Phosphatidylethanolamine:dolichol acyltransferase. Characterization and partial purification of a novel rat liver enzyme.

Incubation of rat or human post-heparin plasma with [3H]dolichol incorporated in liposomes consisting of dioleoyl phosphatidylcholine:dioleoyl phosphatidylethanolamine (3:1) resulted in the formation of radioactive dolichyl oleate. Non-heparinized plasma did not esterify dolichol, and, hence, the enzyme involved is probably associated with the cell surface and released into the blood by heparin. The major location of this activity was the liver, and, therefore, a partial purification of the enzyme from heparinized rat liver perfusates was performed using DEAE-Sephacel and heparin-Sepharose chromatography. The dolichol acyltransferase activity copurified with hepatic lipase activity in a lipid-protein complex of 350 kDa. Optimal acylation is achieved at pH 7.5 in the presence of 5% plasma and 20 mM Ca2+. Esterification can only be obtained when dolichol is present in a phospholipid bilayer, and the reaction is strongly stimulated by unsaturated phosphatidylethanolamine or phosphatidylserine. Radiolabeling experiments demonstrated that the primary acyl donor is phosphatidylethanolamine from which the fatty acid is transferred exclusively from position 1. Neither cholesterol nor retinol are esterified by the enzyme, and the reaction is not stimulated by acyl-CoA. Both the extracellular localization and the mechanism of transacylation clearly distinguish this new enzyme from the acyl-CoA:dolichol acyltransferase described earlier in microsomes.     

The role of calmodulin in the regulation of dolichol kinase

A calcium ion-requiring CTP-dependent kinase that phosphorylates dolichol was found in particulate enzyme preparations from the protozoa Tetrahymena pyriformis. This enzyme and an analogous enzyme present in rat brain microsomes were both shown to be inactivated following washing with EGTA-containing buffers. The activity could be restored by the addition of calcium and the calcium-binding protein calmodulin. In addition, both enzymes were strongly inhibited by trifluoperazine, chlorpromazine, and antiserum against brain calmodulin. These results are evidence that the dolichol kinase from these two sources is regulated by a system involving calmodulin. Dolichol kinase is the enzyme that is believed to be important in the maintenance of the cellular levels of dolichyl phosphate, the factor which is likely to exert the most control over the rate of glycoprotein biosynthesis. On the other hand, microsomal preparations from rat liver which were shown to contain a dolichol kinase that does not require Ca2+ for activity showed no inactivation by EGTA treatment, trifluoperazine, chlorpromazine, or preincubation with antiserum against calmodulin. These findings indicate that the liver enzyme and thus the level of dolichol phosphate is controlled by a different mechanism than that of brain and T. pyriformis.     

Dolichyl phosphate phosphatase from Tetrahymena pyriformis.

A soluble dolichyl phosphate phosphatase from Tetrahymena pyriformis was purified about 68-fold. The enzyme appeared to be specific for dolichyl phosphate and existed in two interrelated forms, one of mol.wt. about 500000 and the other of mol.wt. about 63000. The enzyme was strongly inhibited by 5 mM-Mn2+ and was strongly stimulated by Mg2+. Tetrahymena in the exponential growth phase contained more of this enzymic activity than cells in stationary or lag phase. The dolichyl phosphate phosphatase may be loosely bound to mitochondrial membranes. Two roles proposed for this enzyme are (1) that of releasing dolichol from its phosphorylated biosynthetic form for its use in the cell as unesterified dolichol or dolichyl ester and/or (2) that of regulation of synthesis of glycoproteins or some other glycosylated compound.     

