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Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy and movement disorder. We evaluated a large cohort of patients (n = 25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor and ataxia. Later in the disease course, they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibres and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders.

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.

Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for posttranslational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans and male sterility in plants. Until now, the mechanisms underlying Dol accumulation remain elusive. In this report, we have analyzed the natural variation of the accumulation of Dols and six other isoprenoids among more than 120 Arabidopsis thaliana accessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone, and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis - the AT2G17570 gene encoding a long searched for cis-prenyltransferase (CPT3) and the AT1G52460 gene encoding an α/β-hydrolase (ABH) - is experimentally confirmed. These data will help to generate Dol-enriched plants which might serve as a remedy for Dol-deficiency in humans. This article is protected by copyright. All rights reserved.

Living things produce secondary metabolites, one of which is abundant in plants, namely polyisoprenoid alcohol compounds. Polyisoprenoids are also found in plants with different chain lengths. The distribution of polyisoprenoid compounds (dolichol and polyprenol) was found in the leaf and stem tissues of Pinus merkusii strains derived from Aceh, Tapanuli and Kerinci. The extracted samples were analyzed by the two-dimensional thin-layer chromatography (2D-TLC) method. The distribution of dolichol and polyprenol compounds on the stems and leaves of Aceh, Tapanuli and Kerinci pines provided a type II categorization, both dolichol and polyprenol were traced in pine tissues. Total lipids in stem tissue ranged from 564.4-58.9 mg/g dw, polyisoprenoid values ranged from 1.34-2.44 mg/g dw. In the leaves, total lipids ranged from 590 to 669.43 mg/g dw while polyisoprenoid values ranged from 1.29 to 5.70 mg/g dw. The dolichol carbon chain length in the Pine stem of strain composed C70-C90, C65-C90, and C65-C95. Meanwhile, carbon chain-length of C85-C105, C70-C95, and C50-C65 were found in leaf tissues. The length of the polyprenol carbon chain in the stem was C60-C90, C50-C90, and C70-C90 respectively, while the chain lengths on the leaves were C60-C115, C50-C95, and C30-C90. The present study suggested the presence of both dolichol and polyprenols in Pinus merkusii without predomination either dolichol or polyprenol.

Mangrove plants produce polyisoprenoid alcohol. The polyisoprenoid consists of polyprenol and dolichol, which in pharmacological activity act as anticancer agents. The major polyisoprenoid compound of mangrove plants Ceriops tagal and Rhizophora mucronata was reported as dolichol. The present study was conducted to examine the anticancer effects of dolichol from C. tagal and R. mucronata leaves on WiDr cells and cell cycle-related cancer for 24 h and to evaluate the regulation of five genes, p53, EGFR, PI3K, Akt, and mTOR. The inhibited cell cycle was analysed by flow cytometry and the gene expression of p53, EGFR, PI3K, Akt, and mTOR was determined using reverse transcription-polymerase chain reaction (RT-PCR) method. Dolichol from C. tagal was more effective than that from R. mucronata,where it worked on the G0/G1 cycle for 87.94% and 82.36%, respectively, and regulated positive control 5-FU on the G0/G1 cycle (88.12%), S (9.52%) and G2-M (6.42%). The upregulation (p53) and downregulation (EGFR)contributed to the contracting cell cycle of colon cancer cells (WiDr) in PI3K, Akt and mTOR genes. To summarise, the current study suggests significant pharmacological properties of dolichols in C. tagal and R. mucronata leaves, which worked explicitly in the G0/G1 phase.

A primary virulence-associated trait of the opportunistic fungal pathogen Cryptococcus neoformans is the production of melanin pigments that are deposited into the cell wall and interfere with the host immune response. Previously, our solid-state NMR studies of isolated melanized cell walls (melanin "ghosts") revealed that the pigments are strongly associated with lipids, but their identities, origins, and potential roles were undetermined. Herein, we exploited spectral editing techniques to identify and quantify the lipid molecules associated with pigments in melanin ghosts. The lipid profiles were remarkably similar in whole C. neoformans cells, grown under either melanizing or nonmelanizing conditions; triglycerides (TGs), sterol esters (SEs), and polyisoprenoids (PPs) were the major constituents. Although no quantitative differences were found between melanized and nonmelanized cells, melanin ghosts were relatively enriched in SEs and PPs. In contrast to lipid structures reported during early stages of fungal growth in nutrient-rich media, variants found herein could be linked to nutrient stress, cell aging, and subsequent production of substances that promote chronic fungal infections. The fact that TGs and SEs are the typical cargo of lipid droplets suggests that these organelles could be connected to C. neoformans melanin synthesis. Moreover, the discovery of PPs is intriguing because dolichol is a well-established constituent of human neuromelanin. The presence of these lipid species even in nonmelanized cells suggests that they could be produced constitutively under stress conditions in anticipation of melanin synthesis. These findings demonstrate that C. neoformans lipids are more varied compositionally and functionally than previously recognized.

