Structure of the phosphorylated N-linked oligosaccharides from the mnn9 and mnn10 mutants of Saccharomyces cerevisiae

The N-linked oligosaccharides, from Saccharomyces cerevisiae mnn1 mnn9 mutant mannoprotein extracted from the cells in hot citrate buffer, were separated by ion exchange into a monophosphate diester, a monophosphate monoester, a diphosphate diester, and a diphosphate monoester diester. The structures of the major components with diesterified phosphate were assigned as follows (where M = mannose), according to a recently revised oligosaccharide structure for the mnn mutants (Hernandez, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). formula; see text The monoester derivatives were mixtures of the possible isomers produced by removal of one or the other phosphoglycosyl-linked mannose units, and they were shown to arise by chemical degradation during isolation. The mnn1 mnn2 mnn10 acidic oligosaccharide fraction contained a mono- and a diphosphate ester. The monophosphate consisted predominantly of a single isomer with a mannosyl phosphate unit located at the end of the outer chain in an oligosaccharide with the following structure, where x may range from 2 to 12. The diphosphate had a mannosyl phosphate in this formula; see text position as well as one on the terminal alpha 1----6-linked mannose in the core. The presence in the mnn1 mnn9 or mnn1 mnn2 mnn10 background of the mnn4 or mnn6 mutations, which are known to regulate phosphorylation in yeast, reduced phosphorylation by 90% but did not eliminate it. AI-12522     

A new Saccharomyces cerevisiae mnn mutant N-linked oligosaccharide structure

We find that the N-linked Man8GlcNAc2- core oligosaccharide of Saccharomyces cerevisiae mnn mutant mannoproteins is enlarged by the addition of the outer chain to the alpha 1----3-linked mannose in the side chain that is attached to the beta 1----4-linked mannose rather than by addition to the terminal alpha 1----6-linked mannose. This conclusion is derived from structural studies on a phosphorylated oligosaccharide fraction and from mass spectral fragment analysis of neutral core oligosaccharides.     

Oligosaccharide structures of the major exoglucanase secreted by Saccharomyces cerevisiae.

We have determined the structures of the N-linked carbohydrate chains, released by endo H, of exoglucanase II that are secreted by wild-type Saccharomyces cerevisiae and by the mnn1 mnn9 and mnn1 glycosylation mutants. The mnn9 mutation does not significantly affect N-linked oligosaccharides of exoglucanase II since we found almost identical structures in both mutant strains consisting of a slightly enlarged core with the basic structure shown in A (where M = mannose). Most of the molecules (77%) were phosphorylated on one of the starred mannoses (34%) or on both (43%) with a diesterified (alpha M-->P-->) or monoesterified phosphate group. In addition, some of the molecules apparently escape normal processing and retain the alpha-(1-->2)-linked mannose (italicized) and/or the three glucoses that are characteristic of the lipid-linked precursor (structure B). In the wild type, we found the same basic structure but more [formula; see text] than 90% of the molecules were modified with one to four alpha-(1-->3)-linked mannoses, which were absent in the strains bearing the mnn1 mutation (structure C). The proportion of acidic components was similar to that found in the mutants (78%), although, in this case, the monophosphorylated forms were more abundant (50%) than the diphosphorylated ones (28%). Most of the phosphate groups (69%) were diesterified by a disaccharide (alpha M-->3 alpha M-->P-->) instead of the single mannose found when the mnn1 mutation was present. In both mnn1 and wild type 10-15% of the oligosaccharides had an extra alpha-(1-->6)-linked mannose in the outer chain, a structure described in the recently isolated vrg1 mutant [Ballou, L., Hitzeman, R.A., Lewis, M. S., & Ballou, C. E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3209-3212].     

