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C E Ballou

Publications and source records attributed to C E Ballou.

At least 55 records · Page 3Linked to original sources
PubMed1984

Biosynthesis of the mycobacterial methylmannose polysaccharide. Identification of a 3-O-methyltransferase.

The methylmannose polysaccharide (MMP), found in the cytoplasm of Mycobacterium smegmatis, is composed of 10 to 13 3-O-methylmannoses joined by alpha 1----4 linkages. Each molecule is terminated by an unmethylated mannose and the reducing end is blocked by an alpha-linked methyl aglycon. Two enzymes involved in MMP biosynthesis have been identified in cell extracts, an alpha 1----4 mannosyltransferase (described in the previous paper) and a 3-O-methyltransferase reported here. Studies of substrate specificity and characterization of the products formed demonstrate that MMP elongation occurs via sequential mannosylation and methylation. The 3-O-methyltransferase, unlike the mannosyltransferase, is readily solubilized. It catalyzes transfer of a methyl group from S-adenosylmethionine to position 3 of a terminal alpha 1----4-linked mannose. The labeled product formed from S-[methyl-3H]adenosylmethionine and Man-MeMan5-OCH3 was characterized both by its resistance to periodate oxidation and by the release of labeled 3-O-methylmannose upon acid hydrolysis. Like the mannosyltransferase, the 3-O-methyltransferase utilizes shorter oligomeric acceptors preferentially. The Km values of the methyltransferase for Man-MeMan4-OCH3 and S-adenosylmethionine are 0.7 and 0.4 mM, respectively. Because MMP homologs isolated from the cell are terminated by an unmethylated mannose, the methyl-transferase appears to be responsible for MMP chain termination. Moreover, palmitoyl-CoA selectively inhibits methylation of Man-MeMan12-OCH3 when Man-MeMan4-OCH3 and Man-MeMan12-OCH3 are incubated together with the methyltransferase, which suggests that complex formation between the longer homologs and lipids may play a role in the termination process.

Chromatography, Gel↗
PubMed1984

Affinity purification of mycobacterial polymethyl polysaccharides and a study of polysaccharide-lipid interactions by 1H NMR.

Mycobacterial polymethyl polysaccharides, which bind long-chain fatty acids tightly [Ballou, C.E. (1981) Pure Appl. Chem. 53, 107-112], have been purified on a preparative scale by use of an affinity column packing consisting of (palmitoylamino)alkylsilyl silicate. The relatively large amount of material obtained in this way has allowed a study of the polysaccharide-lipid interactions at millimolar concentrations. The anomeric protons for all of the alpha 1----4-linked hexose units in the mycobacterial methylglucose polysaccharide occur in an envelope centered at delta 5.40, and, on titration with hexadecyltrimethylammonium bromide, the majority of these resonances move upfield to about delta 5.15. This shift is consistent with a change in the polysaccharide from a less ordered chain to one that has a significant proportion of helical conformation, and it is probable that the alkyl chain is included in the coiled portion of the polysaccharide in a manner analogous to the interaction of methylmannose polysaccharide with palmitic acid [Yabusaki, K. K., Cohen, R. E., & Ballou, C. E. (1979) J. Biol. Chem. 254, 7282-7286]. The native methylglucose lipopolysaccharide, which contains several short-chain acyl groups as well as an esterified octanoyl group, has an anomeric proton nuclear magnetic resonance spectrum similar to that of the methylglucose polysaccharide-hexadecyltrimethylammonium bromide complex. This suggests that the acylation stabilizes the polysaccharide chain in the same conformation it assumes when complexed to a long-chain lipid. Thus, acylation of the methylglucose polysaccharide could have an important role in regulating its shape and lipid-binding properties.

Cetrimonium↗
PubMed1984

Isolation of glucose-containing high-mannose glycoprotein core oligosaccharides.

