Revisiting N-glycosylation in Halobacterium salinarum: Characterizing a dolichol phosphate- and glycoprotein-bound tetrasaccharide.

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.

N-glycosylation in the thermoacidophilic archaeon Sulfolobus acidocaldarius involves a short dolichol pyrophosphate carrier.

N-glycosylation is a post-translational modification that occurs across evolution. In the thermoacidophilic archaea Sulfolobus acidocaldarius, glycoproteins are modified by an N-linked tribranched hexasaccharide reminiscent of the N-glycans assembled in Eukarya. Previously, hexose-bearing dolichol phosphate was detected in a S. acidocaldarius Bligh-Dyer lipid extract. Here, we used a specialized protocol for extracting lipid-linked oligosaccharides to detect a dolichol pyrophosphate bearing the intact hexasaccharide, as well as its biosynthetic intermediates. Furthermore, evidence for N-glycosylation of two S. acidocaldarius proteins by the same hexasaccharide and its derivatives was collected. These findings thus provide novel insight into archaeal N-glycosylation.

Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle.

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.

A predicted geranylgeranyl reductase reduces the ω-position isoprene of dolichol phosphate in the halophilic archaeon, Haloferax volcanii.

In N-glycosylation in both Eukarya and Archaea, N-linked oligosaccharides are assembled on dolichol phosphate prior to transfer of the glycan to the protein target. However, whereas only the α-position isoprene subunit is saturated in eukaryal dolichol phosphate, both the α- and ω-position isoprene subunits are reduced in the archaeal lipid. The agents responsible for dolichol phosphate saturation remain largely unknown. The present study sought to identify dolichol phosphate reductases in the halophilic archaeon, Haloferax volcanii. Homology-based searches recognize HVO_1799 as a geranylgeranyl reductase. Mass spectrometry revealed that cells deleted of HVO_1799 fail to fully reduce the isoprene chains of H. volcanii membrane phospholipids and glycolipids. Likewise, the absence of HVO_1799 led to a loss of saturation of the ω-position isoprene subunit of C(55) and C(60) dolichol phosphate, with the effect of HVO_1799 deletion being more pronounced with C(60) dolichol phosphate than with C(55) dolichol phosphate. Glycosylation of dolichol phosphate in the deletion strain occurred preferentially on that version of the lipid saturated at both the α- and ω-position isoprene subunits.

The thermoacidophilic archaeon Sulfolobus acidocaldarius contains an unusually short, highly reduced dolichyl phosphate.

Polyprenoids, polymers containing varied numbers of isoprene subunits, serve numerous roles in biology. In Eukarya, dolichyl phosphate, a phosphorylated polyprenol bearing a saturated α-end isoprene subunit, serves as the glycan carrier during N-glycosylation, namely that post-translational modification whereby glycans are covalently linked to select asparagine residues of a target protein. As in Eukarya, N-glycosylation in Archaea also relies on phosphorylated dolichol. In this report, LC-ESI/MS/MS was employed to identify a novel dolichyl phosphate (DolP) in the thermoacidophilic archaeon, Sulfolobus acidocaldarius. The unusually short S. acidocaldarius DolP presents a degree of saturation not previously reported. S. acidocaldarius DolP contains not only the saturated α- and ω-end isoprene subunits observed in other archaeal DolPs, but also up to five saturated intra-chain isoprene subunits. The corresponding dolichol and hexose-charged DolP species were also detected. The results of the present study offer valuable information on the biogenesis and potential properties of this unique DolP. Furthermore, elucidation of the mechanism of α-isoprene unit reduction in S. acidocaldarius dolichol may facilitate the identification of the alternative, as yet unknown polyprenol reductase in Eukarya.

Glycoprotein maturation and the UPR.

