D-mannose Units Research Articles

Structural and Immunochemical Studies on D-Arabino-D-Mannans and D-Mannans of Mycobacterium tuberculosis and Other Mycobacterium Species1

Serologically active D-arabino-D-mannas ([alpha]D, +82 degrees approximately 89 degrees; ratio of D-arabinose to D-mannose, 1-2:1) were isolated from the soluble fraction of disintegrated cells of M. tuberculosis, M. smegmatis, and several other Mycobacterium species. These arabinomannans had similar structures, consisting of alpha-(1 leads to 5)-linked D-arabinose residues and alpha-(1 leads to 6)-, and (1 leads to 2)-linked D-mannose residues. Methylation and enzymic degradation studies using Arthrobacter sp. alpha-D-mannosidase and M-2 enzyme (D-arabinan hydrolase) indicated that the arabinomannan of M. tuberculosis Aoyama B possesses short side chains built up from alpha-(1 leads to 2)-D-mannosidic linkages which are attached to an alpha-(1 leads to 6)-linked mannan back-bone chain. The alpha-(1 leads to 5)-linked D-arabinose residues located in the side chains were shown, by comparison of the immunochemical activities of the native and enzyme-degraded polysaccharides, to be the main immunodeterminants, as in the cell-wall arabinogalactan. There appeared to be variations in the ratio of arabinose and mannose residues, and also in the proportion of (1 leads to 2)-linked D-mannose units, depending on the individual strain; no (1 leads to 2)-mannosidic linkage was found in M. smegmatis arabinomannan. In addition to arabinomannan, a serologically inactive alpha-D-mannan ([alpha)D, +65 degrees approximately 68 degrees), whose structure may resemble that of the core mannan of the arabinomannan, was isolated as a copper hydroxide complex from the soluble fraction of disintegrated mycobacterial cells.

Chemical and biological studies on the lipopolysaccharide (O-antigen) of Anacystis nidulans

The O-antigen (lipopolysaccharide) of Anacystis nidulans, strain KM, has been isolated from whole cells and from cell wall preparations by phenolwater extraction. The polysaccharide moiety consists of a D-mannose polymer accompanied by smaller amounts of 3- and 4-O-methyl-D-mannoses, D-galactose, D-glucose, L-fucose, D-glucosamine, mannosamine and 2-keto-3-deoxyoctonate. Aldoheptoses are lacking. The degraded polysaccharide is split from lipid A by acid hydrolysis (10% acetic acid, 100°C, 3 h) whereby 2-keto-3-deoxyoctonate is released in small amounts. Degraded polysaccharide forms only one major fraction by Sephadex G-50 gel-filtration. This fraction includes all the sugars mentioned above except L-fucose, which is released during the acetic acid degradation. Periodate studies and methylation analysis revealed that the poly-mannose chain consists of about 75% 1→3 linked and of 25% 1→4 linked D-mannose units. Lipid A of A. nidulans is phosphate-free. The main fatty acid, β-hydroxypalmitic acid, is exclusively amide-bound, presumably to the amino group of D-glucosamine. Other fatty acids, found as minor constituents, are β-hydroxymyristic, palmitic and stearic acids. Lipopolysaccharide of A. nidulans KM exhibits high anticomplementary activity in guineapig serum. It is about 800 times less toxic for adrenalectomized mice than endotoxin from Salmonella typhimurium. The isolated lipopolysaccharide reacts with rabbit antisera against living or heat-killed cells of A. nidulans in passive hemagglutination, when untreated or heated, but not when alkali-treated lipopolysaccharide is used for red blood cell sensibilization. It is concluded that lipopolysaccharide of A. nidulans KM is exposed on the surface of the cell.

Plant mucilages. XIII. Isolation and characterization of a mucous polysaccharide, "Lilium-S-glucomannan," from the bulbs of Lilium speciosum.

A mucous polysaccharide, named Lilium-J-glucomannan, has been isolated from the bulbs of Lilium japonicum THUNB. It was homogeneous on glass-fiber paper electrophoresis, by ultracentrifugal analysis, and on gel chromatography. The component sugars of it were D-mannose and D-glucose in the molar ratio of 5 : 2, and its molecular weight was measured to be 318000. The O-acetyl groups in the glucomannan were identified and determined as the content of 5.0%. They were located in positions 2, 3, and 6 of a part of D-mannose units. Methylation, partial acetolysis, and periodate oxidation studies suggested that the glucomannan is mainly composed of β-1→4 linked aldohexopyranose residues and it contains about seventeen aldohexose units per one non-reducing group on the average. D-Mannose units occupy non-reducing terminal positions and branching points linked through positions 2 or 3. A few D-glucose residues are also present as branching points linked through position 3. The comparison of the structure and the property of the glucomannan with those of the other five Lilium-glucomannans was described.