The total cell wall mannoprotein has been isolated from a mutant of Saccharomyces cerevisiae that fails to remove the glucose units of the dolichol-linked precursor after transfer of the oligosaccharide to asparagine units in the protein. The oligosaccharides released from this mannoprotein by endoglucosaminidase H digestion show 1H NMR signals assignable to three alpha-linked glucose units as delta 5.52, 5.27, and 5.17, and a comparison with the chemical shifts of reference compounds shows that these signals are consistent with the structure alpha Glc----2 alpha Glc----3 alpha Man----2. This provides a direct confirmation for the structure previously assigned to the lipid-linked precursor. Analysis of the larger oligosaccharides confirms that the presence of the glucose units does not prevent elongation of the alpha 1----6-linked polymannose backbone or addition of alpha 1----3-linked mannose to the core.

Glucose↗
PubMed1983

Fast-atom-bombardment, negative-ion mass spectrometry of the mycobacterial O-methyl-D-glucose polysaccharide and lipopolysaccharides.

The mycobacterial O-methyl-D-glucose polysaccharide (MGP) and its acylated derivatives, the O-methyl-D-glucose lipopolysaccharides (MGLP), have been analyzed by fast-atom-bombardment mass spectrometry (F.a.b.m.s.). The molecular-ion peaks for MGP confirmed the degree of polymerization and, in addition, revealed a slight heterogeneity in the degree of methylation. Sequence ions were observed that are consistent with all known features of MGP structure. Spectra of the MGLP isomers confirmed the general distribution of the neutral and acidic acyl groups, and provided additional information regarding the specific location of individual acyl groups. The spectra of some MGLP preparations revealed that they were contaminated by lysophosphatidylinositol dimannoside. The study further documents the utility of F.a.b.m.s. for characterization of relatively large and complex carbohydrate derivatives.

Carbohydrate Sequence↗
PubMed1983

Cell-cell recognition in yeast. Characterization of the sexual agglutination factors from Saccharomyces kluyveri.

The cell surface molecules responsible for sexual agglutination between haploid cells of opposite mating type from Saccharomyces kluyveri have been purified and characterized. The 17-factor, released from 17-cells by beta-glucanase digestion (Zymolyase), is a glycoprotein of 6 X 10(4) Da. Its binding activity is heat- and protease-labile, but it is stable to reducing agents and exo-alpha-mannosidase digestion. The 16-factor, released from 16-cells by Zymolyase digestion, has a molecular weight of 5 X 10(5) and is over 95% carbohydrate. An active binding fragment can be released from 16-factor, from the factor purified from a mutant of 16-cells (16(mnn1)-factor), and from the surfaces of the cells themselves by dithiothreitol treatment. The 16(mnn1)-binding fragment has a molecular weight of 2 X 10(4) and is 30% carbohydrate. Its binding activity is stable to heat and some proteases, but it is labile to pronase, carboxypeptidases A and Y, alpha-mannosidases, and mild periodate treatment. 125I-16(mnn1)-binding fragment adheres specifically to 17-cells but does not bind to 16-cells or cells of other yeast strains. The binding of the labeled fragment to 17-cells is characterized by a KA of 10(8) M-1, and 5 X 10(5) binding sites are present per cell. The purified intact factors are monovalent and appear to interact in a lock and key fashion to cause the specific agglutination of S. kluyveri 16- and 17-cells.

Amino Acids↗
PubMed1983

Pyruvylated glycolipids from Mycobacterium smegmatis. Structures of two oligosaccharide components.