Glycosylation is a complex form of protein modification occurring in the secretory pathway. The addition of N- and O-glycans affects intracellular processes like the folding and trafficking of most glycoproteins. To better understand the impact of glycosylation in protein folding and maturation, parameters like glycosylation site occupancy and oligosaccharide structure must be measured quantitatively. In this chapter, we describe current methods enabling the determination of N-glycosylation by assessment of cellular dolichol phosphate levels, dolichol-linked oligosaccharides, and the occupancy of N-glycosylation sites. We also provide detailed methods for the analysis of O-glycosylation, whose role in intracellular protein maturation is often overlooked.

Distinct glycan-charged phosphodolichol carriers are required for the assembly of the pentasaccharide N-linked to the Haloferax volcanii S-layer glycoprotein.

In Archaea, dolichol phosphates have been implicated as glycan carriers in the N-glycosylation pathway, much like their eukaryal counterparts. To clarify this relation, highly sensitive liquid chromatography/mass spectrometry was employed to detect and characterize glycan-charged phosphodolichols in the haloarchaeon Haloferax volcanii. It is reported that Hfx. volcanii contains a series of C(55) and C(60) dolichol phosphates presenting saturated isoprene subunits at the α and ω positions and sequentially modified with the first, second, third and methylated fourth sugar subunits comprising the first four subunits of the pentasaccharide N-linked to the S-layer glycoprotein, a reporter of N-glycosylation. Moreover, when this glycan-charged phosphodolichol pool was examined in cells deleted of agl genes encoding glycosyltransferases participating in N-glycosylation and previously assigned roles in adding pentasaccharide residues one to four, the composition of the lipid-linked glycans was perturbed in the identical manner as was S-layer glycoprotein N-glycosylation in these mutants. In contrast, the fifth sugar of the pentasaccharide, identified as mannose in this study, is added to a distinct dolichol phosphate carrier. This represents the first evidence that in Archaea, as in Eukarya, the oligosaccharides N-linked to glycoproteins are sequentially assembled from glycans originating from distinct phosphodolichol carriers.

HPLC and mass spectrometry analysis of dolichol-phosphates at the cell culture scale.

Dolichols (Dol) are polyprenol lipids that are essential structural components of eukaryotic membranes. In addition, the phosphorylated derivatives of Dol function as lipid anchors of mono- and oligosaccharide precursors involved in protein glycosylation. The biological importance of Dol phosphates (Dol-P) is illustrated by the severe outcome of human disorders linked to Dol biosynthetic defects, such as Dol-kinase deficiency. For characterization of inherited human diseases and evaluation of therapeutic trials, cultured cells often serve as a sole possible source for experimentation. Limited amounts of cell culture material render the quantitative analysis of Dol a challenging task. Here, we present HPLC- and mass spectrometry-based approaches to analyze and quantitate Dol-P from cultured human cells. The composition of naturally occurring Dol-P and the saturation state of the alpha-isoprene units was identified by negative-ion electrospray ionization mass spectrometry. Furthermore, fluorescently labeled Dol-P were separated by HPLC and quantified by comparison to known amounts of the internal standard polyprenol-P. The effect of pravastatin, a 3-hydroxy-3-methyl-glutaryl coenzyme-A reductase inhibitor, on the formation of Dol-P in HeLa cells was investigated. As expected, this treatment led to a decrease of Dol-P down to 35% of normal levels.

Chemical synthesis of dolichyl phosphates, their analogues and derivatives and application of these compounds in biochemical assays.

Several methods for simple and efficient chemical synthesis of dolichyl phosphates and their analogues and derivatives are briefly summarized with a special emphasis on chemical modification of phosphoryl group and preparation of dolichyl phosphates labelled at the omega-end and at the gamma-isoprene unit of the isoprene chain by fluorescent groups, 2-aminopyridine and 1-aminonaphtalene residues. Additionally, data on biochemical assays with application of the compounds mentioned above are presented.

Application of fluorophore-assisted carbohydrate electrophoresis for the study of the dolichol pyrophosphate-linked oligosaccharides pathway in cell cultures and animal tissues.

