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&nbsp, Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the &omega, 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.

The congenital disorders of glycosylation (CDG) are inborn errors of metabolism with a great genetic heterogeneity. Most CDG are caused by defects in the N-glycan biosynthesis, leading to multisystem phenotypes. However, the occurrence of tissue-restricted clinical symptoms in the various defects in dolichol-phosphate-mannose (DPM) synthesis remains unexplained. To deepen our understanding of the tissue-specific characteristics of defects in the DPM synthesis pathway, we investigated N-glycosylation and O-mannosylation in skeletal muscle of three DPM3-CDG patients presenting with muscle dystrophy and hypo-N-glycosylation of serum transferrin in only two of them. In the three patients, O-mannosylation of alpha-dystroglycan (αDG) was strongly reduced and western blot analysis of beta-dystroglycan (βDG) N-glycosylation revealed a consistent lack of one N-glycan in skeletal muscle. Recently, defective N-glycosylation of βDG has been reported in patients with mutations in guanosine-diphosphate-mannose pyrophosphorylase B (GMPPB). Thus, we suggest that aberrant O-glycosylation of αDG and N-glycosylation of βDG in skeletal muscle is indicative of a defect in the DPM synthesis pathway. Further studies should address to what extent hypo-N-glycosylation of βDG or other skeletal muscle proteins contribute to the phenotype of patients with defects in DPM synthesis. Our findings contribute to our understanding of the tissue-restricted phenotype of DPM3-CDG and other defects in the DPM synthesis pathway.

N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins, Theileria equi and Cytauxzoon felis add (NAcGlc)2-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)2 without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.

Increasing numbers of congenital disorders of glycosylation (CDG) have been reported recently resulting in an expansion of the phenotypes associated with this group of disorders. SRD5A3 codes for polyprenol reductase which converts polyprenol to dolichol. This is a major pathway for dolichol biosynthesis for N-glycosylation, O-mannosylation, C-mannosylation, and GPI anchor synthesis. We present the features of five individuals (three children and two adults) with mutations in SRD5A3 focusing on the variable eye and skin involvement. We compare that to 13 affected individuals from the literature including five adults allowing us to delineate the features that may develop over time with this disorder including kyphosis, retinitis pigmentosa, and cataracts. © 2016 Wiley Periodicals, Inc.

We reported an oligosaccharide diphosphodolichol (DLO) diphosphatase (DLODP) that generates dolichyl-phosphate and oligosaccharyl phosphates (OSPs) from DLO in vitro. This enzyme could underlie cytoplasmic OSP generation and promote dolichyl-phosphate recycling from truncated endoplasmic reticulum (ER)-generated DLO intermediates. However, during subcellular fractionation, DLODP distribution is closer to that of a Golgi apparatus (GA) marker than those of ER markers. Here, we examined the effect of brefeldin A (BFA), which fuses the GA with the ER on OSP metabolism. In order to increase the steady state level of truncated DLO while allowing formation of mature DLO (Glc3Man9GlcNAc2-PP-dolichol), dolichyl-P-mannose Man7GlcNAc2-PP-dolichol mannosyltransferase was partially downregulated in HepG2 cells. We show that BFA provokes GA endomannosidase trimming of Glc3Man9GlcNAc2-PP-dolichol to yield a Man8GlcNAc2-PP-dolichol structure that does not give rise to cytoplasmic Man8GlcNAc2-P. BFA also strikingly increased OSP derived from mature DLO within the endomembrane system without affecting levels of Man7GlcNAc2-PP-dolichol or cytoplasmic Man7GlcNAc2-P. The BFA-provoked increase in endomembrane-situated OSP is sensitive to nocodazole, and BFA causes partial redistribution of DLODP activity from GA- to ER-containing regions of density gradients. These findings are consistent with BFA-provoked microtubule-dependent GA-to-ER transport of a previously reported DLODP that acts to generate a novel endomembrane-situated OSP population.

Oligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co(2+)-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [(3)H]OSP release from [(3)H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc2-PP-solanesol is hydrolyzed to yield GlcNAc2-P and inhibits [(3)H]OSP release from [(3)H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction.

