Free Dolichol Research Articles

Recycling of Dolichyl Monophosphate to the Cytoplasmic Leaflet of the Endoplasmic Reticulum after the Cleavage of Dolichyl Pyrophosphate on the Lumenal Monolayer

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.

1,1,2,2-TETRACHLOROETHANE-INDUCED EARLY DECREASE OF DOLICHOL LEVELS IN RAT LIVER MICROSOMES AND GOLGI APPARATUS

Dolichols are long-chain polyprenols containing 14-22 isoprene units, present in mammalian tissues as free dolichol (Free-Dol), fatty acyl dolichyl esters (Dol-FA), and dolichyl phosphate (Dol-P). The hepatic level of Dol-P seems to be a rate-limiting factor for glycosylation processes. Previous studies from our laboratory demonstrated the susceptibility of the dolichol molecule to undergo radical attacks. Since the toxicity of 1,1,2,2-tetrachloroethane (TTCE)is dependent on the free-radical production during hepatic biotrasformation, it was of interest to determine whether this haloalkane might affect glycosylation mechanisms by changing dolichol levels and distribution in rat liver microsomes and Golgi apparatus (GA) . Male Sprague-Dawley rats received a single dose of TTCE (574 mg/kg body weight) and were then sacrificed at different times (5,15, 30, or 60 min). In the TTCE-treated rats both serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and hepatic triglycerides (TG) were significantly higher than control, while microsomal glucose 6-phosphatase (G6Pase) activity was decreased. In total microsomes Dol-P levels considered rate-limiting for the biosynthesis of the N -glycosylated proteins were significantly lower than in the control group 15 min after TTCE treatment. In normal rat liver, F secretory fraction of GA is 60-1 fold enriched in total dolichol content with respect to microsomes. In this compartment the total dolichol content, essential for the increase in membrane fluidity and permeability required for glycoprotein maturation and secretion, decreased significantly 5 min after TTCE treatment. Our results suggest that TTCE may affect dolichol functions in rat liver.

Effect of Ethanol Administration on the Level of Dolichol in Rat Liver Microsomes and Golgi Apparatus

Data obtained in our laboratory had suggested that acute ethanol administration (6 g/kg body weight) selectively and rapidly affects the intracellular system of protein glycosylation at the level of the Golgi apparatus. Dolichols are important membrane components, and dolichyl phosphate is a glycosyl sugar carrier for N-glycosylation of proteins in endoplasmic reticulum and is considered rate-limiting for this process. In this study, modifications in the concentration and distribution of liver microsomal dolichols after acute ethanol administration were investigated. Between 3 and 24 hr after ethanol administration, the microsomal dolichyl phosphate concentration was significantly lower than in control animals. The highest reduction was observed at 12 hr (-52%). An earlier and more marked reduction of total dolichol was observed in the Golgi apparatus, and, in particular, in the secretory fraction F1 (-70% at 6 hr). Ethanol treatment of isolated hepatocytes led to a significant reduction of the de novo synthesis of both dolichyl phosphate and free dolichol. Moreover, in vitro experiments have demonstrated that pro-oxidant agents lead to a significant decrease of both free dolichol and dolichyl phosphate. Our results suggest that acute ethanol administration induces a marked decrease of dolichols, probably by increasing the degradation and impairing the biosynthetic pathway of these molecules.

Effects of 1,2-dichloroethane intoxication on dolichol levels and glycosyltransferase activities in rat liver microsomes and Golgi apparatus

Rat intoxication with a single dose of 1,2-dichloroethane (DCE) (50 μl/100 g b.w) is able to induce a significant modification of protein glycosylation in the liver endoplasmic reticulum and Golgi apparatus. HPLC analysis shows that within 5–60 min after DCE-intoxication, the levels of total dolichol, free dolichol and dolichyl phosphate strongly decreased in the microsomes and Golgi apparatus. Particularly in total microsomes, dolichyl phosphate, which is ratelimiting for the biosynthesis of the N-linked oligosaccharide chains, drops to values significantly lower than in the control group 15 min after DCE poisoning. In the Golgi apparatus, the total dolichol, essential to enhance the fluidity and permeability of these membranes, early and significantly decreases already 5 min after DCE poisoning. Moreover, in the Golgi apparatus galactosyl- and sialyltransferase activities, the main enzymatic activities of terminal protein glycosylation, are significantly reduced, as measured 15 min after DCE intoxication. These data suggest that the impairment of glycoprotein synthesis, maturation and secretion may be involved in the pathogenesis of liver injury induced by acute DCE-intoxication.

