dolichols and prenol

The reduction pathway leading to the formation of dolichol was clarified in 2010 with the identification of SRD5A3, which is the polyprenol reductase. The finding inspired us to reanalyze the length of the major chain of polyprenol and dolichol from several plant leaves, including mangrove plants, as well as from animal and fish livers by 2D-TLC. Polyprenol- and dolichol-derived metabolites such as polyprenylacetone and epoxydolichol were found together with rubber-like prenol. This review focuses on analyses of polyprenol and its derivatives, including recently found epoxypolyprenol and polyprenylacetone. Attention has also been paid to the chromatographic behavior of rubber-like prenol on TLC.

Topics: Animals; Chromatography, Thin Layer; Dolichols; Hemiterpenes; Liver; Pentanols; Plant Leaves

Monolithic silica columns have very low back-pressures and offer several advantages over conventional columns packed with spherical particles, such as high separation efficiency and rapid analysis. In this review, we report the applicability of monolithic silica columns for the analysis of complex hydrophobic metabolites. We have used monolithic columns in HPLC and developed a separation technique for the high-speed and high-resolution analysis of the geometric analogs of natural polyprenols. We also used monolithic columns in supercritical fluid chromatography for the successful separation of the structural isomers of carotenoids after deciding the analytical conditions that were suitable for this separation and have developed a method for profiling biological samples containing complex matrices. We have proved that excellent resolution can be obtained by connecting a number of monolithic columns in series.

Topics: Carotenoids; Chromatography, High Pressure Liquid; Chromatography, Supercritical Fluid; Dolichols; Hemiterpenes; Hydrophobic and Hydrophilic Interactions; Molecular Structure; Pentanols; Silicon Dioxide

We have investigated dolichol synthesis in yeast Pichia pastoris. Growth of these cells on methanol causes peroxisome proliferation and induction of peroxisomal enzymes. Twenty-four hours methanol treatment was sufficient for the appearance of longer-chain dolichols. Less specific oleic acid induction needed 48 h for the synthesis of longer dolichol family with typical one still present. Cells cultured in non-inducing conditions for 48 h did not reveal the presence of additional dolichol family. Peroxisomes purified from oleic acid treated cells synthesize in vitro polyprenols longer by two isoprene residues than those synthesized by microsomal fraction from glucose culture. These observations lead us to suggest that chain length of dolichols synthesized in yeast cell may depend on the carbon and energy source supply which mobilizes metabolic pathways localized to different cellular compartments.

Topics: Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dolichols; Hemiterpenes; Pentanols; Peroxisomes; Pichia; Transferases

The level of cis-prenyl transferase activity has been implicated in controlling the level of biosynthesis of dolichol and dolichol intermediates. In this study, we isolated a cDNA encoding a human CPT (GenBank Accession No. ), which had substantial homology to other CPT isolated from human brain, bacteria, Arabidopsis, and Saccharomyces cerevisiae. Expression of this cDNA in two different insect cell lines confirmed the functionality of the protein in an in vitro assay. Western blot analysis revealed an expressed protein of approximately 38 kDa in HEK293 cells. Overexpression of the protein in HEK293 cells resulted in an increase in the level of total prenol in vivo. Furthermore, product characterization by thin layer chromatography (TLC) confirmed that the major product was a long-chain prenol with a chain length of 95 carbons. These results suggest a regulatory relationship between CPT activity and dolichol biosynthesis, and may implicate CPT in the levels of dolichol-oligosaccharide intermediate biosynthesis.

Topics: Animals; Cell Line; Chromatography, Thin Layer; Cloning, Molecular; DNA, Complementary; Dolichols; Hemiterpenes; Humans; Lipid Metabolism; Lipids; Molecular Weight; Pentanols; Recombinant Proteins; Spodoptera; Transferases

Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

Topics: Animals; Concanavalin A; Dolichols; Drug Resistance; Glycolipids; Glycosylation; Hemiterpenes; Mutation; Oligosaccharides; Oxidoreductases; Pentanols; Trypanosoma brucei brucei; Trypanosomiasis, African

