Inhibition of Dephosphorylation of Dolichyl Diphosphate Alters the Synthesis of Dolichol and Hinders Protein N-Glycosylation and Morphological Transitions in Candida albicans.

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.

Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation.

The glycosylation of asparagine residues is the predominant protein modification in all three domains of life. An oligosaccharide chain is preassembled on a lipid-phospho carrier and transferred onto asparagine residues by the action of a membrane-bound enzyme, oligosaccharyltransferase. The oligosaccharide donor for the oligosaccharyl transfer reaction is dolichol-diphosphate-oligosaccharide in Eukaryota and polyprenol-diphosphate-oligosaccharide in Eubacteria. The donor in some archaeal species was reportedly dolichol-monophosphate-oligosaccharide. Thus, the difference in the number of phosphate groups aroused interest in whether the use of the dolichol-monophosphate type donors is widespread in the domain Archaea. Currently, all of the archaeal species with identified oligosaccharide donors have belonged to the phylum Euryarchaeota. Here, we analyzed the donor structures of two species belonging to the phylum Crenarchaeota, Pyrobaculum calidifontis and Sulfolobus solfataricus, in addition to two species from the Euryarchaeota, Pyrococcus furiosus and Archaeoglobus fulgidus The electrospray ionization tandem mass spectrometry analyses confirmed that the two euryarchaeal oligosaccharide donors were the dolichol-monophosphate type and newly revealed that the two crenarchaeal oligosaccharide donors were the dolichol-diphosphate type. This novel finding is consistent with the hypothesis that the ancestor of Eukaryota is rooted within the TACK (Thaum-, Aig-, Cren-, and Korarchaeota) superphylum, which includes Crenarchaea. Our comprehensive study also revealed that one archaeal species could contain two distinct oligosaccharide donors for the oligosaccharyl transfer reaction. The A. fulgidus cells contained two oligosaccharide donors bearing oligosaccharide moieties with different backbone structures, and the S. solfataricus cells contained two oligosaccharide donors bearing stereochemically different dolichol chains.

Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.

Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs.

Glycoprotein biosynthesis in a eukaryote lacking the membrane protein Rft1.

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.

Specific transbilayer translocation of dolichol-linked oligosaccharides by an endoplasmic reticulum flippase.

The oligosaccharide donor for protein N-glycosylation, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, is synthesized via a multistep pathway that starts on the cytoplasmic face of the endoplasmic reticulum (ER) and ends in the lumen where the glycosylation reaction occurs. This necessitates transbilayer translocation or flipping of the lipid intermediate Man(5)GlcNAc(2)-PP-dolichol (M5-DLO) across the ER membrane. The mechanism by which M5-DLO-or any other lipid-is flipped across the ER is unknown, except that specific transport proteins or flippases are required. We recently demonstrated M5-DLO flipping activity in proteoliposomes reconstituted from detergent-solubilized ER membrane proteins and showed that it was ATP-independent and required a trypsin-sensitive protein that sedimented at approximately 4S. By using an activity-enriched fraction devoid of glycerophospholipid flippase activity, we now report that M5-DLO is rapidly flipped in the reconstituted system with a time constant tau <2 min, whereas its triantennary structural isomer is flipped slowly with tau >200 min. DLOs larger than M5-DLO are also poorly translocated, with tau ranging from approximately 10 min to >200 min. We conclude that (i) the number and arrangement of mannoses in the DLO glycan has a profound effect on the ability of the DLO to be translocated by the flippase, (ii) glycan size per se does not dictate whether a DLO will be flipped, and (iii) the flippase is highly specific for M5-DLO. Our results suggest a simple structural model for the interaction between the DLO head group and the flippase.

Addition of truncated oligosaccharides to influenza virus hemagglutinin results in its temperature-conditional cell-surface expression.