Metabolic interconversion of dolichol and dolichyl phosphate during development of the sea urchin embryo

The in vivo and in vitro synthesis and turnover of dolichol and dolichyl phosphate have been studied over the course of early development in sea urchin embryos. Synthesis of dolichol and dolichyl phosphate was studied in vivo and in vitro using [3H]acetate and [14C] isopentenylpyrophosphate, respectively, as precursors. Both the in vivo and in vitro results indicate that the principal labeled end product of de novo synthesis is the free alcohol, and that this alcohol is subsequently phosphorylated to produce dolichyl phosphate. The presence of 30 microM compactin inhibits the de novo synthesis of dolichol from [3H]acetate by greater than 90%, but has no effect on the incorporation of 32Pi into dolichyl phosphate for more than 6 h, thus suggesting that during this time interval the major source of dolichyl phosphate is preformed dolichol. The rate of turnover of the [3H]acetate-labeled polyisoprenoid backbone of dolichyl phosphate is very slow (t1/2 = 40-70 h). In contrast, the rate of loss of the [32P]phosphate headgroup is more rapid (t1/2 = 5.7-7.7 h) and increases over the course of development. Finally, dolichyl phosphate phosphatase activity has been measured in vitro. The activity of this enzyme, which can be distinguished from phosphatidic acid phosphatase, was found to increase as a function of development, in qualitative agreement with the increased turnover of 32P from dolichyl phosphate observed in vivo. These results suggest that the phosphate moiety of dolichyl phosphate is in a dynamic state, and that dolichol kinase and dolichyl phosphate phosphatase play key roles in regulating the cellular level of dolichyl phosphate.     

Hydrolysis of dolichyl esters by rat liver lysosomes

Dolichyl ester hydrolase activity is broadly distributed among the organs of the rat. The highest activity was found in spleen, brain, lung, and thyroid tissues, whereas this activity is very low in stomach and intestine. The esterase involved is localized to the lumen of lysosomes and, to some extent, in the plasma membranes. Hydrolysis occurs with both alpha-saturated and alpha-unsaturated polyisoprenes esterified with different fatty acids, but the rate of hydrolysis is strongly dependent on the nature of the substrate. The enzyme involved is inhibited by divalent cations, EDTA and EGTA and also by one of the products, dolichol. The esterase is activated by 3-[(3-cholamidopropyl) dimethylammonio]-1-propranesulfonic acid and taurodeoxycholate and inhibited by Triton X-100. Dolichyl esterase activity is completely inhibited by alpha- and beta-naphthyl acetate, phenylmethylsulfonyl fluoride, and beta-chloromethylmercurisulfate. These inhibitors, as well as the pH optimum for dolichyl ester hydrolysis, clearly differentiate the enzyme involved from cholesteryl esterase and triglyceride lipase. Microsomal phospholipase A hydrolyzes dolichyl esters at a slow rate only. In vivo labeling experiments with [3H]mevalonate demonstrated that newly synthesized dolichol is transported in esterified form to the lysosomes, where this lipid is slowly hydrolyzed by the esterase. The possibility is raised that the role of the fatty acyl moiety may be to target dolichol to its final location in the cell.     

The subcellular localization of enzymes of dolichol metabolism in rat liver

Dolichyl phosphate is an intermediate in the glycosylation of N-glycosamidic linked glycoproteins in mammalian systems, and its availability may be a limiting factor in glycoprotein biosynthesis. The basic kinetics and subcellular distribution of enzymes which may influence the concentration of dolichyl phosphate in rat liver have therefore been investigated. These include dolichyl phosphate phosphatase, dolichol phosphokinase, dolichyl fatty acyl ester synthetase, GDP-mannose dolichyl phosphate mannosyl transferase, and UDP-glucose dolichyl phosphate glucosyl transferase. The specific activity of the enzymes was highest in the microsomes, except for dolichyl phosphate phosphatase and dolichyl fatty acyl ester synthetase, which were most concentrated in the plasma membrane and the cytosol fraction, respectively. The nuclei contained all of the enzyme activities while the mitochondria and cytoplasm were generally less active. The presence of both dolichol phosphokinase and dolichyl phosphate phosphatase in microsomes and nuclei, which contain the highest glycosyl transferase activities, may provide a means for direct enzymatic control of levels of dolichyl phosphate.     