A crude glycolipid fraction was isolated from Mycobacterium smegmatis ATCC 356 by ethanolic extraction and silica gel chromatography. After deacylation of the glycolipid fraction, a dipyruvylated pentasaccharide (acidic oligosaccharide A) and a monopyruvylated tetrasaccharide (acidic oligosaccharide B1) were purified by ion exchange and gel filtration chromatography. Methylation analysis, proton nuclear magnetic resonance, mass spectrometry, and periodate oxidation suggested that oligosaccharide A was 4,6-(1-carboxyethylidene)-3-O-methyl-beta-D Glcp-(1-3)-4,6-(1-carboxyethylidene)-beta-D-Glcp-(1-4)-beta-D-Glcp-(1-6)-alpha- D Glcp-(1-1)-alpha-D-Glcp and that oligosaccharide B1 was 4,6-(1-carboxyethylidene)-beta-D-Glcp-(1-4)-beta-D-Glcp-(1-6)-alpha-D-Glcp-(1-1 ) -alpha-D-Glcp. Both compounds contain a trehalose unit as a part of the oligosaccharide structure, and B1 appears to be a biosynthetic precursor of A because the two differ only in a pyruvylated 3-O-methylglucose unit. A third component, acidic oligosaccharide B2, differs from oligosaccharide A only in lacking 1 of the 2 pyruvate residues.

Carbohydrate Conformation↗
PubMed1982

Effects of mannoprotein mutations on Saccharomyces cerevisiae core oligosaccharide structure.

By the combined actions of an endo-alpha-1 leads to 6-mannanase and an endo-beta-N-acetylglucosaminidase, the core oligosaccharides can be released from Saccharomyces cerevisiae X2180 mnn2 mannoproteins. The effects of various mannoprotein mutations were evaluated by structural comparison of these core oligosaccharides with those prepared from double mutant strains with the genotypes mnn1 mnn2, mnn2 mnn3, mnn2 mnn4, and mnn2 mnn5. The results indicate that only the mnn1 lesion has a major effect on the mannoprotein core structure. Whereas the mnn2 mannoprotein yields a core composed of 6 fragments that differ in size from each other by single mannose units, only the two smallest species predominate in the mnn1 mnn2 preparation. This change is correlated with a loss of terminal alpha 1 leads to 3-mannosyl residues, an effect on the mnn1 lesion that is found also in the polysaccharide outer chain and hydroxyamino acid-linked mannooligosaccharides. The mnn3 and mnn5 mutations also had slight effects on the core size, but clear differences in linkage composition were not apparent. The results suggest that core oligosaccharides have an average composition of Man11GlcNAc, whereas Man9GlcNAc is the major oligosaccharide in strains containing the mnn1 defect. These values are 2 to 3 sugars less than those estimated previously (Nakajima, T., and Ballou, C. E. (1975) Biochem. Biophys. Res. Commun. 66, 870-879). Detailed analysis of the major core oligosaccharide from the mnn1 mnn2 mutant revealed that the two mannoses in alpha 1 leads to 3 linkage to the backbone were adjacent to each other and that the oligosacccharide is nearly identical with one isolated from chinese hamster ovary cell membranes (Li, E., and Kornfeld, S. (1979) J. Biol. Chem. 254, 1600-1605). This finding provides strong evidence for the evolutionary conservation of this structural feature of the high mannose core oligosaccharides.

Acetylglucosaminidase↗
PubMed1982

Purification of an alpha-N-acetylglucosaminyltransferase from the yeast Kluyveromyces lactis and a study of mutants defective in this enzyme activity.

An enzyme activity in Kluyveromyces lactis that catalyzes the transfer of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine to alpha Man(1 leads to 3) alpha Man ( 1 leads to 2) alpha Man (1 leads to 2)Man to yield alpha Man(1 leads to 3) [alpha GlcNAc(1 leads to 2)] alpha Man(1 leads to 2) alpha Man (1 leads to 2)Man, a mannoprotein side-chain unit, has been solubilized by Triton X-100 and purified 18000-fold by a combination of ion-exchange chromatography, gel filtration, hydrophobic chromatography, and adsorption to a lectin column. The enzyme activity from a K. lactis mutant (mnn2-2) that made mannoprotein lacking N-acetylglucosamine in its side chains, but that possessed a normal level of transferase activity in cell extracts, was purified and compared with the enzyme from the wild-type strain. Both transferase activities are integral membrane proteins found in particles associated with endoplasmic reticulum. The two purified enzymes had the same apparent size, heat stability, Mn2+ requirement, and Km for donor and acceptor and a similar Vmax. Wild-type and mutant cells had similar pool sizes of sugar nucleotide donor, and they incorporated labeled N-acetylglucosamine into chitin at similar rates. No evidence was obtained for an inactive enzyme precursor in mutant cells that was activated upon breaking the cells, nor did the mutant cells contain a transferase inhibitor or a hexosaminidase that could remove the sugar from the mannoprotein during processing and secretion. The mnn2-2 locus appears to be allelic with a second mutant, mnn2-1, that has the same phenotype but that lacks transferase activity in cell extracts. This suggests that the two mutations affect the structural gene for the transferase, and we conclude that the mnn2-2 mutant could contain an altered enzyme that fails to function because it is improperly localized or oriented in the membrane.