The mnn2 mutant of Saccharomyces cerevisiae is affected in phosphorylation of N-linked oligosaccharides

We studied the phosphorylation of the inner core region of N-linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain. We performed structural studies of the N-oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 [Hernández, L.M., Ballou, L., Alvarado, E., Tsai, P.-K. and Ballou, C.E. (1989) J. Biol. Chem. 264, 13648-13659]. We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A(3) (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C(1)) is allowed, although it is also reduced. *Phosphorylation sites in mnn1mnn9. (see structure below)     

Localization of alpha 1----3-linked mannoses in the N-linked oligosaccharides of Saccharomyces cerevisiae mnn mutants

Neutral and phosphorylated N-linked oligosaccharides were isolated from Saccharomyces cerevisiae mnn9 and mnn9 gls1 mutant mannoproteins and separated into homologues that differed in the number of terminal alpha 1----3-linked mannoses. In each type of oligosaccharide, the addition of such mannose was shown to occur in an ordered rather than a random fashion. The results confirm and extend an earlier report that dealt with the N-linked oligosaccharides from yeast invertase [Trimble, R.B., & Atkinson, P.H. (1986) J. Biol. Chem. 261, 9815-9824], and they suggest that the postulated processing pathway can be generalized to include phosphorylated and glucose-containing N-linked oligomannosides. We conclude that this processing pathway is identical for the analogous oligosaccharides from the mnn9 and wild-type strains of S. cerevisiae. Analysis of the mnn2 mnn10 mannoprotein revealed that a similar modification occurred at the branched terminus of the outer chain as well as in the core in this mutant.     

Separation and characterization of two alpha 1,2-mannosyltransferase activities from Saccharomyces cerevisiae

Two GDP-mannose-dependent mannosyltransferase activities (designated M1MT-I and M2MT-I) from Triton X-100 extracts of Saccharomyces cerevisiae mnn1 microsomes were separated by concanavalin A lectin chromatography and partially purified. The two transferases were distinguished by differences in concanavalin A affinity and in carbohydrate acceptor specificity. Analyses of the reaction products indicate that both enzymes are alpha 1,2-mannosyltransferases. M1MT-I utilizes mannose or methyl-alpha-mannoside as acceptor while M2MT-I catalyzes the transfer of mannose from GDP-mannose to unsubstituted nonreducing alpha 1,6-linked mannose residues in the acceptor molecule. M2MT-I activity correlates with the presence of a single alpha 1,2-linked mannose residue at the nonreducing terminus of mnn2mnn9 and mnn2mnn10 outer chain oligosaccharides, and the enzyme may be involved in regulating outer chain elongation.     

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).     

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.     

Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris.

Saccharomyces SUC2 invertase, secreted by the methylotrophic yeast Pichia pastoris and purified to homogeneity from the growth medium by DE-52 chromatography, appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a diffuse ladder of species at 85-90 kDa, while the secreted Saccharomyces form migrated as a broad band from 100 to 150 kDa. Endo-beta-N-acetylglucosaminidase H released the Pichia invertase carbohydrate generating a 60-kDa protein with residual Asn-linked GlcNAcs and oligosaccharides separated on Bio-Gel P-4 into Man8-11GlcNAc. Nearly 75% of the oligosaccharides were equally distributed between Man8,9GlcNAc, while 17% were Man10GlcNAc and 8% were Man11GlcNAc. Oligosaccharide pools were analyzed for homogeneity by high-pH anion-exchange chromatography, and structures were assigned using 500 MHz one- and two-dimensional 1H NMR spectroscopy. Pichia Man8GlcNAc was the same isomer as found in Saccharomyces, which arises by removing the alpha 1,2-linked terminal mannose from the middle arm of the lipid-oligosaccharide Man9GlcNAc (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). The Man9GlcNAc pool was 5% lipid-oligosaccharide precursor and 95% Man8GlcNAc isomer with a terminal alpha 1,6-linked mannose on the lower-arm alpha 1,3-core-linked residue (Hernández, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). An alpha 1,2-linked mannose on the new alpha 1,6-linked branch in Man9GlcNAc provided 80% of the Man10GlcNAc, which is the structure on Saccharomyces invertase (Trimble, R. B., and Atkinson, P. H. (1986) J. Biol. Chem. 261, 9815-9824). A minor Man10GlcNAc (12%) and the principal Man11GlcNAc (82%) were the major Man9,10GlcNAc with novel alpha 1,2-linked mannoses on the preexisting alpha 1,2-linked termini. Although Pichia glycans did not have terminal alpha 1,3-linked mannoses as found on Saccharomyces core oligosaccharides, over 60% of the structures were isometric configurations unique to lower eukaryotes.     

