Enzymes that recognize dolichols participate in three glycosylation pathways and are required for protein secretion.

We have explored the structure, function, and membrane topography of enzymes that recognize dolichols and participate in glycosylation pathways in the endoplasmic reticulum. Enzymes that interact with dolichols, including dolichyl phosphate mannose (Dol-P-Man) synthase and UDP-GlcNAc:Dol-P-transferase, revealed a conserved amino acid sequence in membrane-spanning regions. The consensus is Phe-Ile/Val-Xaa-Phe/Try-Xaa-Xaa-Ile-Pro-Phe-Xaa-Phe/Tyr, and we propose it is involved in dolichol recognition. We have used yeast mutants to demonstrate the role of dolichols in three glycosylation pathways. At its nonpermissive temperature, a Dol-P-Man synthase mutant (dpm1) was blocked in N-glycosylation, O-mannosylation, and glycosyl phosphoinositol membrane anchoring of protein, most likely because Dol-P-Man serves as mannosyl donor in all three pathways. The secretion mutant sec59 has a similar phenotype to dpm1, and the presence of a dolichol recognition sequence in the SEC59 protein gave a clue to its defect, which is in dolichol kinase. Comparison of yeast glycosylation mutant suggests that the ability to carry out N-glycosylation alone is sufficient to allow yeast to secrete glycoproteins and that an N-linked saccharide of a minimum size must be attached to proteins for cells to be able to secrete them and maintain a functional secretory pathway.     

Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation

Thermosensitive mutants of Saccharomyces cerevisiae, affected in the endoplasmic reticulum (ER) located glycosylation, i.e. in Dol-P-Man synthase (dpm1), in beta-1,4 mannosyl transferase (alg1) and in alpha-1,3 mannosyltransferase (alg2), were used to assess the role of GDP-Man availability for the synthesis of dolichol-linked saccharides. The mutants were transformed with the yeast gene MPG1 (PSA1/VIG9) encoding GDP-Man pyrophosphorylase catalyzing the final step of GDP-Man formation. We found that overexpression of MPG1 allows growth at non-permissive temperature and leads to an increase in the cellular content of GDP-Man. In the alg1 and alg2 mutants, complemented with MPG1 gene, N-glycosylation of invertase was in part restored, to a degree comparable to that of the wild-type control. In the dpm1 mutant, the glycosylation reactions that depend on the formation of Dol-P-Man, i.e. elongation of Man(5)GlcNAc(2)-PP-Dol, O-mannosylation of chitinase and synthesis of GPI anchor were normal when MPG1 was overexpressed.Our data indicate that an increased level of GDP-Man is able to correct defects in mannosylation reactions ascribed to the ER and to the Golgi.     

Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.     

In vitro biosynthesis of glycosylphosphatidylinositol in Aspergillus fumigatus

Glycosylphosphatidylinositol (GPI) represents a mechanism for the attachment of proteins to the plasma membrane found in all eukaryotic cells. GPI biosynthesis has been mainly studied in parasites, yeast, and mammalian cells. Aspergillus fumigatus, a filamentous fungus, produces GPI-anchored molecules, some of them being essential in the construction of the cell wall. An in vitro assay was used to study the GPI biosynthesis in the mycelium form of this organism. In the presence of UDP-GlcNAc and coenzyme A, the cell-free system produces the initial intermediates of the GPI biosynthesis: GlcNAc-PI, GlcN-PI, and GlcN-(acyl)PI. Using GDP-Man, two types of mannosylation are observed. First, one or two mannose residues are added to GlcN-PI. This mannosylation, never described in fungi, does not require dolichol phosphomannoside (Dol-P-Man) as the monosaccharide donor. Second, one to five mannose residues are added to GlcN-(acyl)PI using Dol-P-Man as the mannose donor. The addition of ethanolamine phosphate groups to the first, second, and third mannose residue is also observed. This latter series of GPI intermediates identified in the A. fumigatus cell-free system indicates that GPI biosynthesis in this filamentous fungus is similar to the mammalian or yeast systems. Thus, these biochemical data are in agreement with a comparative genome analysis that shows that all but 3 of the 21 genes described in the Saccharomyces cerevisiae GPI pathways are found in A. fumigatus.     

Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid

The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.     

Investigations of the active site of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase using fluorescent labeled dolichyl-phosphate derivatives

Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme-phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity.     

