Polyprenyl Phosphate Research Articles

On the specificity of dolichol kinase and DolPMan synthase towards isoprenoid alcohols of different chain length in rat liver microsomal membrane

1. 1. A wide range of dolichols differing in the length of hydrocarbon chain (from 11 to 32 isoprene residues) were found to be phosphorylated in the presence of CTP in rat liver microsomes. 2. 2. Fully unsaturated polyprenols of the same chain length as dolichols were poor substrates for dolichol kinase at low detergent (Nonidet P-40) concentration. At higher concentration of detergent, both dolichols and polyprenols were equally effective. 3. 3. In the transfer of mannosyl residues from GDPMan, the dolichyl phosphates generated in rat liver microsomes were all good lipid acceptors, while fully unsaturated polyprenyl phosphates were not.

Biosynthesis of membrane-derived oligosaccharides. Membrane-bound glucosyltransferase system from Escherichia coli requires polyprenyl phosphate

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) of Escherichia coli and the cyclic glucans of the Rhizobiaceae, are now recognized to be a family of closely related substances with important functions in osmotic adaptation and cell signaling. The synthesis of the beta-1,2-glucan backbone of MDO is catalyzed by a membrane-bound glucosyltransferase system previously shown to require UDP-glucose and (surprisingly) acyl carrier protein (Therisod, H., Weissborn, A. C., and Kennedy, E. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7236-7240). In the present study, no glucan intermediates bound to acyl carrier protein or to UDP could detected. The enzyme system, however, was found to be strongly inhibited by bacitracin and by amphomycin. Because the two antibiotics function by forming specific complexes with polyprenyl phosphates, their inhibitory effect suggests a prenol requirement for MDO biosynthesis. Furthermore, the activity of the glucosyltransferase was greatly stimulated by the addition of polyprenyl phosphates such as decaprenyl-P and dihydroheptaprenyl-P, but not by farnesyl-P. The same membrane preparations carry out the synthesis of polyprenyl-P-glucose, which is also stimulated by added polyprenyl-P, including farnesyl-P, the most active of those tested. Pulse chase experiments, however, indicate that the endogenous pool of polyprenyl-P-glucose cannot be an obligate intermediate in the MDO glucosyltransferase system.

Covalent binding of dolichyl phosphate to proteins in rat liver

Rats were injected via the portal vein with (RS)-[5-3H]-mevalonolactone and the lipids were extracted. From fractions of liver homogenate, all labeled dolichol, cholesterol and ubiquinone could be extracted, but about 40% of microsomal and lysosomal dolichyl phosphate was only released after alkaline hydrolysis. Only a small amount of the non-extractable radioactivity was found to be associated with alpha-unsaturated polyprenyl phosphate. There was no difference in the polyisoprenoid pattern when the two pools of dolichyl phosphate were compared. On the other hand, the specific activity of the bound lipid was only half that of the extractable form. After phenyl-Sepharose chromatography, a peak of protein was isolated exhibiting a 25-fold enrichment in bound radioactive dolichyl phosphate. Treatment with a non-specific protease, followed by chromatography, gave polypeptide fragments associated with bound lipids. On SDS/PAGE a major protein band at 23 kDa and some minor bands with higher molecular masses were found to be associated with this lipid. The results indicate the presence of covalently bound dolichyl phosphate in rat liver.

Polyprenyl phosphates: synthesis and structure-activity relationship for a biosynthetic system of Salmonella anatum O-specific polysaccharide

A series of polyprenyl phosphates with modified structure of polyprenyl residue was prepared through phosphorylation of polyprenyl trichloroacetimidates with phosphoric acid. Interaction of polyprenols with tetra- n-butylammonium dihydrogen phosphate and trichloroacetonitrile was found to represent a very efficient, simple and general method for the synthesis of polyprenyl phosphates. A procedure was developed for smooth conversion of polyprenyl pyrophosphates into the monophosphates through hydrolysis in the presence of 4-dimethylaminopyridine. The polyprenyl phosphates prepared were studied as substrates for the enzymes of Salmonella anatum O-spectific polysaccharide biosynthesis. Correct stereochemistry of α- and β-isoprenic units was found to be essential for substrate efficiency. At the more remote positions of the hydrocarbon chain just the presence of isoprenic units of any configuration seems necessary. Some changes in position of the phosphate group may be permissible without significant loss of substrate properties.

Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase.