In the preceding paper (Hearing, J., E. Hunter, L. Rodgers, M.-J. Gething, and J. Sambrook. 1989. J. Cell Biol. 108:339-353) we described the isolation and initial characterization of seven Chinese hamster ovary cell lines that are temperature conditional for the cell-surface expression of influenza virus hemagglutinin (HA) and other integral membrane glycoproteins. Two of these cell lines appeared to be defective for the synthesis and/or addition of mannose-rich oligosaccharide chains to nascent glycoproteins. In this paper we show that at both 32 and 39 degrees C in two mutant cell lines accumulate a truncated version, Man5GlcNAc2, of the normal lipid-linked precursor oligosaccharide, Glc3Man9GlcNAc2. This is possibly due to a defect in the synthesis of dolichol phosphate because in vitro assays indicate that the mutant cells are not deficient in mannosylphosphoryldolichol synthase at either temperature. A mixture of truncated and complete oligosaccharide chains was transferred to newly synthesized glycoproteins at both the permissive and restrictive temperatures. Both mutant cell lines exhibited altered sensitivity to cytotoxic plant lectins when grown at 32 degrees C, indicating that cellular glycoproteins bearing abnormal oligosaccharide chains were transported to the cell surface at the permissive temperature. Although glycosylation was defective at both 32 and 39 degrees C, the cell lines were temperature conditional for growth, suggesting that cellular glycoproteins were adversely affected by the glycosylation defect at the elevated temperature. The temperature-conditional expression of HA on the cell surface was shown to be due to impairment at 39 degrees C of the folding, trimerization, and stability of HA molecules containing truncated oligosaccharide chains.

Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic.

Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing the plasma membrane. These peptides generally do not act via specific receptors and are active in the micromolar range. Here it is shown that vancomycin and the antibacterial peptide nisin Z use the same target: the membrane-anchored cell wall precursor Lipid II. Nisin combines high affinity for Lipid II with its pore-forming ability, thus causing the peptide to be highly active (in the nanomolar range).

Biosynthesis and characterization of large dolichyl diphosphate-linked oligosaccharides in Saccharomyces cerevisiae.

Yeast membranes incorporate radioactivity from GDP[14C]mannose into various glycolipids. These can be separated by thin layer chromatography into at least seven components. The major component has been identified previously as dolichyl monophosphate mannose. Only one additional component is not sensitive to mild alkaline saponification, but is hydrolyzed instead under mild acidic conditions. This latter glycolipid has all the characteristics of a polyprenyl diphosphate oligosaccharide with a sugar moiety of more than 12 hexose units. It runs like dolichyl diphosphate derivatives on a DEAE column and evidence is presented that the lipid moiety is a polyprenol. When radioactive Dol-PP-di-N-acetylchitobiose is incubated with yeast membranes in the presence of non-radioactive GDPmannose a small amount of a larger lipid oligosaccharides is formed besides the previously-described Dol-PP-(GlcNAc)2 mannose. This oligosaccharide has all the properties of the glycolipid described above. Its formation is greatly increased when Triton is omitted from the incubation. Radioactivity of the polyprenyl diphosphate [14C]oligosaccharide is transferred to ethanol-insoluble material, most likely endogenous membrane glycoproteins.

Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides. I. Mannosylated units.

In order to evaluate the possibility in a pig thyroid rough microsomal system of a transfer of pre-assembled sugar cores from sugar-lipids to protein, we have examined after incubation with GDP-[14C]Man the compounds bearing labeled saccharides and have determined some properties of their released saccharide moieties. The [14C]Man material specifically soluble in CHCl3/CH3OH/H2O, 10:10:3, behaved on DEAE-cellulose and when treated with hot alkali and alkaline phosphatase as a lipid pyrophosphate (sometimes accompanied by some dolichol-P-[14C]Man). Its saccharide moiety, released by mild acid, exhibited properties (molecular size, sensitivity to alpha-mannosidase, affinity for concanavalin A and charge modification introduced by a strong reductive alkaline treatment) pointing to a polymannosylated N,N'-diacetylchitobiose containing an average of nine monosaccharide units (from six to twelve). The [14C]mannosylated glycoproteins have represented all the polymeric label remaining after lipid extraction. From the susceptibility of their pronase glycopeptides to a differential reductive alkaline hydrolysis, it was concluded that their label belonged mainly to N-glycosidically linked units. Released saccharides exhibited the same properties as those from lipids, a result substantiating the possibility raised from previous studies of a transfer of pre-assembled moieties.

Phosphatidylcholine requirement for the N-glycosylation of synthetic peptides by detergent-solubilized oligosaccharyltransferase.