BACKGROUND: Mangroves secondary metabolites are mostly consisting of sterols, ubiquinones, isoprenoids, and polyisoprenoids. Polyisoprenoid is divided into two types, namely, polyprenol and dolichol, which has been reported to have biological and pharmacological activities. AIM: This research was aimed to analyze apoptosis 48 h with double staining and immunocytochemistry (ICC) 48 h of P53 and cyclooxygenase-2 (COX-2) gene expression from chemical constituents of dolichol in three mangrove leaves of Ceriops tagal, Nypa fruticans, and Rhizophora mucronata. METHODS: Apoptosis with the double-staining method was employed to analyze the genes expression in growth and development of cancer cells, P53, and COX-2 with ICC and flow cytometry method. The data were statistically analyzed using one-way ANOVA parametric statistical analysis followed by Duncan’s test. RESULTS: The result revealed that the increased apoptosis of samples C. tagal was 70% fluorescence orange, while N. fruticans and R. mucronata were 35% and 30% fluorescence orange, respectively. However, it was compared with the positive control; it produced orange fluorescent as much as 75%, suggesting that C. tagal have a position similar to 5-FU. Predominance dolichol in N. fruticans and C. tagal leaves led the expression gene of p53 to have 1.57% M1 phase, indicating the domination in G0-G1 phase (70–80%). Inhibit the expression for 48 h in p53 and COX-2 showing that n-hexane extract of C. tagal had the most percentage (80.733 ± 0.11%) to upregulate the p53 and less percentage (20.16 ± 1.19%) to downregulate the COX-2, indicating positive extract belong to N. fruticans and R. mucronata leaves. CONCLUSION: The present study confirmed the pharmacological properties of dolichol from three mangrove leaves as an anticancer of tumor suppressor genes and significantly proliferated of cancer cell growth from mangrove leaves.

Haloferax volcanii AglD is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AglD and Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AglD catalytic domains were revealed. At the same time, AglD includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose synthases. To determine whether this extension affords AglD functions in addition to generating mannose-charged DolP, a series of Hfx. volcanii strains expressing truncated versions of AglD was generated. Mass spectrometry revealed that a version of AglD comprising the catalytic domain and only two of the six to nine predicted membrane-spanning domains could mediate mannose addition to DolP. However, in cells expressing this or other truncated versions of AglD, mannose was not transferred from the lipid to the protein-bound tetrasaccharide precursor of the N-linked pentasaccharide normally decorating Hfx. volcanii glycoproteins. These results thus point to AglD as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AglD, possibly in coordination with AglR, translocates DolP-mannose across the plasma membrane is discussed. IMPORTANCE In the archaeon Haloferax volcanii, the dolichol phosphate (DolP)-mannose synthase AglD charges the lipid DolP with mannose, which is delivered to a protein-bound tetrasaccharide to generate the pentasaccharide decorating glycoproteins in this organism. Structural studies demonstrated the similarity of AglD to Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase with a solved three-dimensional structure. Truncated AglD containing the catalytic domain and only two of the predicted six to nine membrane-spanning regions catalyzed mannose-charging of DolP. Yet, no mannose was delivered to protein-linked tetrasaccharide in cells expressing AglD mutants including only up to five membrane-spanning regions, pointing to a role for the extended C-terminal region in a subsequent step of Hfx. volcanii N-glycosylation, such as DolP-mannose translocation across the plasma membrane.