The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-lon. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30 degrees C and 5% of that at 40 degrees C. At 42 degrees C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30 degrees C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30 degrees C and almost the same in both strains at 42 degrees C. The mutant had no obvious phenotype regarding its growth properties.

A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions.

Previous results suggested that F2A8, a glycosylation mutant of Chinese hamster ovary cells, had a lower amount of dolichyl phosphate available for asparagine-linked glycosylation reactions relative to parental cells. The steady-state amounts and identities of polyisoprenoid lipids were determined by incubating F2A8, its parental cell line B4-2-1, and wild-type Chinese hamster ovary cells for 24 h with [2-3H]mevalonate. The neutral lipids, ubiquinone, cholesterol, and cholesteryl esters, which were the most highly labeled from [3H]mevalonate, were labeled equally in all three cell types. In wild-type and B4-2-1 cells, mevalonate incorporation into the anionic glycosylated and phosphorylated derivatives of dolichol was 10-fold higher than into the neutral free dolichol and dolichyl esters. In contrast, in F2A8 cells, label accumulated in neutral polyisoprenol lipids, so that the ratio of neutral to anionic lipids was 1:1 rather than 1:10. In wild-type and B4-2-1 cells, the polyisoprenoid found as free alcohol and in phosphorylated and glycosylated forms was shown by high pressure liquid chromatography using a silica column to be primarily dolichol, a polyisoprenol that has a saturated terminal isoprene unit. In contrast, in F2A8 cells the polyisoprenoid found primarily as the free alcohol and in phosphorylated and glycosylated forms appeared to be completely unsaturated polyprenol. The distribution of chain lengths of the labeled polyisoprenols of F2A8, B4-2-1, and wild-type cells was the same as determined by high pressure liquid chromatography using a reverse-phase column, with the predominant chain length being 19 isoprene units. These results combined with our previous studies on the phenotype of the F2A8 mutant indicate that the unsaturated polyprenyl phosphate derivatives do not function as well as dolichyl phosphate derivatives in cellular glycosylation reactions.

Glycosyltransfer by pea membranes from sugar nucleotides to added prenyl phosphates

Pea membranes supplied with GDP-[ 14C]mannose, UDP-N-[ 14 C] acetylglucosamine or UDP-[ 14C]glucose catalyze the transfer of 14C-labeled sugars or sugar phosphates to endogeneous lipid acceptors as well as to exogenously added dolichyl phosphates. Fully unsaturated polyprenyl phosphates were not used as effective acceptors by this system. Mannosyl- P-dolichol was formed most rapidly in the presence of long-chained dolichyl- P while mannosyl- PP-, glucosyl- PP- and GlcNAc- PP-dolichol were preferentially formed from relatively short-chained dolichyl phosphate acceptors. Glucosyl- PP- and mannosyl- PP-dolichol accumulated in the preparation without further metabolism, but GlcNAc- PP-dolichol was lengthened by addition of a second GlcNAc plus several [ 14C]mannose units to form an oligosaccharide fraction susceptible to the action of endoglycosidase H. This lipid-linked oligosaccharide could then be glucosylated in the presence of UDP-[ 14C]glucose to form a longer oligosaccharide. It is concluded that levels of endogenous dolichyl phosphates in pea membranes are rate-limiting for several of the key glycosyltransferases required for oligosaccharide assembly.

The alpha-saturation and terminal events in dolichol biosynthesis.

Incubations of 10,000 X g supernatant from rat liver with [3H]mevalonate were performed and the labeling of polyprenols was studied. It was demonstrated that factors like pH, substrate concentration, and presence of detergent not only greatly influence the total incorporation but also the relative distribution of radioactivity among the isoprenologues. The synthesis was shown to be extremely sensitive to Triton X-100. Substrate concentrations of 1 and 100 microM mostly gave polyprenols with 18 and 20 isoprenes, respectively. At a given substrate concentration, pH 6.5 resulted in shorter polyprenols than did pH 7.5. Ozonolytic fragmentation demonstrated that in the initial phase of incubation, polyprenols are elongated by 1 isoprene residue and saturated to give dolichols. No substantial dephosphorylation of polyprenyl phosphates to the free alcohol occurred. The production of dolichol in vitro was shown to utilize NADH for the saturation event. This seemed to occur concomitantly with the synthesis. alpha-Saturation of polyprenyl-P could not be achieved with the procedures employed. It is proposed that the synthesis of dolichol and dolichyl-P do not share the same terminal steps; saturation and terminal isoprene condensation occur in cooperation; and substrate concentration and pH influence the terminal enzyme(s) and the nature of the final product in the polyprenol biosynthesis.