Defects in the synthesis of dolichol-linked oligosaccharide (or lipid-linked oligosaccharide [LLO]) cause severe, multisystem human diseases called type 1 congenital disorders of glycosylation (CDG type 1). LLOs are also involved in another disease, neuronal ceroid lipofuscinosis. Because of the low abundance of LLOs, almost all studies of LLO synthesis have relied upon metabolic labeling of the oligosaccharides with radioactive sugar precursors such as [3H]mannose or [14C]glucosamine, and therefore have been limited almost entirely to cell cultures and tissue slices. A procedure is presented for a facile, accurate, and sensitive non-radioactive method for LLO pathway analysis based on fluorophore-assisted carbohydrate electrophoresis (FACE). It is feasible to analyze almost any component in the LLO pathway with the application FACE, from sugar precursors to mature LLO (Glc3Man9GlcNAc2-P-P-dolichol).

Fluorophore-assisted carbohydrate electrophoresis: a sensitive and accurate method for the direct analysis of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues.

Lipid-linked oligosaccharides (LLOs) such as Glc3Man9GlcNAc2-P-P-dolichol are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I Congenital Disorders of Glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, almost all studies of LLO synthesis have relied upon metabolic labeling of the oligosaccharides with radioactive sugar precursors such as [3H]mannose or [14C]glucosamine. In this article, a procedure is presented for a facile, accurate, and sensitive non-radioactive method for LLO analysis based on fluorophore-assisted carbohydrate electrophoresis (FACE). First, LLOs are extracted and partially purified. Next, oligosaccharides released from LLOs are labeled with negatively charged fluorophores: 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) or 7-amino-1,3-naphthalenedisulfonic acid (ANDS). A specialized form of polyacrylamide gel electrophoresis is then used to resolve and measure ANTS or ANDS labeled oligosaccharides. Finally, the resolved oligosaccharides are detected and quantified by fluorescence imagers using CCD cameras.

Effects of peroxisome proliferators gemfibrozil and clofibrate on syntheses of dolichol and cholesterol in rat liver.

The effects of two peroxisome proliferators, gemfibrozil and clofibrate, on syntheses of dolichol and cholesterol in rat liver were investigated. Gemfibrozil did not affect the overall content of dolichyl phosphate, but it changed the chain-length distribution of dolichyl phosphate, increasing the levels of species with shorter isoprene units. Gemfibrozil suppressed synthesis of dolichyl phosphate from [(3)H]mevalonate and [(3)H]farnesyl pyrophosphate in rat liver. In contrast, clofibrate increased the content of dolichol (free and acyl ester forms). It remarkably enhanced dolichol synthesis from mevalonate, but did not affect dolichol synthesis from farnesyl pyrophosphate. Gemfibrozil elevated cholesterol synthesis from [(14)C]acetate, but did not affect the synthesis from mevalonate. Clofibrate suppressed cholesterol synthesis from acetate, but did not affect cholesterol synthesis from mevalonate. These results suggest that gemfibrozil suppresses synthesis of dolichyl phosphate by inhibiting, at the least, the pathway from farnesyl pyrophosphate to dolichyl phosphate. As a result, the chain-length pattern of dolichyl phosphate may show an increase in shorter isoprene units. Clofibrate may increase the content of dolichol by enhancing dolichol synthesis from mevalonate. Gemfibrozil may increase cholesterol synthesis by activating the pathway from acetate to mevalonate. Unlike gemfibrozil, clofibrate may decrease cholesterol synthesis by inhibiting the pathway from acetate to mevalonate.

[A synthesis of dolichyl fluorophosphate]. / Sintez dolikholftorfosfata.

An improved method for the synthesis of dolichyl H-phosphonate was developed using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (salicyl chlorophosphite) as a reagent. Dolichyl phosphorofluoridate was for the first time synthesized from dolichyl H-phosphonate by its treatment with chlorotrimethylsilane, oxidation with iodine, and subsequent interaction with fluoride ion in pyridine.