A human lysosomal alpha-mannosidase specific for the core of complex glycans.

A novel lysosomal alpha-mannosidase, with unique substrate specificity, has been partially purified from human spleen by chromatography through concanavalin A-Sepharose, DEAE-Sephadex, and Sephacryl S-300. This enzyme can catalyze the hydrolysis of only 1 mannose residue, that which is alpha(1----6)-linked to the beta-linked mannose in the core of N-linked glycans, as found in the oligosaccharides Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc and Man alpha(1----6)Man beta(1----4) GlcNAc. The newly described alpha-mannosidase does not catalyze the hydrolysis of mannose residues outside of the core, even if they are alpha(1----6)-linked, and is not active on the other alpha-linked mannose in the core, which is (1----3)-linked. The narrow specificity of the novel mannosidase contrasts sharply with that of the major lysosomal alpha-mannosidase, which is able to catalyze the degradation of oligosaccharides containing diverse linkage and branching patterns of the mannose residues. Importantly, although the major mannosidase readily catalyzes the hydrolysis of the core alpha(1----3)-linked mannose, it is poorly active towards the alpha(1----6)-linked mannose, i.e. the very same mannose residue for which the newly characterized mannosidase is specific. The novel enzyme is further differentiated from the major lysosomal alpha-mannosidase by its inability to catalyze the efficient hydrolysis of the synthetic substrate p-nitrophenyl alpha-mannoside, and by the strong stimulation of its activity by Co2+ and Zn2+. Similarly to the major mannosidase, it is strongly inhibited by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, but not by deoxymannojirimycin. The presence of this novel alpha-mannosidase activity in human tissues provides the best explanation, to date, for the structures of the oligosaccharides stored in human alpha-mannosidosis. In this condition the major lysosomal alpha-mannosidase activity is severely deficient, but apparently the alpha(1----6)-mannosidase is unaffected, so that the oligosaccharide structures reflect the unique specificity of this enzyme.     

Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes

Sialylated fucosyl lactosaminoglycan was isolated from human neutrophilic granulocytes and its structure was elucidated. The lactosaminoglycan glycopeptides were digested by endo-beta-galactosidase and "the core portion" and released oligosaccharides were analyzed by permethylation, fast atom bombardment mass spectrometry, and exoglycosidases. In addition, lactosaminoglycan saccharides were obtained by hydrazinolysis and the structures of fractionated sialyl oligosaccharides were analyzed by fast atom bombardment mass spectrometry and permethylation coupled with exoglycosidase treatment. The structure of one of the major components was found to be: (Formula: see text). This structure is unique in that 1) four linear polylactosaminyl side chains are attached to the core portion, 2) the side chain arising from position 4 of 2,4-linked mannose contains one or more alpha 1----3 fucosyl residues, 3) the side chain arising from position 6 of 2,6-linked mannose is terminated with NeuNAc alpha 2----3Gal(Fuc alpha 1----3)GlcNAc, sialyl Lex, and 4) the side chain arising from position 2 of 2,4-linked mannose is terminated with sialic acid through alpha 2----6 linkage.     

Specificity of the mannosyltransferase which initiates outer chain formation in Saccharomyces cerevisiae.