Genetic control of yeast mannan structure: mapping genes mnn2 and mnn4 in Saccharomyces cerevisiae.

Two mutations concerned with mannan biosynthesis in the yeast Saccharomyces cerevisiae have been mapping. The mnn2 mutation, which affects the addition to the polysaccharide backbone of the first side-chain D-mannose unit in alpha1-leads to2 linkage, was located on chromosome II linked to the centromere and the gall locus. The mn4 locus, which regulates the synthesis of mannosylphosphate groups on the mannan side chains, was placed on chromosome XI near trp3 and ural and a locus previously reported to regulate the ability of a S. diastaticus strain to bind alcian blue (Friis and Ottolenghi, 1970). The mnn4 mutant also fails to bind alcain blue, but the gene responsible for alcian blud binding in this strain segregates independently from the dye-binding locus of S. diastaticus, and therefore must be a different gene. A diploid heterozygous for mnn4 fails to bind dye, indicating dominance of this mutant genotype. The alcian blue dye binding locus dbll, reported to Friis and Ottolenghi (1970), is also dominant. Thus, there are at least two independent genes that control the formation of the mannosylphosphate units in the mannan side chains, and both have the property of dominance in the mutant form.

11] Methylated polysaccharide activators of fatty acid synthase from Mycobacterium phlei

This chapter discusses the methylated polysaccharide activators of fatty acid synthase from Mycobacterium phlei. Mycobacterium species contain in the cytosol two highly methylated polysaccharides, both of them are shown to activate the fatty acid synthase complex of Mycobacterium phlei in a manner leading to a reduction in the Km for acetyl-CoA. The most active substance is composed of 10 3-O-methyl-D-mannose units in α(1→ 4) linkage, forming a chain to which is attached at position 6 of one of the 3-O-methyhnannose units a side chain of two D-mannose units in α(1 → 2) linkage. It is called MMP for methylmannose polysaccharide. The other substance is a lipopolysaccharide containing 7 moles of D-glucose, 10 moles of 6-O-methyl-D-glucose and 1 mole of 3-O-methyl-D-glucose. The polymer is linked to D-glyceric acid and is acylated by acetyl, propionyl, isobutyryl, octanoyl, and succinyl groups. Four forms of the lipopolysaccharide can be isolated depending on the net negative charge, which reflects different contents of succinic acid. These are called MGLP-I, II, III, and IV for methylglucose lipopolysaccharide.

The Structure of the Capsular Polysaccharide of Klebsiella K-type 24

Methylation, periodate oxidation, and partial hydrolysis studies on the capsular polysaccharide, and on the carboxyl reduced polymer, of Klebsiella K24 show the structure to consist of a repeating unit[Formula: see text]The anomeric linkages were determined in isolated oligosaccharides by p.m.r. spectroscopy which also showed the presence in the polysaccharide of one O-acetyl group per seven or eight sugar residues. The O-acetyl is tentatively assigned to one of the D-mannose units.

Identification of linkages of a galactomannan isolated from seed of Caesalpinia pulcherima

A galactomannan, purified by repeated complex formation with copper acetate, was shown to be composed of D-mannose and D-galactose in a molar ratio of 3:1. From methylation experiments, the following partially methylated sugars were identified: 2,3,4,6-tetra- O-methyl- D-mannose, 2,3,4,6-tetra- O-methyl- D-galactose, 2,3,6,-tri- O-methyl- D-mannose, 2,4,6,-tri- O-methyl- D-mannose, 3,4,6-tri- O-methyl- D-mannose, and 2,3-di- O-methyl- D-mannose (molar ratio 1:27:60:4:2:32). Ordinary and sequential (Smith) periodate degradation indicated that the D-galactose units occupied single unit branch points linked (1→6); approximately one in every twenty D-mannose units was resistant to periodate attack; the occurrence of small and equimolar quantities of 2- O-β- D-mannopyranosyl- D-erythritol and O-β- D-mannopyranosyl-(1→3)- O-β- D-mannopyranosyl-(1→2)- D-erythritol indicates a low frequency of isolated (1→3) linkages and consecutive (1→3) linkages. A relatively simple structure may be assigned, based upon the experimental results.