Isoprenoids are the largest group of natural products, found in all living organisms and play an essential role in numerous cellular processes. These compounds are synthesized by prenyltransferases, catalyzing the condensation reaction between an allylic diphosphate primer and a variable number of isopentenyl diphosphate (C5) units. This superfamily of enzymes can be subdivided into trans- or cis-prenyltransferases according to the stereoisomerism of the product. The cis branch can be further classified according to product length. While the active site volume was suggested to determine the final length in enzymes synthesizing short- and medium-chain products (up to C60), long-chain enzymes (up to C120) and rubber synthases (>C10,000) fail to conform to this paradigm. Here, to resolve the structural basis for long-chain isoprenoid synthesis, we focused on the human cis-prenyltransferase complex (hcis-PT). This enzyme, peripheral to the endoplasmic reticulum membrane, produces the precursor for dolichol phosphate, a membrane residing glycosyl carrier. In line with its crucial role in the cellular protein glycosylation machinery, disease-causing mutations in hcis-PT were shown to result in a wide spectrum of clinical phenotypes. The crystallographic structures of hcis-PT in four different substrate/product-bound conformations revealed an outlet enabling product elongation into the bulk solvent. Moreover, hydrogen-deuterium exchange mass spectrometry analysis in solution showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Finally, using a fluorescent substrate analog and a fluorescently-labeled lipid nanodiscs, we show that product elongation and membrane association are closely correlated. Together, our results support directional product synthesis in long-chain enzymes and rubber synthases, with a distinct substrate inlet and product outlet, allowing direct membrane insertion of the elongating isoprenoid during catalysis. This mechanism uncouples active site volume from product length and circumvents the need to expulse hydrophobic product into a polar environment prior to membrane insertion.

Contains fulltext : 215635.pdf (Publisher’s version ) (Open Access) Congenital disorders of glycosylation type I (CDG-I) are inborn errors of metabolism, generally characterized by multisystem clinical manifestations, including developmental delay, hepatopathy, hypotonia, and skin, skeletal, and neurological abnormalities. Among others, dolichol-phosphate-mannose (DPM) is the mannose donor for N-glycosylation as well as O-mannosylation. DOLK-CDG, DPM1-CDG, DPM2-CDG, and DPM3-CDG are defects in the DPM synthesis showing both CDG-I abnormalities and reduced O-mannosylation of alpha-dystroglycan (alphaDG), which leads to muscular dystrophy-dystroglycanopathy. Mannose-phosphate-dolichol utilization defect 1 (MPDU1) plays a role in the utilization of DPM. Here, we report two MPDU1-CDG patients without skin involvement, but with massive dilatation of the biliary duct system and dystroglycanopathy characteristics including hypotonia, elevated creatine kinase, dilated cardiomyopathy, buphthalmos, and congenital glaucoma. Biochemical analyses revealed elevated disialotransferrin in serum, and analyses in fibroblasts showed shortened lipid linked oligosaccharides and DPM, and reduced O-mannosylation of alphaDG. Thus, MPDU1-CDG can be added to the list of disorders with overlapping biochemical and clinical abnormalities of CDG-I and dystroglycanopathy. Synopsis: Mannose-phosphate-dolichol utilization defect 1 patients can have overlapping biochemical and clinical abnormalities of congenital disorders of glycosylation type I and dystroglycanopathy.

The asparagine (N)-linked Man9GlcNAc2 is required for glycoprotein folding and secretion. Understanding how its structure contributes to these functions has been stymied by our inability to produce this glycan as a homogenous structure of sufficient quantities for study. Here, we report the high yield chemoenzymatic synthesis of Man9GlcNAc2 and its biosynthetic intermediates by reconstituting the eukaryotic lipid-linked oligosaccharide (LLO) pathway. Endoplasmic reticulum mannosyltransferases (MTases) are expressed in E. coli and used for mannosylation of the dolichol mimic, phytanyl pyrophosphate GlcNAc2. These recombinant MTases recognize unique substrates and when combined, synthesize end products that precisely mimic those in vivo, demonstrating that ordered assembly of LLO is due to the strict enzyme substrate specificity. Indeed, non-physiological glycans are produced only when the luminal MTases are challenged with cytosolic substrates. Reconstitution of the LLO pathway to synthesize Man9GlcNAc2 in vitro provides an important tool for functional studies of the N-linked glycoprotein biosynthesis pathway., Attachment of the oligosaccharide Man9GlcNAc2 is required for glycoprotein folding and secretion but synthesizing this compound for structural and functional studies has remained challenging. Here, the authors achieve efficient Man9GlcNAc2 synthesis by reconstituting its biosynthetic pathway in vitro.