The influence of dolichol, dolichol esters, and dolichyl phosphate on phospholipid polymorphism and fluidity in model membranes

The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichyl phosphate on the structure and fluidity of model membranes was studied using 31P NMR, small-angle x-ray scattering, differential scanning calorimetry, and freeze-fracture electron microscopy. These studies suggest that dolichol and dolichol derivatives destabilize unsaturated phosphatidylethanolamine containing bilayer structures and promote hexagonal II phase formation; high concentrations of dolichol induce lipid structures characterized by "isotropic" 31P NMR and particulate fracture faces; dolichol, contrary to cholesterol, has no effect on the thermotropic behavior of membranes consisting of phosphatidylcholine, while dolichyl-P incorporation abolishes the transition from the gel to liquid crystalline phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine; both dolichol and dolichyl-P increase the fatty acid fluidity in phosphatidylethanolamine mixtures; the effect of dolichol on bilayer structure and fluidity is more pronounced with increasing number of isoprene residues; dolichol esters are only soluble to a limited extent in the bilayer and segregates into domains at low concentrations; the results are consistent with a localization of dolichyl-P in which the phosphate group is oriented to the water interphase. The induction of hexagonal II phase by dolichyl-P may elicit the transmembrane movement of glycosylated lipid intermediate.     

Variations of hepatic dolichols during rat development

The content and the percent distribution of dolichol and dolichyl phosphate homologues were measured by high-performance liquid chromatography in perinatal rat livers. Short dolichol chains and no dolichyl phosphate are detectable in the liver at foetal stages; dolichol content progressively increases during liver development. A good correlation is observable between the changes of the dolichol, dolichyl phosphate and the activity of dolichyl-phosphate phosphatase.     

Separation and purification of dolichol and dolichyl phosphate by anion-exchange paper chromatography: application to cultured cells.

Dolichyl phosphate, dolichol C80-105 (dolichol 17:dihydroheptadecaprenol-dolichol 21:dihydrohexeicosaprenol), and dolichol C55 (dolichol 11:dihydroundecaprenol) were separated by anion-exchange paper chromatography. Squalene, sterols, phospholipids, anionic glycolipids, and glycerol did not migrate as dolichyl phosphate, dolichol C80-105, and dolichol C55 under our elution conditions. However, since the Rf of triglycerides was similar to that of dolichol C80-105, saponification, prior to chromatography, removed traces of triglycerides. Silica gel thin-layer chromatography (TLC) allowed the separation of dolichol C80-105 from dolichol C55, whereas dolichyl phosphate was eluted with other lipids. Incubation of spontaneously transformed cells derived from rat astrocytes primary cultures with [2-14C]acetate, saponification of the extracted lipids, and anion-exchange paper chromatography revealed the presence of radioactive dolichyl phosphate and dolichol C80-105 (15 pmol/mg protein). Extraction of labeled dolichyl phosphate followed by acid phosphatase treatment and subsequent analysis on TLC confirmed the identity of dolichyl phosphate since all the radioactivity was associated with dolichol C55. Treatment of the transformed cells with 30 microM 7-ketocholesterol or 7 beta-hydroxycholesterol stimulated markedly (two- to threefold) the incorporation of [2-14C]-acetate in both dolichol C80-105 and dolichyl phosphate. These data demonstrate that anion-exchange paper chromatography is technically suitable for the separation and analysis of dolichol C55, dolichol C80-105, and dolichyl phosphate in cultured cells prelabeled with radioactive precursors.     

Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A

Two N-acetylglucosamine (GlcNAc) transferases which catalyze the incorporation of GlcNAc into GlcNAc-P-P-dolichol (dolichol enzyme) and into bovine pancreatic ribonuclease A (RNAseA enzyme) were solubilized from the rat liver microsomes in a non-ionic detergent, Triton X-100. Both enzyme activities were adsorbed on activated CH-Sepharose 4B, and could be eluted with a linear KCl gradient. Two enzyme activities were separated by this column with the dolichol enzyme eluting before the RNAseA enzyme. A 49-fold and 136-fold purification was achieved for the dolichol and the RNAseA enzyme, respectively. The addition of exogeneous dolichyl phosphate resulted in a 3-5-fold stimulation of the purified dolichol enzyme, but did not affect the purified RNAseA enzyme. The addition of RNAseA stimulated only the RNAseA enzyme. Whereas, tunicamycin could inhibit only the dolichol enzyme. The purified dolichol enzyme had a Km of 14 X 10(-6) M for UDP-GlcNAc and the reaction was saturated with about 0.25 M dolichyl phosphate. The purified RNAseA enzyme had a Km of 4.55 X 10(-6) M for UDP-GlcNAc and was saturated with about 0.36 mM RNAseA. The pH optima and the metal ion requirement for the two enzymes were different. These results suggest that because of the different properties of these two enzymes they may have distinct functions regarding the core glycosylation of N-linked glycoproteins. It is well established that the dolichol enzyme catalyzes the formation of the first dolichol-linked intermediate GlcNAc-P-P-dolichol, whereas according to the present finding, the RNAseA enzyme may catalyze the transfer of GlcNAc directly from UDP-GlcNAc into acceptor protein.     

Effects of peroxisome proliferators gemfibrozil and clofibrate on syntheses of dolichol and cholesterol in rat liver

The effects of two peroxisome proliferators, gemfibrozil and clofibrate, on syntheses of dolichol and cholesterol in rat liver were investigated. Gemfibrozil did not affect the overall content of dolichyl phosphate, but it changed the chain-length distribution of dolichyl phosphate, increasing the levels of species with shorter isoprene units. Gemfibrozil suppressed synthesis of dolichyl phosphate from [(3)H]mevalonate and [(3)H]farnesyl pyrophosphate in rat liver. In contrast, clofibrate increased the content of dolichol (free and acyl ester forms). It remarkably enhanced dolichol synthesis from mevalonate, but did not affect dolichol synthesis from farnesyl pyrophosphate. Gemfibrozil elevated cholesterol synthesis from [(14)C]acetate, but did not affect the synthesis from mevalonate. Clofibrate suppressed cholesterol synthesis from acetate, but did not affect cholesterol synthesis from mevalonate. These results suggest that gemfibrozil suppresses synthesis of dolichyl phosphate by inhibiting, at the least, the pathway from farnesyl pyrophosphate to dolichyl phosphate. As a result, the chain-length pattern of dolichyl phosphate may show an increase in shorter isoprene units. Clofibrate may increase the content of dolichol by enhancing dolichol synthesis from mevalonate. Gemfibrozil may increase cholesterol synthesis by activating the pathway from acetate to mevalonate. Unlike gemfibrozil, clofibrate may decrease cholesterol synthesis by inhibiting the pathway from acetate to mevalonate.     

Separation, quantitation and distribution of dolichol and dolichyl phosphate in rat and human tissues

Two procedures for quantitative determination of dolichol were studied and these were applied to analyze tissue and subcellular distribution. In the first procedure the dolichols were oxidized with Cr2O3 and reduced with NaB3H4. The radioactivity in the individual dolichols was measured using reversed-phase thin-layer chromatography. In the second procedure, dolichols were analyzed by high-pressure liquid chromatography. For determination of dolichyl phosphates the lipid extract was subjected to acid and alkaline hydrolysis, and after hydrolysis with acid phosphatase the distribution was determined by high-pressure liquid chromatography. Recovery was monitored by the addition of dolichol D15 and D23 phosphate to the homogenate. Rat spleen had the highest dolichol content (114 micrograms/g) followed by lower content in rat liver and brain. The distribution pattern was similar in all organs, with 18 and 19 isoprene residues as dominating components. Human organs contain considerably higher concentrations of dolichol, with the 19 and 20 isoprene residues as the main components. In rat liver, outer mitochondrial and Golgi membranes, lysosomes and plasma membranes contain considerable amounts of dolichol. A drastic increase in dolichol content was observed in rat liver hyperplastic nodules while human liver cirrhosis and hepatocarcinoma showed a marked decrease in dolichol. In the latter case, the distribution pattern was also changed. Of the total amount of dolichol present in the tissues, 2% was phosphorylated in human liver, 10% in human testis and 18% in rat liver. In rat liver mitochondria and in microsomes 4 and 31%, respectively, of the polyprenols were in activated form. The results demonstrated that dolichyl phosphate and dolichol concentrations were regulated by different mechanisms and that the two forms possessed an independent distribution.     