The trappc11 mutation in zebrafish causes a stressed UPR, leading to fatty liver disease. This phenotype is not due to a trafficking defect but instead results from N-glycosylation defects. Hypoglycosylation and lipid accumulation are also found in patient cells with knockdown or mutated TRAPPC11 genes, suggesting a common etiology with zebrafish., Activation of the unfolded protein response (UPR) can be either adaptive or pathological. We term the pathological UPR that causes fatty liver disease a “stressed UPR.” Here we investigate the mechanism of stressed UPR activation in zebrafish bearing a mutation in the trappc11 gene, which encodes a component of the transport protein particle (TRAPP) complex. trappc11 mutants are characterized by secretory pathway defects, reflecting disruption of the TRAPP complex. In addition, we uncover a defect in protein glycosylation in trappc11 mutants that is associated with reduced levels of lipid-linked oligosaccharides (LLOs) and compensatory up-regulation of genes in the terpenoid biosynthetic pathway that produces the LLO anchor dolichol. Treating wild-type larvae with terpenoid or LLO synthesis inhibitors phenocopies the stressed UPR seen in trappc11 mutants and is synthetically lethal with trappc11 mutation. We propose that reduced LLO level causing hypoglycosylation is a mechanism of stressed UPR induction in trappc11 mutants. Of importance, in human cells, depletion of TRAPPC11, but not other TRAPP components, causes protein hypoglycosylation, and lipid droplets accumulate in fibroblasts from patients with the TRAPPC11 mutation. These data point to a previously unanticipated and conserved role for TRAPPC11 in LLO biosynthesis and protein glycosylation in addition to its established function in vesicle trafficking.

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 ?-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 ?-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 ?-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.

Dolichyl phosphate-linked mono- and oligosaccharides (DLO) are essential intermediates in protein N-glycosylation, C- and O-mannosylation and GPI anchor biosynthesis. While many membrane proteins in the endoplasmic reticulum (ER) involved in the assembly of DLOs are known, essential proteins believed to be required for the transbilayer movement (flip-flopping) and proteins potentially involved in the regulation of DLO synthesis remain to be identified.The synthesis of a series of Dol-P derivatives composed of citronellyl-based photoprobes with benzophenone groups equipped with alkyne moieties for Huisgen "click" chemistry is now described to utilize as tools for identifying ER proteins involved in regulating the biosynthesis and transbilayer movement of lipid intermediates. In vitro enzymatic assays were used to establish that the photoprobes contain the critical structural features recognized by pertinent enzymes in the dolichol pathway. ER proteins that photoreacted with the novel probes were identified by MS.The potential of the newly designed photoprobes, m-PAL-Cit-P and p-PAL-Cit-P, for identifying previously unidentified Dol-P-interacting proteins is supported by the observation that they are enzymatically mannosylated by Man-P-Dol synthase (MPDS) from Chinese Hamster Ovary (CHO) cells at an enzymatic rate similar to that for Dol-P. MS analyses reveal that DPM1, ALG14 and several other yeast ER proteins involved in DLO biosynthesis and lipid-mediated protein O-mannosylation photoreacted with the novel probes.The newly-designed photoprobes described in this paper provide promising new tools for the identification of yet to be identified Dol-P interacting ER proteins in yeast and mammalian cells, including the Dol-P flippase required for the "re-cycling" of the glycosyl carrier lipid from the lumenal monolayer of the ER to the cytoplasmic leaflet for new rounds of DLO synthesis.

Mangrove plants are known to produce secondary metabolites including isoprenoid and long chain polyisoprenoid. SonneratiaalbaandS. caseolaris, are true mangroves that contain alkane (C25-C33), triterpenoid, phytosterol, dolichol, polyprenol, and polyprenyl acetone. Here we reported new compounds of probably epoxy polyprenol and epoxy dolichol from S. albaandS. caseolarisold leaves using two-dimensional thin layer chromatography (2D-TLC). In the S. albaold leaves, having the occurrence of polyprenol, dolichol, epoxy polyprenol, and epoxy dolichol with a dominating pattern of polyprenol. A similar result obtained in the S. caseolarisold leaves, displaying the presence of polyprenol, dolichol, epoxy polyprenol, and epoxy dolichol with a dominating carbon chain length of dolichol. By contrast, no epoxy polyprenol or epoxy dolichol were detected in S. alba, and S. caseolarisleaves. The present study indicated a new type of polyisoprenoid distribution and further investigation is needed to clarify the chemical structure of this newcompound.

