Abnormal synthesis of mannose 1-phosphate derived carbohydrates in carbohydrate-deficient glycoprotein syndrome type I fibroblasts with phosphomannomutase deficiency

In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] mannose into mannose phosphates, GDP-mannose, GDP-fucose, dolichol-P-mannose, lipid- linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into mannose 1-phosphate, GDP-mannose, GDP-fucose, dolichol-P-mannose, and nascent glycoproteins. The incorporation of radioactivity into mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (</=20%), but their size was severely reduced, mostly containing five or fewer residues. As a consequence, truncated oligosaccharides were transferred to newly synthesized glycoproteins. The metabolic changes can be explained by a deficiency of phosphomannomutase activity, which was reduced to </=10% of control.      

Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment

We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-alpha-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-alpha-D-mannosyl-D-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-alpha-D-mannosyl-D-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-alpha-D-mannosyl-D-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.     

A mutant of Chinese hamster ovary cells with a reduction in levels of dolichyl phosphate available for glycosylation

F2A8, a glycosylation mutant of Chinese hamster ovary cells, was isolated without prior enrichment or selective procedures by screening colonies for reduced [3H]mannose incorporation into macromolecules. F2A8 cells incubated with [3H]mannose synthesized 70% the amount of labeled GDP-mannose found in parental cells, and the same oligosaccharides attached to lipid and protein as did parental cells, but in reduced amounts. Incorporation of radioactivity from labeled mannose into saccharide-lipids and into total glycopeptides of F2A8 was reduced 7-fold compared to parental cells. In addition, glycosylation of the vesicular stomatitis virus glycoprotein was reduced in F2A8 cells as assessed by a mobility intermediate between normally glycosylated and unglycosylated protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vitro assays using membrane preparations showed that F2A8 had parental levels of glucosylphosphoryldolichol synthase and of UDP-GlcNAc:dolichyl phosphate:GlcNAc-phosphotransferase when the enzymatic determinations were done in the presence of exogenous dolichyl phosphate. However, 5-fold less glucosylphosphoryldolichol synthase activity was detected in membranes of F2A8 compared to membranes of parental cells in assays relying on endogenous lipid substrate. F2A8 appears to have reduced amounts of dolichyl phosphate available for its glycosylation reactions.     

Reduced mannose incorporation into GDP-mannose and dolichol-linked intermediates of N-glycosylation in hamster liver during vitamin A deficiency

Summary The molecular mechanism of reduced incorporation of radioactively labeled mannose into hamster liver glycoconjugates during the progression of vitamin A deficiency was investigated. In particular the in vivo incorporation of [2-³H]mannose into GDP-mannose, dolichyl phosphate mannose (Dol-P-Man), lipid-linked oligosaccharides, and glycopeptides of hamster liver was examined. Hamsters maintained on a vitamin A-free diet showed a reduction in the incorporation of mannose into GDP-mannose about 10 days before clinical signs of vitamin A deficiency could be observed. The decrease in [2-³H]mannose incorporated into GDP-mannose was accompanied by a reduction in label incorporated into Dol-P-Man, lipid linked oligosaccharides and glycopeptides, which became more severe with the progression of vitamin A deficiency. By the time they reached a plateau stage of growth, hamsters fed the vitamin A-free diet showed a 50% reduction in the amount of [2-³H]mannose converted to GDP-mannose, and the radioactivity associated with Dol-P-Man and glycopeptides was reduced by approximately 60% as compared to retinoic acid-supplemented controls. These results strongly indicate that the reduced incorporation of mannose into lipidic intermediates and glycoproteins observed during vitamin A deficiency is due to impaired GDP-mannose synthesis.Abbreviations Dol-P-Man Dolichyl Phosphate Mannose - Dol-P Dolichyl Phosphate     

Parameters interacting with mannose selection employed for the production of transgenic sugar beet

