Poly-Saturated Dolichols from Filamentous Fungi Modulate Activity of Dolichol-Dependent Glycosyltransferase and Physical Properties of Membranes.

 Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the ω-end, have been identified in A. fumigatus and A. niger. Here we confirm using an LC-ESI-QTOF-MS analysis, that poly-saturated dolichols are abundant in other filamentous fungi, Trichoderma reesei, A. nidulans and Neurospora crassa, while the yeast Saccharomyces cerevisiae only contains the typical mono-saturated dolichols. We also show, using differential scanning calorimetry (DSC) and fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) that the structure of dolichols modulates the properties of membranes and affects the functioning of dolichyl diphosphate mannose synthase (DPMS). The activity of this enzyme from T. reesei and S. cerevisiae was strongly affected by the structure of dolichols. Additionally, the structure of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) model membranes was more strongly disturbed by the poly-saturated dolichols from Trichoderma than by the mono-saturated dolichols from yeast. By comparing the lipidome of filamentous fungi with that from S. cerevisiae, we revealed significant differences in the PC/PE ratio and fatty acids composition. Filamentous fungi differ from S. cerevisiae in the lipid composition of their membranes and the structure of dolichols. The structure of dolichols profoundly affects the functioning of dolichol-dependent enzyme, DPMS.

Candida albicans cis-prenyltransferase Rer2 is required for protein glycosylation, cell wall integrity and hypha formation.

cis-Prenyltransferase is the first enzyme of the mevalonate pathway committed to the biosynthesis of dolichol in eukaryotes. The RER2 gene encoding cis-prenyltransferase (Rer2p) in the human fungal pathogen Candida albicans was characterized. In addition, the ORF19.5236 encoding the second cis-prenyltransferase, which putatively is responsible for the synthesis of longer polyisoprenoids chains, was identified. When cultivated under repressive conditions, the conditional mutant strain expressing the RER2 gene from the regulatable MET3 promoter contained only 4% of cis-prenyltransferase activity and markedly diminished amounts of dolichols, as compared to the wild-type strain. Moreover, transcriptomal analyses revealed changes in the expression of 300 genes, mainly involved in transport, response to stress, filamentous growth and organelle organization. Growth of the conditional strain was blocked completely at 37 °C. The strain was hypersensitive to a wide range of inhibitors, which suggested glycosylation defects and compromised cell wall integrity. Moreover, the rer2 conditional mutant grown in the repressive conditions, unlike the same strain in the absence of repressor, failed to form hyphae. The results indicate that dolichols are essential not only for protein glycosylation and cell wall integrity but also for growth and development of C. albicans.

Liquid chromatography/tandem mass spectrometry of dolichols and polyprenols, lipid sugar carriers across evolution.

Across evolution, dolichols and polyprenols serve as sugar carriers in biosynthetic processes that include protein glycosylation and lipopolysaccharide biogenesis. Liquid chromatography coupled with electrospray ionization mass spectrometry offers a powerful tool for studying dolichols and polyprenols in their alcohol or glycan-modified forms in members of all three domains of life. In the following, recent examples of the how different versions of this analytical approach, namely reverse phase liquid chromatography-multiple reaction monitoring, normal phase liquid chromatography/tandem mass spectrometry and normal phase liquid chromatography-precursor ion scan detection have respectively served to address novel aspects of dolichol or polyprenol biology in Eukarya, Archaea and Bacteria.

[Biomarkers of alcohol abuse. Part II. New biomarkers and their interpretation]. / Biomarkery naduzywania alkoholu. Czesc II. Nowe biomarkery oraz ich interpretacja.

An increasing number of new biomarkers of alcohol abuse appear in the literature. The most commonly used biomarkers (5-hydroxytryptophol, fatty acid ethyl esters, ethyl glucuronide, phosphatidyl ethanol, ethyl sulphate, mitochondrial aspartate aminotransferase, carbohydrate deficient transferrin, acetaldehyde adducts, beta-hexosaminidase, and sialic acid) were described. Then other known and less known biomarkers associated with alcohol abuse were described in brief (e.g. acetaldehyde, acetate, methanol, alpha-amino-n-butyric acid, dolichol, proteomics). Their sensitivity and specificity is generally higher than that of traditional biomarkers. The time of detection in biological fluids occur from one day to few months after alcohol consumption. Hence, their usefulness in clinical practice as well as in experimental studies is increasing.