Sequential microanalyses of free dolichol, dolichyl fatty acid ester and dolichyl phosphate levels in human serum

Sequential microanalyses of free dolichol, dolichyl fatty acid ester and dolichyl phosphate in human serum were made. To determine the level of each dolichol, samples were pretreated using three different methods prior to fluorescent derivatization. To estimate the concentrations of free dolichol, samples were added to alkaline methanol and kept at room temperature for 1 h. In case of dolichyl fatty acid ester, samples were saponified at 100°C for 1 h. To estimate dolichyl phosphate, saponified lipid extracts were treated with acid phosphatase. Each pretreated dolichol was reacted with anthracene-9-carboxylic acid and amounts of 9-anthroyl derivatives were determined fluorometrically by HPLC. This method is simple and three types of dolichols can be estimated using the same HPLC system. This analysis is also sufficiently sensitive for measurement of serum dolichol levels. The contents of free dolichol, dolichyl fatty acid ester and dolichyl phosphate in human serum were found to be 44.9 ± 10.5 ng/ml ( n = 32), 76.4 ± 24.2 ng/ml ( n = 32) and 43.5 ± 15.1 ng/ml ( n = 13), respectively. These levels had apparently no correlation to age or serum total cholesterols. A linear correlation between dolichols and high-density lipoprotein cholesterols reflects the fact that the dolichols are associated with the high-density lipoprotein fraction.

The deficiency of sterol biosynthesis in Saccharomyces cerevisiae affects the synthesis of glycosyl derivatives of dolichyl phosphates

Mutants deficient in sterol (thermosensitive ergosterol auxotrophs) erg 8, 9, 12 and heme synthesis hem 1, 12 were screened for the level of free dolichol and dolichyl phosphate synthesized in the mevalonate pathway as well as for the activity of dolichyl phosphate-dependent glycosyl transferases. The amount of DolP synthesized via CTP-dependent phosphorylation was the same in mutants and parental strains. However, mannosylation and glucosylation of endogenous dolichyl phosphates in ergosterol mutants was about four times lower compared to parental strains, while the same reactions carried out with exogenous Dol24P reached 80% of the level observed in parental strains indicating that activities of DolPMan and DolPGlc synthases are not the rate-limiting factors. It is postulated that the de novo synthesis of DolP is impaired in the ergosterol mutants. Moreover, a block in the ergosterol branch of the metabolic pathway (erg 9) causes an increase in the de novo synthesis of dolichyl phosphate.

The deficiency of sterol biosynthesis in Saccharomyces cerevisiae affects the synthesis of glycosyl derivatives of dolichyl phosphates.

Mutants deficient in sterol (thermosensitive ergosterol auxotrophs) erg 8, 9, 12 and heme synthesis hem 1, 12 were screened for the level of free dolichol and dolichyl phosphate synthesized in the mevalonate pathway as well as for the activity of dolichyl phosphate-dependent glycosyl transferases. The amount of DolP synthesized via CTP-dependent phosphorylation was the same in mutants and parental strains. However, mannosylation and glucosylation of endogenous dolichyl phosphates in ergosterol mutants was about four times lower compared to parental strains, while the same reactions carried out with exogenous Dol24P reached 80% of the level observed in parental strains indicating that activities of DolPMan and DolPGlc synthases are not the rate-limiting factors. It is postulated that the de novo synthesis of DolP is impaired in the ergosterol mutants. Moreover, a block in the ergosterol branch of the metabolic pathway (erg 9) causes an increase in the de novo synthesis of dolichyl phosphate.

Blood dolichol in lysosomal diseases.