The mannose selection system employs the phosphomannose isomerase (PMI) gene as selectable gene and mannose, converted to mannose-6-phosphate by endogenous hexokinase, as selective agent. The transgenic PMI-expressing cells have acquired the ability to convert mannose-6-phosphate to fructose-6-phosphate, while the non-transgenic cells accumulate mannose-6-phosphate with a concomitant consumption of the intracellular pools of phosphate and ATP. Thus, certain steps of mannose selection depend on the cells’ own metabolism which may be affected by a number of factors, some of which are studied here using Agrobacterium tumefaciens-mediated gene transfer to sugar beet cotyledonary explants. Four frequently employed saccharides (sucrose, glucose, fructose, and maltose) were tested at various concentrations and were found to interact strongly with the phytotoxic effect of mannose, glucose being able to counteract nearly 100% of an almost complete mannose-induced growth inhibition. Sucrose, maltose, and fructose also alleviated significantly the mannose-induced growth inhibition, but were 4-, 5-, and 7-fold less potent than glucose, respectively (calculated as hexose equivalents). The transformation frequencies were also dependent on the nature and concentration of the added carbohydrates, but in this respect sucrose resulted in the highest transformation frequencies, about 1.0%, while glucose and fructose gave significantly lower frequencies. The selection efficiencies were highest in the presence of maltose where no non-transgenic escapes were found over a range of concentrations. The effect of the light intensity was also investigated and the transformation frequencies were positively correlated to light intensity, although the relative impact of light on growth in the presence of mannose appeared not to be dependent on the mannose concentration. Additional phosphate in the selection media had a strong positive effect on the transformation frequencies, suggesting phosphate limitation during selection. The mannose selection system was found to be relatively genotype-independent, provided a slight optimization of the mannose concentrations during selection. Analysis of F₁-offspring showed that all studied primary transformants resulted in PMI-expressing plantlets and that the segregational patterns were in accordance with expectations in at least 50% of the transformants, confirming the stable and active inheritance of the PMI-gene.     

The yeast SEC53 gene encodes phosphomannomutase

Yeast sec53 cells incubated at a restrictive temperature (37 degrees C) accumulate inactive and incompletely glycosylated forms of secretory proteins within the lumen of the endoplasmic reticulum. A defect in glycosylation of alpha-factor precursor has been reproduced in vitro using membranes and cytosol isolated from sec53 mutant cells. Normal glycosylation is restored in reactions supplemented with a cytosolic fraction from wild type cells, with GDP-mannose, or with mannose 1-phosphate and GTP, but not with mannose 6-phosphate and GTP. This pattern of stimulation suggests that extracts of sec53 cells are deficient in phosphomannomutase activity or in the production of a precursor of mannose 1-phosphate. Several lines of evidence demonstrate that SEC53 encodes the yeast phosphomannomutase. Direct assay of soluble fractions from independent alleles of sec53 shows low to negligible phosphomannomutase, but nearly normal levels of phosphomannoisomerase activity. The residual phosphomannomutase activity in mutant cell lysates is thermolabile in proportion to the severity of the sec53 cell growth defect. Introduction of the SEC53 gene on a multicopy plasmid into sec53 or wild type yeast and into Salmonella typhimurium results in an increase in phosphomannomutase activity that correlates with elevated expression of the Sec53 protein. Finally, the Sec53 protein and phosphomannomutase activity cofractionate exactly in a 70-fold partial purification involving gel filtration and DEAE chromatography. The secretory defect in sec53 cells may now be explained by a deficit in GDP-mannose production.     

Properties of mannosyl- and N-acetylglucosamine-1-phosphate transferases towards dolichol phosphate in liver microsomes from pig embryos

1. The activity of mannosyl- and N-acetylglucosamine-1-phosphate transferases in microsomes from pig embryonic liver was linear to 1 min of incubation at 37 degrees C. 2. The activity of both enzymes was higher in the presence of Mg2+ as compared to Mn2+. A maximal stimulatory effect of Mn2+ was obtained at 2 mM concentration and greater concentrations of it inhibited the activities of both enzymes. 3. The activity of mannosyl transferase was found to be highest after treatment of microsomes with Nonidet P-40 while the activity of N-acetylglucosamine-1-phosphate transferase was greatest in the presence of sodium deoxycholate. 4. The Km for acceptor substrate was 1.6 x 10(-5)M in the reaction for dolichol phosphate mannose synthesis and 2.2 x 10(-5)M in the reaction for dolichol pyrophosphate N-acetylglucosamine formation. 5. The Km for GDP-mannose was 1.4 x 10(-5)M and for UDP-N-acetylglucosamine-6.2 x 10(-5)M. At saturating concentrations of donor substrates V values (pmol/min/mg) were 1330 and 150, respectively.     

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.     