Quantification of dolichol in the human lens with different types of cataracts.

PURPOSE: To quantify and characterize dolichol species in cataractous and clear human lenses. METHODS: Whole lenses were collected from cadaver eyeballs from the C.H. Nagri Eye Bank and Red Cross Society Eye Bank (Ahmedabad, India). Cataractous nuclei were collected after extracapsular cataract extraction (ECCE). Wet weight for all the lenses was taken and were stored at -50 degrees C until used. Dolichol was extracted using a standard protocol and then analyzed using High Performance Liquid Chromatography (HPLC) on a 4.6 mmx60 mm Hypersil-Octadecylsilane (ODS; 3 microm) reversed phase column using a Waters dual pump apparatus, a Waters gradient programmer, and an ultraviolet (UV) detector set at 210 nm. Dolichol 13 was used as an internal standard, and dolichol mixture from the liver was used as an external qualitative standard. RESULTS: The highest dolichol concentration was found in nuclear cataract (2.54+/-0.6 microg) followed by posterior subcapsular cataract (1.4+/-0.35 microg), and the lowest levels were observed in cortical cataract (0.37+/-0.06 microg). The level of dolichol concentration in cataractous lenses was statistically significantly higher than the levels in clear lenses (1.0+/-04.3 microg; p<0.01). CONCLUSIONS: The dolichol concentration was significantly higher in lenses with nuclear cataract. A significant difference in dolichol concentration was observed between the different types of cataract. It suggests that dolichol and other isoprenoids may be associated with cataractogenesis.

Analysis of ubiquinones, dolichols, and dolichol diphosphate-oligosaccharides by liquid chromatography-electrospray ionization-mass spectrometry.

Prenols, a class of lipids formed by the condensation of five carbon isoprenoids, have important roles in numerous metabolic pathways of the eukaryotic cell. Prenols are found in the cell as free alcohols, such as dolichol, or can be attached to vitamins, as with the fat soluble vitamins. In addition, prenols such as farnesyl- and geranylgeranyl-diphosphate are substrates for the transfer of farnesyl and geranylgeranyl units to proteins with important implications for signal transduction within the cell. Dolichol phosphate- and dolichol diphosphate-linked sugars are central to the formation of the lipid-linked branched oligosaccharide, Dol-PP-(GlcNAc)2(Man)9(Glc)3, used for co-translational en bloc protein N-glycosylation in the lumen of the endoplasmic reticulum. Toward furthering our understanding of the role of prenol lipids in the cell, we have developed a method for the detection and quantification of dolichol and coenzyme Q by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). These methods, developed using the mouse macrophage RAW 264.7 tumor cells, are broadly applicable to other cell lines, tissues, bacteria, and yeast. We also present a new MS-based method for the detection and structural characterization of the intact dolichol diphosphate oligosaccharide Dol-PP-(GlcNAc)2 (Man)9(Glc)3 from porcine pancreas.

Lipid content determines aggregation of neuromelanin granules in vitro.

The neuromelanin pigment of the substantia nigra of the human brain is closely associated with lipids and other non-melanogenic compounds which appear to contribute to the unique and complex morphology of neuromelanin pigment granules. In this work we show that insoluble granules isolated from the human substantia nigra associate in vitro to form pigment aggregates similar to those present in the human brain. Extraction of neuromelanin-associated polar lipids by methanol and/or hexane significantly enhanced melanin aggregate size. A marked (10-fold) increase in granule size was seen after methanol treatment, whereas the application of hexane after methanol reduced this pro-aggregation effect. We have previously reported that hexane and methanol remove the neuromelanin-associated polyisoprenoids dolichol and cholesterol respectively. Thus, the current data suggests that pigment-associated lipids may be a factor regulating pigment aggregation and neuromelanin granule size in vivo.