Structure of Saccharomyces cerevisiae alg3, sec18 mutant oligosaccharides

Asparagine-linked oligosaccharides are synthesized by transfer of Glc3Man9GlcNAc2 from dolichol pyrophosphate to nascent polypeptides. Assembly of the precursor proceeds by highly ordered sequential addition of mannose and glucose to form Glc3Man9GlcNAc2-P-P-dolichol. Yeast mutants in asparagine-linked glycosylation (alg), generated by an 3H-Man suicide technique, were assigned to eight complementation groups which define steps in oligosaccharide-lipid synthesis (Huffaker, T.C., and Robbins, P.W. (1982) J. Biol. Chem. 257, 3203-3210). Alg3 invertase oligosaccharides are resistant to endo-beta-N-acetylglucosaminidase H, and the lipid-oligosaccharide pool yields Man5Glc-NAc2, suggesting its structure may be that from mammalian cells lacking Man-P-dolichol (Chapman, A., et al. (1980) J. Biol. Chem. 255, 4441-4446). To test this supposition, the endoplasmic reticulum form of invertase derepressed in alg3,sec18 yeast at 37 degrees C was isolated as a source of oligosaccharides whose processing beyond glucose and/or mannose trimming, if involved, would be prevented. Man8GlcNAc2 and Man5GlcNAc2 were released by peptide-N-glycosidase F from alg3,sec18 invertase in a 1:5 molar ratio. 1H NMR spectroscopy revealed Man8GlcNAc2 to be the alpha 1,2-mannosidase-trimming product described earlier (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666), while Man5GlcNAc2 was Man alpha 1, 2Man alpha 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc beta 1, 4GlcNAc. This provides a structural proof for the lipid-linked Man5GlcNAc2 originally proposed from enzymatic and chemical analyses of the radiolabeled mammalian precursor. Experimental evidence indicates that, unlike the mammalian cell mutants which are unable to synthesize Man-P-dolichol, alg3 yeast accumulate Man5GlcNAc2-P-P-dolichol due to a defective alpha 1,3-mannosyltransferase required for the next step in oligosaccharide-lipid elongation.     

Human Smp3p adds a fourth mannose to yeast and human glycosylphosphatidylinositol precursors in vivo

Yeast and human glycosylphosphatidylinositol (GPI) precursors differ in the extent to which a fourth mannose is present as a side branch of the third core mannose. A fourth mannose addition to GPIs has scarcely been detected in studies of mammalian GPI synthesis but is an essential step in the Saccharomyces cerevisiae pathway. We report that human SMP3 encodes a functional homolog of the yeast Smp3 GPI fourth mannosyl-transferase. Expression of hSMP3 in yeast complements growth and biochemical defects of smp3 mutants and permits in vivo mannosylation of trimannosyl (Man(3))-GPIs. Immunolocalization shows that hSmp3p resides in the endoplasmic reticulum in human cells. Northern analysis of mRNA from human tissues and cell lines indicates that hSMP3 is expressed in most tissues, with the highest levels in brain and colon, but its mRNA is nearly absent from cultured human cell lines. Correspondingly, increasing expression of hSMP3 in cultured HeLa cells causes abundant formation of three putative tetramannosyl (Man(4))-GPIs. Our data indicate that hSmp3p functions as a mannosyltransferase that adds a fourth mannose to certain Man(3)-GPIs during biosynthesis of the human GPI precursor, and suggest it may do so in a tissue-specific manner.     

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.     

The role of C-4-substituted mannose analogues in protein glycosylation. Effect of the guanosine diphosphate esters of 4-deoxy-4-fluoro-D-mannose and 4-deoxy-D-mannose on lipid-linked oligosaccharide assembly.