The ability of dolichyl-P-P-oligosaccharide:peptide oligosaccharyltransferase to use exogenous substrates (a previously labeled oligosaccharide lipid and an Asn-X-Thr containing heptapeptide) is shown to require phospholipid. The enzyme was extracted from porcine thyroid rough microsomes using NaCl-Nonidet P-40. When measured at low concentration, in a neutral detergent-containing medium, it undergoes a rapid loss of activity, which renders impossible quantitative estimates in the range of 0-50 micrograms microsomal protein/50 microliters assay. We observed that inactivation could be prevented by supplementing the assay with a previously heat-treated suspension of microsomes in neutral detergent, or with the corresponding extract. Further investigation revealed that phospholipids are responsible for this enzyme stabilization, since phospholipase A2 and phospholipase C treatments were both able to abolish this effect. When individual phospholipids were compared for their protective efficiency, egg yolk phosphatidylcholine was found to be by far the most efficient. Phosphatidylglycerol, phosphatidylinositol and phosphatidylserine were only slightly effective, while phosphatidylethanolamine and lysophosphatidylcholine had no effect at all. Of those tested, partly unsaturated phosphatidylcholines with 16-18 carbon atom acyl chains were the most active, at an optimal concentration of 1-2 mM. Under these conditions a Km of 15 microM was measured for the acceptor, a synthetic ribonuclease heptapeptide, and a Km of 0.55 microM for the donor, dolichyl-P-P-GlcNAc2-Man9-Glc2-3. These findings were confirmed by subjecting a sodium deoxycholate extract to depletion of endogenous lipids by gel filtration. Enzyme activity was totally abolished and then restored (up to now only partially) by addition of phosphatidylcholine.

Mannosylation of glycoproteins and dolichol derivatives in fibroblasts from patients with cystic fibrosis.

Increased incorporation of mannose into endogenous glycoprotein fractions has been found in whole cell lysates and crude membrane preparations of cultured skin fibroblasts from patients with cystic fibrosis (1.3-2.3-times normal) when GDP[14C]mannose served as the mannosyl donor. In contrast, the incorporation of mannose from GDPmannose into lipid fractions containing dolichol phosphate and dolichol pyrophosphate oligosaccharides as well as the incorporation of mannose from dolichol phospho[3H]mannose into both glycoproteins and dolichol derivatives were not significantly different among cell preparations from patients with cystic fibrosis and normal controls. Mannosyltransferase activity toward exogenous glycoproteins as well as the activities of soluble and membranous alpha-mannosidase and beta-mannosidase appeared to be normal and could not account for the observed differences. The altered incorporation of mannose into endogenous glycoprotein may reflect changes in glycosylation processes other than mannosylation.

Solubilization of an alpha-(1 leads to 3)-D-mannosyltransferase from pancreas which utilizes synthetic dolichyl pyrophosphate trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc as substrate.

Calf pancreas microsomes were treated with 0.5-1% Triton X-100 and the resulting soluble enzyme preparation was incubated with GDP-D-[14C]mannose. The addition of synthetic Dol-PP derivative of the trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc stimulated the synthesis of labeled lipid-bound tetrasaccharide 50-100-fold. The labeled tetrasaccharide thus formed was identified as alpha-Man-(1 leads to 3)-beta-Man-(1 leads to 4)-beta-GlcNAc- (1 leads to 4)GlcNAc by its chromatographic properties and by its sensitivity to alpha-mannosidase and to endo-beta-N-acetylglucosaminidase D. The solubilized alpha-(1 leads to 3)mannosyltransferase did not require divalent cation and was active in the presence of 10 mM EDTA.

Stored dolichyl pyrophosphoryl oligosaccharides in Batten disease.

