Structural study of phosphomannan of yeast-form cells of Candida albicans J-1012 strain with special reference to application of mild acetolysis

Structural analysis of the phosphomannan isolated from yeast-form cells of a pathogenic yeast, Candida albicans J-1012 strain, was conducted. Treatment of this phosphomannan (Fr. J) with 10 mM HCl at 100 degrees C for 60 min gave a mixture of beta-1,2-linked manno-oligosaccharides, from tetraose to biose plus mannose, and an acid-stable mannan moiety (Fr. J-a), which was then acetolyzed by means of an acetolysis medium, 100:100:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4, at 40 degrees C for 36 h in order to avoid cleavage of the beta-1,2 linkage. The resultant manno-oligosaccharide mixture was fractionated on a column of Bio-Gel P-2 to yield insufficiently resolved manno-oligosaccharide fractions higher than pentaose and lower manno-oligosaccharides ranging from tetraose to biose plus mannose. The higher manno-oligosaccharide fraction was then digested with the Arthrobacter GJM-1 alpha-mannosidase in order to cleave the enzyme-susceptible alpha-1,2 and alpha-1,3 linkages, leaving manno-oligosaccharides containing the beta-1,2 linkage at their nonreducing terminal sites, Manp beta 1----2Manp alpha 1----2Manp alpha 1----2Manp alpha 1----2Man, Manp beta 1----2Manp beta 1----2Manp alpha 1----2Manp alpha 1---- 2Manp alpha 1----2Man, and Manp beta 1----2Manp beta 1----2Manp beta 1----2Manp alpha 1---- 2Manp alpha 1----2Manp alpha 1----2Man. However, the result of acetolysis of Fr. J-a by means of a 10:10:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4 at 40 degrees C for 13 h was significantly different from that obtained by the mild acetolysis method; i.e., the amount of mannose was apparently larger than that formed by the mild acetolysis method. In summary, a chemical structure for Fr. J as a highly branched mannan containing 14 different branching moieties was proposed.     

Acetolysis of Pichia pastoris IFO 0948 strain mannan containing alpha-1,2 and beta-1,2 linkages using acetolysis medium of low sulfuric acid concentration

To obtain manno-oligosaccharides containing beta-1,2-linked nonreducing terminal groups from the mannan of Pichia pastoris IFO 0948 strain by acetolysis, an attempt was made to establish the reaction conditions under which cleavage of the alpha-1,6 linkage took place preferentially leaving manno-oligosaccharides composed largely of beta-1,2 linkages. By the action of an ordinary acetolysis medium, a 10/10/1 (v/v) mixture of acetic anhydride, acetic acid, and sulfuric acid at 40 degrees C for 13 h or at 25 degrees C for 120 h, the O-acetyl derivative of this mannan gave mannose, mannobiose, mannotriose, and mannopentaose. However, treatment of the same O-acetyl mannan with a 50/50/1 (v/v) acetolysis medium at 40 degrees C for 15 h gave a mannotetraose in addition to mannose, mannobiose, mannotriose, and mannopentaose. Use of a 100/100/1 (v/v) acetolysis medium at 40 degrees C for 36 h gave a more satisfactory result, a mixture of oligosaccharides, from mannose to mannopentaose, which contained more mannotetraose than mannopentaose. Because both mannotetraose and mannopentaose contained alpha-1,2 and beta-1,2 linkages, it was concluded that an acetolysis medium containing a low concentration of sulfuric acid, up to 0.5% (v/v), facilitates the preferential cleavage of the alpha-1,6 linkage, leaving manno-oligosaccharides containing the beta-1,2 linkage which was found to be labile to the action of the 10/10/1 (v/v) acetolysis medium.     