The effects of the guanosine diphosphate esters of 4-deoxy-4-fluoro-D-mannose (GDP-4FMan) and 4-deoxy-D-mannose (GDP-4dMan) on reactions of the dolichol pathway in chick-embryo cell microsomal membranes were investigated by studies with chick-embryo cell microsomal membranes in vitro and in baby-hamster kidney (BHK) cells in vivo. Each nucleotide sugar analogue inhibited lipid-linked oligosaccharide biosynthesis in a concentration-dependent manner. GDP-4FMan blocked in vitro the addition of mannose to Dol-PP-(GlcNAc)2Man from GDP-Man (where Dol represents dolichol), but did not interfere with the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2. Although GDP-4FMan and Dol-P-4FMan were identified as metabolites of 4FMan in BHK cells labelled with [1-14C]4FMan, GDP-4FMan was a very poor substrate for GDP-Man:Dol-P mannosyltransferase and Dol-P-4FMan could only be synthesized in vitro if the chick-embryo cell membranes were primed with Dol-P. It therefore appears that the inhibition of lipid-linked oligosaccharide formation in BHK cells treated with 4FMan [Grier & Rasmussen (1984) J. Biol. Chem. 259, 1027-1030] is due primarily to a blockage in the formation of Dol-PP-(GlcNAc)2Man2 by GDP-4FMan. In contrast, GDP-4dMan was a substrate for those mannosyltransferases that catalyse the transfer of the first five mannose residues to Dol-PP-(GlcNAc)2. In addition, GDP-4dMan was a substrate for GDP-Man:Dol-P mannosyltransferase, which catalysed the formation of Dol-P-4dMan. As a consequence of this, the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2 may be inhibited through competition for Dol-P. In BHK cells treated with 10 mM-4dMan, Dol-PP-(GlcNAc)2Man9 was the major lipid-linked oligosaccharide detected. Nearly normal extents of protein glycosylation were observed, but very little processing to complex oligosaccharides occurred, and the high-mannose structures were smaller than in untreated cells.     

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 α-mannosidase and β-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.     

Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).     

Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway

Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a [3H]mannose suicide enrichment. Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro. When UDP-[3H]Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose. When dolicholphospho[3H]glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides. These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide. We also present evidence that both mutants transfer Man9GlcNAc2 to protein.     

In vivo characterization of the GPI assembly defect in yeast mcd4-174 mutants and bypass of the Mcd4p-dependent step in mcd4Delta cells

Yeast mcd4-174 mutants are blocked in glycosylphosphatidylinositol (GPI) anchoring of protein, but the stage at which GPI biosynthesis is interrupted in vivo has not been identified, and Mcd4p has also been implicated in phosphatidylserine and ATP transport. We report that the major GPI that accumulates in mcd4-174 in vivo is Man(2)-GlcN-(acyl-Ins)PI, consistent with proposals that Mcd4p adds phosphoethanolamine to the first mannose of yeast GPI precursors. Mcd4p-dependent modification of GPIs can partially be bypassed in the mcd4-174/gpi11 double mutant and in mcd4Delta; mutants by high-level expression of PIG-B and GPI10, which respectively encode the human and yeast mannosyltransferases that add the third mannose of the GPI precursor. Rescue of mcd4Delta; by GPI10 indicates that Mcd4p-dependent addition of EthN-P to the first mannose of GPIs is not obligatory for transfer of the third mannose by Gpi10p.     

Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein

Dolichol phosphate mannose (Dol-P-Man) synthase (EC 2.4.1.83) catalyzes the formation of Dol-P-Man from Dol-P and GDP-Man. The structural gene for yeast Dol-P-Man synthase (DPM1) was isolated by screening a yeast genomic DNA library for colonies that overexpressed Dol-P-Man synthase activity. This approach relied on a method to screen for Dol-P-Man synthase activity in lysed yeast colonies and used a yeast mutant with very low Dol-P-Man synthase activity in colony lysates. Transformants isolated using this technique expressed Dol-P-Man synthase activity 9-14-fold higher than that of a wild type strain, and all seven plasmids conferring this overproduction had a common region in their yeast genomic DNA insert. DPM1 is the structural gene for yeast Dol-P-Man synthase since Escherichia coli transformants harboring this gene express Dol-P-Man synthase activity in vitro. DNA sequencing of the DPM1 gene revealed an open reading frame of 801 bases. The 30-kDa size of the predicted protein is in excellent agreement with the size of the purified yeast enzyme (Haselbeck, A., and Tanner, W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1520-1524). Analysis of the predicted amino acid sequence reveals the protein has a potential membrane spanning domain of 25 amino acids at its COOH terminus. The protein's NH2 terminus, though not hydrophobic, meets existing criteria for yeast signal sequences, but there is no site for cleavage by signal peptidase. If the NH2 terminus is a functional signal sequence, the protein is predicted to be oriented toward the lumen of the endoplasmic reticulum with both NH2 and COOH termini serving as membrane anchors. If there is no signal sequence, the enzyme is predicted to face the cytoplasm and be anchored only by its COOH terminus. The DPM1 gene is essential for viability in yeast since disruption of the gene is lethal. We suspect Dol-P-Man synthase is not an essential protein due to its role in N-glycosylation since mutations in other genes that affect the late steps in lipid-linked oligosaccharide synthesis do not affect cell growth. Instead, DPM1 may be an essential gene because its product is required for O-glycosylation in yeast or because Dol-P-Man synthase is needed in some unidentified pathway.     