Kartika EBA, Siregar LAM, Basyuni M. 2021. Distribution of polyprenol and dolichol in oil palms from Pisifera parents and mature plants from tissue culture propagation. Biodiversitas 22: 3423-3436. Oil palm tissue culture is carried out through indirect embryogenesis, which causes somaclonal diversity to occur at the in vitro propagation stage, especially in the callus growth phase. In the cells of all living organisms can be found a group of polyisoprenoid compounds. This study aims to determine variations in oil palm plants resulting from tissue culture based on the presence of polyisoprenoid compounds. Oil palm leaf samples (Elaeis guineensis Jacq.) were collected from the plantation of PT. Socfin Indonesia, North Sumatra, Indonesia. Sample extraction, saponification, isolation of polyisoprenoid compounds, and two-dimensional thin-layer chromatography analysis were carried out to obtain data related to total lipids, polyprenol and dolichol profile, and Carbon (C) chain length polyprenol and dolichol from oil palm leaves of Pisifera parents and their propagated derivatives by indirect embryogenesis. The results showed that the amount of polyisoprenoid mother S24 was 2.41 mg/g higher than that of the parentS8 at 2.17 mg/g dry weight. Total polyisoprenoid fromS8 plants in vitro ranged from 0.71 - 8.53 mg/g dry weight, with the lowest total polyisoprenoid found atS894/22 and the highest was found atS893/2. While for total polyisoprenoid from plant tissue culture of Pisifera S24 ranged from 0.73 - 8.05 mg/g dry weight, with the lowest total polyisoprenoid found at S24H14 and the highest was found at S24H16. The parent plants of Pisifera S8 and S24, as well as plants resulting from tissue culture, were categorized as having lipid pattern type II, which showed a balanced distribution of polyprenol with dolichol. The longest carbon chain was found in vitro plantsS8 93/4 ranged from C50-C110, while the shortest was found in plants produced in vitro S24H7 starting from C45-C55. There were variations in the carbon chain length of polyprenol and dolichol in the leaf samples derived from in vitro propagation of the Pisifera S8 and S24 parents.

Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic archaea remains either unclear or unknown. The composition of an N-linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40 years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N-linked glycan as corresponding to a tetrasaccharide comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasaccharide. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Hbt. salinarum, will allow delineation of the N-glycosylation pathway in this organism.

Kopasanda (Chromolaena odorata (L.) R.M. Rob, Asteraceae) is a, distributed in the tropical, and subtropical, countries including Indonesia. C. odorata have been reported to show biological and pharmacological activities. This work purposed to determine the profile and composition of polyprenols and dolichols from C. odorata leaves senescence (bud, young, and old leaves), investigated by two-dimensional thin layer chromatography (2D-TLC) technique. In these tissues, one-type, type-II of a profile of presence polyprenols and dolichols was observed. In contrast, Type-I and Type-III was not detected. In the buds, the shorter carbon chain length and the longer one belongs to old leaves with a carbon chain length of polyprenol C70-C95 and dolichol of C70-C95. The pattern of polyisoprenoids in this senescence leaves stage indicated the chemotaxonomic significance of polyisoprenoids in this species. The current report confirmed the occurrence of polyisoprenoids in kopasanda buds, young and old leaves.

Habsyah TS, Basyuni M, Siregar LAM, Afandi D, Syahputra I. 2021. Distribution of polyprenol and dolichol in oil palm genotype (Elaeis guineensis) involving lipase activity. Biodiversitas 22: 830-837. The aim of this study was to determine the pattern of chemical compounds from the polyisoprenoid group in oil palm plants that were contributing to the lipase activity. The study was conducted in two locations. A sampling of leaves and fruit mesocarp from 90 progenies was conducted at PT. Socfin Indonesia Aek Loba Plantation, Asahan District, North Sumatra, Indonesia. Biochemical analysis of polyisoprenoid was carried out at the Forest Biotechnology Laboratory, Faculty of Forestry, University of Sumatera Utara, Medan. The results showed that the total lipids in leaves tissue ranged from 0.40 to 1.032 mg/g, with the lowest total lipid found in SL-4966 progeny and the highest total lipid found in SL-9737 progeny. Polyisoprenoid values ​​ranged from 1.63 to 10.82 mg/g dry weight. The highest polyisoprenoid content was obtained in SL-10682 progeny, and the lowest was in SL-5045 progeny leaf tissue. The polyisoprenoid ​​in fruit mesocarp tissue ranged from 1.79 to 4.14 mg/g. The lowest polyisoprenoid content (1.79 mg/g) of fruit mesocarp was found in SL-4233 progeny, while the highest (4.14 mg/g) was found in SL-5010 progeny. Dolichol and polyprenol are equally dominant in palm leaves. Carbon polyprenol chain lengths ranged from C45-C105, and dolichol ranged from C50-C110. The polyprenol carbon chain lengths in the mesocarp tissues were C65-C120 and dolichol was C65-C125. Polyisoprenoid analysis on oil palm leaf and fruit mesocarp tissue showed that lipid patterns of oil palm leaf and mesocarp were categorized as type II lipid patterns. It provides important information for selecting genotypes of low lipase activity of oil palm plants.

Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for posttranslational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans. Until now, the regulatory mechanisms underlying Dol accumulation remain elusive. In this report, we have analyzed the natural variation of the accumulation of Dols and six other isoprenoids between 120Arabidopsis thalianaaccessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone, and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis – the AT2G17570 gene encoding a long searched forcis-prenyltransferase (CPT3) and the AT1G52460 gene encoding an alpha-beta hydrolase (ABH) – is experimentally confirmed. These data will help to generate Dol-enriched plants which might serve as a remedy for Dol-deficiency in humans.

Cis-prenyltransferase (cis-PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here, we report the crystal structure of the human NgBR/DHDDS complex, which represents an atomic resolution structure for any heterodimeric cis-PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme's active site through its C-terminal -RXG- motif, and how phospholipids markedly stimulate cis-PTase activity. Comparison of NgBR/DHDDS with homodimeric cis-PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme's active site tunnel, and an insert within the α3 helix helps to stabilize this energetically unfavorable state to enable long-chain synthesis to occur. These data provide unique insights into how heterodimeric cis-PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long-chain polyprenol synthesis.

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite’s development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite.

A primary virulence-associated trait of the opportunistic fungal pathogenCryptococcus neoformansis the production of melanin pigments that are deposited into the cell wall and interfere with the host immune response. Previously, our solid-state NMR studies of isolated melanized cell walls (melanin ‘ghosts’) revealed that the pigments are strongly associated with lipids, but their identities, origins, and potential roles were undetermined. Herein, we exploited spectral editing techniques to identify and quantify the lipid molecules associated with pigments in melanin ghosts. The lipid profiles were remarkably similar in wholeC. neoformanscells, grown under either melanizing or non-melanizing conditions; triglycerides (TGs), sterol esters (SEs) and polyisoprenoids (PPs) were the major constituents. Although no quantitative differences were found between melanized and non-melanized cells, melanin ghosts were relatively enriched in SEs and PPs. In contrast to lipid structures reported during early stages of fungal growth in nutrient-rich media, variants found herein could be linked to nutrient stress, cell aging, and subsequent production of substances that promote chronic fungal infections. The fact that TGs and SEs are the typical cargo of lipid droplets suggests that these organelles could be connected toC. neoformansmelanin synthesis. Moreover, the discovery of PPs is intriguing because dolichol is a well-established constituent of human neuromelanin. The presence of these lipid species even in non-melanized cells suggests that they could be produced constitutively under stress conditions in anticipation of melanin synthesis. These findings demonstrate thatC. neoformanslipids are more varied compositionally and functionally than previously recognized.

Like both eukaryotes and bacteria, archaea can decorate proteins with N- and O-linked glycans. Whereas pathways and roles of N-glycosylation have been studied in several model archaeal organisms, little is known of O-glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S-layer glycoproteins are modified by an N-linked glucose-α-(1, 2)-[sulfoquinovosamine-β-(1, 6)-]galactose trisaccharide and an O-linked glucose-α-(1, 4)-galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose-DolP onto galactose-DolP to form the glucose-α-(1, 4)-galactose-DolP precursor of the N-glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase-associated protein responsible for flipping of hexose-DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N- and O-linked glycans onto target protein occurs on the outer surface of the cell using hexose-DolPs as sugar donors. Deletion mutant showed that N- and O-glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.

Dolichol is an essential polyisoprenoid within the endoplasmic reticulum of all eukaryotes. It serves as a membrane bound anchor onto which N-glycans are assembled prior to being transferred to nascent polypeptides, many of which enter the secretory pathway. Historically, it has been posited that the accumulation of dolichol represents the 'rate-limiting' step in the evolutionary conserved process of N-glycosylation, which ultimately affects the efficacy of approximately one fifth of the entire eukaryotic proteome. Therefore, this study aimed to enhance dolichol accumulation by manipulating the enzymes involved in its biosynthesis using an established Nicotiana benthamiana platform. Co-expression of a Solanum lycopersicum (tomato) cis-prenyltransferase (CPT) and its cognate partner protein, CPT binding protein (CPTBP), that catalyze the antepenultimate step in dolichol biosynthesis led to a 400-fold increase in the levels of long-chain polyprenols but resulted in only modest increases in dolichol accumulation. However, when combined with a newly characterized tomato polyprenol reductase, dolichol biosynthesis was enhanced by approximately 20-fold. We provide further evidence that in the aquatic macrophyte, Lemna gibba, dolichol is derived exclusively from the mevalonic acid (MVA) pathway with little participation from the evolutionary co-adopted non-MVA pathway. Taken together these results indicate that to effectively enhance the in planta accumulation of dolichol, coordinated synthesis and reduction of polyprenol to dolichol, is strictly required.