Each of the 3 childhood forms of Batten disease, juvenile (JB), late-infantile (LIB), and infantile (IB), have abnormally high brain concentrations of dolichyl pyrophosphoryl oligosaccharides (Dol-PP-OS). In this study, the carbohydrate portions of Dol-PP-OS were analysed: in JB and LIB, they range in size from Man2GlcNAc2 to Glc3Man9GlcNAc2, predominant components being Man5-7GlcNAc2 and Glc3Man7GlcNAc2. In IB, they range from Man6-9GlcNAc2, no glucose containing oligosaccharides being identified. In Batten disease, the main subcellular location of Dol-PP-OS is within storage material, where it represents up to 7% of the dry weight. [3H]-Mannose incorporation experiments with cultured fibroblasts show that synthesis of Dol-PP-OS in JB is normal. We infer that the glycosylation intermediate Glc3Man9GlcNAc2-PP-dolichol is synthesised normally within the endoplasmic reticulum in Batten disease, but that catabolic derivatives accumulate within the lysosomes. It is unclear whether this process is central to the pathogenesis of the disease, though in IB a defect in the release of mannose residues from Dol-PP-OS is a distinct possibility.

Evidence for processing of dolichol-linked oligosaccharides in patients with neuronal ceroid-lipofuscinosis.

In agreement with reports from other laboratories, we have shown that patients with the juvenile or late infantile forms of neuronal ceroid-lipofuscinosis (NCL) have greatly increased levels (5-fold to 20-fold) of dolichyl pyrophosphoryl oligosaccharides in their cerebral gray matter. Oligosaccharides containing 2 GlcNAc residues and 3 to 9 mannose residues were liberated by mild acid hydrolysis. The oligosaccharide profile given by brain tissue from 2 patients with infantile NCL was markedly different from that of late infantile and juvenile NCL brain, with Man9GlcNAc2 as the most abundant component and decreasing amounts of Man8- Man7- and Man6GlcNAc2. By contrast, Man5GlcNAc2 was the most abundant oligosaccharide present in all juvenile NCL brain samples analyzed. Both the susceptibility of the isolated Man5GlcNAc2 to endoglucosaminidase H digestion and permethylation analysis clearly indicated that it is not an intermediate in the biosynthesis of Glc3Man9GlcNAc2-PP-dolichol but has undergone catabolism, probably either in the endoplasmic reticulum or in the Golgi apparatus. Treatment of cultured skin fibroblasts for 7 days with N-methyldeoxynojirimycin, a potent inhibitor of the endoplasmic reticulum processing enzymes glucosidase I and II, resulted in an accumulation of the same Man5GlcNAc2-PP-dolichol species that was elevated in juvenile NCL brain. The level in untreated fibroblasts was undetectable, suggesting that inhibition of processing glucosidases has interfered with the regulation and compartmentalization of lipid-linked oligosaccharides.

In situ assay for identifying inhibitors of bacterial transglycosylase.

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.

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.

Biosynthesis, in calf pancreas microsomes, of three lipid-linked oligosaccharide diphosphates from a synthetic dolichyl diphosphate tetrasaccharide.

Incubation of calf pancreas microsomes with synthetic alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1 ----4)-alpha-D- GlcpNAc-PP-Dol and GDP-D-[14C]-mannose gave three major lipid-linked oligosaccharide diphosphates. After release of the phospholipid residue by mild acid hydrolysis, the corresponding [14C]oligosaccharides were analyzed by gel-filtration, liquid chromatography, degradation by endo-N-acetyl-beta-D-glucosaminidases D and H, by jack bean alpha-D-mannosidase and Aspergillus oryzae (1----2)-alpha-D-mannosidase, acetolysis, and binding to concanavalin A-Sepharose. From the results it could be inferred that the following reaction took place in calf pancreas microsomes: alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1 ----4)-alpha-D- GlcpNAc-PP-Dol + GDP-D-Man gave GDP + alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)- beta-D-GlcpNAc-(1----4)-alpha-D-GlcpNAc-PP-Dol. The next products to be formed were alpha-D-Manp-(1----2)-alpha-D-Manp-(1----3)-[alpha-D-Manp -(1----6)]-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-alpha-D-GlcpNAc-PP-Dol, followed by alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----3)- [alpha-D-Manp-(1----6)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-alpha- D-GlcpNAc-PP-Dol. The mannose incorporation was enhanced by Triton X-100 and inhibited by Mn2+, and it occurred in the presence of either Mg2+ or EDTA. It is likely that the mannose donor was GDP-mannose since, under the conditions used, the formation of dolichyl mannosyl phosphate was negligible and the dolichyl heptasaccharide diphosphate accumulated.