Inhibition of lipid-linked saccharide synthesis by bacitracin.

The antibiotic bacitracin was found to inhibit the incorporation of mannose and GlcNAc from their respective sugar nucleotides into lipid-linked saccharides. The inhibition of both systems was apparent in the aorta particulate enzyme system but it was much more pronounced with the solubilized enzyme system. In both cases, GlcNAc incorporation into Dol-P-P-GlcNAc was more sensitive than mannose incorporation into Dol-P-Man, with 50% inhibition being seen at about 0.1–0.2 mm antibiotic. Bacitracin inhibition of mannose incorporation appeared to be overcome at high concentrations of dolichyl phosphate but, in these cases, an unexplained stimulation was observed. However, GlcNAc inhibition could not be overcome by high concentrations of dolichol phosphate, metal ion, or both together. Thus, the mechanism of inhibition by bacitracin is not clear. Bacitracin also inhibited the transfer of mannose from GDP-mannose to lipid-linked oligosaccharides and to glycoprotein in the particulate enzyme, as well as the transfer of radioactivity from Dol-P-Man or from lipid-linked oligosaccharides to glycoprotein. Thus, bacitracin apparently blocks each of the steps in the lipid-linked pathway. In yeast spheroplasts, bacitracin inhibited the incorporation of [¹⁴C]mannose into Dol-P-Man, into lipid-linked oligosaccharides, and into glycoprotein. However, in this case, the antibiotic also blocked the incorporation of leucine into protein. Bacitracin also inhibited the cell-free synthesis of mannosyl-phosphoryl-decaprenol in Mycobacterium smegmatis with 50% inhibition being observed at a concentration of about 0.5 mm.     

The effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.     

Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase

The formation of N-glycosidic linkages of glycoproteins involves the ordered assembly of the common Glc3Man9GlcNAc2 core-oligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as donor, the final reactions from Man5GlcNAc2-PP-Dol to Man9GlcNAc2-PP-Dol on the lumenal side use Dol-P-Man. We have investigated these later stages in vitro using a detergent-solubilized enzyme extract from yeast membranes. Mannosyltransfer from Dol-P-Man to [3H]Man5GlcNAc2-PP-Dol with formation of all intermediates up to Man9GlcNAc2-PP-Dol occured in a rapid, time- and protein-dependent fashion. We find that the initial reaction from Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol is independent of metal ions, but further elongations need Mn2+ that can be partly replaced by Mg2+ or Ca2+. Zn2+ or Cd2+ ions were found to inhibit formation of Man(7-9)GlcNAc2-PP-Dol, but do not affect synthesis of Man6GlcNAc2-PP-Dol. Extension did not occur when the acceptor was added as a free Man5GlcNAc2 oligosaccharide or when GDP-Man was used as mannosyl donor. The alg3 mutant was described to accumulate Man5GlcNAc2-PP-Dol. We expressed a functional active HA-epitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.     

Partial purification and characterization of a mannosyl transferase involved in O-linked mannosylation of glycoproteins in Candida albicans

Incubation of a mixed membrane fraction of C. albicans with the nonionic detergents Nonidet P-40 or Lubrol solubilized a fraction that catalyzed the transfer of mannose either from endogenously generated or exogenously added dolichol-P-[14C]Man onto endogenous protein acceptors. The protein mannosyl transferase solubilized with Nonidet P-40 was partially purified by a single step of preparative nondenaturing electrophoresis and some of its properties were investigated. Although transfer activity occurred in the absence of exogenous mannose acceptors and thus depended on acceptor proteins isolated along with the enzyme, addition of the protein fraction obtained after chemical de-mannosylation of glycoproteins synthesized in vitro stimulated mannoprotein labeling in a concentration-dependent manner. Other de-mannosylated glycoproteins, such as yeast invertase or glycoproteins extracted from C. albicans, failed to increase the amount of labeled mannoproteins. Mannosyl transfer activity was not influenced by common metal ions such as Mg(2+), Mn(2+) and Ca(2+), but it was stimulated up to 3-fold by EDTA. Common phosphoglycerides such as phosphatidylglycerol and, to a lower extent, phosphatidylinositol and phosphatidylcholine enhanced transfer activity. Interestingly, coupled transfer activity between dolichol phosphate mannose synthase, i.e., the enzyme responsible for Dol-P-Man synthesis, and protein mannosyl transferase could be reconstituted in vitro from the partially purified transferases, indicating that this process can occur in the absence of cell membranes.     

Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker’s yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.