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FEBS Letters Nov 2006We performed reverse-phase thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of polyisoprenoids released by...
We performed reverse-phase thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of polyisoprenoids released by sulfonium-salt cleavage with methyl iodide from Plasmodium falciparum proteins labeled with [3H]FPP or [3H]GGPP and showed that a dolichol of 11 isoprene units is bound to 21-28-kDa protein clusters from trophozoite and schizont stages. The dolichol structure was confirmed by electrospray-ionization mass spectrometry analysis. Treatment with protein synthesis inhibitors and RP-HPLC analysis of the proteolytic digestion products from parasite proteins labeled with [35S]cysteine and [3H]FPP showed that the attachment of dolichol to protein is a post-translational event and probably occurs via a covalent bond to cysteine residues.
Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cysteine; Dolichols; Plasmodium falciparum; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Protozoan Proteins; Spectrometry, Mass, Electrospray Ionization
PubMed: 17084391
DOI: 10.1016/j.febslet.2006.10.042 -
The Biochemical Journal Apr 1988
Review
Topics: Animals; Biological Transport; Diterpenes; Dolichol Phosphates; Dolichols; Humans
PubMed: 3291859
DOI: 10.1042/bj2510001 -
The Journal of Biological Chemistry Mar 1987Incubations 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... (Comparative Study)
Comparative Study
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.
Topics: Animals; Diterpenes; Dolichol Phosphates; Dolichols; Hydrogen-Ion Concentration; Liver; Male; Mevalonic Acid; Microsomes, Liver; NAD; Octoxynol; Ozone; Phosphoric Monoester Hydrolases; Polyethylene Glycols; Rats; Rats, Inbred Strains
PubMed: 3031061
DOI: No ID Found -
Journal of Lipid Research Dec 2013We observed a characteristic shortening of plasma and urinary dolichols in retinitis pigmentosa (RP) patients carrying K42E and T206A mutations in the dehydrodolichol...
We observed a characteristic shortening of plasma and urinary dolichols in retinitis pigmentosa (RP) patients carrying K42E and T206A mutations in the dehydrodolichol diphosphate synthase (DHDDS) gene, using liquid chromatography-mass spectrometry. Dolichol-18 (D18) became the dominant dolichol species in patients instead of dolichol-19 (D19) in normal individuals. The D18/D19 ratio was calculated and used as an index of dolichol length distribution. K42E/K42E and K42E/T206A patients have significantly higher plasma and urinary D18/D19 ratios than K42E and T206A carriers. The ratios of carriers are significantly higher than normal individuals. Receiver operating characteristic (ROC) analysis shows that plasma and urinary D18/D19 ratios can unambiguously discriminate patients from carriers, and carriers from normal individuals. Dolichol analysis also provides evidence that the T206A mutation is RP-causative. The methodologies and procedures used for dolichol profiling are reliable, high throughput, and cost effective. Dolichol profiling, complementary to genotyping, can be readily adapted as a test in the clinic not only for the diagnosis of patients but also for identification of carriers with DHDDS or other genetic mutations that may impair dolichol biosynthesis.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alkyl and Aryl Transferases; Biomarkers; Child; Dolichols; Female; Humans; Male; Middle Aged; Mutation; Retinitis Pigmentosa; Young Adult
PubMed: 24078709
DOI: 10.1194/jlr.M043232 -
Journal of Lipid Research Jul 2007Neuromelanin (NM) isolated from the substantia nigra of the human brain is found to contain a series of dolichoic acids (dol-CA) containing 14-20 isoprene units. This is...
Neuromelanin (NM) isolated from the substantia nigra of the human brain is found to contain a series of dolichoic acids (dol-CA) containing 14-20 isoprene units. This is the first observation of dol-CA in a natural system. Using internally spiked nor-dolichol and nor-dolichoic acid standards, the concentrations of dolichol (dol) and dol-CA present in NM were determined. Remarkably, dol was only four times as abundant as dol-CA in NM. The distribution of dol-CA chains lengths in NM also differed from that of dol, suggesting that the enzyme(s) responsible for the conversion of dol to dol-CA prefer a dolichol substrate containing 19 isoprene units.
Topics: Adult; Animals; Chromatography, High Pressure Liquid; Dolichols; Humans; Melanins; Substantia Nigra; Sus scrofa; Tandem Mass Spectrometry; Terpenes
PubMed: 17446624
DOI: 10.1194/jlr.C700008-JLR200 -
Nature Mar 2020In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer...
In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.
Topics: Biocatalysis; Catalytic Domain; Conserved Sequence; Cryoelectron Microscopy; Dolichol Monophosphate Mannose; Dolichol Phosphates; Endoplasmic Reticulum; Glucose; Glycosyltransferases; In Vitro Techniques; Lipids; Membrane Proteins; Models, Molecular; Mutation; Polyisoprenyl Phosphate Monosaccharides; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 32103179
DOI: 10.1038/s41586-020-2044-z -
The Journal of Biological Chemistry Jul 1981Results are presented here which demonstrate that the rates of [14C]acetate incorporation into cholesterol and dolichol increased 4- to 5-fold as mouse spermatocytes...