Acetylglucosamine↗
PubMed1982

Sexual agglutination factors from the yeast Pichia amethionina.

Pichia amethionina is a heterothallic yeast isolated from necrotic cactus tissue. Haploid cells of opposite mating type, designated alpha and alpha, agglutinate strongly when mixed. The agglutination factors of the two cell types have been solubilized from the cell walls by beta-glucanase digestion and then partially purified by affinity adsorption to the opposite cell type and by gel filtration. From alpha-cells was obtained a large, heat-stable glycoprotein with the ability to agglutinate alpha-cells. This alpha-agglutinin was inactivated by mercaptoethanol, probably because the recognition sites are linked to the glycoprotein core by disulfide bonds. Digestion of alpha-cells with beta-glucanase released a large heat-labile glycoprotein that did not agglutinate alpha-cells but did inhibit agglutination of alpha-cells by alpha-agglutinin. Subtilisin digestion of this alpha-factor released a carbohydrate-free protein of 27,000 daltons that retained the biological activity of the factor. These agglutination factors are sex- and species-specific and are not found on the surface of heterozygous diploid cells.

Agglutinins↗
PubMed1982

Yeast invertase polymorphism is correlated with variable states of oligosaccharide chain phosphorylation.

Saccharomyces cerevisiae invertase (EC 3.2.1.26) isolated from wild-type strain X2180 can be resolved by isoelectric focusing into at least seven bands revealed by an activity stain. Most of this polymorphism is eliminated in mutants that are defective in phosphorylation of the mannoprotein carbohydrate chains (mnn4 and mnn6). In contrast to strain X2180, invertase from the mnn9 mutant, which makes mannoprotein lacking the outer portion of the polymannose chains, shows only two major bands on isoelectric focusing. Although mnn2 mannoprotein is though not to have any branches in its outer chain, the invertase of this mutant shows at least six bands on isoelectric focusing, and digestion of this invertase with an endo-alph aI leads to 6-mannanase that removes the unbranched outer chain produces an invertase with two bands that are similar to those from the mnn9 mutant. The invertase from mnn2 cells, grown with [32P]orthophosphate and precipitated with specific antiserum, gives at least five radioactive bands on isoelectric focusing, and after digestion with the endomannanase the radioactivity no longer migrates with the residual invertase. Mutants with shortened and unbranched outer chains (mnn2 mnn7, mnn2 mnn8, and mnn2 mnn10) give invertase patterns similar to mnn2. The results suggest that multiple states of outer chain phosphorylation lead to isoelectric polymorphism of S. cerevisiae external invertase and, because invertase has nine carbohydrate chains, no more than one phosphate group per chain would be required to account for this property.

Glycoproteins↗
PubMed1981

Saccharomyces kluyveri mannoprotein mutants.