Synthesis of an unnatural N-glycan-linked dolichyl pyrophosphate precursor.

An unnatural alpha-D-mannopyranose-linked chitobiosyl dolichyl pyrophosphate, a stereoisomer of the N-glycan biosynthesis intermediate, was synthesized. The protected trisaccharide, alpha-D-Man-(1-->4)-beta-D-GlcNAc-(1-->4)-D-GlcNAc, carrying a 4-methylbenzoyl group was prepared for the convenience of a TLC analysis. 1-O-Phosphorylation, condensation with dolichyl phosphate, and subsequent deprotection afforded the title compound.

Characterization by NMR and molecular modeling of the binding of polyisoprenols and polyisoprenyl recognition sequence peptides: 3D structure of the complexes reveals sites of specific interactions.

The objective of these studies was to test the hypothesis that proteins that contain potential polyisoprenyl recognition sequences (PIRSs) in their transmembrane-spanning domain can bind to the polyisoprenyl (PI) glycosyl carrier lipids undecaprenyl phosphate (C55-P) and dolichyl phosphate (C95-P). A number of prokaryotic and eukaryotic glycosyltransferases that utilize PI coenzymes contain a conserved PIRS postulated to be the active PI binding domain. To study this problem, we first determined the 3D structure of a PIRS peptide, NeuE, by homonuclear 2D 1H-nuclear magnetic resonance (NMR) spectroscopy. Experimentally generated distance constraints derived from nuclear Overhauser enhancement and torsion angle constraints derived from coupling constants were used for restrained molecular dynamics and energy minimization calculations. Molecular models of the NeuE peptide were built based on calculations of energy minimization using the DGII program NMRchitect. 3D models of dolichol (C95) and C95-P were built based on our 2D 1H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) results and refined by energy minimization with respect to all atoms using the AMBER (assisted modeling with energy refinements) force field. Our energy minimization studies were carried out on a conformational model of dolichol that was originally derived from small-angle X-ray scattering and molecular mechanics methods. These results revealed that the PIs are conformationally nearly identical tripartite molecules, with their three domains arranged in a coiled, helical structure. Analyses of the intermolecular cross-peaks in the 2D NOESY spectra of PIRS peptides in the presence of PIs confirmed a highly specific interaction and identified key contact amino acids in the NeuE peptide that constituted a binding motif for interacting with the PIs. These studies also showed that subtle conformational changes occurred within both the PIs and the NeuE peptide after binding. 3D structures of the resulting molecular complexes revealed that each PI could bind more than one PIRS peptide. These studies thus represent the first evidence for a direct physical interaction between specific contact amino acids in the PIRS peptides and the PIs and supports the hypothesis of a bifunctional role for the PIs. The central idea is that these superlipids may serve as a structural scaffold to organize and stabilize in functional domains PIRS-containing proteins within multiglycosyltransferase complexes that participate in biosynthetic and translocation processes.

Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis.

Lipid-linked oligosaccharides (LLOs) are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I congenital disorders of glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, simple and sensitive nonradioactive methods for LLO analysis are lacking. Thus, almost all studies of LLO synthesis have relied on metabolic labeling of the oligosaccharides with radioactive sugar precursors. We report that LLOs in cell cultures and tissues can be easily detected and quantified with a sensitivity of 1-2 pmol by fluorophore-assisted carbohydrate electrophoresis (FACE). These analyses required efficient removal of contaminants, most likely trace quantities of glycogen breakdown products, that interfered with FACE. Studies with CHO-K1 cells showed that LLOs detected by FACE and by metabolic labeling had similar turnover rates. Glc(3)Man(9)GlcNAc(2)-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc(0-2)Man(5-9)GlcNAc(2)-P-P-dolichol intermediates in tissues, such as liver and kidney, were unexpectedly greater than for cultured cells. IV injection of D-mannose, raising the circulatory concentration by three- to fourfold, did not affect LLO composition. Thus, the relative accumulation of LLO intermediates in mouse liver and kidney is not likely due to inadequate D-mannose in the circulation. In summary, FACE is a facile, accurate, and sensitive method for LLO analysis, permitting investigations not feasible by metabolic labeling.