Highly elevated serum total dolichol (free dolichol + dolichyl ester) concentrations have recently been found in two lysosomal storage diseases, aspartylglucosaminuria (AGU) and mannosidosis. The present study demonstrates that the increase of serum dolichol in AGU patients is caused by an increase of serum free dolichol. In 15 patients the mean serum level of free dolichol (227 +/- 16 ng/mL) was 1.9 times higher (p < 0.001) than that in healthy controls (120 +/- 6 ng/mL), while the amounts of dolichol fatty acid esters were similar in the patients and controls (110 +/- 9 vs. 118 +/- 6 ng/mL). In contrast, 10 patients with neuronal ceroid-lipofuscinosis (NCL) (three with infantile, four with juvenile, and three with variant late infantile NCL) had significantly (p < 0.01) lower mean serum levels of both free (79 +/- 5 ng/mL) and total (159 +/- 6 ng/mL) dolichol than age-adjusted healthy controls (free, 100 +/- 6 ng/mL; total, 206 +/- 14 ng/mL). Decreased blood dolichol has not been reported earlier for any other disease. We conclude that the increased serum free dolichol in AGU reflects disturbed lysosomal function and that the decreased free and esterified dolichols in NCLs speak against their presumed primary lysosomal nature.

Changes of dolichol and dolichyl fatty acyl esters during the germination and development of soybeans.

The amounts of dolichol and dolichyl fatty acyl esters and their composition in various parts of soybean seedlings were determined during germination and development. The dolichol content of cotyledons decreased during germination. Dolichyl fatty acyl esters were identified in cotyledons and the amount was estimated by high performance liquid chromatography. The relative amounts of short-chain dolichols of 15, 16, and 17 isoprene units increased during development of the seedlings. The homologue distribution of free dolichol was different from that of dolichyl fatty acyl esters. The relative amounts of dolichols with 16, 17, and 18 isoprene units were greater in free dolichol than in dolichyl fatty acyl esters. The percentages of long-chain saturated fatty acids in dolichyl fatty acyl esters, specifically 21:0, 22:0, 23:0, 24:0, and 25:0, increased during development. These fatty acids represented more than 40% of the fatty acids in dolichyl fatty acyl esters in stems. These results suggest that dolichyl fatty acyl esters are not a storage form of dolichol. The large accumulation of dolichol and dolichyl fatty acyl esters in the leaves, where photosynthesis takes place, suggests some other function.

Partition of free dolichol in human urine.

We have demonstrated that dolichol is present in the urinary supernatant. Most of the dolichol present in the supernatant seems to be associated with cellular debris or membrane fragments. The amount of sediment in healthy subjects correlate well with the volume of urine. Although it is illogical to express urinary dolichol relative to urine volume, a good correlation between the amount of sediment and urine volume has made its use justifiable. Because of the presence of a substantial amount of dolichol in the supernatant, it seems better to use uncentrifuged whole urine as the sample for measurement of dolichol.

Dolichol levels during development and ageing of Drosophila melanogaster

1. 1. Dolichol levels in the fruit fly, Drosophila melanogaster were determined at the larva and pupa stages and in 1, 10, 20 and 30-day-old flies. 2. 2. Free dolichol increased from 1.2 μg/g wet weight in the larvae to 14.9 μg/g in 30-day-old flies, while total dolichol increased from 3.4 μg/g in the larvae to 21.2 μg/g at 30-days-old. 3. 3. Dolichol released after saponification is primarily from dolichyl fatty acid ester, which accounts for up to 65% of the total dolichol. 4. 4. The major dolichol homologs, which remain relatively constant throughout development and ageing in D. melanogaster, are C-80, C-85 and C-90, which represent approximately 7%, 60% and 33%, respectively.