We synthesized three water-soluble biotin-tagged compounds with different prenyl chain lengths, biotinylated farnesal (BF), biotinylated C(55)-polyprenal (BP55), and biotinylated C(80)-polyprenal (BP80), and examined their effects on in vitro dolichol synthesis from farnesyl diphosphate. BF and BP55 did not affect the dolichol synthesis, whereas BP80 inhibited the reduction pathway from polyprenol to dolichol, accompanied by a decrease in the entire polyprenol and dolichol synthesis. Comparison of BP80 with eighteen detergents, including Triton X-100, CHAPS, octylglucoside, deoxycholate, and Tween 80, revealed the specific effect of BP80 on the reduction pathway. On SDS-polyacrylamide gel electrophoresis, BP80 was detected in an associated form with a 50 kDa protein. These results suggest that the reduction of polyprenol to dolichol in the dolichol biosynthetic pathway proceeds with the recognition of the polyprenol chain length by a 50 kDa protein.

Topics: Biotinylation; Carrier Proteins; Dolichols; Hemiterpenes; Oxidation-Reduction; Oxidoreductases; Pentanols; Polyisoprenyl Phosphates; Saccharomyces cerevisiae; Sesquiterpenes

Recently we reported that CHB11-1-3, a Chinese hamster ovary cell mutant defective in glycosylation of asparagine-linked proteins, is defective in the synthesis of dolichol [Quellhorst et al., 343:19-26, 1997: Arch Biochem Biophys]. CHB11-1-3 was found to be in the Lec9 complementation group, which synthesizes polyprenol rather than dolichol. In this paper, levels of various polyprenyl derivatives in CHB11-1-3 are compared to levels of the corresponding dolichyl derivatives in parental cells. CHB11-1-3 was found to maintain near normal levels of Man5GlcNAc2-P-P-polyprenol and mannosylphosphorylpolyprenol, despite reduced rates of synthesis, by utilizing those intermediates at a reduced rate. The Man5GlcNAc2 oligosaccharide attached to prenol in CHB11-1-3 cells and to dolichol in parental cells is the same structure, as determined by acetolysis. Man5GlcNAc2-P-P-polyprenol and Man5GlcNAc5-P-P-dolichol both appeared to be translocated efficiently in an in vitro reaction. Glycosylation of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was normally glycosylated in CHB11-1-3 cells, a large portion of G was underglycosylated, resulting in the addition of either one or no oligosaccharide to G. Addition of a single oligosaccharide occurred randomly rather than preferentially at one of the two sites.

Topics: Animals; Biological Transport; Carbohydrate Sequence; Cell Membrane; CHO Cells; Cricetinae; Dolichols; Electrophoresis, Polyacrylamide Gel; Glycosylation; GTP-Binding Proteins; Hemiterpenes; Lipid Metabolism; Mannose; Methionine; Molecular Sequence Data; Mutation; Pentanols; Polyisoprenyl Phosphate Sugars; Vesicular stomatitis Indiana virus; Viral Proteins

Exposure of rat glioma C6 cells to dolichyl phosphate resulted in cell shrinkage followed by nuclear fragmentation and internucleosomal cleavage of genomic DNA, yielding ladder patterns of oligonucleosomal fragments, all characteristics of apoptosis. This phenomenon occurred in a dose and time dependent manner. Dolichol and prenol failed to induce apoptosis. The inhibitors of N-glycosylation, tunicamycin and swainsonine had no apparent effect on dolichyl phosphate-induced apoptosis. Apoptotic changes were also observed in HL-60 cells, SIRC cells and HeLa cells. Thus, dolichyl phosphate functions as a potential apoptosis inducer as well as an essential carrier lipid in the biosynthesis of N-linked glycoprotein.

Topics: Animals; Apoptosis; DNA; Dolichol Phosphates; Dolichols; Electrophoresis, Agar Gel; Glioma; Glycoproteins; Glycosylation; HeLa Cells; Hemiterpenes; Humans; Kinetics; Pentanols; Rats; Swainsonine; Tumor Cells, Cultured; Tunicamycin

Topics: Chemical Phenomena; Chemistry, Physical; Dolichols; Hemiterpenes; Pentanols; Stereoisomerism; Terminology as Topic; Terpenes