Increased synthesis and concentration of dolichyl phosphate in mouse spleens during phenylhydrazine-induced erythropoiesis

Erythropoietic spleens from mice treated with phenylhydrazine synthesized dolichol + dolichyl acyl esters at a higher rate than did normal spleens, and this increased synthesis occurred 1–2 days after the peak of cholesterol synthesis. We have further characterized this dolichol synthesis and have found that at 4 days following phenylhydrazine treatment, dolichyl phosphate accounted for 30% of total synthesis, and at this time 60% of tissue dolichol was phosphorylated. In contrast, treatment with erythropoietin caused simultaneous increases in dolichol and cholesterol synthesis, with very low levels of dolichyl phosphate synthesis. The present results show that the synthesis and the mass of dolichyl phosphate increased in the spleens of phenyl-hydrazine- but not erythropoietin- treated mice.     

Dolichol metabolism in Chinese hamster ovary cells.

The addition of oligosaccharide to asparagine residues of soluble and membrane-associated proteins in eukaryotic cells involves a polyisoprenoid lipid carrier, dolichol. In Chinese hamster ovary cells, the major isomer of this polyisoprenol has 19 isoprenyl units, the terminal one being saturated. Our laboratory has developed a procedure to analyze the levels and nature of the cell's dolichyl derivatives. Chinese hamster ovary cells contain predominately activated, anionic dolichol derivatives, such as oligosaccharyl pyrophosphoryldolichol, monoglycosylated phosphoryldolichols, and dolichyl phosphate. Our studies show that in growing cells there is continual synthesis of total dolichol. Also, preliminary data suggest there is no catabolism or secretion of this lipid. The level of dolichyl phosphate did not change significantly under a variety of conditions where the levels of enzyme activities utilizing dolichyl phosphate did change. These results suggested that these enzymes had access to the same pool of dolichyl phosphate and had similar Km values for this lipid.     

Lipolytic cleavage of dolichyl oleate catalyzed by calf brain membranes

Calf brain membranes catalyze the lipolytic cleavage of dolichyl [¹⁴C]oleate added as an aqueous dispersion in Triton X-100. The enzymatic release of [¹⁴C]oleate from the dolichyl ester is not affected by divalent cations or EDTA, but the lipase activity is inhibited by iodoacetamide and pHMB. The amount of [¹⁴C]oleate released is dependent on the time of incubation, the amount of membrane protein added and the concentration of the radiolabeled lipid substrate. Dolichyl ester hydrolase activity exhibits a pH optimum of 7.5, distinguishing this lipase activity from cholesteryl ester hydrolase (5.0–5.5) and triolein hydrolase (5.0) activity associated with the same membrane preparations. The enzymatic hydrolysis of dolichyl [¹⁴C]oleate is also partially inhibited by oleate and free dolichol, possibly by end-product inhibition.     

Dolichol kinase activity: a key factor in the control of N-glycosylation in inner mitochondrial membranes

Inner mitochondrial membranes from liver contain a dolichol kinase which required CTP as a phosphoryl donor. Kinase activity was linear with protein concentration and unlike other reported kinases, activated almost equally well by Mg2+, Mn2+ or Ca2+. Thin-layer chromatography showed that the reaction product co-migrated with authentic dolichyl monophosphate. The phosphorylation of dolichol did not occur in presence of ATP, GTP or UTP but required exogenous dolichol for maximal activity. Newly synthesized [3H]dolichyl monophosphate has been shown to be glycosylated in the presence of GDP[14C]mannose or UDP[14C]glucose. The double labeled lipids formed by the sugar nucleotide-dependent reactions were identified respectively as [14C]mannosylphosphoryl[3H]dolichol and [14C]glucosylphosphoryl [3H]dolichol. These results are discussed in terms of regulation of N-glycosylation processes in inner mitochondrial membranes from liver.     

