Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.     

Phosphomannose isomerase inhibitors improve N-glycosylation in selected phosphomannomutase-deficient fibroblasts

Congenital disorders of glycosylation (CDG) are rare genetic disorders due to impaired glycosylation. The patients with subtypes CDG-Ia and CDG-Ib have mutations in the genes encoding phosphomannomutase 2 (PMM2) and phosphomannose isomerase (MPI or PMI), respectively. PMM2 (mannose 6-phosphate → mannose 1-phosphate) and MPI (mannose 6-phosphate ⇔ fructose 6-phosphate) deficiencies reduce the metabolic flux of mannose 6-phosphate (Man-6-P) into glycosylation, resulting in unoccupied N-glycosylation sites. Both PMM2 and MPI compete for the same substrate, Man-6-P. Daily mannose doses reverse most of the symptoms of MPI-deficient CDG-Ib patients. However, CDG-Ia patients do not benefit from mannose supplementation because >95% Man-6-P is catabolized by MPI. We hypothesized that inhibiting MPI enzymatic activity would provide more Man-6-P for glycosylation and possibly benefit CDG-Ia patients with residual PMM2 activity. Here we show that MLS0315771, a potent MPI inhibitor from the benzoisothiazolone series, diverts Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation. Finally, we show that MLS0315771 increases mannose metabolic flux toward glycosylation in zebrafish embryos.     

Identification of intercellular cell adhesion molecule 1 (ICAM-1) as a hypoglycosylation marker in congenital disorders of glycosylation cells

Many human inherited disorders cause protein N-glycosylation defects, but there are few cellular markers to test gene complementation for such defects. Plasma membrane glycoproteins are potential biomarkers because they may be reduced or even absent in plasma membranes of glycosylation-deficient cells. We combined stable isotope labeling by amino acids in cell culture (SILAC) with linear ion trap mass spectrometry (LTQ Orbitrap(TM)) to identify and quantify membrane proteins from wild-type CHO and glycosylation-deficient CHO (Lec9) cells. We identified 165 underrepresented proteins from 1447 unique quantified proteins, including 18 N-glycosylated plasma membrane proteins. Using various methods, we found that intercellular cell adhesion molecule 1 (ICAM-1) was reduced in Lec9 cells and in fibroblasts from 31 congenital disorder of glycosylation (CDG) patients compared with normal controls. Mannose supplementation of phosphomannose isomerase-deficient CDG-Ib (MPI-CDG) cells and complementation with PMM2 in PMM2-deficient CDG-Ia (PMM2-CDG) cells partially corrected hypoglycosylation based on increased ICAM-1 presence on the plasma membrane. These data indicate that ICAM-1 could be a useful hypoglycosylation biomarker to assess gene complementation of CDG-I patient cells and to monitor improved glycosylation in response to therapeutic drugs.     

The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells

Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding approximately 13 microm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A(1), an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 microm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.     

Analysis of glycosylation in CDG-Ia fibroblasts by fluorophore-assisted carbohydrate electrophoresis: implications for extracellular glucose and intracellular mannose 6-phosphate

Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc(3)Man(9)GlcNAc(2)-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [(3)H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [(3)H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, fluorophore-assisted carbohydrate electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [(3)H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc(3)Man(9)GlcNAc(2)-P-P-dolichol, without hypoglycosylation, CDG-Ia fibroblasts grown with physiological glucose. This suggested a "missing link" to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, M6P was a specific activator for cleavage of Glc(3)Man(9)GlcNAc(2)-P-P-dolichol. This led to futile cycling the LLO pathway, exacerbated by GDP-mannose/PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.     

Congenital disorders of glycosylation type Ig is defined by a deficiency in dolichyl-P-mannose:Man7GlcNAc2-PP-dolichyl mannosyltransferase

Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for protein N-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast gene ALG12 that encodes the dolichyl-P-Man:Man(7)GlcNAc(2)-PP-dolichyl alpha6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man(7)GlcNAc(2) from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum.     

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.     

Phosphomannomutase activity in congenital disorders of glycosylation type Ia determined by direct analysis of the interconversion of mannose-1-phosphate to mannose-6-phosphate by high-pH anion-exchange chromatography with pulsed amperometric detection

Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP(+) to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0-15.3% of control, average 4.9+/-4.7%) and also decreased in seven obligate heterozygotes (range 33.0-72.0% of control, average 52.2+/-14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated.     

Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type 1A.

Carbohydrate-deficient-glycoprotein syndrome type 1 (CDG1; also known as "Jaeken syndrome") is an autosomal recessive disorder characterized by defective glycosylation. Most patients show a deficiency of phosphomannomutase (PMM), the enzyme that converts mannose 6-phosphate to mannose 1-phosphate in the synthesis of GDP-mannose. The disease is linked to chromosome 16p13, and mutations have recently been identified in the PMM2 gene in CDG1 patients with a PMM deficiency (CDG1A). The availability of the genomic sequences of PMM2 allowed us to screen for mutations in 56 CDG1 patients from different geographic origins. By SSCP analysis and by sequencing, we identified 23 different missense mutations and 1 single-base-pair deletion. In total, mutations were found on 99% of the disease chromosomes in CDG1A patients. The R141H substitution is present on 43 of the 112 disease alleles. However, this mutation was never observed in the homozygous state, suggesting that homozygosity for these alterations is incompatible with life. On the other hand, patients were found homozygous for the D65Y and F119L mutations, which must therefore be mild mutations. One particular genotype, R141H/D188G, which is prevalent in Belgium and the Netherlands, is associated with a severe phenotype and a high mortality. Apart from this, there is only a limited relation between the genotype and the clinical phenotype.     

Phosphomannose isomerase and phosphomannomutase gene disruptions in Streptomyces nodosus: impact on amphotericin biosynthesis and implications for glycosylation engineering

Streptomycetes synthesise several bioactive natural products that are modified with sugar residues derived from GDP-mannose. These include the antifungal polyenes, the antibacterial antibiotics hygromycin A and mannopeptimycins, and the anticancer agent bleomycin. Three enzymes function in biosynthesis of GDP-mannose from the glycolytic intermediate fructose 6-phosphate: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMPP). Synthesis of GDP-mannose from exogenous mannose requires hexokinase or phosphotransferase enzymes together with PMM and GMPP. In this study, a region containing genes for PMI, PMM and GMPP was cloned from Streptomyces nodosus, producer of the polyenes amphotericins A and B. Inactivation of the manA gene for PMI resulted in production of amphotericins and their aglycones, 8-deoxyamphoteronolides. A double mutant lacking the PMI and PMM genes produced 8-deoxyamphoteronolides in good yields along with trace levels of glycosylated amphotericins. With further genetic engineering these mutants may activate alternative hexoses as GDP-sugars for transfer to aglycones in vivo.     

New and potential strategies for the treatment of PMM2-CDG

BackgroundMutations in the PMM2 gene cause phosphomannomutase 2 deficiency (PMM2; MIM# 212065), which manifests as a congenital disorder of glycosylation (PMM2-CDG). Mutant PMM2 leads to the reduced conversion of Man-6-P to Man-1-P, which results in low concentrations of guanosine 5′-diphospho-D-mannose, a nucleotide-activated sugar essential for the construction of protein oligosaccharide chains. To date the only therapeutic options are preventive and symptomatic.Scope of reviewThis review covers the latest advances in the search for a treatment for PMM2-CDG.Major conclusionsTreatments based on increasing Man-1-P levels have been proposed, along with the administration of different mannose derivates, employing enzyme inhibitors or repurposed drugs to increase the synthesis of GDP-Man. A single repurposed drug that might alleviate a severe neurological symptom associated with the disorder is now in clinical use. Proof of concept also exists regarding the use of pharmacological chaperones and/or proteostatic regulators to increase the concentration of hypomorphic PMM2 mutant proteins.General significanceThe ongoing challenges facing the discovery of drugs to treat this orphan disease are discussed.     

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.     