Although there are similarities in the core steps of the secretion pathway from yeast to higher eukaryotes, significant functional differences exist even among diverse yeast species. Here, we used next-generation sequencing to identify two mutations in the Kluyveromyces lactis KlSEC59 gene, encoding dolichol kinase (DK), which are responsible for an enhanced secretion phenotype in a previously isolated mutant, MD2/1-9. Compared with the temperature-sensitive Saccharomyces cerevisiae sec59-1 mutant, which exhibits reduced N-glycosylation and decreased secretory efficacy, the identified K. lactis DK mutations had fewer effects on glycosylation, as well as on survival at high temperature and cell wall integrity. Moreover, despite some glycosylation defects, double DK mutations (G405S and I419S) in the K. lactis mutant strain demonstrated three times the level of recombinant α-amylase secretion as the wild-type strain. Overexpression of potential suppressors KlMNN10, KlSEL1, KlERG20, KlSRT1, KlRER2, KlCAX4, KlLPP1 and KlDPP1 in the DK-mutant strain restored carboxypeptidase Y glycosylation to different extents and, with the exception of KISRT1, reduced α-amylase secretion to levels observed in wild-type cells. Our results suggest that enhanced secretion related to reduced activity of mutant DK in K. lactis results from mild glycosylation changes that affect activity of other proteins in the secretory pathway.

The occurrence of polyisoprenoids has been well documented over the past 60 years. Polyisoprenoids are lipophilic polymers of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) which are linked via a class of enzymes known as cis-prenyltransferases (CPTs). In Solanum lycopersicum, only dolichols of 75-85 carbons are known to exist, as they play a role in the post-translational modification of proteins. Polyprenols, the other major class of polyisoprenoids has yet to be identified in tomato. Using S. lycopersicum, which contains a seven member CPT gene family, two CPTs were studied for their role in the biosynthesis of polyprenols and dolichols. One such CPT (SlCPT5) was identified for its role in catalyzing the formation of polyprenols of 45-55 carbons. Using a combination of in vitro and in vivo analyses, it was determined that SlCPT5 resides in the plastid stroma where it catalyzes the biosynthesis of medium-chain polyprenols using geranylgeranyl diphosphate (GGPP) as a substrate. Furthermore, the use of RNAi-mediate knockdown of SlCPT5 tomato plants demonstrated that an absence of polyprenols can lead to membrane and protein instability in the plastid and can hinder photosynthetic efficiency. When establishing a heterologous expression system for the biosynthesis of polyprenols, it was discovered that SlCPT5 exhibits moonlighting activity as it is also involved in the biosynthesis of glycinoprenols. The glycinoprenols were of similar medium-chain lengths (40-50 carbons) but contained three saturated IPP units. Lastly, the above heterologous expression system was configured for the synthesis of dolichols by over-expressing the SlCPT3 gene. It was shown that SlCPT3 together with its partner protein, CPT binding protein (SlCPTBP), catalyze the formation of polyprenols of 75-85 carbons in length. The addition of a polyprenol reductase (SlPPRD) results in the biosynthesis of dolichols. In summary, this study demonstrates that two S. lycopersicum CPTs, namely SlCPT5 and SlCPT3, are required for the biosynthesis of medium- and long-chain polyprenols. NSERC PGS-D, OMAFRA 2022-05

Discovered 40 years ago, the Lec5 glycosylation mutant cell line has a complex recessive genotype and is characterized by accumulation of lipid-linked oligosaccharide assembly intermediates, reduced conversion of polyprenols to dolichols, and an unusual phenotypic dependence upon cell culture conditions such as temperature, plating density and medium quality. The heritable defect in Lec5 is unknown. Here we demonstrate an unexpected epigenetic basis for Lec5, with a surprising linkage to increased expression of homeobox genes, which in turn is associated with increased transcription of cholesterol biosynthesis genes. These results suggest testable hypotheses for the biochemical abnormalities of the Lec5 mutant.

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high-molecular-weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis-1,4-isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis-prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT-like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis-1,4-isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1-RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low-expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS-based comparison of latex proteomes from TkCPTL1-RNAi plants and T. koksaghyz wild-types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.