The in vitro specificity of the alpha 1-6 mannosyltransferase that initiates outer chain formation in Saccharomyces cerevisiae (Romero and Herscovics, J. Biol. Chem., 264, 1946-1950, 1989) was reassessed by fast atom bombardment mass spectrometry (FAB-MS). A particulate fraction from the mnn1 mutant was incubated with GDP-mannose and either Man9GlcNAc (M9T) isolated from thyroglobulin or Man8GlcNAc (M8Y) obtained by treatment of the M9T with the yeast specific mannosidase. The Man10GlcNAc (M10Y) and Man9GlcNAc (M9Y) oligosaccharides thus obtained, and the substrate oligosaccharides, were peracetylated or perdeuteroacetylated and submitted to FAB-MS using meta-nitrobenzylalcohol as the matrix. The latter was chosen as the matrix because it enhances the abundance of high-mass-fragment ions of peracetylated oligosaccharides and thereby facilitates the assignment of branching patterns. The results indicate that the alpha 1-6 mannosyltransferase catalyses the addition of mannose to the alpha 1-3 mannose residue, and thus provide additional new evidence to support the revised structure of yeast mannoproteins proposed by Hernandez et al. (J. Biol. Chem., 264, 11849-11856, 1989). [formula: see text] where Gn is N-acetylglucosamine, M is mannose and M is mannose added by the enzyme.     

The processing of N-linked glycans in yeast. Mutually exclusive steps in the processing of a Man6 derivative by yeast membrane preparations

When a derivatized oligosaccharide isolated from ovalbumin and containing 6 mannose residues was incubated with yeast membranes and GDP-mannose, two sets of products were obtained, a high molecular weight one containing about 25 mannose residues and a low molecular weight one consisting of compounds with 7, 8, and 9 mannose residues, respectively. When the low molecular weight products were reincubated with the yeast membranes and GDP-mannose, no further mannose incorporation was observed, showing that these compounds must be of the wrong structure as substrates for yeast glycan processing enzymes. The structures were investigated by 1H NMR spectroscopy. The high molecular weight products contained an outer chain of an average length of 18 1----6-linked mannose residues attached to a core structure made up of the original 6 mannose residues with one additional 1----2-linked mannose added. The low molecular weight product with 8 mannose residues was deduced to contain a terminal 1----6-linked mannose (on the 1----6 arm) substituted by mannose at the 2-position, and the ones with 7 and 9 mannose residues were identified as having an additional 1----3-linked mannose on the starting Man6 substrate and on the Man8 product, respectively. The results lend further support to the picture that the processing steps must occur in proper sequence for specific products to form.     

The genes for the Golgi apparatus N-acetylglucosaminyltransferase and the UDP-N-acetylglucosamine transporter are contiguous in Kluyveromyces lactis

The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha(1-->2)-linked N-acetylglucosamine. Previously, Smith et al. (Smith, W. L. Nakajima, T., and Ballou, C. E. (1975) J. Biol. Chem. 250, 3426-3435) characterized two mutants, mnn2-1 and mnn2-2, which lacked terminal N-acetylglucosamine in their mannoproteins. The former mutant lacks the Golgi N-acetylglucosaminyltransferase activity, whereas the latter one was recently found to be deficient in the Golgi UDP-GlcNAc transporter activity. Analysis of extensive crossings between the two mutants led Ballou and co-workers (reference cited above) to conclude that these genes were allelic or tightly linked. We have now cloned the gene encoding the K. lactis Golgi membrane N-acetylglucosaminyltransferase by complementation of the mnn2-1 mutation and named it GNT1. The mnn2-1 mutant was transformed with a 9.5-kilobase (kb) genomic fragment previously shown to contain the gene encoding the UDP-GlcNAc transporter; transformants were isolated, and phenotypic correction was monitored after cell surface labeling with fluorescein isothiocyanate-conjugated Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescence-activated cell sorter. The above 9.5-kb DNA fragment restored the wild-type lectin binding phenotype of the transferase mutant; further subcloning of this fragment yielded a smaller one containing an opening reading frame of 1,383 bases encoding a protein of 460 amino acids with an estimated molecular mass of 53 kDa, which also restored the wild-type phenotype. Transformants had also regained the ability to transfer N-acetylglucosamine to 3-0-alpha-D-mannopyranosyl-D-mannopyranoside. The gene encoding the above transferase was found to be approximately 1 kb upstream from the previously characterized MNN2 gene encoding the UDP-GlcNAc Golgi transporter. Each gene can be transcribed independently by their own promoter. To our knowledge this is the first demonstration of two Golgi apparatus functionally related genes being contiguous in a genome.     