Stimulation of protein glycosylation in cultured hepatoma cells by polyprenyl phosphates.

The aim of this work was to selectively stimulate N-glycosylation of proteins in cultured AH 70Btc hepatoma cells. Dolichyl phosphate, alpha-dihydrodecaprenyl phosphate and solanesyl phosphate were entrapped in egg lecithin liposomes and supplied to the cells. After treatment with 0.6-15 nmol of the polyprenyl phosphates/3 ml/dish for 48 hr, the incorporations of [14C]glucosamine into N-linked saccharide chains of glycoproteins and into lipid intermediates were increased to various extents. The stimulation by alpha-dihydrodecaprenyl phosphate was most significant and 15 nmol of the lipid/dish increased the incorporation into N-linked saccharide chains of glycoproteins by about 80%. The incorporations of [14C]glucosamine into O-linked saccharide chains and of [14C]leucine into proteins were not changed significantly by alpha-dihydrodecaprenyl phosphate. When the N-linked glycopeptides prepared from the cells and those from the cells treated with the polyprenyl phosphate were compared by Sephadex G-50 column chromatography, no significant difference was observed. Analysis of reduced cellular glycoproteins by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis showed that alpha-dihydrodecaprenyl phosphate increased the incorporation of [14C]glucosamine into N-linked saccharide chains of certain high-molecular-weight glycoproteins. In addition, the polyprenyl phosphate enhanced the adhesiveness of the cells to the substratum, probably as a result of the stimulation of N-glycosylation of proteins.

13] Photolabile analogs of the allylic pyrophosphate substrate of prenyltransferases

This chapter designs, prepares and tests photolabile analogs of the allylic pyrophosphate substrate of prenyltransferases as substrates, inhibitors, or covalent modifying agents for the long-chain prenyltransfcrase, C 55 PP synthetase. The synthesis of photolabile analogs of isoprenoid compounds provides a tool for probing the structure of the substrate binding sites of a variety of prenyltransferases. Recently, ones have synthesized two photolabile analogs of the allylic pyrophosphate substrates, geranyl and trans,trans-farnesyl pyrophosphate, which bear the 2-diazo-3-trifluoropropionyloxy (DATFP) moiety. DATFP containing lipids have been used subsequently as effective probes of lipid-lipid and lipid-protein interactions. The availability of photolabile prenyltransferase substrates now offers a number of other opportunities for their application in studying prenyltransferases. The ability to prepare long chain photolabile polyprenyl phosphates such as photoactivatable undecaprenyl- and dolichylmonophosphates would be of significant value in providing a tool to probe enzymes or enzyme complexes, which use the parent prenyl phosphates as glycosyl carriers in bacterial cell wall biosynthesis or mammalian glycoprotein biosynthesis.

Convenient Syntheses of Vitamin K and Coenzyme Q by the Coupling of Grignard Reagents with Allylic Phosphate

Abstract Convenient syntheses of vitamin K and coenzyme Q are described. Grignard coupling of polyprenyl diethyl phosphates with 1,4-dimethoxy-3-methyl-2-naphthylmagnesium bromide gave high yields of the polyprenylated hydroquinone dimethyl ethers regioselectively with the preservation of the stereochemistry. The precursors of coenzyme Q were similarly obtained by the coupling of 2,3,4,5-tetramethoxy-6-methylphenylmagnesium bromide and the polyprenyl phosphates. The synthesized (polyprenyl)hydroquinone dimethyl ethers are known to be oxidized to the biologically active quinones.

Oviduct dolichyl phosphate phosphatase: Estrogen effects and a possible biosynthetic role