Enzymatic synthesis of beta-mannosyl phosphates on solid support.

Synthetic bifunctional analogues 4a, b and 14 of dolichol phosphate 1 were attached to solid support and were shown to be substrates for Dol-P-Man synthase.

Substrate specificity of the dolichol phosphate mannose: glucosaminyl phosphatidylinositol alpha1-4-mannosyltransferase of the glycosylphosphatidylinositol biosynthetic pathway of African trypanosomes.

The biosynthesis of glycosylphosphatidylinositol (GPI) precursors in Trypanosoma brucei involves the D-mannosylation of D-GlcN alpha 1-6-D-myo-inositol-1-PO4-sn-1,2-diacylglycerol (GlcN-PI). An assay for the first mannosyltransferase of the pathway, Dol-P-Man:GlcN-PI alpha 1-4-mannosyltransferase, is described. Analysis of the acceptor specificity revealed (a) that the enzyme requires the myo-inositol residue of the GlcN-PI substrate have the D configuration; (b) that the enzyme requires the presence of the NH2 group of the D-GlcN residue; (c) that GlcNAc-PI is more efficiently presented to the enzyme than GlcN-PI, suggesting a degree of substrate channelling via the preceding GlcNAc-PI de-N-acetylase enzyme; (d) that the fatty acid and phosphoglycerol components of the phosphatidyl moiety are important for enhancing substrate presentation and substrate recognition, respectively; and (e) that D-GlcN alpha 1-6-D-myo-inositol is the minimum structure that can support detectable acceptor activity. Analysis of the donor specificity revealed that short chain (C5 and C15) analogues of dolichol phosphate can act as substrates for the trypanosomal dolichol-phosphomannose synthetase, whereas the corresponding mannopyranosides cannot act as donors for the Dol-P-Man:GlcN-PI alpha 1-4-mannosyltransferase.

A recent approach to the study of dolichyl monophosphate topology in the rough endoplasmic reticulum.

Amphomycin though withdrawn as an antibiotic against the gram-positive bacterial infection, can certainly serve as an excellent tool for determination of the topology of Dol-P in the endoplasmic reticulum membranes which has been otherwise impossible.

Mass spectrometric analysis of prenyl phosphates and their glycosylated forms.

Three different mass spectrometric method suitable for the analysis of polyprenyl and dolichyl phosphates and their glycosylated forms are described. Fast atom bombardment mass spectrometry (FAB MS) of glycosyl monophosphopolyprenols produces negative ions characteristic of the intact molecule. Tandem mass spectrometry of (M-H)- anions allows the determination of masses of both glycosyl and lipid moieties. Thus, for example, FAB-MS/MS of a mixture of native glycosyl monophosphopolyprenols isolated from ethambutol-treated Mycobacterium smegmatis enabled us to detect two novel pentosyl monophosphopolyprenols. Two other methods are proposed for the analysis of prenyl phosphates, as these compounds do not produce fragments in FAB-MS/MS at low collisional energy. By Desorption Electron Impact ionization (DEI) an intense (M-H3PO4)+ ion as well as fragments corresponding to the successive loss of isoprene residues (68 Da) can be observed. Alternatively, Desorption Chemical Ionization yields ions corresponding to the loss of 66, 78 and 98 Da (i.e. of a part or the entire phosphate moiety) of a prenyl phosphate molecule. Tandem mass spectrometry of the (M-H-98)- ion gives a series of intense fragments differing by 68 mass units over the whole mass range.