Glycoprotein biosynthesis in Saccharomyces cerevisiae. Characterization of alpha-1,6-mannosyltransferase which initiates outer chain formation

A particulate fraction from the Saccharomyces cerevisiae mnn1 mutant was obtained after extracting a 115,000 x g pellet with 0.75% Triton X-100. Incubation of this preparation with labeled Man8GlcNAc and Man9GlcNAc in the presence of GDP-mannose followed by high pressure liquid chromatography showed the formation of Man9GlcNAc and Man10GlcNAc, respectively. Analysis by high resolution 1H NMR of the products indicates that, in each case, the mannose residue added is alpha-1,6-linked to the alpha-1,6-mannose residue of the substrate as follows (where M represents mannose and Gn represents N-acetylglucosamine): (Formula: see text). The mannosyltransferase therefore catalyzes the first step specific to the biosynthesis of the outer chain of yeast mannoproteins. The apparent Km values for both substrates are similar: 0.39 mM for Man8GlcNAc and 0.35 mM for Man9GlcNAc. The alpha-1,6-mannosyltransferase exhibits maximum activity between pH 7.1 and 7.6 in Tris maleate buffer, has an absolute requirement for Mn2+, and also requires Triton X-100. These results indicate that removal of the alpha-1,2-linked mannose residue from Man9GlcNAc is not essential for the alpha-1,6-mannosyltransferase which initiates outer chain synthesis, at least when oligosaccharides are used as substrates in a cell-free system.     

Biosynthesis of yeast mannan. Isolation of Kluyveromyces lactis mannan mutants and a study of the incorporation of N-acetyl-D-glucosamine into the polysaccharide side chains.

One side chain in the cell wall mannan of the yeast Kluyveromyces lactis has the structure (see article). (Raschke, W. C., and Ballou, C. E. (1972) Biochemistry 11, 3807). This (Man)4GNAc unit (the N-acetyl-D-glucosamine derivative of mannotetroase) and the (Man)4 side chain, aMan(1 yields 3)aMan(1 yields 2)aMan(1 yields 2)Man, are the principle immunochemical determinants on the cell surface. Two classes of mutants were obtained which lack the N-acetyl-D-glucosamine-containing determinant. The mannan of one class, designated mmnl, lacks both the (Man)4GNAc and (Man)4 side chains. Apparently, it has a defective alpha-1 yields 3-mannosyltransferase and the (Man)4 unit must be formed to serve as the acceptor before the alpha-1 yields 2-N-acetyl-glucosamine transferase can act. The other mutant class, mnn2, lacks only the (Man)4GNAc determinant and must be defective in adding N-acetylglucosamine to the mannotetrasose side chains. Two members of this class were obtained, one which still showed a wild type N-acetylglucosamine transferase activity in cell-free extracts and the other lacking it. They are allelic or tightly linked, and were designated mnn2-1 mnn2-2. Protoplast particles from the wild type cells catalyzed a Mn2+-dependent transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the mannotetraose side chain of endogenous acceptors. Exogenous mannotetraose also served as an acceptor in a Mn2+-dependent reaction and yielded (Man)4GNAc. Related oligosaccharides with terminal alpha (1 yields 3)mannosyl units were also good acceptors. The product from the reaction with alphaMan(1 yields 3)Man had the N-acetylglucosamine attached to the mannose unit at the reducing end, which supports the conclusion that the cell-free glycosyltransferase activity is identical with that involved in mannan synthesis. The reaction was inhibited by uridine diphosphate. Protoplast particles from the mmnl mutants showed wild type N-acetylglucosamine transferase activity with exogenous acceptor, but they had no endogenous activity because the endogenous mannan lacked acceptor side chains. Particles from the mnn2-1 mutant failed to catalyze N-acetylglucosamine transfer. In contrast, particles from the mnn2-2 mutant were indistinguishable from wild type cells in their transferase activity. Some event accompanying cell breakage and assay of the mnn2-2 mutant allowed expression of a latent alpha-1 yields 2-N-acetylglucosamine transferase with kinetic properties similar to those of the wild type enzyme.     