Basyuni M, Sagami H, Baba S, Oku H. 2019. Response of polyisoprenoid concentration and profile in three groups of mangrove seedlings of coping with long-term salinity. Biodiversitas 20: 320-326. The response of polyisoprenoid (polyprenol and dolichol) concentration and distribution was investigated with three groups mechanism of coping with salinity: a secreting mangrove species of Avicennia officinalis, a non-secreting species (excluder) of Bruguiera cylindrica, and a salt-accumulating species of Xylocarpus granatum. The seedlings of three mangroves were grown under 0 and 3% salinity concentration for five months. Polyisoprenoids in the lipid extracts were examined by two-dimensional thin layer chromatography (2D-TLC). The pattern of the polyprenols and dolichols in the leaves and roots were categorized as two types (I and II). In category I, dolichols dominated over polyprenols, however, in category II, the existence of both polyprenols and dolichols was found. In the leaves, type-I was observed in A. officinalis under 0 and 3% salinity. On the other hand, type-II was found in B. cylindrica and X. granatum under 0 and 3% salt concentrations. A similar pattern was found in the roots, A. officinalis (type-I), and that in B. cylindrica and X. granatum was type-II. This finding depicted that the seedlings of A. officinalis, B. cylindrica, and X. granatum leaves and roots imply no change in the distribution type: the categories were distributed as type I or II under 0% salt concentrations, as well as type-I or II under 3% salt concentrations. This study implied that polyisoprenoids may play a protective function against salinity in the mangrove leaves and roots of three groups scheme (secreting, excluding, and accumulating) of salt management.

Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concentrations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.

Missense mutations of the human mitochondrial citrate carrier, encoded by the SLC25A1 gene, lead to an autosomal recessive neurometabolic disorder characterised by neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development, often resulting in early death. Here, we have measured the effect of all twelve known pathogenic mutations on the transport activity. The results show that nine mutations abolish transport of citrate completely, whereas the other three reduce the transport rate by >70%, indicating that impaired citrate transport is the most likely primary cause of the disease. Some mutations may be detrimental to the structure of the carrier, whereas others may impair key functional elements, such as the substrate binding site and the salt bridge network on the matrix side of the carrier. To understand the consequences of impaired citrate transport on metabolism, the substrate specificity was also determined, showing that the human citrate carrier predominantly transports citrate, isocitrate, cis-aconitate, phosphoenolpyruvate and malate. Although D-2- and L-2 hydroxyglutaric aciduria is a metabolic hallmark of the disease, it is unlikely that the citrate carrier plays a significant role in the removal of hydroxyglutarate from the cytosol for oxidation to oxoglutarate in the mitochondrial matrix. In contrast, computer simulations of central metabolism predict that the export of citrate from the mitochondrion cannot be fully compensated by other pathways, restricting the cytosolic production of acetyl-CoA that is required for the synthesis of lipids, sterols, dolichols and ubiquinone, which in turn explains the severe disease phenotypes.

Dolichols are isoprenoid lipids of varying length that act as sugar carriers in glycosylation reactions in the endoplasmic reticulum. In Saccharomyces cerevisiae, there are two cis-prenyltransferases that synthesize polyprenol—an essential precursor to dolichol. These enzymes are heterodimers composed of Nus1 and either Rer2 or Srt1. Rer2-Nus1 and Srt1-Nus1 can both generate dolichol in vegetative cells, but srt1∆ cells grow normally while rer2∆ grows very slowly, indicating that Rer2-Nus1 is the primary enzyme used in mitotically dividing cells. In contrast, SRT1 performs an important function in sporulating cells, where the haploid genomes created by meiosis are packaged into spores. The spore wall is a multilaminar structure and SRT1 is required for the generation of the outer chitosan and dityrosine layers of the spore wall. Srt1 specifically localizes to lipid droplets associated with spore walls, and, during sporulation there is an SRT1-dependent increase in long-chain polyprenols and dolichols in these lipid droplets. Synthesis of chitin by Chs3, the chitin synthase responsible for chitosan layer formation, is dependent on the cis-prenyltransferase activity of Srt1, indicating that polyprenols are necessary to coordinate assembly of the spore wall layers. This work shows that a developmentally regulated cis-prenyltransferase can produce polyprenols that function in cellular processes besides protein glycosylation.

