Further characterization of carbohydrate-containing fractions from Trypanosoma mega

Epimastigotes of Trypanosoma mega were submitted to phenol extraction after lipid extraction, providing an extract whose carbohydrate portion (30%) contained fucose, ribose, xylose, mannose, galactose, and glucose. The purified fraction recovered in the void volume of Bio Gel P-150 gave on SDS-PAGE a band of Mr approximately equal to 55,000 positive for protein and carbohydrate and a diffuse band strongly positive for carbohydrate and lipids (Mr approximately equal to 22,000). The structural analysis of the carbohydrate moiety of this fraction by GLC-MS indicated the presence of nonreducing end groups of fucopyranose, mannopyranose, and galactopyranose, 3-O- and 4-O-substituted and 2,3- and 2,4-di-O-substituted galactopyranosyl units. Extraction of this fraction with chloroform/methanol/water provided a soluble fraction that on SDS-PAGE gave rise to a carbohydrate and lipid-positive band (Mr approximately equal to 22,000). This fraction contained fucose, mannose, and galactose (1:1:1). As main branch points, 2,3-di-O-substituted galactopyranosyl units were present according to methylation data. Similar proportions of fucopyranosyl, mannopyranosyl, galactopyranosyl end units were present. The presence of lipids in this fraction was confirmed by methanolysis following isolation and characterization of the corresponding fatty acid methyl esters. Palmitic acid (16:0) and an 18:1 fatty acid were the predominant fatty acids.     

Comparative study of the asparagine-linked oligosaccharide structures of normal and acute-phase rat plasma thiostatin

Rat plasma thiostatin is a 68 kDa glycoprotein with kinin donor and cysteine proteinase inhibitor properties. Thiostatin is an acute-phase plasma protein (APPP) with dramatically elevated plasma levels in response to inflammatory stimuli. APPPs have been shown to possess altered glycan structures in inflammation. This study compares the carbohydrate structure of normal thiostatin with that expressed during the acute-phase response. Thiostatin from both normal and acute-phase plasma was purified by carboxymethyl-papain Sepharose 4B affinity chromatography. Sugar composition analysis by gas chromatography and the Warren method yielded similar mean values for both proteins on a mole sugar per mole protein basis (normal/acute phase): fucose, 2.4/1.7; mannose, 7.5/8.0; galactose, 11.2/10.6; and sialic acid, 14.2/13.0. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot identified a homogeneous 68–70 kDa molecular species for normal and acute-phase thiostatin. Inter-sugar linkage analysis was carried out for permethylated oligosaccharides released by hydrazinolysis. Gas chromatography yielded the following partially methylated alditol acetates relative to 1.0 mole of 1,3,6-tri-O-linked mannose (mean normal/mean acute phase): galactose: 1,3-di-O-, 1.44/1.01; 1,6-di-O-, 1.02/0.68; mannose: 1,2-di-O-, 1.64/1.42; 1,2,4-tri-O-, 0.24/0.13; 1,3,6-tri-O-, 1.0/1.0; 2-deoxy-2-N-methylacetamidoglucose: 1,4-di-O-, 1.42/1.12. These analytical studies indicated that corresponding carbohydrate structures are present in normal and acute-phase thiostatin. Crossed affinoimmunoelectrophoresis (CAIE) further confirmed the structural similarity between the glycan moieties.     

Isolation and characterization of glycoprotein lectins from the bark of three species of elder,Sambucus ebulus,S. nigra and S. racemosa

Lectins have been isolated from the bark of three members of the family Caprifoliaceae, Sambucus nigra (elder), S. racemosa (red-berried elder) and S. ebulus (dwarf elder), by affinity chromatography on fetuin-agarose, ion-exchange and gel-filtration chromatography. They are all glycoproteins of M _r 140 000 made up of at least four subunits. The lectin have similar but not identical amino-acid compositions and the carbohydrate content varies between 12% and 19% (w/w), the main sugars being (N-acetyl)glucosamine, mannose, fucose and xylose. Inhibition studies of hemagglutination with various mono- and oligosaccharides have shown that N-acetylgalactosamine and galactose together with galactose-containing oligosaccharides are the most effective inhibitors. There are some differences in specificity, in particular S. ebulus agglutinin is inhibited to the same degree by galactosamine, N-acetylgalactosamine and by galactose.Abbreviations PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SEA S. ebulus agglutinin - SNA S. nigra agglutinin - SRA S. racemosa agglutinin     

Carbohydrate content and monosaccharide composition of Rapana thomasiana grosse (Gastropoda) hemocyanin and its structural subunits. Comparison with gastropodan hemocyanins

The hemocyanin of Rapana thomasiana grosse (marine snail, gastropod) is a glycoprotein with a carbohydrate content of 8.9% (w/w) and monosaccharide constituents xylose, fucose, 3-OOmethylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. The two structural subunits of this oxygen carrier, RHSS1 and RHSS2, are unevenly glycosylated. On subtracting the carbohydrate contribution from the M_r values of 250 and 450 kDa attributed to the two subunits, values of 2.18 × 10⁵ daltons and 4.30 × 10⁵ daltons were calculated for the polypeptide part of the “light” and “heavy” subunits, respectively. Comparison of the monosaccharide compositions of gastropodan hemocyanins revealed qualitative similarities, as well as relationships between the quantities, of the individual monosaccharides: Man 3MeGal > GlcNAc GalNAc and Fuc Xyl     