The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum

When pig liver microsomal preparations were incubated with GDP-[¹⁴C]mannose, 10–40% of the ¹⁴C was transferred to mannolipid and 1–3% to mannoprotein. The transfer to mannolipid was readily reversible and GDP was one of the products of the reaction. It was possible to reverse the reaction by adding excess of GDP and to show the incorporation of [¹⁴C]GDP into GDP-mannose. When excess of unlabelled GDP-mannose was added to a partially completed incubation there was a rapid transfer back of [¹⁴C]mannose from the mannolipid to GDP-mannose. The other product of the reaction, the mannolipid, had the properties of a prenol phosphate mannose. This was illustrated by its lability to dilute acid but stability to dilute alkali, and by its chromatographic properties. Dolichol phosphate stimulated the incorporation of [¹⁴C]mannose into both mannolipid and into protein, although the former effect was larger and more consistent than the latter. The incorporation of exogenous [³H]dolichol phosphate into the mannolipid, and its release, accompanied by mannose, on treatment of the mannolipid with dilute acid, confirmed that exogenous dolichol phosphate can act as an acceptor of mannose in this system. It was shown that other exogenous polyprenol phosphates (but not farnesol phosphate or cetyl phosphate) can substitute for dolichol phosphate in this respect but that they are much less efficient than dolichol phosphate in stimulating the transfer of mannose to protein. Since pig liver contained substances with the chromatographic properties of both dolichol phosphate and dolichol phosphate mannose, which caused an increase in transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to mannolipid, it was concluded that endogenous dolichol phosphate acts as an acceptor of mannose in the microsomal preparation. The results indicate that the mannolipid is an intermediate in the transfer of mannose from GDP-mannose to protein. Some 4% of the mannose of a sample of mannolipid added to an incubation was transferred to protein. A scheme is proposed to explain the variations with time in the production of radioactive mannolipid, mannoprotein, mannose 1-phosphate and mannose from GDP-[¹⁴C]mannose that takes account of the above observations. ATP, ADP, UTP, GDP, ADP-glucose and UDP-glucose markedly inhibited the transfer of mannose to the mannolipid.     

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.     

Characterisation of hexokinase in Toxoplasma gondii tachyzoites

We have cloned the hexokinase [E.C. 2.7.1.1] gene of Toxoplasma gondii tachyzoite and obtained an active recombinant enzyme with a calculated molecular mass of 51,465Da and an isoelectric point of 5.82. Southern blot analysis indicated that the hexokinase gene existed as a single copy in the tachyzoites of T. gondii. The sequence of T. gondii hexokinase exhibited the highest identity (44%) to that of Plasmodium falciparum hexokinase and lower identity of less than 35% to those of hexokinases from other organisms. The specific activity of the homogeneously purified recombinant enzyme was 4.04 micromol/mg protein/min at 37 degrees C under optimal conditions. The enzyme could use glucose, fructose, and mannose as substrates, though it preferred glucose. Adenosine triphosphate was exclusively the most effective phosphorus donor, and pyrophosphate did not serve as a substrate. K(m) values for glucose and adenosine triphosphate were 8.0+/-0.8 microM and 1.05+/-0.25mM, respectively. No allosteric effect of substrates was observed, and the products, glucose 6-phosphate and adenosine diphosphate, had no inhibitory effect on T. gondii hexokinase activity. Other phosphorylated hexoses, fructose 6-phosphate, trehalose 6-phosphate which is an inhibitor of yeast hexokinase, and pyrophosphate, also did not affect T. gondii hexokinase activity. Native hexokinase activity was recovered in both the cytosol and membrane fractions of the whole lysate of T. gondii tachyzoites. This result suggests that T. gondii hexokinase weakly associates with the membrane or particulate fraction of the tachyzoite cell.     

Biosynthesis of the mannose 6-phosphate recognition marker in transport-impaired mouse lymphoma cells. Demonstration of a two-step phosphorylation

The biosynthesis of the mannose 6-phosphate recognition marker has been studied in transport-impaired mouse lymphoma cells to determine the subcellular location of the processing enzymes and to characterize the biosynthetic intermediates. Cells were labeled with [2-3H]mannose and chased at a low temperature (15 or 20 degrees C) or at 37 degrees C in the presence of m-chlorocarbonylcyanide phenylhydrazone to disrupt transport of the pulse-labeled molecules within the secretory apparatus. Both treatments inhibited the migration of the pulse-labeled glycoproteins to the Golgi apparatus as measured by the production of complex-type asparagine-linked oligosaccharides. Despite this inhibition in protein transport, acid hydrolases were phosphorylated. Structural analysis of the phosphorylated oligosaccharides indicated that the transport-impaired cells produced a single species of phosphorylated high mannose oligosaccharide; essentially all of the molecules contain a single phosphodiester group that is restricted to the alpha 1,6 branch of the oligosaccharide. The results suggest that synthesis of mannose 6-phosphate-bearing high mannose oligosaccharides occurs in an ordered, compartmentalized posttranslational process. The initial phosphorylation of newly synthesized acid hydrolases occurs at a pre-Golgi site and results in the production of high mannose-type units that contain a single phosphodiester group. In a subsequent compartment, probably within the Golgi apparatus, the monophosphorylated units may be converted to diphosphorylated forms. Finally, at a site distal to the phosphorylation reactions the diesters are hydrolyzed to reveal the mannose 6-phosphate recognition marker.     