Guayule (Parthenium argentatum) is a perennial shrub in the Asteraceae family and synthesizes a high quality, hypoallergenic cis-1,4-polyisoprene (or natural rubber; NR). Despite its potential to be an alternative NR supplier, the enzymes for cis-polyisoprene biosynthesis have not been comprehensively studied in guayule. Recently, implications of the protein complex involving cis-prenyltransferases (CPTs) and CPT-binding proteins (CBPs) in NR biosynthesis were shown in lettuce and dandelion, but such protein complexes have yet to be examined in guayule. Here we identified four guayule genes – three PaCPTs (PaCPT1-3) and one PaCBP, whose protein products form PaCPT/PaCBP complexes. Co-expression of both PaCBP and each of the PaCPTs could complemented the dolichol (a short cis-polyisoprene)-deficient yeast, whereas the individual expressions could not. Microsomes from the PaCPT/PaCBP-expressing yeast efficiently incorporated 14C-isopentenyl diphosphate into dehydrodolichyl diphosphates. Furthermore, co-immunoprecipitation and split-ubiquitin yeast 2-hybrid assays using PaCPTs and PaCBP confirmed the formation of protein complexes. Of the three PaCPTs, transcriptomics analysis indicated that the protein complex formed by PaCPT3 and PaCBP is likely to be the key component in guayule NR biosynthesis. The comprehensive analyses of these PaCPTs and PaCBP here provide the foundational knowledge to generate a high NR-yielding guayule.

Bintaro (Cerbera manghas L, Apocynaceae) is a mangrove plant that has been described to have several important biological properties and pharmacological activities. In this study, the vegetative organs (leaves, roots, and stems) and the generative tissues (flowers and fruits) were analysed using two-plate thin layer chromatography to investigate the polyisoprenoid (polyprenol or dehydrodolichol and dolichol) profiles. It was found that in the vegetative tissues, there was two types (I and II) of polyprenols and dolichols detected. Type-I having a prevalence of dolichols over dehydrodolichols, was traced in the roots and stems, and type-II, having an occurrence of both polyprenols and dolichols, was found in the leaves. In contrast, in the generative organs only type-I was detected. Taken together, four out of five tissues tested showed a predominance of dolichols over polyprenols, suggesting the existence of polyprenol reductase in C. manghas. The varying polyisoprenoids in the different tissues of C. manghas indicating that these compounds have chemotaxonomic significance in C. manghas.Bintaro (Cerbera manghas L, Apocynaceae) is a mangrove plant that has been described to have several important biological properties and pharmacological activities. In this study, the vegetative organs (leaves, roots, and stems) and the generative tissues (flowers and fruits) were analysed using two-plate thin layer chromatography to investigate the polyisoprenoid (polyprenol or dehydrodolichol and dolichol) profiles. It was found that in the vegetative tissues, there was two types (I and II) of polyprenols and dolichols detected. Type-I having a prevalence of dolichols over dehydrodolichols, was traced in the roots and stems, and type-II, having an occurrence of both polyprenols and dolichols, was found in the leaves. In contrast, in the generative organs only type-I was detected. Taken together, four out of five tissues tested showed a predominance of dolichols over polyprenols, suggesting the existence of polyprenol reductase in C. manghas. The varying polyisoprenoids in the different tissues of C. man...

Polyisoprenoid (polyprenols or dehydrodolichols and dolichols) content and composition of four true mangrove plants namely Excoecaria agallocha, Lumnitzera racemosa, Rhizophora apiculata, and R. lamarkii were investigated with particular emphasis on leaf senescence. The distribution and occurrence of polyisoprenoids was analyzed using the two-plate thin layer chromatography (2P-TLC) method. The age of the leaves was divided into young leaf, leaf, and yellow leaf. In the leaves, the distribution of polyisoprenoid was found to show the presence of both polyprenols and dolichols. The occurrence of ficaprenols (C50–C55) was observed in young leaves and leaves of the mangrove samples studied. On the other hand, dolichol contents were not altered in the leaves with senescence. By contrast, longer polyprenols were found in yellow leaves of E. agallocha, L. racemosa, and R. apiculata. In addition, it should be noted that there were changes to the polyprenols in leaf senescence identified. Furthermore, the formation of ficaprenols, longer polyprenols, and dolichols was modulated in the mangrove plants. These findings suggest that the accumulated polyprenols in the yellow leaves may be due to the catalyzation process that converts ficaprenols into longer polyprenols.