Keto groups in cellulose and mannan oxidized by dinitrogen tetroxide

Mannan B and cellulose were oxidized with nitrogen tetroxide to transform the C-6 hydroxyls into carboxyl groups. In both products keto groups had formed on secondary carbons in side-reactions. After reduction of the oxidized polysaccharides with sodium borohydride- t 4, determination of the radioactivity indicated that the ratio of keto groups in oxycellulose to those in oxymannan was 2:1. Comparison of the count of radioactivity with that of diphenylmethanol- t 2 prepared from a similarly reduced sample of diphenyl ketone showed that the oxymannan contained one keto group for every 10 anhydro- d-mannose units.

CONSTITUTION OF AN EXTRACELLULAR ACIDIC GLUCOMANNAN FROM SERRATIA MARCESCENS

An acidic glucomannan isolated from culture filtrate of Serratia marcescens contained D-glucose and D-mannose (3.5:1), D-glucuronic acid (5.5%), and "bound" fatty acids (7.4%). The polysaccharide, on examination by moving boundary electrophoresis and by sedimentation in the ultracentrifuge, was homogeneous. Degree of polymerization was estimated to be ca. 20 by vapor pressure osmometry. Isolation of nigerose (3-O-α-D-glucopyranosyl-D-glucopyranose) established the glycosidic linkage between the D-glucose units as α (1 → 3). Methylation and periodate oxidation data showed that the D-glucose and D-mannose units were joined by (1 → 3) linkages in a straight chain structure terminated at the non-reducing end by a unit of D-glucuronic acid which was attached to C3 of a D-mannose residue. The reducing end of the molecule was terminated by a D-glucose unit. The fatty acids, consisting mainly of hydroxymyristic acid and myristic acid, were attached to the molecule mainly by ester linkages.

The location of 2-O-(β-D-glucopyranosyluronic acid)-D-mannose units in leiocarpan A

The location of 2-O-(β-<scp>D</scp>-glucopyranosyluronic acid)-<scp>D</scp>-mannose units in leiocarpan A

A NEW TYPE OF CARBOHYDRATE ORTHOESTER: SIDE REACTIONS OF THE KÖNIGS-KNORR SYNTHESIS

A trisaccharide orthoester (IV) is one of the products formed by decomposition of 2,3,4,6-tetra-O-acetyl α-D-mannopyranosyl bromide in the presence of silver oxide. This new type of carbohydrate derivative contains three units of D-mannose linked together through the functional groups of a molecule of acetoacetic acid. The carboxyl group of the latter takes part in the orthoester structure of IV, with one D-mannose unit closing a 1,2-orthoester ring, and another unit comprising the —OR group through its anomeric center; the keto group of the acetoacetate forms a 1,2-ketal structure with the third D-mannose unit. The n.m.r. spectra characteristics of the trisaccharide and some related compounds are described. The following sequence of reactions is consistent with formation of the trisaccharide: 1,2-trans-acetoxyl bromide [Formula: see text] 1,2-cyclic acetoxonium ion (IX)[Formula: see text] 1,2-cyclic ketene-acetal; ion IX and 2,3,4,6-tetra-O-acetyl D-mannose then added to the ketene derivative.Selective degradation of trisaccharide IV yields a novel type of ketal, i.e., 1,2,O-[1-(carbomethoxymethyl)-ethylidene]-β-D-mannose.

THE WATER-SOLUBLE POLYSACCHARIDES OF DERMATOPHYTES: I. A GALACTOMANNAN FROM TRICHOPHYTON GRANULOSUM

A galactomannan containing D-galactose (16%) and D-mannose (84%) has been isolated from the water-soluble polysaccharides of Trichophyton granulosum. Methylation studies showed that the structure was highly branched, the predominant structural feature being a chain of α-D-mannopyranose units joined together by an equal number of 1 → 2 and 1 → 6 linkages. About 22% of the D-mannose units were doubly substituted at positions 2,6; 2,4; and 2,3 to form branch points. The branches were terminated by D-mannopyranose and D-galactofuranose residues, the latter accounting for all of the D-galactose present in the polysaccharide.

PERIODATE-OXIDIZED PHOSPHOMANNAN Y-2448: STRUCTURAL SIGNIFICANCE OF ITS REACTION WITH ALKALI

The diesterphosphomannan produced by Hansenula holstii NRRL Y-2448 was shown by periodate-oxidation analysis to have 20% of its D-mannose units linked through carbon-1 only (or carbons-1 and -6), 33% through carbons-1 and -2, and 47% through carbons-1 and -3. Information regarding branching in this macromolecular polysaccharide derivative was not established. The periodate-oxidation product neutralized dilute alkali rapidly at pH 8.5–10.0, with elimination of about 30% of the phosphorus as inorganic orthophosphate. About 80%, inorganic orthophosphate was obtained by longer treatment at pH 10.1–10.5. These observations are shown to support the hypothesis that diesterorthophosphate groups (0.20 mole/mole mannose unit) are held between carbon-6 of units otherwise linked only through carbon-1, and carbon-1 of 60% of the carbon-2-linked units. After periodate oxidation, both phosphoester bonds may undergo β-aldehydo elimination by alkali; the unit having phosphate in hemiacetal linkage and a mannosidic attachment at carbon-2 is presented as the more reactive site. Periodate oxidation also produces sites for β-dealkoxylation by alkali.

CONSTITUTION OF A GLUCOMANNAN FROM THE SAPWOOD OF SUGAR MAPLE (ACER SACCHARUM)

A glucomannan containing D-mannose and D-glucose in a molar ratio of 7:3 has been isolated from sugar maple sapwood. Electrophorectic examination in acetate and borate buffers showed that the polysaccharide was essentially homogeneous. Methylation and hydrolysis of the glucomannan yielded the following O-methyl ethers:2,3,4,6-tetra-O-methyl-D-glucose (1.1%),2,3,4,6-tetra-O-methyl-D-mannose (2.3%),2,3,6-tri-O-methyl-D-glucose (23.4%),2,3,6-tri-O-methyl-D-mannose (65.9%),2,3-di-O-methyl-D-glucose (2.6%),2,3-di-O-methyl-D-mannose (2.7%).These data in conjunction with periodate oxidation results and estimates of the degree of polymerization indicated that the glucomannan was composed of 25–27 hexose units linked β(1 → 4) in a linear structure. The chains were terminated predominately by D-mannose units; traces of D-xylose were present in terminal positions only and appeared to be part of the glucomannan molecule.

AN EXTRACELLULAR POLYSACCHARIDE FROM GIBBERELLA FUJIKUROI (FUSARIUM MONILIFORME)

Growth of Gibberellafujikuroi (Fusariummoniliforme) on a glucose medium produced au extracellular polysaccharide containing D-glucose, D-mannose, D-galactose, D-glucuronic acid (molar ratio 1.0:1.1:1.3:0.6), and possibly D-mannuronic acid. Protein was retained tenaciously by the polysaccharide and several deproteinization methods reduced the nitrogen content only slightly. Methylation studies showed that the polysaccharide was highly branched with D-glucose and D-mannose forming the non-reducing ends in the molecule. Most of the D-mannose and D-galactose units were joined by 1 → 2 and 1 → 6 linkages with some branching also at C2and C6positions; D-galactose occurred exclusively in the furanose form. D-Glucose units were joined by 1 → 2 and 1 → 3 linkages. The D-glucuronic acid residues were mainly non-terminal and were attached to both D-galactose and D-mannose units. Periodate oxidation studies supported the foregoing conclusions.

BIOCHEMISTRY OF THE USTILAGINALES: XIII. OBSERVATIONS ON THE STRUCTURE AND BIOSYNTHESIS OF 4-O-β-D-MANNOPYRANOSYL-D-ERYTHRITOL

D-Glucose-6-C14, D-erythrose-4-C14, and D-erythritol-4-C14were used as precursors in the biosynthesis of 4-O-β-D-mannopyranosyl erythritol by Ustilago sp. PRL 627 and the patterns of C14labelling in the products determined. The disaccharide appears to be formed by direct transformation of D-glucose to a D-mannose unit which is then attached to C-4 of D-erythritol.* The labelling of the D-erythritol † portion shows that 80% of C-4 of the molecule is derived from the 6-position of D-glucose.

BIOCHEMISTRY OF THE USTILAGINALES: XIII. OBSERVATIONS ON THE STRUCTURE AND BIOSYNTHESIS OF 4-O-β-D-MANNOPYRANOSYL-D-ERYTHRITOL

D-Glucose-6-C14, D-erythrose-4-C14, and D-erythritol-4-C14were used as precursors in the biosynthesis of 4-O-β-D-mannopyranosyl erythritol by Ustilago sp. PRL 627 and the patterns of C14labelling in the products determined. The disaccharide appears to be formed by direct transformation of D-glucose to a D-mannose unit which is then attached to C-4 of D-erythritol.* The labelling of the D-erythritol † portion shows that 80% of C-4 of the molecule is derived from the 6-position of D-glucose.