Structural study of phosphomannan-protein complex of Citeromyces matritensis containing beta-1,2 linkage. Application of partial acid degradation and acetolysis techniques under mild conditions

The phosphomannan-protein complex of Citeromyces matritensis IFO 0651 strain was investigated for its chemical structure by a sequential degradation procedure, partial acid degradation followed by acetolysis under mild conditions. Upon treatment with 10 mM HCl at 100 degrees C for 1 h, this complex released mannotriose and mannotetraose consisting solely of 1,2-linked beta-D-mannopyranosyl residues, ca. 20% on weight basis of the parent complex. The acid-degraded complex was then subjected to acetolysis using an acetolysis medium of low sulfuric acid concentration, a 100:100:1 (v/v) mixture of acetic anhydride, acetic acid, and sulfuric acid at 40 degrees C for 36 h. A phosphate-containing manno-oligosaccharide fraction eluted in the void-volume region of a Bio-Gel P-2 column was found to consist of Manp beta 1----2Manp beta 1----2Manp alpha 1----2Man to which 1 mol of phosphate group was attached, while a manno-oligosaccharide fraction eluted in the diffusable region was a mixture of Manp beta 1----2Manp beta 1----2Manp beta 1----2Manp alpha 1----2Man, Manp beta 1----2Manp beta 1----2Manp alpha 1----2Man, Manp beta 1----2Manp alpha 1----2Man, Manp alpha 1----2Man, and mannose in the molar ratio of 0.08:0.33:0.19:0.32:1.00. Therefore, the structural analysis of the polysaccharide moiety of a beta-1,2 linkage-containing phosphomannan-protein complex of fungal origin can be achieved by means of a sequential degradation procedure, partial acid degradation followed by acetolysis under mild conditions.     

Existence of novel beta-1,2 linkage-containing side chain in the mannan of Candida lusitaniae, antigenically related to Candida albicans serotype A.

The antigenicity of Candida lusitaniae cells was found to be the same as that of Candida albicans serotype A cells, i.e. both cell wall mannans react with factors 1, 4, 5, and 6 sera of Candida Check. However, the structure of the mannan of C. lusitaniae was significantly different from that of C. albicans serotype A, and we found novel beta-1,2 linkages among the side-chain oligosaccharides, Manbeta1-->2Manbeta1--> 2Manalpha1-->2Manalpha1-->2Man (LM5), and Manbeta1-->2Man-beta1-->2Manbeta1-->2Manalpha1-->2Manalpha1-->2Man (LM6). The assignment of these oligosaccharides suggests that the mannoheptaose containing three beta-1,2 linkages obtained from the mannan of C. albicans in a preceding study consisted of isomers. The molar ratio of the side chains of C. lusitaniae mannan was determined from the complete assignment of its H-1 and H-2 signals and these signal dimensions. More than 80% of the oligomannosyl side chains contained beta-1,2-linked mannose units; no alpha-1,3 linkages or alpha-1,6-linked branching points were found in the side chains. An enzyme-linked immunosorbent inhibition assay using oligosaccharides indicated that LM5 behaves as factor 6, which is the serotype A-specific epitope of C. albicans. Unexpectedly, however, LM6 did not act as factor 6.     

Purification and characterization of a novel alpha-mannosidase from Aspergillus saitoi

An alpha-mannosidase differing from 1,2-alpha-mannosidase was found to occur in Aspergillus saitoi. By a series of column chromatographies the enzyme was purified up to 1,000-fold, and its properties were studied in detail. The enzyme preparation, which was practically free from other exoglycosidases, showed a pH optimum of 5.0. In contrast to 1,2-alpha-mannosidase, the enzyme was strongly activated by Ca2+ ions. p-Nitrophenyl alpha-mannopyranoside was not hydrolyzed by the enzyme. Accordingly, the substrate specificity of the new alpha-mannosidase was studied by using a variety of tritium-labeled oligosaccharides. Studies with linear oligosaccharides revealed that the enzyme cleaves the Man alpha 1----3Man linkage more than 10 times faster than the Man alpha 1----6Man and the Man alpha 1----2Man linkages. Furthermore, it cleaves the Man alpha 1----6Man linkage of the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT only after its Man alpha 1----3 residue is removed. Because of this specificity, the enzyme can be used as an effective reagent to discriminate R----Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT from its isomeric counterparts, Man alpha 1----6(R----Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, in which R represents sugars.     