Major polyisoprenoid alcohols of mangrove plants were dolichols. Polyprenol reductase, therefore, may be active in mangrove plant leaves to catalyze the reduction of polyprenol to dolichol. Genomic sequence and homology-based PCRs method of mangrove plant Kandelia obovata have resulted in the cloning of predicted polyprenol reductase. The partial gene termed KoPPRD1 (1 192 bp, coding for 382 amino acids). The deduced amino acid sequence of KoPPRD1 showed significant similarity to known plant polyprenol reductase with the highest homology to Ricinus communis polyprenol reductase 2 (62 %). This result showed that the fragment gene of KoPPRD1 might encode polyprenol reductase gene. To clarify the relationship of KoPPRD1 to other polyprenol reductase genes, the phylogenetic analysis was constructed using ClustalW ver. 2.0 followed by drawing with TreeView based on a neighbor-joining method. Phylogenetic tree shows that KoPPRD1 join with R. communis polyprenol reductase 2, Ipomea nil polyprenol reductase 2, and Populus euphratica polyprenol reductase 2 branches, suggesting that KoPPRD1 gene is a new member of plant polyprenol reductase.Major polyisoprenoid alcohols of mangrove plants were dolichols. Polyprenol reductase, therefore, may be active in mangrove plant leaves to catalyze the reduction of polyprenol to dolichol. Genomic sequence and homology-based PCRs method of mangrove plant Kandelia obovata have resulted in the cloning of predicted polyprenol reductase. The partial gene termed KoPPRD1 (1 192 bp, coding for 382 amino acids). The deduced amino acid sequence of KoPPRD1 showed significant similarity to known plant polyprenol reductase with the highest homology to Ricinus communis polyprenol reductase 2 (62 %). This result showed that the fragment gene of KoPPRD1 might encode polyprenol reductase gene. To clarify the relationship of KoPPRD1 to other polyprenol reductase genes, the phylogenetic analysis was constructed using ClustalW ver. 2.0 followed by drawing with TreeView based on a neighbor-joining method. Phylogenetic tree shows that KoPPRD1 join with R. communis polyprenol reductase 2, Ipomea nil polyprenol reductase 2, an...

Dolichol is a membrane lipid, which carries monnosaccharides and glycans for N-linked protein glycosylation and glycosylphosphatidylinositol-anchor biosynthesis occuring in endoplasmic reticulum. Its structure is composed of isoprenoid units. Dolichol is present in all tissues and in most of the membrane organelles of eukaryotic cells. Recently some types of congenital disorders of glycosylation have been described as a consequence of dolichol biosynthesis and metabolism defects, which are not detectable by standard methods. The aim of this diploma thesis was to analyze dolichol content in urine and in different tissues from patients with deficiency in dolichol biosynthesis by mass spectrometry and to study the impact of these defects on energetic metabolism. Biological material for this study consisted of urine samples from 76 controls with age ranging from 1 months to 81 years, 6 patients with congenital disorders of glycosylation and 43 patients with suspicion of congenital disorder of glycosylation; samples of frontal cortex, liver, muscle and heart tissues from 2 patients with mutation in NUS1 gene and controls. Urine samples were stored at -20 řC and tissue homogenates were stored in -80 řC until analysis. Lipid fraction after extraction was separated by liquid chromatography. Dolichols were...

Dolichol phosphate mannose synthase (DPMS) is a key enzyme in N- and O-linked glycosylations and glycosylphosphatidylinositol (GPI)-anchor synthesis. DPMS generates DPM, the substrate for mentioned processes, by the transfer of mannosyl residue from GDP-Man to dolichol phosphate. Here we describe the role of DPMS for Candida albicans physiology with emphasis on the cell wall composition and morphogenesis.C. albicans genes for DPMS subunits were cloned, tagged and expressed in Saccharomyces cerevisiae. The C. albicans strains with controlled expression of DPM genes were constructed and analyzed. Gene expression and enzyme activities were measured using RT-PCR and radioactive substrate. Sensitivities against chemical agents were tested with microdilution method. The composition of the cell wall was estimated by HPLC. Glycosylation status of the marker protein was analyzed by Western blot. Morphological differentiation of the strains was checked on the media promoting hyphae and chlamydospore formation.We demonstrate that C. albicans DPMS consists of three interacting subunits, among which Dpm1 and Dpm3 are indispensable, whereas Dpm2 increases enzymatic activity. Lowered expression of DPMS genes results in decreased DPMS activity, increased susceptibility to cell wall perturbing agents and in altered cell wall composition. Mutants Tetp-DPM1 and Tetp-DPM3 show defective protein glycosylation and are impaired in hyphae and chlamydospore formation.DPMS from C. albicans, opposite to S. cerevisiae, belongs to the family of DPMS with multimeric protein structure.This work provides important data about factors required for a proper protein glycosylation and for morphogenesis of pathogenic yeast C. albicans.