Estrogen-induced chick oviduct differentiation is accompanied by an increased capacity for protein glycosylation. A portion of this increase has been attributed to increased levels of dolichyl phosphate (Dol-P). Hormone withdrawal leads to an apparent decrease in Dol-P. Dol-P metabolism in the oviduct has been studied, and one of the enzymes having a direct effect on Dol-P, Dol-P phosphatase is herein described. Dol-P phosphatase has a pH optimum of 6.0, does not require a metal ion, and is inhibited by Mn 2+ at concentrations greater than 5 m m. Inhibitor studies indicate that Dol-P hydrolysis is inhibited by polyprenyl phosphates having both saturated and unsaturated α-isoprene residues, but not by the corresponding alcohols. The enzyme is also inhibited by phosphatidic acid unless 2 m m Mn 2+ is included in the incubations. Under these conditions Dol-P hydrolysis is only slightly inhibited (<10%), but phosphatidate inhibition is totally eliminated. Oviduct membranes also possess phosphatidate phosphatase, but this enzyme is distinct from Dol-P phosphatase based on thermolability, metal ion sensitivity, and sulfhydryl reagent sensitivity. Studies of enzyme activity in response to estrogen treatment reveal that both Dol-P phosphatase and phosphatidate phosphatase have maximal specific activity early in the differentiation process (peaking after 3 days of treatment), and low specific activity in fully differentiated oviducts, including laying hen oviduct. Hormone withdrawal elicits a small increase in specific activity of both phosphatases. The hormone effects suggest that Dol-P phosphatase may be a biosynthetic enzyme,

Novel mannose carrier in the trypanosomatid Crithidia fasciculata behaving as a short α-saturated polyprenyl phosphate

Crithidia fasciculata cells incubated with [14C]glucose or membranes derived from the same protozoan incubated with GDP-[14C]mannose were found to synthesize a lipid monophosphate mannose. No glucosylated mild acid-labile compound was formed in vivo or in vitro when UDP-[14C]glucose was used instead of GDP-[14C]mannose. The lipid moiety of the mannosyl derivative formed behaved as a polyprenol having 11 isoprene residues as judged by t.l.c. and be gel filtration in sodium deoxycholate-containing buffers. The mannolipid was not broken on treatment with hot phenol, suggesting the existence of an alpha-saturated isoprene unit. This is the first case reported in which a mannosyl phospholipid involved in sugar transfer in a eukaryotic cell behaves as if it was similar to that of bacterial polyprenols, although having its putative alpha-isoprene unit saturated to the same extent as dolichols from higher organisms.

In vivo biosynthesis of the saturated isoprene unit of dolichyl phosphate.

Reduction of the alpha-isoprene unit of polyprenols to form dolichols was studied in vivo using 3H-polyprenol derivatives as substrates and liposomes as carriers. Liposomes containing labeled polyprenol, polyprenyl phosphate, or polyprenyl pyrophosphate were injected through the portal vein into the livers of rats under anesthesia. Uptake and conversion of the labeled compounds to dolichol derivatives was studied at different intervals. The greatest conversion to dolichol derivatives was found with polyprenyl pyrophosphate and polyprenyl monophosphate, with 31% and 8% of the absorbed dose converted respectively. Less than 0.2% of the absorbed polyprenol was converted to dolichol derivatives. These results suggest that the substrate for the alpha-isoprene reductase involved in dolichol biosynthesis is either polyprenyl monophosphate or polyprenyl pyrophosphate, or both.

Subcellular distribution of phospholipids and of polyprenyl phosphate in Aspergillus niger van tieghem

Growth of Aspergillus niger Van Tieghem in presence of 32p 1 was followed by cell fractionation; the subcellular fractions were identified using marker enzymes and their phospholipid content was analyzed. Microsomes are characterized by high PC and PS values, whereas the largest amount of cardiolipin is recovered in the mitochondria. Polyprenyl phosphate was found mainly in mitochondria (46%) (microsomal content: 38%) and, more precisely, the highest ratio was in the outer membrane. This situation raises the problem of polyprenyl phosphate transfer from mitochondria to microsomes where the enzyme responsible for polyprenyl phosphate mannosylation is located.

Uptake and metabolism of polyprenols by animal cells cultured in vitro.

Several mammalian, chicken, and mosquito cells grown in vitro take up tritiated dolichol supplied to the incubation medium. The extent of labelling varied markedly between different cell cultures. After 20 h incubation most of the dolichol taken up was unchanged and the major product of metabolism of dolichol was identified as its fatty acid esters. Green-monkey kidney cells were tested with 8 fully unsaturated and 6 alpha-saturated polyprenols ranging from C35 to C105. In general the uptake of alpha-saturated polyprenols (dolichol type, was higher. Considerable differences were found between the uptake of polyprenols of differing chain lengths. Less than 1% of the polyprenols taken up was converted into more polar product, mainly polyprenyl phosphates and polyprenyl phosphate sugars. The short-chain polyprenols, from C35 to C65, were metabolized more rapidly than the long-chain polyprenols, as judged from the amount of polar products and fatty acid esters of polyprenols.