Saccharomyces kluyveri cells were mutagenized with ethylmethane sulfonate and, after the cells had grown a few divisions to express any altered cell-surface antigenic structure, the culture was treated with rabbit antiserum directed against the wild type cells in order to enrich for mutants that failed to precipitate with the serum. Several mutant clones were obtained that proved to be altered in the carbohydrate component of the cell-wall mannoprotein. Whereas the wild type strain produces mannoprotein with carbohydrate side chains up to 8 mannose units in length (Zhang, W.-J., and Ballou, C. E. (1981) J. Biol. Chem. 256, 10073-10079), one of the mutants (designated mnn1) has side chains no longer than 3 mannoses. From a comparison of the carbohydrate structures of the mutant and wild type mannoproteins by beta-elimination, acetolysis, and methylation, it appears that this mutant is unable to add mannose in alpha 1 leads to 3 linkage to the alpha 1 leads to 2-linked di- and trisaccharide side chains, thus preventing elongation and branching of the chains that occur in the wild type. Another mutant, designated mnn2, was unable to make the octasaccharide chain, whereas a third class made oligosaccharides of all sizes but did so in ratios that differed from the wild type. These three classes of mutants involve different loci because they complemented each other in the heterozygous diploids.

Carbohydrate Conformation↗
PubMed1981

Field desorption mass spectrometry of oligosaccharides.

Field desorption mass spectrometry has been used to analyze carbohydrate polymers with 5 to 14 hexose units without prior derivatization. In all examples, the molecular weight of the oligosaccharide could be determined by means of the abundant quasimolecular ions of the type MNa(+), MH(+), MNa(2) (2+), and MNa(3) (3+). Fragmentation at glycosidic linkages was observed in varying extents. The reduced oligosaccharide Man(8)GlcNAcH(2), obtained from IgM [Cohen, R. E. & Ballou, C. E. (1980) Biochemistry 19, 4345-4358], gave quasimolecular ion signals MNa(+) at m/z 1544, MH(+) at m/z 1522, MNa(2) (2+) at m/z 784, and MNa(3) (3+) at m/z 530, all corresponding to its assumed molecular weight of 1519.5. Mycobacterial methylmannose polysaccharides with the general structure Man(x)MeMan(y)-OCH(3) [Yamada, H., Cohen, R. E. & Ballou, C. E. (1979) J. Biol. Chem. 254, 1972-1979] were also successfully analyzed. Man(1)MeMan(13)-OCH(3), the largest homolog, gave the expected signal of the quasimolecular ion MNa(+) at m/z 2506. The larger polysaccharides were analyzed by using a KRATOS MS-50 mass spectrometer with a high-field magnet enabling full sensitivity to be maintained up to 3000 atomic mass units. Polysaccharides up to m/z 1978 were analyzed by using a KRATOS MS-9 mass spectrometer operated at 4 Kv. The signal-to-noise ratio, which becomes a serious problem in field desorption mass spectrometry at low accelerating voltages, and the low instrument sensitivity were improved considerably by our use of a method of adding scans with low total ion currents obtained over a longer desorption time. In this way, we obtained complete sequence information on methylmannose polysaccharides up to Man(1)MeMan(9)-OCH(3)(MNa(+) at m/z 1802). Analysis of a presumed Man(1)MeMan(7)-OCH(3), gave a spectrum consistent only with the structure Man(2)MeMan(6)-OCH(3), revealing the existence of a methylmannose homolog with 2 unmethylated mannoses at the nonreducing end of the chain.

Carbohydrate Sequence↗
PubMed1981

Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated.