Changes in murine tissue concentrations of dolichol and dolichol derivatives associated with age

The concentrations of the three major cellular forms of dolichol (free, esterified and phosphorylated) were determined in murine liver, kidney and heart. The tissue levels of these forms of dolichol were studied in detail as a function of age. Changes in the activities of dolichyl phosphate phosphatase and dolichol kinase were also determined. In liver, the concentration of unesterified dolichol, fatty acyl dolichol and dolichyl phosphate increased markedly over a period of 6 to 25 months (four-fold, 5.5-fold and nine-fold, respectively). In kidney only, free dolichol and phosphorylated dolichol increased (approximately four-fold in each case). However, this tissue consistently showed the highest concentrations of all forms of dolichol as compared to liver and heart. In heart, both free and esterified dolichol concentrations increased (approximately 3.25-fold in each case); dolichyl phosphate levels were not determined in this tissue. In all tissues studied, the activity of the dolichyl phosphate phosphatase enzyme was considerably higher than that of the dolichol kinase enzyme. In liver, there was no evidence to suggest that either enzyme was critical in determining the relative concentrations of dolichol and dolichyl phosphate. Evidence for such a role for the kinase in the kidney was stronger. Treatment of aging mice with meclofenoxate, a drug that is reported to cause dissolution of lipofuscin, failed to prevent accumulation of dolichol and dolichyl phosphate with age. These observations suggest that not all accumulated dolichol is associated with lipofuscin. Meclofenoxate treatment had no consistent effect on the activities of the enzymes studied.

Effects of CC14Poisoning on Metabolism of Dolichol in RAT Liver Microsomes and Golgi Apparatus

Carbon tetrachloride (CCl4) poisoning affects glycoprotein processing and maturation at the level of rat liver microsomes and Golgi apparatus. HPLC analysis showed that within 5-60 min after CCl4 administration the levels of total dolichol, free dolichol and dolichyl-phosphate strongly decreased both in total microsomes and in Golgi apparatus. The most marked and early reduction of total dolichol was observed in the secretory membranes of Golgi area already 15 min after CCl4 poisoning. The incubation of CCl4-pretreated isolated hepatocytes with [3H]-mevalonate showed a significant slowing down of the label incorporation into both free-dolichol and dolichyl-phosphate. Moreover, lipid peroxidation might cause alterations in the molecular structure of both free-dolichol and dolichyl-phosphate. A notable prevention of dolichol decrease was observed in animals pretreated with vitamin E. The results suggest that the prooxidant activity of CCl4 is able to affect the metabolism of dolichol either by increasing the oxidative degradation or impairing the biosynthetic pathway.

Effect of dolichyl monophosphate on the permeability properties of lipid membranes

The effect of dolichyl monophosphate on the permeability properties of dimyristoylphosphatidylcholine bilayers to alkaline cations, Ca 2+ and glucose has been determined by stop-flow spectrophotometry. The results show that, in con trast to free dolichol effects, the monophosphate derivative increased the permeability following a decreasing order of the permeating particle size. Phase diagrams indicate that dolichyl monophosphate is fully incorporated into the phosphatidylcholine bilayer around 0.75% weight/weight ratio. For these ratios, the permeation of ions is higher in the gel than in the liquid crystalline state.

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.

Oligosaccharyl diphosphodolichols in the ceroid-lipofuscinoses.

Autopsy brain samples from patients with late-infantile, juvenile and adult forms of ceroid-lipofuscinosis (CL) and cultured skin fibroblasts from juvenile CL were analyzed for the content of phosphodolichol (P-Dol) related compounds. The levels of P-Dol obtained on treatment with hot dilute acid of the chloroform-methanol (CM 2:1) extract and the chloroform-methanol-water (CMW 1:1:3) extract of the residue were estimated by high performance liquid chromatography. Compared to age-matched control individuals, the levels of P-Dol obtained in both the extracts were increased more than 6.6 times in all the patient samples. Further analysis of the CMW extract indicates that the increased P-Dol is primarily due to oligosaccharyl diphosphodolichol. Cultured skin fibroblasts from the juvenile form of CL show normal level of free dolichol and elevated level of phosphorylated dolichols. Glycoprotein synthesis measured by incorporation of labeled glucosamine show no deficit in the transfer of oligosaccharides from lipids to proteins. A hypothesis is presented to explain the accumulation of oligosaccharyl diphosphodolichol and deficiency of lysosomal proteases in ceroid-lipofuscinosis.

Depression of hepatic dolichol levels by cholesterol feeding.