cis-Prenyltransferases (cis-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. In eukaryotes and archaea, cis-PT is the first enzyme committed to the synthesis of dolichyl phosphate, an obligate lipid carrier in protein glycosylation reactions. The homodimeric bacterial enzyme, undecaprenyl diphosphate synthase, generates 11 isoprene units and has been structurally and mechanistically characterized in great detail. Recently, we discovered that unlike undecaprenyl diphosphate synthase, mammalian cis-PT is a heteromer consisting of NgBR (Nus1) and hCIT (dehydrodolichol diphosphate synthase) subunits, and this composition has been confirmed in plants and fungal cis-PTs. Here, we establish the first purification system for heteromeric cis-PT and show that both NgBR and hCIT subunits function in catalysis and substrate binding. Finally, we identified a critical RXG sequence in the C-terminal tail of NgBR that is conserved and essential for enzyme activity across phyla. In summary, our findings show that eukaryotic cis-PT is composed of the NgBR and hCIT subunits. The strong conservation of the RXG motif among NgBR orthologs indicates that this subunit is critical for the synthesis of polyprenol diphosphates and cellular function.

Background Prenyltransferases catalyze the sequential addition of isopentenyl diphosphate units to allylic prenyl diphosphate acceptors and are classified as either trans -prenyltransferases (TPTs) or cis -prenyltransferases (CPTs). The functions of CPTs have been well characterized in bacteria, yeast, and mammals compared to plants. The characterization of CPTs also has been less studied than TPTs. In the present study, molecular cloning and functional characterization of a CPT from a medicinal plant, Panax ginseng Mayer were addressed. Methods Gene expression patterns of PgCPT1 were analyzed by quantitative reverse transcription polymerase chain reaction. In planta transformation was generated by floral dipping using Agrobacterium tumefaciens . Yeast transformation was performed by lithium acetate and heat-shock for rer2Δ complementation and yeast-two-hybrid assay. Results The ginseng genome contains at least one family of three putative CPT genes. PgCPT1 is expressed in all organs, but more predominantly in the leaves. Overexpression of PgCPT1 did not show any plant growth defect, and its protein can complement yeast mutant rer2Δ via possible protein–protein interaction with PgCPTL2. Conclusion Partial complementation of the yeast dolichol biosynthesis mutant rer2Δ suggested that PgCPT1 is involved in dolichol biosynthesis. Direct protein interaction between PgCPT1 and a human Nogo-B receptor homolog suggests that PgCPT1 requires an accessory component for proper function.

The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (Arabidopsis thaliana). The contribution of the MEP pathway increased significantly (reaching 100%) exclusively for the dominating Dols, while for long-chain Dols, the relative input of the MEP and MVA pathways remained unchanged, suggesting divergent sites of synthesis for dominating and long-chain Dols. The analysis of numerically modeled Dol mass spectra is a novel method to follow modulation of the concomitant activity of isoprenoid-generating pathways in plant cells; additionally, it suggests an exchange of isoprenoid intermediates between plastids and peroxisomes.

Lipid-linked oligosaccharides (LLOs) play an important role in the N-glycosylation pathway as the donor substrate of oligosaccharyltransferases (OSTs), which are responsible for the en bloc transfer of glycan chains onto a nascent polypeptide. The lipid component of LLO in both eukarya and archaea consists of a dolichol, and an undecaprenol in prokarya, whereas the number of isoprene units may change between species. Given the potential relevance of LLOs and their related enzymes to diverse biotechnological applications, obtaining reliable LLO models from distinct domains of life could support further studies on complex formation and their processing by OSTs, as well as protein engineering on such systems. In this work, molecular modeling techniques, such as quantum mechanics calculations, molecular dynamics simulations, and metadynamics were employed to study eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol), bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol), and archaeal (Glc1-Man1-Gal1-Man1-Glc1-Gal1-Glc1-P-Dolichol) LLOs in membrane bilayers. Microsecond molecular dynamics simulations and metadynamics calculations of LLOs revealed that glycan chains are more prone to interact with the membrane lipid head groups, while the PP linkages are positioned at the lipid phosphate head groups level. The dynamics of isoprenoid chains embedded within the bilayer are described, and membrane dynamics and related properties are also investigated. Overall, there are similarities regarding the structure and dynamics of the eukaryotic, the bacterial, and the archaeal LLOs in bilayers, which can support the comprehension of their association with OSTs. These data may support future studies on the transferring mechanism of the oligosaccharide chain to an acceptor protein.