4-Nerolidylcatechol ( 1 ) is an abundant antiplasmodial metabolite that is isolated from Piper peltatum roots. O -Acylation or O -alkylation of compound 1 provides derivatives exhibiting improved stability and significant in vitro antiplasmodial activity. The aim of this work was to study the in vitro inhibition of hemozoin formation, inhibition of isoprenoid biosynthesis in Plasmodium falciparum cultures, and in vivo antimalarial activity of several 4-nerolidylcatechol derivatives. 1,2- O , O -Diacetyl-4-nerolidylcatechol ( 2 ) inhibited in vitro hemozoin formation by up to 50%. In metabolic labeling studies using [1-( n )- 3 H]geranylgeranyl pyrophosphate, diester 2 significantly inhibited the biosynthesis of isoprenoid metabolites ubiquinone 8 , menaquinone 4 , and dolichol 12 in cultures of P. falciparum 3D7. Similarly, 2- O -benzyl-4-nerolidylcatechol ( 3 ) significantly inhibited the biosynthesis of dolichol 12 . P. falciparum in vitro protein synthesis was not affected by compounds 2 or 3 . At oral doses of 50 mg per kg of body weight per day, compound 2 suppressed Plasmodium berghei NK65 in infected BALB/c mice by 44%. This in vivo result for derivative 2 represents marked improvement over that obtained previously for natural product 1 . Compound 2 was not detected in mouse blood 1 h after oral ingestion or in mixtures with mouse blood/blood plasma in vitro . However, it was detected after in vitro contact with human blood or blood plasma. Derivatives of 4-nerolidylcatechol exhibit parasite-specific modes of action, such as inhibition of isoprenoid biosynthesis and inhibition of hemozoin formation, and they therefore merit further investigation for their antimalarial potential.

A defect in the assembly of the oligosaccharide donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)) for N-linked glycosylation causes hypoglycosylation of proteins by the oligosaccharyltransferase (OST). Mammalian cells express two OST complexes that have different catalytic subunits (STT3A or STT3B). We monitored glycosylation of proteins in asparagine-linked glycosylation 6 (ALG6) deficient cell lines that assemble Dol-PP-GlcNAc(2)Man(9) as the largest oligosaccharide donor. Based upon pulse labeling experiments, 30-40% of STT3A-dependent glycosylation sites and 20% of STT3B-dependent sites are skipped in ALG6-congenital disorders of glycosylation fibroblasts supporting previous evidence that the STT3B complex has a relaxed preference for the fully assembled oligosaccharide donor. Glycosylation of STT3B-dependent sites was more severely reduced in the ALG6 deficient MI8-5 cell line. Protein immunoblot analysis and RT-PCR revealed that MI8-5 cells express 2-fold lower levels of STT3B than the parental Chinese hamster ovary cells. The combination of reduced expression of STT3B and the lack of the optimal Dol-PP-GlcNAc(2)Man(9)Glc(3) donor synergize to cause very severe hypoglycosylation of proteins in MI8-5 cells. Thus, differences in OST subunit expression can modify the severity of hypoglycosylation displayed by cells with a primary defect in the dolichol oligosaccharide assembly pathway.