Carboxypeptidase Y, a vacuolar enzyme from Saccharomyces cerevisiae, was digested with endo-beta-N-acetyl-D-glucosaminidase H to release the four oligosaccharide chains that are linked to asparagine in the glycoprotein. The oligosaccharides were fractionated into a neutral and acidic component, and the latter proved to phosphorylated. From its gel filtration pattern, the neutral fraction was shown to be a mixture of at least four homologs, the smallest of which had a proton NMR spectrum almost identical to that given by an IgM oligosaccharide with eight mannoses and one N-acetylglucosamine [Cohen, R. E. & Ballou, C. E. (1980) Biochemistry 19, 4345--4358]. The yeast oligosaccharide has one additional mannose unit in an alpha 1 leads to 3 or alpha 1 leads to 6 linkage, whereas the larger homologs appear to have two, three, and four more mannose units. One phosphorylated oligosaccharides with a mannose/phosphate ratio of 12.5 was reduced with NaB3H4 and then subjected to mild acid hydrolysis. This released mannose and mannobiose that were glycosidically linked to the phosphate group, whereas complete acid hydrolysis yielded D-mannose 6-phosphate. The recovered oligosaccharide phosphomonoester, which contained 11 or 12 mannose units, was digested exhaustively with alpha-mannosidase, and the product of this reaction was treated with alkaline phosphatase, which yielded radioactive Man3GlcNAcH2. These results suggest that the mannosidase-resistant phosphorylated oligosaccharide has the structure Man leads to P leads to 6 alpha Man leads to alpha Man leads to 6 beta Man leads to 4GlcNAcH2, in which some of the phosphate groups are substituted with mannobiose instead of mannose. A second phosphorylated oligosaccharide with a mannose/phosphate ratio of 6.5 probably contains two phosphodiester groups, but its structure has not been investigated in detail.

Carboxypeptidases↗
PubMed1981

Temperature-sensitive glucosamine auxotroph of Saccharomyces cerevisiae.

Temperature-sensitive revertants were isolated from Saccharomyces cerevisiae D-glucosamine auxotrophs previously obtained in this laboratory (W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). The auxotrophs lack the enzyme 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (EC 5.3.1.19), and the revertants appear to be temperature sensitive in the formation of enzyme activity. The enzyme they produce under permissive conditions decays in activity at a rate comparable to that of the wild-type enzyme, and it has similar kinetic properties. The homozygous diploid mutant fails to sporulate at the nonpermissive temperature. Temperature shift experiments were carried out in an effort to determine what effect glucosamine deficiency had on mannoprotein secretion as reflected in the formation of external asparaginase. Although the results were complicated by the slow decay of the residual ketol-isomerase activity, they did show that mannoprotein synthesis or secretion was altered when the internal pool of D-glucosamine was depleted.

Asparaginase↗
PubMed1980

Characterization of phosphorylated oligomannosides from Hansenula wingei mannoprotein.

Yeast cell wall mannoproteins often contain phosphate esterified to the oligosaccharide side chains, and partial acetolysis yields both neutral and phosphorylated fragments [Thieme, T. R., & Ballou, C. E. (1971) Biochemistry 10, 4121--4129]. We have isolated the acidic acetolysis fragments from Hansenula wingei mannoprotein [Yen, P. H., & Ballou, C. E. (1974) Biochemistry 13, 2420--2427] and have separated them into a mannopentaose monophosphate (Man5P) and a mannotriose monophosphate (Man3P). On the basis of selective enzymatic and partial acid hydrolysis and 1H and 31P NMR studies, Man5P was shown to have the structure P leads to 6 alpha Man1 leads to 3 alpha Man1 leads to 2 alpha Man1 leads to 2 alpha Man1 leads to 2Man (where Man = D-mannopyranose). The 31P NMR spectrum of the Man3P showed it to be a mixture of a phosphate monoester and a phosphate diester, which was separated by ion-exchange chromatography. The monoester had the structure P leads to 6 alpha Man1 leads to 3 alpha Man1 leads to 3Man whereas the diester had the properties of a cyclic phosphate. Although native H. wingei mannoprotein contains phosphodiester linkages, the starting mannoprotein preparation was isolated under alkaline conditions that hydrolyze such bonds, and it did not show a phosphodiester signal in the 31P NMR spectrum. We conclude that the cyclic phosphate was an artifact formed during the acetolysis reaction. Because acetolysis of H. wingei mannoprotein yields only phosphorylated mannotriose and mannopentaose whereas the mannoprotein contains mannotetraose side chains as well, the phosphorylation process must be a very specific event in the biosynthesis of the glycoprotein.

Ascomycota↗