Results are presented here which demonstrate that the rates of [14C]acetate incorporation into cholesterol and dolichol increased 4- to 5-fold as mouse spermatocytes matured from the preleptotene to prepuberal pachytene stages. The rate of acetate incorporation into cholesterol then decreased in late pachynema, remained low at all subsequent stages of meiosis, and was very low in mature sperm. In contrast, the rate of acetate incorporation into dolichol remained elevated in late pachytene spermatocytes and round spermatids, then decreased and remained low in mature sperm. The ratio of the rate of [14C]acetate incorporation into dolichol to the rate of incorporation into cholesterol increased during late meiotic prophase and remained high in round spermatids; this altered ratio is further evidence of independent regulation of dolichol and cholesterol synthesis in testes. It was shown previously that normal adult mouse testes incorporated acetate into dolichol at a much higher rate (1.8 to 2.4% of the rate of incorporation into cholesterol) than did testes from sterile W/Wv mice (0.02%) or X-irradiated mice (0.24%). This high rate of acetate incorporation into dolichol in adult testes is now attributed to differentiating spermatocytes, with particularly high rates being observed during pachynema.
Topics: Acetates; Aging; Animals; Cholesterol; Diterpenes; Dolichols; Hydroxymethylglutaryl CoA Reductases; Male; Mice; Sexual Maturation; Spermatocytes; Spermatozoa; Testis
PubMed: 7251590
DOI: No ID Found -
Molecules (Basel, Switzerland) Dec 2023Dolichols are isoprenoid end-products of the mevalonate and 2-methyl-D-erythritol-4-phosphate pathways. The synthesis of dolichols is initiated with the addition of...
Dolichols are isoprenoid end-products of the mevalonate and 2-methyl-D-erythritol-4-phosphate pathways. The synthesis of dolichols is initiated with the addition of several molecules of isopentenyl diphosphate to farnesyl diphosphate. This reaction is catalyzed by a -prenyltransferase and leads to the formation of polyprenyl diphosphate. Subsequent steps involve the dephosphorylation and reduction of the α-isoprene unit by a polyprenol reductase, resulting in the generation of dolichol. The size of the dolichol varies, depending on the number of isoprene units incorporated. In eukaryotes, dolichols are synthesized as a mixture of four or more different lengths. Their biosynthesis is predicted to occur in the endoplasmic reticulum, where dolichols play an essential role in protein glycosylation. In this study, we have developed a selection of aptamers targeting dolichols and enhanced their specificity by incorporating fatty acids for negative selection. One aptamer showed high enrichment and specificity for linear polyisoprenoids containing at least one oxygen atom, such as an alcohol or aldehyde, in the α-isoprene unit. The selected aptamer proved to be a valuable tool for the subcellular localization of polyisoprenoids in the malaria parasite. To the best of our knowledge, this is the first time that polyisoprenoids have been localized within a cell using aptamer-based imaging techniques.
Topics: Animals; Parasites; Diagnostic Imaging; Dolichols; Malaria; Butadienes; Hemiterpenes
PubMed: 38202761
DOI: 10.3390/molecules29010178 -
Biochimie Nov 1988It is possible to interfere with different steps in the dolichol pathway of protein glycosylation and in the processing of asparagine-linked oligosaccharides. Thus some... (Review)
Review
It is possible to interfere with different steps in the dolichol pathway of protein glycosylation and in the processing of asparagine-linked oligosaccharides. Thus some clues about the role of protein-bound carbohydrate can be obtained by comparing the biochemical fates and functions of glycosylated proteins with their non-glycosylated counterparts, or with proteins exhibiting differences in the type of oligosaccharide side chains. Cells infected with enveloped viruses are good systems for studying both aspects of protein glycosylation, since they contain a limited number of different glycoproteins, often with well-defined functions. Tunicamycin, an antibiotic, as well as several sugar analogues have been found to act as inhibitors of protein glycosylation by virtue of their anti-viral properties. They interfere with various steps in the dolichol pathway resulting in a lack of functional lipid-linked oligosaccharide precursors. Compounds that interfere with oligosaccharide trimming represent a second generation of inhibitors of glycosylation. They are glycosidase inhibitors that interfere with the processing glucosidases and mannosidases and, as a result, the conversion of high-mannose into complex-type oligosaccharides is blocked. Depending upon the compound used, glycoproteins contain glucosylated-high-mannose, high-mannose or hybrid oligosaccharide structures instead of complex ones. The biological consequences of the alterations caused by the inhibitors are manifold: increased susceptibility to proteases, improper protein processing and misfolding of polypeptide chains, loss of biological activity and alteration of the site of virus-budding, to name but a few.
Topics: Animals; Antiviral Agents; Carbohydrates; Dolichols; Glycoproteins; Glycosylation; Oligosaccharides; Protein Synthesis Inhibitors; Tunicamycin; Viral Proteins
PubMed: 3149521
DOI: 10.1016/0300-9084(88)90290-8 -
British Journal of Experimental... Oct 1986The content of dolichol and dolichyl phosphate in various human organs was analysed using autopsy samples. The reliability of these measurements was demonstrated by...
The content of dolichol and dolichyl phosphate in various human organs was analysed using autopsy samples. The reliability of these measurements was demonstrated by comparison with values for fresh biopsy material. Dolichol was present in all tissues investigated and the content was highest in the adrenal gland, pancreas, pituitary gland, testis and thyroid gland, ranging between 1.5 and 7.1 mg/g tissue. Dolichyl-P was detected in the various organs in highly variable amounts, ranging between 1 and 9% of the total dolichol content. While the main pattern of isoprene composition for dolichol and dolichyl-P was similar in individual organs, some variation was observed between tissues. Dolichol represents the largest lipid component in the pituitary gland, exceeding the total phospholipid content. The high concentrations of dolichol and dolichyl-P in human organs indicate that these lipids may play important roles in physiological and pathological cellular functions.
Topics: Adrenal Glands; Aged; Diterpenes; Dolichol Phosphates; Dolichols; Humans; Male; Pituitary Gland; Polyisoprenyl Phosphates; Testis; Thyroid Gland
PubMed: 3641633
DOI: No ID Found