Contains fulltext : 155350.pdf (Publisher’s version ) (Open Access) Congenital disorders of glycosylation (CDG) comprise a group of inborn errors of metabolism with abnormal glycosylation of proteins and lipids. Patients with defective protein N-glycosylation are identified in routine metabolic screening via analysis of serum transferrin glycosylation. Defects in the assembly of the dolichol linked Glc(3)Man(9)GlcNAc(2) glycan and its transfer to proteins lead to the (partial) absence of complete glycans on proteins. These defects are called CDG-I and are located in the endoplasmic reticulum (ER) or cytoplasm. Defects in the subsequent processing of protein bound glycans result in the presence of truncated glycans on proteins. These defects are called CDG-II and the enzymes involved are located mainly in the Golgi apparatus. In recent years, human defects have been identified in dolichol biosynthesis genes within the group of CDG-I patients. This has increased interest in dolichol metabolism, has resulted in specific recognizable clinical symptoms in CDG-I and has offered new mechanistic insights in dolichol biosynthesis. We here review its biosynthetic pathways, the clinical and biochemical phenotypes in dolichol-related CDG defects, up to the formation of dolichyl-P-mannose (Dol-P-Man), and discuss existing evidence of regulatory networks in dolichol metabolism to provide an outlook on therapeutic strategies.

To verify the idea that extracellular free oligosaccharides might be able to reflect the functional status of the endoplasmic reticulum (ER) and lysosomal-endosomal system, HPLC-profiles of serum-derived free oligosaccharides (FOS) in human healthy aging, acute myeloproliferative neoplasms, and cardiovascular pathologies were compared with intracellular glycans. After plasma deproteinization and FOS purification the oligosaccharides were labelled with anthranilic acid, separated into the neutral and charged with QAE Sephadex (Q25-120) chromatography and analysed using high-performance liquid chromatography (HPLC). The charged FOS were digested with a sialidase and compared with free oligosaccharides from transferrin for structural decoding. HPLC-profiles of serum-derived FOS revealed mild delay of the dolichol phosphate cycle in ER, moderate intensification of ER-associated degradation (ERAD) and degradation in endosomal-lysosomal system with aging; an inhibition of the dolichol phosphate cycle, intensification of ERAD and increasing of lysosomal exocytosis in acute myeloproliferative neoplasms; intensification of ERAD and glycocojugate degradation with endosomal-lysosomal system in cardiovascular diseases. As serum free oligosaccharides are able to reflect specifically perturbations in ER and endosomal-lysosomal system under wide range of stressors they can serve as extracellular markers of functionality of these organelles.

All ER protein glycosylation reactions occur in the lumen and often involve lumenal mannosylation and glucosylation steps in which mannose and glucose residues are sourced from the glycolipids mannosyl- and glucosyl-phosphoryl dolichol. Paradoxically, these two lipids are synthesized on the cytoplasmic face of the ER and must therefore be flipped across the ER membrane to provide a source of lumenal mannose and glucose. The molecular identities of the specific transporters that are needed to facilitate the transbilayer movement of MPD and GPD across the ER membrane at a physiological rate remain unknown. Taking advantage of the fact that not all N-glycosylation-competent organisms have glucose in their N-glycan precursor, we implemented a bioinformatics approach for assignment of protein function, namely phylogenetic profiling. Using this procedure, we identified a number of polytopic ER membrane proteins as GPD scramblase candidates in yeast.

The widespread occurrence of polyprenols throughout the plant kingdom is well documented. These compounds are believed to be assembled by a class of enzymes designated as cis-prenyltransferases (CPTs), which are encoded by small, yet largely uncharacterized, CPT gene families in plants. This research aimed to identify and characterize the CPT from Solanum lycopersicum (tomato) responsible for polyprenol synthesis. RNAi-mediated knockdown of one member of the tomato CPT family (SlCPT5) reduced polyprenols in leaves by ~70%. Assays with recombinant SlCPT5 determined that the enzyme synthesizes polyprenols of approximately C50-C55 in length and accommodates a variety of trans-prenyl diphosphate precursors as substrates. Introduction of SlCPT5 into the polyprenol-deficient yeast rer2∆ mutant resulted in the accumulation of C55 polyprenols in yeast cells, restored proper protein N-glycosylation, and rescued the temperature sensitive growth phenotype associated with its polyprenol deficiency. Finally, site-directed mutagenesis of SlCPT5 identified a suite of amino acids that are catalytically essential. Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA). Natural Sciences and Engineering Research Council of Canada (NSERC). Canada Foundation for Innovation (CFI).