The mechanism of synthesis of the polysaccharide part of mannan in Saccharomyces cerevisiae

Saccharomyces cerevisiae wild-type and mutant cells affected in the structure of mannan outer chain were shown to possess in vivo one major dolichol-P-P-bound oligosaccharide. The size, monosaccharide composition, and pattern obtained upon acetolysis and paper chromatography of the oligosaccharide were the same for all strains and for the main corresponding compound isolated from animal tissues. Evidence is presented indicating that the dolichol-P-P derivative occurring in vivo, and containing 2-N-acetylglucosamine, 9-mannose, and 3-glucose residues, is the intermediate involved in yeast protein glycosylation. The transfer of the oligosaccharide to protein was followed in vivo by the excision of the glucose and at the most one mannose residue. Mannoses were then added to the trimmed saccharide moiety. No difference between the first stages (i.e., excision of monosaccharides) of the processing of the protein-bound oligosaccharides by wild-type and mutant cells was found. However, mutants carrying the mnn 1 mutation, which are known to be devoid of terminal α(1–3)-linked mannose residues in the mannan outer chain and inner core, were found not to add such mannose residues to the already glucose-free protein-bound oligosaccharide.     

Coflocculation of Escherichia coli and Schizosaccharomyces pombe

Several yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, Saccharomyces cerevisiae and Schizosaccharomyces pombe, were found to coaggregate with Escherichia coli, but S. pombe showed much less coflocculation than the other yeasts (Peng et al. 2001)). S. pombe is known to have galactose-rich cell walls and we investigated whether this might be responsible for its different behavior by studying the wild-type TP4-1D, with a mannose to galactose ratio of 1 to 1.2, and the glycosylation mutant gms1delta (Man:Gal=1:0). The wild-type induced very low levels of coflocculation (3%) while gms1delta induced a remarkable amount of coflocculation (48%). Coflocculation of the mutant was inhibited by mannose but not affected by galactose or glucose. The S. cerevisiae mnn2 mutant, with a mannan structure similar to gms1delta, also showed a high degree of coflocculation (40%). However, S. cerevisiae mutant mnn9, with a mature core similar to S. pombe, showed decreased coflocculation (21.3%). Both these S. cerevisae mutants were sensitive to mannose inhibition. Coflocculation of E. coli and gms1delta also could be inhibited by gms1delta mannan and plant lectins, such as HHA, GNA and NPA, specific to either alpha-1-3- or alpha-1-6-linked mannosyl units. From these results we conclude that the E. coli lectins may have specificity for alpha-1-6- and alpha-1-3-linked mannose residues either in the outer chain or in the core of S. pombe, but in wild-type strains these mannose residues are shielded by galactose residues.     

Partial deletion of the cloned rfb gene of Escherichia coli O9 results in synthesis of a new O-antigenic lipopolysaccharide.

The rfb gene, involved in the synthesis of the O-specific polysaccharide (a mannose homopolymer) of Escherichia coli O9 lipopolysaccharide (LPS), was cloned in E. coli K-12 strains. The O9-specific polysaccharide covalently linked to the R core of K-12 was extracted from the K-12 strains harboring the O9 rfb gene. All the other genes required for the synthesis of rfe-dependent LPS are therefore considered to be present in the K-12 strains. It was found that bacteria harboring some clones with deletions of the ca. 20-kilobase-pair (kbp) BglII-StuI fragment no longer synthesized the O9-specific polysaccharide. However, bacteria harboring clones del 21, del 22, and del 25, which carry deletions of the 10-kbp PstI-StuI fragment, synthesized an O-specific polysaccharide antigenically distinct from E. coli O9 LPS. Although this new O-specific polysaccharide consisted solely of mannose and the mannose residues were combined only through alpha-1,2 linkage, it was still composed of a repeating oligosaccharide unit, possibly a trisaccharide unit,----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----. It is therefore likely that this new O-specific polysaccharide was derived from a part of the O9-specific polysaccharide----3)alpha Man-(1----3)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----and that the deleted part of the clones was responsible for the synthesis of alpha-1,3 linkages of the O9-specific polysaccharide.     

Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis

The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.