Lipid compositions of intracellular membranes isolated from rat liver nodules in Wistar rats.

The mevalonate pathway gives rise to important end products for the regulation of growth and resistance to oxidative stress and is, consequently, of importance in carcinogenesis. In this study liver nodules were produced in Wistar rats by intermittent feeding with dietary 2-acetylaminofluorene, and the lipid compositions of isolated microsomes, mitochondria, and lysosomes were examined. The phospholipid compositions of these subfractions were unchanged compared to normal hepatic tissue, but the fatty acid patterns were altered, particularly in microsomes. An increase in the content of palmitic acid and a decrease in that of stearic acid were noted. The pattern of fatty acyl moieties on carbon atoms 1 and 2 of the glycerol backbone of phospholipids was unchanged in nodular tissue compared to normal liver. The amount of dolichol was significantly higher in microsomes and mitochondria, but not in lysosomes, and the relative amounts of longer polyisoprenoid compounds were increased in the liver nodules. The relative concentration of esterified dolichol was decreased and an enrichment in saturated fatty acids in this fraction could be observed. The cholesterol concentration was found to be lower in microsomes, but was unchanged in mitochondria and lysosomes, and the normally low concentration of cholesteryl esters was elevated somewhat in microsomes and lysosomes. The ubiquinone content of liver nodular mitochondria was unchanged, but increased 7-fold in microsomes and 2-fold in lysosomes. The alterations found in the lipid composition of liver nodules are significant and have functional implications in many cellular processes of proposed importance for the carcinogenic process, i.e., protein glycosylation cholesterogenesis, regulation of the mevalonate pathway, cellular oxidation-reduction state, and resistance to oxidative stress.

Dolichol: A Component of the Cellular Antioxidant Machinery.

Dolichol, an end product of the mevalonate pathway, has been proposed as a biomarker of aging, but its biological role, not to mention its catabolism, has not been fully understood. UV-B radiation was used to induce oxidative stress in isolated rat hepatocytes by the collagenase method. Effects on dolichol, phospholipid-bound polyunsaturated fatty acids (PL-PUFA) and known lipid soluble antioxidants [coenzyme Q (CoQ) and α-tocopherol] were studied. The increase in oxidative stress was detected by a probe sensitive to reactive oxygen species (ROS). Peroxidation of lipids was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). Dolichol, CoQ, and α-tocopherol were assessed by high-pressure liquid chromatography (HPLC), PL-PUFA by gas-liquid chromatography (GC). UV-B radiation caused an immediate increase in ROS as well as lipid peroxidation and a simultaneous decrease in the levels of dolichol and lipid soluble antioxidants. Decrease in dolichol paralleled changes in CoQ levels and was smaller to that in α-tocopherol. The addition of mevinolin, a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoAR), magnified the loss of dolichol and was associated with an increase in TBARS production. Changes in PL-PUFA were minor. These findings highlight that oxidative stress has very early and similar effects on dolichol and lipid soluble antioxidants. Lower levels of dolichol are associated with enhanced peroxidation of lipids, which suggest that dolichol may have a protective role in the antioxidant machinery of cell membranes and perhaps be a key to understanding some adverse effects of statin therapy.

Microdetermination of dolichol in tissues.

A method is described for the rapid purification and analysis of tissue dolichol on a nanomole scale. The assay is based on a radioisotope dilution technique in which [3H]dolichyl palmitate is used as tracer and acetylation with [14C]acetic anhydride serves as the quantitating reaction. The acetylation conditions were characterized with respect to time, temperature and concentration of reagents. When this method was applied to pig liver, a value was obtained which fell within the range of previously published results based on a gravimetric assay. In chicken, dolichol levels were found to be high in oviduct and low or absent in red blood cells and plasma.

Analysis of dolichyl pyrophosphoryl oligosaccharides in purified storage cytosomes from ovine ceroid-lipofuscinosis.