International audience; Citronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and tested along with undecaprenyl compounds for their ability to inhibit the release of [(3)H]OSP from [(3)H]DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) compounds were 50-500 fold more potent than their citronellyl (C10)-based counterparts, indicating that the alkyl chain length is important for activity. The relative potency of the compounds within the citronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold more potent than citronellyl-PP-GlcNAc2 and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-GlcNAc and solanesyl-PP-GlcNAc2 were 4 and 8 fold more potent, respectively, than solanesyl diphosphate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds, diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP activity.

Congenital disorders of glycosylation (CDGs) are a diverse category of diseases caused by the inability to glycosylate glycoproteins, proteoglycans, and/or glycolipids due to abnormal glycan synthesis, transfer or post-glycosylation processing. Normal glycosylation of protein is a diverse process, typically with glycan chains being added to asparagine (N-linked) or threonine/serine (O-linked). Normal glycosylation is important for intracellular trafficking, protein folding, and protein–protein interactions. These protean disorders manifest relating to the glycoproteins affected. Stereotypic symptoms of CDG are dictated by the most common CDG, PMM2-CDG (CDG-Ia), and include neurologic dysfunction, cerebellar atrophy, nipple inversion, abnormal fat pad distribution, coagulopathy, and hyperinsulinemic hypoglycemia. Because of the diversity of disease, CDG should be suspected in any child with unexplained neurologic or multisystem organ involvement. Diagnostic testing for CDG includes transferrin glycosylation analysis, N- and O-glycan analysis, flow cytometry analysis of cell surface markers, immunohistochemistry staining in muscle biopsy and molecular testing, but no one test is completely sensitive for all subtypes of CDG.

N-glycosylation, the covalent attachment of glycans to select protein target Asn residues, is a post-translational modification performed by all three domains of life. In the halophilic archaea Haloferax volcanii, in which understanding of this universal protein-processing event is relatively well-advanced, genes encoding the components of the archaeal glycosylation (Agl) pathway responsible for the assembly and attachment of an N-linked pentasaccharide have been identified. As elsewhere, the N-linked glycan is assembled on phosphodolichol carriers before transfer to target Asn residues. However, as little is presently known of the Hfx. volcanii Agl pathway at the protein level, the seemingly unique ability of Archaea to use dolichol phosphate (DolP) as the glycan lipid carrier, rather than dolichol pyrophosphate used by eukaryotes, remains poorly understood. With this in mind, a chemoenzymatic approach was taken to biochemically study AglG, one of the five glycosyltransferases of the pathway. Accordingly, a novel regio- and stereoselective reduction of naturally isolated polyprenol gave facile access to S-dolichol via asymmetric transfer hydrogenation under very mild conditions. This compound was used to generate glucose-charged DolP, a precursor of the N-linked pentasaccharide, as well as DolP-glucose-glucuronic acid and DolP-glucuronic acid. AglG, purified from Hfx. volcanii membranes in hypersaline conditions, like those encountered in situ, was subsequently combined with uridine diphosphate (UDP)-glucuronic acid and DolP-glucose to yield DolP-glucose-glucuronic acid. The in vitro system for the study of AglG activity developed here represents the first such tool for studying halophilic glycosyltransferases and will allow for a detailed understanding of archaeal N-glycosylation.

SUMMARY Dolichol phosphate (Dol-P) serves as a carrier of complex polysaccharides during protein glycosylation. DolP is synthesized by the phosphorylation of dolichol or the monodephosphorylation of dolichol pyrophosphate (Dol-PP); however, the enzymes that catalyze these reactions remain unidentified in Arabidopsis thaliana. We performed a genome-wide search for cytidylyltransferase motif-containing proteins in Arabidopsis, and found that At3g45040 encodes a protein homologous with Sec59p, a dolichol kinase (DOK) in Saccharomyces cerevisiae. At3g45040, designated AtDOK1, complemented defects in the growth and N-linked glycosylation of the S. cerevisiae sec59 mutant, suggesting that AtDOK1 encodes a functional DOK. To characterize the physiological roles of AtDOK1 in planta, we isolated two independent lines of T-DNAtagged AtDOK1 mutants, dok1-1 and dok1-2. The heterozygous plants showed developmental defects in male and female gametophytes, including an aberrant pollen structure, low pollen viability, and short siliques. Additionally, the mutations had incomplete penetrance. These results suggest that AtDOK1 is a functional DOK required for reproductive processes in Arabidopsis.

There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae.