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.     

Studies on the Antigenic Activities of Yeasts

In order to determine the antigenic determinant groups of the mannan of Candida albicans by the precipitation-inhibition test, several oligosaccharides were prepared by acetolysis of the polysaccharide. The manno-oligosaccharides, from biose to heptaose were separated by a charcoal-Celite chromatography and a subsequent cellulose column chromatography. The oligosaccharides thus separated were examined on the degrees of polymerization and the mode of the linkages, and evidence was obtained that the biose and triose were joined through α1→2 linkage only, while the tetraose, pentaose and hexaose contained α1→3 linkage in addition to α1→2 linkages. Heptaose was joined entirely through α1→2 linkage. In the precipitation-inhibition test, the inhibitory power of the oligosaccharides of acetolysis product was found to be the following order: hexaose>heptaose>pentaose>tetraose>triose>biose, and the amount for the 50% inhibitions were 0.025, 0.09, 0.12, 0.60, 3.96 and 5.84 μmoles respectively. On the other hand, the biose, triose and tetraose, which were isolated from the acid-hydrolysate of the mannan of Saccharomyces cerevisiae and joined through α1→6 linkage, showed poor or nearly no inhibitory power. The above facts provide an evidence that the consecutive α1→6 linkages were not located in a position that is responsible for antigenic specificity of the mannan of C. albicans.     

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.     

Immunochemical study on the mannans of Candida albicans NIH A-207, NIH B-792, and J-1012 strains prepared by fractional precipitation with cetyltrimethylammonium bromide

The mannans of Candida albicans NIH A-207 (A strain, serotype A), C. albicans NIH B-792 (B strain, serotype B), and C. albicans J-1012 (J strain, serotype C) prepared by fractional precipitation with cetyltrimethylammonium bromide (Cetavlon) were investigated for their immunochemical properties. Upon treatment with 10 mM HCl at 100 degrees C for 60 min, the mannans of A and B strains each released a mixture of manno-oligosaccharides ranging from hexaose to mannose together with (for each one) an acid-modified mannan, while J-strain mannan released lower oligosaccharides, tetraose to mannose. The acid-modified mannan of B strain did not show antibody-precipitating activity against homologous antiserum, whereas acid-modified A- and J-strain mannans retained most of this activity. The acid-released oligosaccharides were assumed to consist of beta-1,2-linked D-mannopyranosyl residues from the results of specific rotation and proton magnetic resonance studies.     

Studies on the Antigenic Activities of Yeasts

The antigenic mannan of Candida albicans was degraded by acid-hydrolysis and the resultant oligosaecharides were fractionated by a carbon-Celite and a subsequent cellulose-powder chromatography to yield four oligosaecharides, pentaose, hexaose, heptaose and octaose, which involved 2,6-di-0- and 6-0-substituted mannopyranosyl residues as the common. feature. These oligosaccharides showed lower precipitation-inhibition activity than that of the hexaose of acetolysate, the strongest inhibitor among the oligosaccharides described in the preceding study. The order of inhibitory powers of oligosaccharides was as follows: hexaose of acetolysate>heptaose>pentaosez=octaose>hexaose. The μmoles requiring for 50%-inhibition were 0.025, 0.15, 0.20, 0.20 and 0.50 respectively. The results clearly indicate that the determinant groups of the mannan of C. albicans employed this study are the hexaose moieties which constitute the branching parts of polysaccharide.     

Studies on the Antigenic Activities of Yeasts

In order to identify the antigenic determinant groups of the mannan of C. albicans serotype A, six kinds of manno-oligosaccharides of up to 7 units in chain-length connected by α1→2 linkages were prepared from the partial acetolysate of the parent mannan. In the precipitation-inhibition test of anti-C. albicans serotype A serum with its homologous mannan, inhibitory power of the oligosaccharides was of the following order: heptaose→: hexaose>pentaose>tetraose>triose> biose, and the amounts for 50%-inhibition of the former four oligosaccharides were 0.08, 0.10, 0.50 and 3.0 μmole respectively, and the inhibitory power of the latter two oligomers at 0.5 μmole were 8 and 5% respectively. On the other hand, the cross-inhibition test of anti-C. albicans serotype A serum with the heterologous mannan of C. albicans serotype B afforded the result that the order of inhibitory activities was hexaose>heptaose>pentaose>tetraose>triose> biose, and that the amounts for 50%-inhibition were 0.05, 0.08, 0.1, 0.45, 0.50 and 3.0μmole respectively. Furthermore, the results of inhibition test on the anti-C. albicans serotype A serum absorbed with the mannan of C. albicans serotype B revealed that the biose, triose and tetraose did not show significant inhibitory power in the range employed, whereas the pentaose, hexaose and heptaose did not significantly affect the inhibitory activities. Thus, it was concluded that the antigenic determinants of the mannan of C. albicans serotype A are α1→2 linked hexaose or heptaose moieties. Based on the above facts, the serological differences between two antigenic mannans of C. albicans serotype A and B may reside at least in the length of the antigenic determinants in which the former is longer than the latter considering the length of the α-D-manno-pyranosyl residue.     

Immunochemical characterization of Candida albicans cell wall antigens: specific determinant of Candida albicans serotype A mannan

We investigated the chemical structure of the specific determinant in the mannan of Candida albicans M-1012 (serotype A) strain. Acetolysis of the mannan, obtained by alkali extraction and purified as the copper complex, gave mannose and six oligosaccharides (from di- to hexasaccharide) and a small amount of a heptasaccharide. We examined the inhibition by these oligosaccharides up to hexaose of the precipitin reaction between anti-factor 6 serum specific for serotype A and homologous mannan, and found that the mannohexaose was the most effective inhibitor. These, and results obtained by proton magnetic resonance (PMR) spectroscopy, methylation analysis, and other structural studies, suggest that the main component of this hexaose consists of one terminal alpha (1-3) linkage in addition to four alpha (1-2) linkages, and that this alpha (1-3)-containing mannohexaose may be responsible for the specificity of antigenic factor 6. Further results obtained by analyses of polarimetry, PMR spectroscopy, and chromium trioxide oxidation-methylation of C. albicans M-1012 mannan has a beta-linkage in addition to alpha-linkages, and that the mode of the beta-linkage is mainly (1-6) linkage. Further evidence obtained by Smith degradation-methylation analysis and by quantitative precipitin reactions of intact and acid-degraded mannan suggests that the antigenic determinant of antigenic factor 6 may be bound, via the beta (1-6) linkage, to C-6 of mannose residues involved in oligosaccharide side chains of serotype A mannan.     

Identification of a novel UDP-Gal:GalNAc beta 1-4GlcNAc-R beta 1-3-galactosyltransferase in the connective tissue of the snail Lymnaea stagnalis

Connective tissue of the freshwater pulmonate Lymnaea stagnalis was shown to contain galactosyltransferase activity capable of transferring Gal from UDP-Gal in beta 1-3 linkage to terminal GalNAc of GalNAc beta 1-4GlcNAc-R [R = beta 1-2Man alpha 1-O(CH2)8COOMe, beta 1-OMe, or alpha,beta 1-OH]. Using GalNAc beta 1-4GlcNAc beta 1-2Man alpha-1-O(CH2)8COOMe as substrate, the enzyme showed an absolute requirement for Mn2+ with an optimum Mn2+ concentration between 12.5 mM and 25 mM. The divalent cations Mg2+, Ca2+, Ba2+ and Cd2+ at 12.5 mM could not substitute for Mn2+. The galactosyltransferase activity was independent of the concentration of Triton X-100, and no activation effect was found. The enzyme was active with GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe (Vmax 140 nmol.h-1.mg protein-1; Km 1.02 mM), GalNAc beta 1-4GlcNAc (Vmax 105 nmol.h-1.mg protein-1; Km 0.99 mM), and GalNAc beta 1-4GlcNAc beta 1-OMe (Vmax 108 nmol.h-1.mg protein-1; Km 1.33 mM). The products formed from GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe and GalNAc beta 1-4GlcNAc beta 1-OMe were purified by high performance liquid chromatography, and identified by 500-MHz 1H-NMR spectroscopy to be Gal beta 1-3GalNAc beta 1-4GlcNAc 1-OMe, respectively. The enzyme was inactive towards GlcNAc, GalNac beta 1-3 GalNAc alpha 1-OC6H5, GalNAc alpha 1--ovine-submaxillary-mucin, lactose and N-acetyllactosamine. This novel UDP-Gal:GalNAc beta 1-4GlcNAc-R beta 1-3-galactosyltransferase is believed to be involved in the biosynthesis of the hemocyanin glycans of L. stagnalis.     

Control of glycoprotein synthesis. Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine:GlcNAc beta 1-6 (GlcNAc beta 1-2)Man alpha-R (GlcNAc to Man) beta-4-N-acetylglucosaminyltransferase VI

Hen oviduct membranes were shown to contain high activity of a novel enzyme, UDP-GlcNac:GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-R (GlcNAc to Man) beta 4-GlcNAc-transferase VI. The enzyme was shown to transfer GlcNAc in beta 1-4 linkage to the D-mannose residue of GlcNAc beta 1-6 (GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or methyl. Radioactive enzyme products were purified by several chromatographic steps, including high performance liquid chromatography, and structures were determined by proton nmr, fast atom bombardment-mass spectrometry, and methylation analysis to be GlcNAc beta 1-6 ([14C]GlcNAc beta 1-4) (GlcNAc beta 1-2) Man alpha-R. The enzyme is stimulated by Triton X-100 and has optimum activity at a relatively high MnCl2 concentration of about 100 mM; Co2+, Mg2+, and Ca2+ could partially substitute for Mn2+. A tissue survey demonstrated high GlcNAc-transferase VI activity in hen oviduct and lower activity in chicken liver and colon, duck colon, and turkey intestine. No activity was found in mammalian tissues. Hen oviduct membranes cannot act on GlcNAc beta 1-6Man alpha-R but have a beta 4-GlcNAc-transferase activity that converts GlcNAc beta 1-2Man alpha-R to GlcNAc beta 1-4(GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or 1-6Man beta methyl. The latter activity is probably due to GlcNAc-transferase IV which preferentially adds GlcNAc in beta 1-4 linkage to the Man alpha 1-3 arm of the GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn core structure of asparagine-linked glycans. The minimum structural requirement for a substrate of beta 4-GlcNAc-transferase VI is therefore the trisaccharide GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-; this trisaccharide is found on the Man alpha 6 arm of many branched complex asparagine-linked oligosaccharides. The data suggest that GlcNAc-transferase VI acts after the synthesis of the GlcNAc beta 1-2Man alpha 1-3-, GlcNAc beta 1-2Man alpha 1-6-, and GlcNAc beta 1-6 Man alpha 1-6-branches by GlcNAc-transferases I, II, and V, respectively, and is responsible for the synthesis of branched oligosaccharides containing the GlcNAc beta 1-6(GlcNAc beta 1-4)(GlcNAc beta 1-2)Man alpha 1-6Man beta moiety.     

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).     

1H-NMR spectroscopy of manno-oligosaccharides of the beta-1,2-linked series released from the phosphopeptidomannan of Candida albicans VW-32 (serotype A).

Manno-oligosaccharides (DP 2 to greater than 15) were released by mild acid hydrolysis from the phosphopeptidomannan of a Candida albicans strain of A serotype (VW-32). Manno-oligosaccharides ranging from biose to heptaose were obtained in appreciable amount. Structural investigation of these oligosaccharides showed them to be of the beta-1,2-linked series. The occurrence of such compounds has already been reported in other strains of Candida albicans. We here report the assignment of the structural reporter groups of each of them, and general rules applicable for the 1H-NMR spectrum analysis of linear manno-oligosaccharide of general structure: Man(beta 1-2) [Man(beta 1-2)]nMan     

Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans.

The substrate specificity of rat liver cytosolic neutral alpha-D-mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-2)Man(alpha 1-6)]Man(alpha 1-6) [Man(alpha 1-2)Man(alpha 1-3)]Man(beta 1-4)GlcNAc; Man5GlcNAc I, Man(alpha 1-3)[Man(alpha 1-6)]-Man(alpha 1-6)Man(alpha 1-3)] Man(beta 1-4)GlcNAc; Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3) [Man(alpha 1-6)]Man(beta 1-4)GlcNAc; Man4GlcNAc, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc. The different oligosaccharide isomers resulting from alpha-D-mannosidase hydrolysis were analyzed by 1H-NMR spectroscopy after HPLC separation. The cytosolic alpha-D-mannosidase activity is able to hydrolyse all types of alpha-mannosidic linkages found in the glycans of the oligomannosidic type, i.e. alpha-1,2, alpha-1,3 and alpha-1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble alpha-D-mannosidase acting on Man5-GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, represents the 'construction' Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The cytosolic alpha-D-mannosidase is activated by Co2+, insensitive to 1-deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal alpha-D-mannosidases [Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]. A possible complementarity between cytosolic and lysosomal alpha-D-mannosidase activities in the catabolism of N-glycosylprotein is proposed.     

Acetolysis and 1H NMR studies on mannans isolated from very flocculent and weakly flocculent cells of Pichia pastoris IFP 206

Growth of the yeast Pichia pastoris IFP 206 in methanol- and glucose-containing media led respectively to very and weakly flocculent cells. Mannans from both kinds of cells were extracted and compared. Chemical analysis and molecular mass estimation showed some differences between the mannans from very and weakly flocculent cells, especially in quantitative amino acid content. 1H NMR analysis showed that both kinds of mannan contained alpha-1,2 and beta-1,2 linkages. Two acetolysis conditions, combined with 1H NMR analysis, revealed that mannans from both kinds of cells were composed of mannose, mannobiose, mannotriose, mannotetraose and mannopentaose side-chains with the following respective structures: Man; Man alpha 1---2Man; Man alpha 1----2Man alpha 1----2Man; Man beta 1----2Man alpha 1----2Man; Man beta 1----2Man beta 1----2Man alpha 1----2Man; Man alpha 1----2Man beta 1----2Man beta 1----2Man alpha 1----2Man. Additionally the beta-1,2 linkages of the non-reducing terminal residues of the mannotetraose were shown to be acetolysis-labile. The mannans from very flocculent cells were richer in mannopentaose than the mannans from weakly flocculent cells. According to these results, the extended conformations in the branching moieties of the mannan could be the basis of the higher degree of flocculation of the methanol-grown cells.     

Characterization of a sulfotransferase responsible for the 4-O-sulfation of terminal beta-N-acetyl-D-galactosamine on asparagine-linked oligosaccharides of glycoprotein hormones

The Asn-linked oligosaccharides on the glycoprotein hormones lutropin (LH) and thyrotropin terminate with the sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2 Man alpha-. Using a chemically synthesized trisaccharide GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOCH3 (GGnM-MCO), we have developed a sensitive assay for the sulfotransferase responsible for the 4-O-sulfation of the terminal beta-D-GalNAc. GGnM-MCO is incubated with a bovine pituitary membrane extract and [35S]3'-phosphoadenosine 5'-phosphosulfate ([35S]PAPS). The sulfated product [35S]SGGnM-MCO is separated from [35S]PAPS, PAPS degradation products and endogenous sulfated products by a two-step procedure utilizing an Ecteola cellulose column and a Sep-Pak (C18) cartridge. Characterization of the [35S]SGGnM-MCO produced in the assay indicates that sulfate is incorporated exclusively on the 4-position of GalNAc. Linear incorporation of sulfate into GGnM-MCO can be maintained for greater than 10 h. GGnM-4-sulfotransferase has a pH optimum of 7.2, requires the presence of a reducing agent, and is stimulated by, but does not require, divalent cations. Initial velocity studies indicate an apparent Km (Henri-Michaelis-Menten equilibrium constant) for PAPS of 4 microM and for GGnM-MCO of 9 microM. Incorporation of sulfate into the trisaccharide is stimulated 3-fold by the presence of basic proteins including deglycosylated LH. The stimulation by deglycosylated LH suggests that the protein component of glycoproteins that bear oligosaccharides terminating with GalNAc-GlcNAc-Man- may modulate GGnM-4-sulfotransferase.     

Comparative study of lysosomal and cytosolic catabolisms of oligomannosidic-type glycans

In order to study the substrate specificities of the enzymes implicated in the catabolism of oligomannosidic-type glycans, the oligosaccharides Man9GlcNAc and Man5GlcNAc were incubated with rat liver lysosomal and cytosolic alpha-D-mannosidases and the hydrolysis products were characterized by 400 MHz 1H-NMR spectroscopy. Although they both occur in an ordered way, the two catabolic pathways are quite different. The lysomal pathway is realized in two stages: the first leads from Man9GlcNAc to Man5GlcNAc by preferential cleavage of the four alpha-1,2-linked mannose residues, and the second, Zn(2+)-dependent, leads from Man5GlcNAc to Man (beta 1-4) GlcN Ac by hydrolysis of alpha-1, 3- and alpha-1,6-linked residues. On the contrary, the cytosolic pattern leads by a pathway quite different to a unique hexasaccharide Man5GlcNAc which has, curiously, the same structure as one of the polyprenolic intermediates occurring in the cytosol during the biosynthesis of N-glycosylprotein glycans: Man (alpha 1-2) Man (alpha 1-2) Man (alpha 1-3) [Man (alpha 1-6)] Man (beta 1-4) GlcN Ac (beta 1-4) GlcNAc alpha 1-P-P-Dol.     

A new mannoheptaose containing alpha and beta-(1-->2) linkages isolated from the mannan of Torulaspora delbrueckii: ELISA inhibition studies

Torulaspora delbrueckii starin IFO 0955 was examined with respect to its structural and serological properties of the cell wall mannan (Td-0955-M). Td-0955-M revealed significant reactivities with sera from a commercially available factor serum kit (Candida Check) in ELISA. Td-0955-M was investigated for its chemical structure by acetolysis under conventional and mild conditions. NMR and GC techniques were used as analytical techniques. The mannooligosaccharide fractions eluted from a Bio-Gel P-2 column were found to consist of Man(alpha1-2)Man, M2, Man(alpha1-2)Man(alpha1-2)Man and Man(beta1-2)Man(alpha1-2)Man, M3, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M5, and a new mannoheptaose, which possesses the structure, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M7. The results of the inhibition ELISA showed that the M7 oligosaccharide significantly inhibited the reactivities in the Td-0955-M-factor serum systems.     

Refined structure of the lipophosphoglycan of Leishmania donovani.

The primary structure of the major surface glycoconjugate of Leishmania donovani parasites, a lipophosphoglycan, has been further characterized. The repeating PO4-6Galp beta 1-4Man disaccharide units, which are a salient feature of the molecule, are shown to terminate with one of several neutral structures, the most abundant of which is the branched trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The phosphosaccharide core of lipophosphoglycan, which links the disaccharide repeats to a lipid anchor, contains 2 phosphate residues. One of the core phosphates has previously been localized on O-6 of the galactosyl residue distal to the lipid anchor; the second phosphate is now shown to be on O-6 of the mannosyl residue distal to the anchor and to bear an alpha-linked glucopyranosyl residue. Also, the anomeric configuration of the unusual 3-substituted Galf residue in the phosphosaccharide core is established as beta. The complete structure of the core is thus PO4-6Galp alpha 1-6Galp alpha 1-3Galf beta 1-3[Glcp alpha 1-PO4-6]Manp alpha 1-3Manp alpha 1-4GlcN alpha 1-. This further clarification of the structure of lipophosphoglycan may prove beneficial in determining the structure-function relationships of this highly unusual glycoconjugate.     

Sulfated N-linked carbohydrate chains in porcine thyroglobulin

N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on Lichrosorb-NH2, and analyzed by 500-MHz 1H-NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide-alditols, the following sulfated carbohydrate chains could be identified: NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3[(SO3Na----3)Gal beta 1----4GlcNAc beta1----2-Mana alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc-ol and NeuAc alpha 2----6Gal beta 1----4(SO3Na----)0-1 GlcNAc beta 1----2-Man alpha 1----3[NeuAc alpha 2----6Gal beta 1----4(SO3Na----6)1-0GlcNAc beta 1----2Man alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc- ol. The sulfated structural elements for porcine thyroglobulin form novel details of N-linked carbohydrate chains. They contribute to the fine structure of these oligosaccharides and are another type of expression of microheterogeneity.     

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.     

Free oligosaccharides with Lewis x structure expressed in the segmentation period of zebrafish embryo

We previously reported that zebrafishalpha1-3fucosyltrasferase 1 (zFT1) was expressed in embryos at the segmentation period, and was capable of synthesizing the Lewis x epitope [Gal beta1-4(Fuc alpha1-3)GlcNAc] [Kageyama et.al, J. Biochem., 125, 838-845 (1999)]. In the current study, we attempted to detect the enzyme products of zFT1 in zebrafish embryos. Oligosaccharides were prepared from the zebrafish embryos at 12, 18 and 48 h after fertilization and labelled with a fluorophore, 2-aminopyridine, for highly sensitive detections. Pyridylamino (PA)-oligosaccharides that were alpha1-3/4fucosidase sensitive and time-dependently expressed at 18 h after fertilization were identified as candidates for the in vivo products synthesized by zFT1. Structures of these oligosaccharides were determined by a combination of exoglycosidase digestions and two-dimensional HPLC sugar mapping to be the biantennary complex-type structures with two Lewis x epitopes: (Gal beta1-4)(0,1,2)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]}Man beta1-4GlcNAc, and (Gal beta1-4)(0,1)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]} Man beta1-4GlcNAc beta1-4GlcNAc. The presence of Lewis x structure of these oligosaccharides together with their expression time suggests that they are products of zFT1. Remarkably, most of these oligosaccharides were free form. Furthermore, we detected an endo-beta-N-acetylglucosaminidase activity in the 18 h embryo. These results suggest that the oligosaccharides synthesized by zFT1 are present in the embryo at the segmentation period in free form, owing to the liberation from glycoproteins with endo-beta-N-acetylglucosaminidase(s) and/or glycoamidase(s).