Structure and Immunochemistry of the Cell Wall Mannans from Saccharomyces chevalieri, Saccharomyces italicus, Saccharomyces diastaticus, and Saccharomyces carlsbergensis

The mannans of Saccharomyces chevalieri, S. italicus, S. diastaticus, and S. carlsbergensis, were acetolyzed, and the fragments were separated by gel filtration. All gave similar acetolysis fingerprints, which were distinguished from S. cerevisiae by the presence of a pentasaccharide component in addition to the mono-, di-, tri-, and tetrasaccharides. All oligosaccharide fragments were composed of mannose in alpha-linkage. From methylation analysis and other structural studies, the disaccharide was shown to be alphaMan(1 --> 2)Man; the trisaccharide was shown to be a mixture of alphaMan(1 --> 2)alphaMan (1 --> 2)Man and alphaMan(1 --> 3)alphaMan(1 --> 2)Man; the tetrasaccharide was alphaMan(1 --> 3)alphaMan(1 --> 2)alphaMan(1 --> 2)Man; and the pentasaccharide was alphaMan(1 --> 3)alphaMan(1 --> 3)alphaMan(1 --> 2)alphaMan(1 --> 2)Man. The ratios of the different fragments varied slightly from strain to strain. Mannanase digestion of two of the mannans yielded polysaccharide residues that were unbranched (1 --> 6)-linked polymers, thus establishing the structural relationship between these mannans and that from S. cerevisiae. Antisera raised against the various yeasts cross-reacted with the mannans from each, and also with S. cerevisae mannan. The mannotetraose and mannopentaose acetolysis fragments gave complete inhibition of the precipitin reactions, which indicated that, in these systems as in the S. cerevisiae system, the terminal alpha(1 --> 3)-linked mannose unit was the principal immunochemical determinant on the cell surface.     

Structure of the phosphopeptidomannans from flocculent and non-flocculent yeast Kluyveromyces lactis

After extraction from whole cells, and purification by gel filtration, the chemical composition and molecular mass estimation of the cell-wall phosphopeptidomannan (PPM) showed no significant difference respectively between flocculent, weakly, very weakly and non-flocculent Kluyveromyces lactis yeast strains. However, when PPMs were tested as ligands of a lectin, extracted from the flocculent strain, the PPM isolated from the flocculent and weakly flocculent strain were recognized to a higher degree than those isolated from the non and very weakly flocculent strains. Acetolysis of PPM extracted from the four strains produced five oligosaccharide fractions corresponding to mono-, di-, tri-, penta-and hexa-saccharides. The flocculent strain was characterised by a high content of di-and penta-saccharides. The ¹H NMR analysis of the oligosaccharides demonstrated that the flocculent strain contained equivalent levels of the two mannobioses: Man( 1 → 2)Man and Man( 1 → 3)Man and of the two mannotrioses Man( 1 → 2)Man( 1 → 2)Man and Man( 1 → 3)Man( 1 → 2)Man. In contrast, the non-flocculent and the very weakly flocculent strains contained a single type of mannobiose Man( 1 → 2)Man and one type of mannotriose Man( 1 → 2)Man( 1 → 2)Man.     

Characterization of phosphomannan-protein complexes isolated from viable cells of yeast and mycelial forms of Candida albicans NIH B-792 strain by the action of Zymolyase-100T

The isolation of phosphomannan-protein complexes from the viable cells of yeast (Y) and mycelial (M) forms of Candida albicans NIH B-792 strain was conducted by treatment with Zymolyase-100T followed by fractional precipitation with cetyltrimethylammonium bromide. The M-form complex was found to contain smaller amount of phosphate (1.3%) than that of the Y-form complex (1.6%). Proton magnetic resonance (PMR) spectra of these complexes indicated that the content of beta-1,2-linked oligomannosyl and nonreducing terminal alpha-1,3-linked mannopyranosyl residues in the M-form complex was lower than that of the Y-form complex. With hot 10 mM HCl, the Y-form complex released a mixture of oligosaccharides ranging from mannose to mannoheptaose, while the M-form complex produced lower oligosaccharides, from mannose to mannotetraose. Upon acetolysis, the acid-modified complex of the M form gave mainly mannotetraose, while that of the Y form produced mainly mannopentaose and mannohexaose in addition to mannotetraose. The average length of branching moieties of the mannan of Y-form cells was therefore longer than that of M-form cells. These results indicate that the Y to M transformation of this C. albicans strain accompanies the suppression of enzyme activity concerning the biosynthesis of mannan such as beta-1,2- and alpha-1,3-mannosyltransferases to synthesize the phosphomannan-protein complex containing mannan moiety with incomplete structure.     

Reassessment of antigenic determinant of Saccharomyces cerevisiae serotype Ia.

We examined the immunochemical structure of the antigenic determinant of S. cerevisiae serotype Ia. The specific factor serum for S. cerevisiae serotype Ia was obtained either from factor 18 serum by adsorption with heat-killed cells of Candida glabrata, or from anti-S. cerevisiae Ia (M 6001) serum by adsorption with heat-killed cells of S. cerevisiae Ib (IFO 0751). We designated this adsorbed serum as factor 18a. Acetolysis of S. cerevisiae cell wall D-mannan gave five oligosaccharides. Signals of 1H-nuclear magnetic resonance spectra of mannooligosaccharides derived from S. cerevisiae mannan were assigned for their linkages by the aid of those of alpha-1,3'-linked mannooligosaccharides derived from glucuronoxylomannan of capsule of Cryptococcus neoformans serotype A-D. Agglutination-inhibition experiments revealed that the mannopentaose from S. cerevisiae mannan was the most effective inhibitor. Moreover, inhibitory activities of alpha-1,3'-linked mannotriose, mannotetraose, and mannopentaose which were derived from glucuronoxylomannan of C. neoformans were shown to be higher than those of mannotetraose with one terminal alpha-(1-3) linkage from homologous S. cerevisiae mannan. These results indicate that mannopentaose with terminal two alpha-(1-3) linkages is responsible for the specificity of S. cerevisiae Ia.     

Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of<Emphasis Type="Italic"> Candida tropicalis</Emphasis>

In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manalpha1-3Manalpha1-(2Manalpha1-)(n)2Man [n> or =2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man and Manalpha1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man [n> or =2]. However, this mannan lacked the phosphate group and the beta-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an alpha-1,3-linked mannose residue previously observed in Candida albicans.     

Bowringia milbraedii agglutinin. Specificity of binding to early processing intermediates of asparagine-linked oligosaccharide and use as a marker of endoplasmic reticulum glycoproteins

We have purified a protein with hemagglutinating activity from the seeds of a West African legume, Bowringia milbraedii. The purified protein, designated BMA, has a native Mr = 38,000 on gel filtration and a subunit size of Mr = 16,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions. Hemagglutination was inhibited most effectively by Man alpha 1----2 linked sugars. Affinity chromatography of oligosaccharides on BMA-Sepharose showed that Man alpha 1----2Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol (where GlcNAcol is N-acetylglucosaminitol) and Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol were retarded on the column, whereas Man alpha 1----3Man beta 1----4GlcNAcol did not bind. Oligomannosidic-type glycans obtained by treatment of [3H] mannose-labeled baby hamster kidney cells with endo-beta-N-acetylglucosaminidase H bound more strongly to BMA-Sepharose and required 10 or 200 mM methyl-alpha-mannoside for elution. Oligosaccharides bearing the sequence Man alpha 1----2Man alpha 1----6Man alpha 1----6Man, i.e. Man9GlcNAc and certain isomers of Man8GlcNAc and Man7GlcNAc, bound more tightly than other Man8 GlcNAc and Man7GlcNAc isomers lacking this sequence. Man6GlcNAc and Man5GlcNAc were weakly bound. These results suggest that BMA binds preferentially to glycoproteins that are subjected to early steps of oligosaccharide processing in the endoplasmic reticulum but not to glycoproteins that are exposed to more extensive processing by Golgi mannosidases. Staining of permeabilized cells with BMA-chromophore conjugates revealed a reticular cytoplasmic pattern consistent with a preferential visualization of the endoplasmic reticulum. BMA staining was less evident in the juxtanuclear regions that were stained brightly with wheat germ agglutinin, a lectin that binds preferentially to sialylated glycoproteins located in Golgi compartments.     

Structural study of cell wall mannan of a Candida albicans (serotype A) strain.

The structure of the mannan of Candida albicans NIH A-207 strain (serotype A) was investigated by adopting mild acetolysis followed by enzymolysis with an Arthrobacter GJM-1 exo-alpha-mannosidase. The resultant oligosaccharides, from pentaose to octaose (where manp = D-mannopyranose), were identified as manp beta (1----2)manp alpha (1----2)manp alpha (1----2)manp alpha (1----2)manp, manp beta (1----2)manp beta (1----2)manp alpha (1----2)manp alpha (1----2)- manp alpha (1----2)manp, manp beta (1----2)manp beta (1----2)manp beta (1----2)manp alpha (1----2)manp alpha (1----2)manp alpha (1----2)manp and manp beta (1----2)manp beta (1----2)manp beta (1----2)manp beta (1----2)manp alpha (1----2)manp alpha (1----2)manp alpha (1----2)manp, respectively. Analyses of alpha-linked oligosaccharides obtained by acetolysis under conventional conditions gave the same oligosaccharides, from biose to heptaose, as those obtained from the mannans of C. albicans NIH B-792 (serotype B) and J-1012 (serotype A, formerly serotype C).     

New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs.

Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose. The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay. ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S. cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T. gropengiesseri and T. lactis-condensi, a linear mannopentaose, and murine IgM. On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S. cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units. Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds. The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.     

Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus

Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-beta-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Man alpha 1----6(Man alpha 1----3)Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Man alpha 1----6(Man alpha 1----3)Man alpha 1----6 and Man alpha 1----3Man alpha 1----6 and Man alpha 1----3Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were found at Asn12, as previously published [Schlüter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. & Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAc alpha 2----3 or Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(NeuAc alpha 2----6 or NeuAc alpha 2----3Gal-beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNac beta 1----4GlcNAc beta 1---- with some bifurcation at ----6Man alpha 1----6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Gal beta 1----3GalNAc alpha 1----, monosialylated or desialylated at position 3 of Gal or/and position 6 of GalNAc.     

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.     

N-acetylglucosaminyltransferase III, IV and V activities in Novikoff ascites tumour cells, mouse lymphoma cells and hen oviduct. Application of a sensitive and specific assay by use of high-performance liquid chromatography

A specific and fast method for the determination of N-acetylglucosaminyltransferase III, IV and V activity in one assay is described. The method is based on the separation by HPLC of the three transferase products formed from the common acceptor oligosaccharide substrate GlcNAc beta 1----2Man alpha 1----3(GlcNAc beta 1----2Man alpha 1---- 6)Man beta 1----4GlcNAc. Assays are not interfered with by substances that result from enzymatic or chemical breakdown of the donor substrate UDP-[14C]GlcNAc. Using this assay system N-acetylglucosaminyltransferase III, IV and V activities were estimated in Novikoff ascites tumour cells, mouse lymphoma BW 5147 cells and hen oviduct.     