Fractionation studies of the enzyme complex involved in teichoic acid synthesis.

The membrane-bound enzymes participating in the syntheses of the teichoic acid main chain and linkage unit have been solubilized with Triton X-100 and fractionated by sucrose density gradient centrifugation. Two main fractions were obtained: a heavy fraction, containing enzymes effecting synthesis of the main chain attached to the linkage unit, which was associated with only a small amount of lipid, and a light fraction which was rich in prenyl phosphate and catalyzed only linkage-unit synthesis. The separation by density was not based entirely on polypeptide chain length, as some of the shortest chains appeared in the denser fractions and some relatively high-molecular-weight peptides occurred in the lightest fraction. High activity for linkage-unit synthesis was observed in a fraction containing only a few peptides. Addition of ficaprenyl phosphate to the enzyme preparations had no stimulatory effect. It is concluded that the enzymes for main-chain and linkage unit syntheses frm one or more fairly tightly associated complexes and that polyprenyl phosphate is an integral firmly bound component of the complex in which the linkage unit is synthesized.

Properties of a soluble polyprenyl phosphate. UDP-D-N-acetylglucosamine N-acetylglucosamine-1-phosphate transferase.

Properties of a soluble polyprenyl phosphate. UDP-D-N-acetylglucosamine N-acetylglucosamine-1-phosphate transferase.

Specificity of solubilized yeast glycosyl transferases for polyprenyl derivatives.

Four glycosyl transferases solubilized by detergents from Saccharomyces cerevisiae membranes have been tested for their specificity towards various polyprenols differing in chain length and saturation. The formation of dolichyl diphosphate‐(N‐acetylglucosamine)1‐2 from uridine diphosphate N‐acetylglucosamine and dolichyl monophosphate showed an obligatory requirement for α‐saturated polyprenols. The apparent affinity for α‐saturated polyprenyl phosphates improves with increasing chain length; those shorter than seven isoprene units were inactive. The rate of transfer of the chitobiosyl unit from dolichyl diphosphate chitobiose to the hexapeptide Tyr‐Asn‐Leu‐Thr‐Ser‐Val followed the sequence: α‐dihydropolyprenyl (C55) &gt;(C80 &gt; (C100). The enzyme transferring the mannosyl group from guanosine diphosphate mannose to dolichyl monophosphate also showed a clear dependence on α‐saturation and chain length. In this case the chain length affects mainly the maximal velocity of the reaction. The highest value was obtained with C55α‐dihydropolyprenyl phosphate (100 nmol × h−1× mg protein−1), whereas the value with C35α‐dihydropolyprenyl phosphate was only 8 nmol × h−1× mg protein.−1. The mannosyl transfer from dolichyl monophosphate mannose to dinitrophenylated tetrapeptide Asn‐Ala‐Thr‐Vol to form on O‐glycosidic bond strongly depended on α‐saturation and chain length of the polyprenyl phosphate. The rate of transfer correlated directly with the length of the polyprenyl residue. With C80α‐dihydropolyprenol less than half the rate of the C100 compound was observed, whereas C55 and C35α‐dihydropolyprenol are only 20 and 10% as active, respectively.

Location of fucosyl transferases in the membrane system of maize root cells.

There are two fucosyl transferase activities present within the endomembranes of the cells of maize root-tips. One transfers fucose to polyprenyl phosphate and occurs in the endoplasmic reticulum, the second transfers fucose probably to polysaccharide or glycoprotein. In order to show an association of this second fucosyl transferase activity with the endoplasmic reticulum as well as the Golgi apparatus, a method of fractionating the membranes in a discontinuous sucrose gradient was used. Membranes were prepared in the presence of Mg2+, which maintained the attachment of ribosomes to the endoplasmic reticulum, and also in the presence of EDTA, which removed most of the ribosome complex. This caused a shift in density of these membranes. Two types of experiments were carried out; either maize roots were incubated in L-[1-3H]fucose and then membranes prepared and the amount of polymer synthesized in vivo determined or isolated membranes were incubated with GDP-L-[U-14C]fucose in vitro and the amount of polymer synthesized was found. The results showed that the Golgi apparatus had the highest amount of this fucosyl transferase activity, but there was a significant amount of activity associated with the endoplasmic reticulum and the latter was shifted in the sucrose gradient depending on the conditions used.