its site and rate of de novo biosynthesis.

Two independent experiments indicated that the major subcellular site of de novo dolichyl phosphate biosynthesis in rat liver is in the microsomes. First, when purified subcellular fractions were assayed for long chain prenyltransferase activity, the profile obtained indicated exclusively a microsomal localization. Second, when liver slices were incubated 60 min with [14C]acetate and subcellular fractions were subsequently isolated, the microsomal fraction was found to contain greater than two-thirds of the total radioactivity incorporated into dolichyl phosphate. Chemical analysis of dolichyl phosphate in subcellular fractions showed substantial quantities in both the microsomal and mitochondrial-lysosomal fractions. In contrast, dolichol was found primarily in the mitochondrial-lysosomal fraction. The absolute in vitro rate of hepatic dolichyl phosphate synthesis was determined in liver slices and isolated hepatocytes by [14C]acetate labeling using [3H] water incorporation into cholesterol to correct for the dilution of the labeled acetate by the endogenous acetyl-CoA pool. From this value, an in vivo rate of de novo synthesis was estimated as 1.7-2.6 nmol of dolichyl phosphate/liver/day. This rate is more than 30 times the maximum possible rate of accumulation in liver of dietary dolichol (Keller, R. K., Jehle, E., and Adair, W. L., Jr. (1982) J. Biol. Chem. 257, 8985-8989). From the previously determined concentration of dolichyl phosphate in rate liver (Keller, R. K. Tamkun, J. M., and Adair, W. L. (1981) Biochemistry 20, 5831-5836) and the de novo rate of synthesis, it appears that the half-life of hepatic dolichyl phosphate is on the order of days.

Studies on the regulation of glycoprotein biosynthesis. An investigation of the rate-limiting steps of dolichyl phosphate biosynthesis.

The possible role of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (the rate-controlling enzyme of cholesterol biosynthesis) in regulating the rate of dolichyl phosphate biosynthesis in rat liver was investigated. Rats were either fasted 48 h or fed diets supplemented with the drug cholestyramine. The activity of HMG-CoA reductase was 5000-fold greater in liver from cholestyramine-fed rats as compared to fasted rats. The activity of dolichyl phosphate synthetase, the prenyl transferase responsible for the biosynthesis of dolichyl phosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate, was similar in both nutritional conditions and was markedly less active than HMG-CoA reductase even in the fasted state. Acetate incorporation into cholesterol was 2200-fold greater in liver slices from cholestyramine-fed rats as compared to fasted rats. By contrast, acetate incorporation into dolichyl phosphate was only 6-fold higher. Further studies suggested that the levels of farnesyl pyrophosphate and isopentenyl pyrophosphate are several hundred-fold greater in liver from cholestyramine-treated rats. From these results, it is concluded that the rate of dolichyl phosphate biosynthesis in rat liver is not regulated by the activity of HMG-CoA reductase but is probably regulated at the level of dolichyl phosphate synthetase.

Studies on the biosynthesis of dolichyl phosphate: Evidence for the [formula omitted] formation of 2,3-dehydrodolichyl phosphate

A particulate enzyme preparation from hen oviduct is shown to carry out the biosynthesis of a long chain polyprenyl phosphate from isopentenyl pyrophosphate and farnesyl pyrophosphate. The compound has the physical and chemical properties of 2,3-dehydrodolichyl phosphate. The enzyme system is inhibited by EDTA and stimulated by Triton X-100 and dithiothreitol. If the product of the reaction is 2,3-dehydrodolichyl phosphate, it may be derived from 2,3-dehydrodolichyl pyrophosphate, a likely intermediate in the biosynthesis of dolichyl phosphate.