Balancing N-linked glycosylation to avoid disease

Complete loss of N-glycosylation is lethal in both yeast and mammals. Substantial deficiencies in some rate-limiting biosynthetic steps cause human congenital disorders of glycosylation (CDG). Patients have a range of clinical problems including variable degrees of mental retardation, liver dysfunction, and intestinal disorders. Over 60 mutations in phosphomannomutase (encoded by PMM2) diminish activity and cause CDG-Ia. The severe mutation R141H in PMM2 is lethal when homozygous, but heterozygous in about 1/70 Northern Europeans. Another disorder, CDG-Ic, is caused by mutations in ALG6, an alpha 1,3glucosyl transferase used for lipid-linked precursor synthesis, yet some function-compromising mutations occur at a high frequency in this gene also. Maintenance of seemingly deleterious mutations implies a selective advantage or positive heterosis. One possible explanation for this is that production of infective viruses such as hepatitis virus B and C, or others that rely heavily on host N-glycosylation, is substantially inhibited when only a tiny fraction of their coat proteins is misglycosylated. In contrast, this reduced glycosylation does not affect the host. Prevalent functional mutations in rate-limiting glycosylation steps could provide some resistance to viral infections, but the cost of this insurance is CDG. A balanced glycosylation level attempts to accommodate these competing agendas. By assessing the occurrence of a series of N-glycosylation-compromising alleles in multi-genic diseases, it may be possible to determine whether impaired glycosylation is a risk factor or a major determinant underlying their pathology.     

The role of phosphomannose isomerase in Leishmania mexicana glycoconjugate synthesis and virulence

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate, which is the first step in the biosynthesis of activated mannose donors required for the biosynthesis of various glycoconjugates. Leishmania species synthesize copious amounts of mannose-containing glycolipids and glycoproteins, which are involved in virulence of these parasitic protozoa. To investigate the role of PMI for parasite glycoconjugate synthesis, we have cloned the PMI gene (lmexpmi) from Leishmania mexicana, generated gene deletion mutants (Delta lmexpmi), and analyzed their phenotype. Delta lmexpmi mutants lack completely the high PMI activity found in wild type parasites, but are, in contrast to fungi, able to grow in media deficient for free mannose. The mutants are unable to synthesize phosphoglycan repeats [-6-Gal beta 1-4Man alpha 1-PO(4)-] and mannose-containing glycoinositolphospholipids, and the surface expression of the glycosylphosphatidylinositol-anchored dominant surface glycoprotein leishmanolysin is strongly decreased, unless the parasite growth medium is supplemented with mannose. The Delta lmexpmi mutant is attenuated in infections of macrophages in vitro and of mice, suggesting that PMI may be a target for anti-Leishmania drug development. L. mexicana Delta lmexpmi provides the first conditional mannose-controlled system for parasite glycoconjugate assembly with potential applications for the investigation of their biosynthesis, intracellular sorting, and function.     

A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis

Deficiency of GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man1GlcNAc2-PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man1GlcNAc2-PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an alpha1,3-mannosyltransferase. The resulting Manalpha1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown alpha1,6-mannosyltransferase.     

Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus

The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan. The major mannolipid has been purified and characterized as alpha-D-mannosyl-(1 --> 3)-alpha-D-mannosyl-(1 --> 3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated with GDP-[3H]mannose (GDP-Man), [3H]mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.     

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.     

Induction of glycoprotein biosynthesis in activated B lymphocytes

Resting murine splenic B lymphocytes (B cells) can be stimulated to proliferate by exposure to a variety of polyclonal activators. To investigate changes in glycoprotein synthesis that occur during the activation process, N-glycosylation activity was assessed by following the incorporation of [2-3H]mannose into dolichol-linked oligosaccharide intermediates and glycoprotein after B cells were exposed to anti-immunoglobulin M (anti-mu). Stimulation of B cells by anti-mu resulted in a dramatic induction of N-glycosylation activity. The incorporation of radiolabeled mannose into oligosaccharide-lipid increased 9-fold while the rate of labeling of glycoprotein increased 27-fold between 18 and 38 h after exposure to anti-mu. Maximal stimulation of N-glycosylation activity was observed at an anti-mu concentration of 20-50 micrograms/ml. Similar results were obtained when B cells were activated by bacterial lipopolysaccharide (LPS), another polyclonal activating agent. The major dolichol-bound oligosaccharide labeled during the induction period was determined to be Glc3Man9GlcNAc2 by HPLC analysis. Nearly full induction of oligosaccharide-lipid synthesis and protein N-glycosylation was also seen when DNA synthesis was suppressed by activating B cells with anti-mu in a serum-free medium, or by activating with anti-mu or LPS in the presence of hydroxyurea. The results suggest that the N-glycosylation pathway is induced during the G0 to G1 transition or during the G1 period, and that entry into S phase is not required. These studies describe a striking developmental increase in N-glycosylation activity and extend the information on biochemical changes occurring during the activation of B cells.     

A Temperature-sensitive mutation in the Arabidopsis thaliana phosphomannomutase gene disrupts protein glycosylation and triggers cell death

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.