A study was conducted to determine whether repression of 3-hydroxy-3-methylglutaryl CoA reductase by a chronic high-cholesterol diet would deplete hepatic dolichol levels. Four-week-old male C57BL/6J mice were maintained on a control diet or a diet supplemented with 5% cholesterol. Animals from both groups were killed at various times and reductase activity and levels of free dolichol, dolichyl acyl ester, dolichyl phosphate, and ubiquinone were measured. The reductase activity was reduced by 90% within 1 week and remained depressed through 56 days. Initially, the levels of the free dolichol, acyl ester, phosphoryl ester, and ubiquinone were 7, 16, 5, and 80 micrograms/g liver, respectively. Early increases in the concentration of dolichyl phosphate and free dolichol were similar in both the cholesterol-fed and control groups. However, in the cholesterol-fed group the concentration of dolichyl acyl esters was only 50% of that in the control group by 7 days and it remained lower throughout the experiment. Total dolichol levels were lower by about 30%. Ubiquinone levels were transiently depressed at 7 days by 33% but returned to control levels by 4 weeks. After 56 days, the control values of dolichol and dolichyl phosphate remained constant whereas the dolichyl acyl ester levels continuously increased to a value of 133 micrograms/g of liver by 156 days. Subcellular fractionation of livers from 4-week-old mice indicated a lysosomal distribution of dolichol and dolichyl acyl ester and a lysosomal and microsomal distribution of dolichyl phosphate.

Dolichol and phosphorylated dolichol content of tissues in ceroid-lipofuscinosis.

Concentrations of free dolichol, total non-phosphorylated dolichol, and total phosphorylated dolichol were measured in autopsy specimens of brain and liver from ceroid-lipofuscinosis (CL) and control cases. Levels of non-phosphorylated dolichol, mainly as free dolichol, were increased approximately two-fold in late-infantile CL brain. No increase was observed for CL liver. In late-infantile and juvenile CL brain, the increase in phosphorylated dolichol was at least ten-fold, but no significant difference was found for CL liver. A substantial part of the increase in phosphorylated dolichol in CL brain appeared to be due to lipid-linked oligosaccharides. The results suggest that CL might involve a defect in the metabolism of dolichol-linked oligosaccharides.

Age-associated increase of free dolichol levels in mice

Free dolichol levels were determined by high-performance liquid chromatography in liver, brain, heart, lung, testis and kidney of C57 B1/6 mice at 3, 6, 12 and 30 months of age. In all the tissues, free dolichol levels were increased with aging. The largest increase in dolichol was in testis and the smallest increase was in the liver. Among the various tissues, aging testis and kidney showed high levels of free dolichol, and represented more than twice the level of dolichol in brain. The major dolichols among the various tissues were of the 17, 18, 19 and 20 isoprene unit containing types and the proportions were unaffected by aging. Tissues such as brain and testis showed a tendency toward high proportions of longer-chain dolichols, whereas lung and heart showed high proportions of shorter-chain dolichols. This study shows that the age-associated increase in dolichol levels is a general phenomenon and the increases vary widely with tissues.

Different tissue concentrations and rates of synthesis of dolichol, dolichyl acyl esters, and dolichyl phosphate in mouse testes and preputial glands

The rates of synthesis and tissue concentrations of dolichol derivatives differ markedly in testes and preputial glands from adult C57BL 6J mice. In testes, the rates of free dolichol, dolichyl acyl esters, and dolichyl phosphate synthesis were approximately 5,000, 4,000 and 2,000 dpm/3h/g, respectively. Comparable rates for preputial glands were 12,000, 60,000 and 0 dpm/3h/g. Thus in testes, dolichyl phosphate represented 15–20% of total dolichol synthesis, whereas no de novo dolichyl phosphate synthesis was detected in preputial glands. In testes, free dolichol was the predominant derivative synthesized; in preputial glands, 80% of dolichol synthesized was esterified to fatty acids. The concentration of total dolichol (free alcohol plus acyl esters) was 160 μg/g tissue in testes and 1270 μg/g tissue in preputial glands. In both tissues, 85–90% of dolichol was esterified to fatty acids, and no dolichyl phosphate was detected.