Role of mannosyl lipid intermediate in the synthesis of Neurospora crassa glycoproteins.

Particulate membrane preparations from Neurospora crassa incorporated mannose from GDP-[14C] mannose into endogenous lipid and particulate protein acceptors. Synthesis of the mannosyl lipid is reversible in the presence of GDP. Chemical and chromatographic characterization of the mannosyl lipid suggest that it is a mannosylphosphorylpolyisoprenol. The other endogenous acceptor was precipitated by trichloracetic acid. Gel filtration and electrophoresis studies before and after treatment with proteolytic enzymes indicate that the second acceptor is a glycoprotein(s). beta Elimination studies on the mannosyl protein formed from GDP-[14C] mannose with Mg2+ in the reaction mixture or formed from mannosyl lipid indicate thad with the peptide chain. Several lines of evidence indicate that in Neurospora crassa the mannosyl lipid is an obligatory intermediate in the in vitro mannosylation of the protein. (a) At 15 degrees C the initial formation of the mannosyl lipid is faster than the initial formation of the mannosyl protein. (b) Exogenous partially purified mannosyl lipid can function as a mannosyl donor for the synthesis of the mannosyl protein. This reaction was also dependent on a divalent metal. The rate of this reaction was optimal at a concentration of Triton X-100 which effectively inhibited the transfer of mannose from GDP-[14C] mannose to lipid and protein, indicating that GDP-mannose was not an intermediate in the transfer of mannose from lipid to protein. The mannosyl protein formed in this reaction was indistinguishable by several criteria from the mannosyl protein formed from GDP-[14C] mannose and Mg2+. (c) The effect of a chase with an excess of unlabeled GDP-mannose on the incorporation of mannose into endogenous acceptors was immediate cessation of the synthesis and subsequent turnover of the mannosyl lipid; in contrast, however, incorporation of mannose into protein continued and was proportional to the loss of mannose from the mannosyl lipid.     

Overexpression of Mycobacterium tuberculosis manB, a phosphomannomutase that increases phosphatidylinositol mannoside biosynthesis in Mycobacterium smegmatis and mycobacterial association with human macrophages

Mycobacterium tuberculosis (M. tb) pathogenesis involves the interaction between the mycobacterial cell envelope and host macrophage, a process mediated, in part, by binding of the mannose caps of M. tb lipoarabinomannan (ManLAM) to the macrophage mannose receptor (MR). A presumed critical step in the biosynthesis of ManLAM, and other mannose-containing glycoconjugates, is the conversion of mannose-6-phosphate to mannose-1-phosphate, by a phosphomannomutase (PMM), to produce GDP-mannose, the primary mannose-donor in mycobacteria. We have identified four M. tb H37Rv genes with similarity to known PMMs. Using in vivo complementation of PMM and phosphoglucomutase (PGM) deficient strains of Pseudomonas aeruginosa, and an in vitro enzyme assay, we have identified both PMM and PGM activity from one of these genes, Rv3257c (MtmanB). MtmanB overexpression in M. smegmatis produced increased levels of LAM, lipomannan, and phosphatidylinositol mannosides (PIMs) compared with control strains and led to a 13.3 +/- 3.9-fold greater association of mycobacteria with human macrophages, in a mannan-inhibitable fashion. This increased association was mediated by the overproduction of higher order PIMs that possess mannose cap structures. We conclude that MtmanB encodes a functional PMM involved in the biosynthesis of mannosylated lipoglycans that participate in the association of mycobacteria with macrophage phagocytic receptors.     

Utilization of mannose by astroglial cells

Uptake and metabolism of mannose were studied in astroglia-rich primary cultures derived from neonatal rat brains. A saturable component of mannose uptake was found with half-maximal uptake at 6.7±1.0 mM mannose. In addition, a non-saturable component dominated the uptake at high concentrations of mannose. Glucose, cytochalasin B, or phloretin in the incubation buffer inhibited the carrier-mediated uptake of mannose. Within the astroglial cells mannose is phosphorylated to mannose-6-phosphate. In cell homogenates, the K_M value of mannose-phosphorylating activity was determined to be 24±7 M. The V_max value of this activity is only 40% that of glucose-phosphorylating activity. Mannose-6-phosphate was converted to fructose-6-phosphate by mannose-6-phosphate isomerase. The specific activity of this enzyme in homogenates of astroglial cultures was higher than that of hexokinase. Two products of mannose utilization in astroglial cells are glycogen and lactate. The amounts of each of these products increased with increasing concentrations of mannose. In contrast to the generation of lactate, that of glycogen from mannose was enhanced in the presence of insulin. In conclusion, we suggest that mannose is taken up into the cells of astroglia-rich primary cultures by the glial glucose transporter and is metabolized to fructose-6-phosphate within the astroglial cells.     