BACKGROUND: Mangrove forest is a typical forest found along the coast or river mouth which is affected by tides and salinity. Although polyisoprenoid was widespread in the plant kingdom, the physiological roles of these compounds are not well understood, especially from mangrove plants. It is therefore essential to characterize the polyisoprenoid content under abiotic stress. AIM: This study aimed to determine the effect of salinity and subsequent fresh water change on polyisoprenoids concentration in Bruguiera cylindrica seedlings. METHODS: Bruguiera cylindrica planted in a greenhouse for three months under various salinity concentrations. After three months grew under variable salinity, these seedlings were then divided into two treatment groups, and grown for another three months: one continuously in a salt solution and another in fresh water to relieve salt stress. The leaves and roots of B. cylindrica seedlings were harvested after six months of cultivation. The leaves and roots of B. cylindrica seedlings were extracted for polyisoprenoids content and composition analyzed using two-dimensional thin layer chromatography. RESULTS: Polyisoprenoids composition under salinity and subsequent fresh water with dominating dolichols (more than 90%) were found in leaves and roots of B. cylindrica seedlings referring type I of polyisoprenoid composition. The carbon chain length of dolichols located in the leaves and roots were ranging from C75–C100 and C75–C105, respectively. CONCLUSION: Dolichol dominated over polyprenol both in B. cylindrical leaves and roots under salinity and subsequent relief supported the previous finding on the predominance dolichols over polyprenols in mangrove plants. The present study suggested the significance of dolichols in the adaptation to cope with salt stress and or water stress.

The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.

Arabidopsis roots accumulate a complex mixture of dolichols composed of three families, (i.e., short-, medium- and long-chain dolichols), but until now none of the cis-prenyltransferases (CPTs) predicted in the Arabidopsis genome has been considered responsible for their synthesis. In this report, using homo- and heterologous (yeast and tobacco) models, we have characterized the AtCPT1 gene (At2g23410) which encodes a CPT responsible for the formation of long-chain dolichols, Dol-18 to -23, with Dol-21 dominating, in Arabidopsis. The content of these dolichols was significantly reduced in AtCPT1 T-DNA insertion mutant lines and highly increased in AtCPT1-overexpressing plants. Similar to the majority of eukaryotic CPTs, AtCPT1 is localized to the endoplasmic reticulum (ER). Functional complementation tests using yeast rer2&Delta, or srt1&Delta, mutants devoid of medium- or long-chain dolichols, respectively, confirmed that this enzyme synthesizes long-chain dolichols, although the dolichol chains thus formed are somewhat shorter than those synthesized in planta. Moreover, AtCPT1 acts as a homomeric CPT and does not need LEW1 for its activity. AtCPT1 is the first plant CPT producing long-chain polyisoprenoids that does not form a complex with the NgBR/NUS1 homologue.

In this study we attempted to verify the hypothesis that the mevalonate pathway affects amyloid beta precursor protein (A&beta, PP) processing and regulates clusterin protein levels. A&beta, PP expression was monitored by green fluorescence (FL) and Western blot (WB). WB showed soluble amyloid protein precursor alpha (sA&beta, PP&alpha, ) presence in A&beta, PP-wt cells and A&beta, expression in A&beta, PP-sw cells. Nerve growth factor (NGF)-differentiated rat neuronal pheochromocytoma PC-12 cells were untreated/treated with statins alone or together with non-sterol isoprenoids. Co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol demonstrated statin-dependent neurotoxicity resulted from the attenuated activity of mevalonate pathway rather than lower cholesterol level. Atorvastatin (50 &mu, M) or simvastatin (50 &mu, M) as well as cholesterol chelator methyl-&beta, cyclodextrin (0.2 mM) diminished cell viability (p &lt, 0.05) and clusterin levels. Interestingly, co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol stimulated (p &lt, 0.05) clusterin expression. Effects of non-sterol isoprenoids, but not water soluble cholesterol (Chol-PEG), were the most significant in mock-transfected cells. Geranylgeraniol (GGOH) overcame atorvastatin (ATR)-dependent cytotoxicity. This effect does not seem to be dependent on clusterin, as its level became lower after GGOH. The novelty of these findings is that they show that the mevalonate (MEV) pathway rather than cholesterol itself plays an important role in clusterin expression levels. In mock-transfected, rather than in A&beta, PP-overexpressing cells, GGOH/farnesol (FOH) exerted a protective effect. Thus, protein prenylation with GGOH/FOH might play substantial role in neuronal cell survival.

&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.

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.

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.

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 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.