Characteristics of asparagine-linked sugar chains of sphingolipid activator protein 1 purified from normal human liver and GM1 gangliosidosis (type 1) liver

Asparagine-linked sugar chains of sphingolipid activator protein 1 (SAP-1) purified from normal human liver and GM1 gangliosidosis (type 1) liver were comparatively investigated. Oligosaccharides released from the two SAP-1 samples by hydrazinolysis were fractionated by paper electrophoresis and by Aleuria aurantia lectin-Sepharose and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of oligosaccharides in each fraction were estimated from data on their effective molecular sizes, behavior on immobilized lectin columns with different carbohydrate-binding specificities, results of sequential digestion by exoglycosidases with different aglycon specificities, and methylation analysis. Sugar chains of SAP-1 purified from normal human liver and from GM1 gangliosidosis (type 1) liver were different from each other, although both of them were derived from complex-type sugar chains. The sugar chains of the former were the following eight degradation products from complex-type sugar chains by exoglycosidases in lysosomes: Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT, Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAcOT, Man alpha 1----6Man beta 1----4GlcNAc beta 1----4GlcNAcOT, Man alpha 1----6Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAcOT, Man beta 1----4GlcNAc beta 1----4GlcNAcOT, Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAcOT, GlcNAc beta 1----4GlcNAcOT, and GlcNAcOT. In contrast to these, the sugar chains of the latter were sialylated and nonsialylated mono- to tetraantennary complex-type sugar chains that were not fully degraded due to a metabolic defect in acid beta-galactosidase activity.     

Two positional isomers of sialylheptasaccharides isolated from the urine of a patient with sialidosis

We describe the structures of two positional isomers of sialylheptasaccharide isolated from the urine of a patient with sialidosis with partial deficiency of beta-galactosidase. Based on structural studies including compositional sugar analysis, exoglycosidase digestion, chemical ionization mass spectrometry, proton nuclear magnetic resonance spectrometry, and methylation analysis, their structures were deduced to be as follows: AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----3(Man alpha 1----6)Man beta 1----4GlcNac; AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNac. Sialyloligosaccharide 1 has previously been found in the urine and liver of patients with mucolipidosis I and II and sialidosis, but sialyloligosaccharide 2 has not been found yet in human urine. These two sialyloligosaccharides could not be completely separated by any chromatographic procedures tested. The analytical techniques, including methylation study and NMR spectroscopy, could not clearly detect the differences between them. However, alpha-mannosidase treatment gave important information for the structural analyses of these sialyloligosaccharides.     

Interaction of linear manno-oligosaccharides with three mannose-specific bulb lectins. Comparison with mannose/glucose-binding lectins.

Three new mannose-binding lectins, isolated from daffodil (NPA), amaryllis (HHA), and snowdrop (GNA) bulbs, are capable of precipitating with a linear mannopentaose (Man alpha 1-3Man alpha 1-3Man alpha 1-3Man alpha 1-2Man). NPA and HHA reacted strongly with the mannopentaose whereas GNA gave a precipitate only at concentrations greater than 500 microM. A phosphate group at C-6 of the nonreducing terminal mannosyl group prevented precipitation in all three cases. The reduced (NaBH4) mannopentaose, Man4Man-ol, did not precipitate with GNA or NPA, but was active with HHA. This activity was lost when Man4Man-ol was converted (NaIO4 then NaBH4; mild acid hydrolysis of the reduced product) into trisaccharide derivatives. With alpha-D-Manp-OMe the three lectins gave UV difference spectra having large positive peaks at 292-293 and 283-284 nm, and a small positive peak at 275 nm, characteristic of tryptophanyl and tyrosyl residues. The association constants for the interaction with alpha-D-Manp-OMe were very low (NPA, 86; HHA, 66; and GNA, 41 M-1), but the lectins bound methyl (1----3)-alpha-mannobioside with increased affinity (K for NPA 540, for HHA 2400, and for GNA 200 M-1). The bulb lectins lack binding sites for hydrophobic ligands, as judged by their failure to interact with the fluorescent probes 8-anilino-1-napthalenesulfonic acid (ANS) and 6-p-toluidino-2-naphthalenesulfonic acid (TNS).     

Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.