Ovine ceroid-lipofuscinosis is an inherited neurodegenerative disorder characterised by the accumulation of storage cytosomes in brain and visceral organs. Phosphorylated dolichol-containing compounds, largely in the form of dolichyl pyrophosphoryl oligosaccharides, have been shown to constitute 1-2% of the dry weight of storage cytosomes isolated from brain and pancreas, and 0.5 and 0.1% respectively of storage cytosomes isolated from liver and kidney. The carbohydrate portion of these glyconjugates in storage cytosomes isolated from brain, pancreas and liver consisted of a series of oligosaccharides of composition Man2-9GlcNAc2, with Man5-8GlcNAc2 predominating. The concentrations of dolichyl pyrophosphoryl oligosaccharides in storage cytosomes from ovine ceroid-lipofuscinosis are much higher than has been reported for endoplasmic reticulum, their normal functional location.

Localization of dolichols in phospholipid membranes. An ESR spin label study.

We have used ESR methods employing spin-labeled stearates to investigate the effects of dolichol on the motion of lipid molecules in phospholipid membranes of phosphatidylethanolamine and phosphatidylcholine. The ESR spectra show that the presence of dolichol affects the motion of the spin probes at carbon-16, but not at carbon-5. Similar results are obtained with phospholipid membranes comprising only phosphatidylcholine. It is suggested that dolichol molecules are present mainly in the lipid core region of phospholipid membranes.

Uptake and modification of dietary polyprenols and dolichols in rat liver.

Short and long dolichols and polyprenols in free form or esterified with fatty acids were incorporated into liposomes and administered to rats through a gastric tube. The free alcohols were taken up by the liver to different extents. While uptake in other organs was less, it also involved the fatty acid esters. The use of systems other than liposomes did not increase the efficiency of uptake. Most of the administered lipids were recovered in the lysosomes. Exogenous dolichols and polyprenols were both partly esterified in the liver and, to some extent, also phosphorylated; a portion of the polyprenols was also alpha-saturated. These results indicate that various polyisoprenes are taken up, to a small extent, from the diet by tissues under normal conditions and in liver these dietary lipids undergo terminal modifications.

Dolichols in brain and urinary sediment in neuronal ceroid lipofuscinosis.

Long-chain polyisoprenoid alcohols (dolichols) increase more than tenfold from age 5 to 80 years in human cerebral cortex. The dolichol content of brain from infantile, late infantile, and juvenile forms of neuronal ceroid lipofuscinosis (NCL) was significantly higher than in age-matched patients with other neurologic diseases. Significant increase of dolichols was also found in the urinary sediment in all three types of NCL patients, and this test is useful in making the diagnosis. Dolichol accumulation is the first biochemical marker of NCL and seems to parallel storage of ceroid lipofuscin.

Studies on the polyisoprenoid composition in hepatocellular carcinomas and its correlation with their differentiation.

The levels of cholesterol, ubiquinone and dolichol and the polyprenol composition of dolichol in human hepatocellular carcinomas (hepatomas) with different degrees of differentiation were analyzed and compared with healthy liver tissue. Dolichols were also analyzed in liver metastases. The total level of cholesterol was increased, while the levels of dolichol and ubiquinone were decreased in all hepatomas, but no correlation between these levels and the degree of differentiation of the hepatomas could be observed. The level of dolichol decreased more in the hepatomas than in the liver metastases. The dolichol fraction from hepatomas with a low degree of differentiation contained higher relative amounts of short polyisoprenols (D17) and slightly lower relative amounts of D21 compared with healthy liver tissue, metastatic liver tumors or hepatomas with a high degree of differentiation. The significance of the lipid values found in the different groups is discussed.

Dolichol deposition in developing mammalian brain: content of free and fatty-acylated dolichol and proportion of specific isoprenologues.

Dolichol serves a critical role in N-linked glycoprotein synthesis as the carrier of saccharide moieties. In this study the deposition of dolichol was investigated in developing rat brain. The contents of both the free and fatty-acylated forms of dolichol as well as the specific isoprenologues of dolichol were determined. Two distinctive phases of rapid dolichol deposition were observed; the first during brain development, and the second, with aging. The predominant form of brain dolichol was shown to be the free derivative at all ages; however, during the two phases of rapid deposition of total dolichol, fatty-acylated dolichol became detectable and increased transiently. The rapid rate of increase of brain dolichol during development occurred later than that of brain cholesterol, which shares a common biosynthetic pathway with dolichol. Thus, the peak rate of increase of dolichol occurred at 25 days postnatal, and of cholesterol, at 18 days postnatal. A distinct ontogenetic alteration in the proportion of the individual molecular species (isoprenologues) of dolichol also was defined. These observations suggest that dolichol is particularly involved in developmental events of the third to sixth postnatal weeks in rat brain, e.g. myelination and synaptogenesis.

Dolichol and vitamin A content in rat liver Ito (perisinusoidal) cells.

Dolichol has been determined in many tissues but to date no data are available on liver Ito (fat storing) cells. In this note dolichol was determined in two subpopulations of liver Ito cells isolated from rats pretreated with vitamin A: Ito-1, vitamin A enriched and Ito-2, relatively poor of vitamin A. Differences were observed in the behaviour of the two fractions after vitamin A pretreatment of rats. In fact, in Ito-1 fraction dolichol increases with the increase of vitamin A, while in Ito-2 fraction it does not change significantly with the increase of vitamin A. These results, while confirming the heterogeneity of fat storing cells, are discussed as to the possible role of dolichol and vitamin A metabolism.

The effect of dolichol on the permeability properties of phosphatidylcholine bilayers.

The permeability of liposomes to water, glucose, Ca2+ and alkaline cations was monitored by recording the change in absorbance at 450 nm using a rapid reaction stopped-flow spectrophotometer. Liposomes were prepared with egg phosphatidylcholine and concentrations of dolichol ranging from 0.1% to 9% (w/w). Net permeability of phosphatidylcholine bilayers to alkaline cations was induced by the incorporation of dolichol. This effect was not observed in the case of non-charged solutes like glucose or in that of alkaline earth cations such as calcium. Permeation of K+ was significantly increased above the phase transition temperature. These results suggest that dolichols may play a role in biological membranes, besides the well-known glycosyl carrier function in the biosynthesis of glycoproteins.

Analysis of dolichol in human tissues by high pressure liquid chromatography.

Dolichol from human liver was shown by reverse-phase high pressure liquid chromatography to consist of a series of homologues ranging in length from 17 to 23 isoprene units. The two major components, corresponding to 19 and 20 isoprene units, respectively, were isolated and identified by mass spectrometry. Dolichyl palmitate, synthesized from liver dolichol, showed an identical series of peaks with longer retention times. Attempts to chromatography dolichyl phosphate under similar conditions were unsuccessful. Dolichol from uterine tissue and several other human tissues showed a shift toward shorter chain length, with a predominance of homologues containing 18 and 19 isoprene units.

Dolichol levels in younger and older rat hearts heterotopically transplanted in younger recipients.

Dolichol (D) levels increase dramatically in older tissue. An understanding of the exchangeability of D between tissues may be essential in order to understand the mechanism of the abnormal accumulation associated with aging. The question was investigated by the use of organ transplantation. D-poor hearts donated by 3-mon-old and D-rich by 22-mon-old male Lewis rats were transplanted heterotopically in 3-mon-old syngenic recipients, whose peripheral tissues and liver were poor in D. Native and transplanted hearts were taken 7 and 21 d after surgery. Native hearts of 3-mon- and 22-mon-old male Lewis rats served as control. D concentration and quantity were higher in older than in younger native hearts as expected. In the transplanted hearts, the quantity of D was unchanged, irrespective of the age of the donor and of the time of transplantation, whereas D concentration increased because of the remarkable disuse atrophy. No changes in D were observed in recipients' tissues. It is concluded that dolichol is not redistributed via circulation from the transplanted heart to the tissues and liver of the younger recipient.