Synthesis of alpha 1----6-mannooligosaccharides in Mycobacterium smegmatis. Function of beta-mannosylphosphoryldecaprenol as the mannosyl donor

Incubation of a membrane fraction from Mycobacterium smegmatis cells with GDP-mannose and free mannose at pH 7 in presence of Mg2+ ions resulted in the formation of a series of alpha 1----6-linked mannooligosaccharides with up to 12 mannoses. The membrane fraction also catalyzed incorporation of mannose from GDP-mannose into a lipid-soluble product with the properties of a mannosyl phospholipid. A similar product was formed by the incubation of the membrane protein with decaprenol phosphate and GDP-mannose, and it was characterized as beta-mannosylphosphoryldecaprenol. A pulse-chase experiment suggested that the mannosyl phospholipid was an intermediate in alpha 1----6-linked mannooligosaccharide synthesis, and the isolated beta-mannosylphosphoryldecaprenol was shown to function as a direct mannosyl donor on incubation with mannose, methyl alpha-D-mannoside, or alpha 1----6-linked mannooligosaccharides as acceptors. The Km values for mannose, methylmannoside, and alpha 1----6-linked mannobiose were 30-90 mM, whereas for alpha 1----6-linked mannotriose, mannotetraose, and mannopentaose the Km dropped to 2 mM. A weak enzymic activity was detected at pH 6 in the presence of both Mg2+ and Mn2+ ions that catalyzed addition of mannose in alpha 1----2 linkage to the longer alpha 1----6-mannooligosaccharides in a reaction that was specific for GDP-mannose as the donor. The membrane preparation also contained an endo-alpha 1----6-mannanase activity that degraded products longer than mannotriose by cleavage of trisaccharide units from the nonreducing end of the alpha 1----6-mannooligosaccharides.     

The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.     

Studies on the effect of mevinolin (lovastatin) and mevastatin (compactin) on the fusion of L6 myoblasts

The effect of mevastatin and mevinolin on the fusion of L6 myoblasts was studied. Both compounds were potent inhibitors of myoblast fusion at concentrations as low as 0.25 M, but fusion was restored when the inhibitors were removed. Both compounds resulted in decreased binding of conA and WGA to cell surface oligosaccharides showing they were causing a reduction in N-linked cell surface glycoproteins. There was a reduction in creatine phosphokinase activities in the presence of both compounds showing that they were affecting biochemical differentiation. The presence of both compounds inhibited the incorporation of labeled mannose from GDP-mannose into lipid-sugar and N-linked glycoprotein, but the inhibition was reversed by addition of exogenous dolichol phosphate to the incorporation mixture. The main conclusion from these studies is that mevinolin and mevastatin are inhibiting myoblast fusion by affecting the synthesis of fusogenic cell surface N-linked glycoproteins probably by affecting the synthesis of dolichol phosphate containing oligosaccharides that are required as intermediates in N-linked glycoprotein biosynthesis.Abbreviations HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A - Dol dolichol - Dol-P dolichol phosphate - Man mannose - GlcNAc N-acetylglucosamine - Glc glucose - conA concanavalin A - WGA wheat germ agglutinin - CPK creatine phosphokinase     

Evidence for the stimulation of dolichol and mannolipid synthesis by glucocorticoids in HeLa cells.

The effects of addition of 1 microM-dexamethasone on the rate of transfer of mannose from GDP-mannose into mannolipid have been studied in HeLa cell cultures. Concurrent with an increase in incorporation of mannose into glycoproteins, the incorporation of mannose from GDP-mannose in vitro into mannolipid and dolichol-linked oligosaccharides was increased after dexamethasone treatment. Stimulation of mannolipid synthesis showed a correlation with the 11 beta, 17 alpha, 21-trihydroxy structure of C21 steroids. Dexamethasone treatment also resulted in an increased incorporation of acetate into dolichol and dolichyl phosphate. The results suggest that the effect of dexamethasone on the cell-surface glycoprotein accumulation is related to increased sugar-linked dolichol synthesis.