Submicrosomal distribution of dolichol kinase and dolichyl phosphate phosphatase in the microsomes of germinating soybeans

The availability of dolichyl phosphate is a major factor in the rate of formation of N-linked glycoproteins in mammalian cells. Recent studies in our laboratory suggested that glycoproteins required for seed germination and early plant development are formed via the dolichyl phosphate pathway. Soybean microsomes contain dolichol kinase and dolichyl phosphate phosphatase, enzymes that regulate dolichyl phosphate levels by interconversion of dolichyl phosphate and dolichol. In the present study, soybean microsomes were fractionated into rough and smooth endoplasmic reticulum and Golgi, and the activities of dolichol kinase and dolichyl phosphate phosphatase were measured in each. Submicrosomal fractions were obtained using a procedure developed for rat liver, and were characterized by marker enzymes, RNA content and electron microscopy. The site of N-glycosylation, the rough endoplasmic reticulum, contained high levels of both dolichol kinase and dolichyl phosphate phosphatase. This makes possible a mechanism whereby glycoprotein formation during seed germination is regulated by availability of dolichyl phosphate.     

Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo

We examined changes in the levels of the dolichol forms in Chinese hamster ovary cells containing alterations in the levels of activity of two enzymes in the oligosaccharyl-P-P-dolichol biosynthetic pathway, namely UDP-GlcNAc:dolichyl phosphate:GlcNAc-phosphotransferase (GlcNAc-1-phosphotransferase) and mannosylphosphoryldolichol (Man-P-Dol) synthase. Under normal conditions in wild type cells, Glc3Man9GlcNAc2-pyrophosphoryldolichol was the most abundant form. Of the other anionic forms of dolichols, dolichyl phosphate, Man-P-Dol, glucosylphosphoryldolichol, and Man5GlcNAc2-pyrophosphoryl dolichol were approximately equally abundant. When 3E11 cells (a tunicamycin-resistant Chinese hamster ovary line containing 15 times more GlcNAc-1-phosphotransferase activity than wild type), B4-2-1 cells (a mutant lacking Man-P-Dol synthase activity), and wild type cells incubated with or without tunicamycin were utilized, significant changes in the levels of most of the anionic dolichol derivatives, with the exception of dolichyl phosphate, were found. Since changes in dolichyl phosphate levels were not detected under a variety of conditions where the levels of enzyme activity utilizing this substrate were varied, all three enzymes appear to have access to the same pool of dolichyl phosphate, and further, to have similar Km values for dolichyl phosphate.     

Involvement of sterol carrier protein-2 in dolichol biosynthesis.

The effect of sterol carrier protein-2 (SCP-2) on dolichol biosynthesis by rat liver microsomes was investigated. cis-Prenyltransferase activity was stimulated 7-fold in the presence of 5 micrograms of purified SCP-2/mg of microsomal protein, which was similar to the increase obtained by adding detergent. The polyisoprenoid pattern obtained in the presence of SCP-2 was the same as that present in rat liver, in contrast to the pattern appearing upon incubation of microsomes with detergent, which gave shorter polyisoprenoids. Like SCP-2, the cytosolic fraction from rat liver also stimulated cis-prenyltransferase. Incubation with cytosol pretreated with anti-SCP-2 showed no stimulatory effect and led to the accumulation of shorter polyisoprenoids. SCP-2 had no appreciable effect on polyprenol alpha-saturase, dolichol kinase, dolichyl phosphate phosphatase, or acyl-CoA:dolichol acyltransferase. The results demonstrate that SCP-2 greatly stimulates and may regulate the condensation reactions mediated by cis-prenyltransferase in the process of dolichol biosynthesis and permits polymerization of the polyisoprenoid to its natural chain length.     

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.