Hemicellulosic polymers from the leaves of coffea arabica

Polysaccharide fractions from leaves of Coffea arabica var. Mundo Novo were obtained by extraction with 24% potassium hydroxide solution and were found to contain rhamnose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid and 4-O-methylglucuronic acid in different proportions. 2-Acetamido-2-deoxygalactose was detected in all fractions. The structures of the carbohydrate portions were analysed by methylation and Smith degradation. A high amount of 2,3,5-tri-O-methylarabinose and 2,3,4-tri-O-methylxylose units, which are related through end groups, suggested a large degree of branching in the polysaccharide fractions. Glucose was present mainly as (1 → 4)-linked residues, as indicated by the presence of 2,3,6-tri-O-methylglucitol in the hydrolysates of the methylated fractions. A greater proportion of monomethylxylitol in acidic fraction B-IV indicated that it was more branched than the others. The glucose and galactose residues are 4,6- and 3,4-di-O-substituted, respectively. Three successive Smith degradations gave mainly glycerol with some erythritol and threitol. In the linkage of carbohydrate—protein, the presence of O-glycosyl linkages between arabinose and hydroxyproline was indicated. A phenolic compound was detected in all polysaccharide fractions from leaves of the coffee tree and is probably derived from chlorogenic acid.     

Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous culture on glucose and fructose

The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l⁻¹ exopolysaccharides with glucose and 30 mg l⁻¹ with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (M _r) of 1.7 × 10⁶ and 4 × 10⁴ in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M _r of 4 × 10⁴. The high-M _r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-M _r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-M _r fractions appeared to be dependent on the carbohydrate source, whereas the low-M _r fractions were produced more continuously. Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997     

A New Lectin from Meadow Saffron (Colchicum automnale)

A lectin has been isolated from tubers of the meadow saffron (Colchicum autumnale). It is an octameric protein (M_r 100,000) composed of 4A- and 4B-subunits of M_r 15,000 and 10,000, respectively. It is a glycoprotein with 4.4% carbohydrate, the main sugars are (N-acetyl-) glucosamine, mannose, fucose, and xylose. Although the Colchicum autumnale agglutinin (CAA) agglutinates human red blood cells, it has a much higher activity with rabbit erythrocytes. With respect to its carbohydrate-binding specificity CAA behaves rather unusually as it is inhibited by lactose, galactose, N-acetylgalactosamine and related sugars when assayed with human red blood cells but not in assays with rabbit erythrocytes.     

Les polysaccharides des graines de quelques liliacees et iridacees

The alkali-soluble polysaccharides have been surveyed in the seeds of 7 species of the Liliaceae and 2 species of the Iridaceae. All appear to contain galactoglucomannans and/or glucomannans. The structure of the water-soluble galactoglucomannan from the endosperm of Asparagus officinalis has been studied in detail. It contains residues of glucose, mannose and galactose in the ratio 43:49:7. Hydrolysis of the fully methylated polysaccharide released 2,3,4,6-tetra-O-methyl-d-hexoses (mannose and glucose), 2,3,4,6-tetra-O-methyl-d-galactose, 2,3,6-tri-O-methyl-d-mannose, 2,3,6-tri-O-methyl-d-glucose, 2,3-di-O-methyl-d-mannose and 2,3-di-O-methyl-d-glucose in the molar proportions of 1:4.5:50:41:2:1·5. The following oligosaccharides were identified on partial hydrolysis of the galactoglucomannan: mannobiose, mannotriose, mannotetraose, cellobiose, glucopyranosylmannose, mannopyranosylglucose and a trisaccharide composed of two mannosyl residues and one glucosyl residue. The galactoglucomannan consists of a linear chain of β(1 → 4)-Iinked d-mannosyl and d-glucosyl residues, to which are attached single-unit galactosyl side chains. The galactose residues are linked 1 → 6, probably α. The terminal, non-reducing residues of the main chain may be either glucosyl or mannosyl units but the former predominate.     

Chemical structures of a galactose-rich glycoprotein of Leishmania tarentolae

1. Aqueous phenol treatment of water extracted disrupted cells of Leishmania tarentolae (LV-414) provided a glycoprotein mixture which was purified by gel filtration chromatography, and Concanavalin A-Sepharose column. 2. The bound fraction on Concanavalin A-Sepharose column (protein 74%, and carbohydrate, 26%) had [alpha]D + 9 degrees and contained mannose (18%), galactose (60%), and glucose (22%), and some of the galactose residues were resistant to periodate oxidation. 3. Treatment of the phenol extract with hot aqueous NaBH4 containing NaOH gave a preparation having mannose (12%), galactose (82%), and glucose (6%). 4. Methylation analysis showed the presence of a mainly linear structure with non-reducing end-units of mannopyranose (6%), 3-O-substituted galactopyranosyl (64%), 2-O- (11%), and 6-O- (5%) substituted mannopyranosyl, and 4-O- (9%), and 4,6-di-O- (3%) substituted glycopyranosyl units. 5. The specific rotation of the preparation, +20 degrees, indicated beta-linked galactopyranosyl units.     

A new lectin from tulip (Tulipa) bulbs

A lectin was isolated from tulip (Tulipa) bulbs by affinity chromatography on fetuin-agarose and partially characterized. The tulip lectin is a tetrameric protein composed of four identical subunits of M_r 28 000, which are not held together by disulphide bonds. It is not glycosylated and has an amino-acid composition typified by a high content of asparagine-aspartic acid, leucine, glycine and serine. Tulip lectin agglutinates human red blood cells, but has a much higher specific activity with rabbit erythrocytes. In hapten-inhibition assays with the latter type of red blood cell the lectin exhibits a complex specificity, whereas its agglutination with human erythrocytes is readily inhibited by N-acetylgalactosamine, lactose, fucose and galactose.Abbreviations DEAE diethylaminoethyl - PBS phosphate-buffered saline - TL Tulipa lectin - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

A high molecular weight diapause-associated protein from the stem-borer Busseola fusca: Purification and properties

The hemolymph of diapausing larvae of the stem borer, Busseola fusca Fuller (Lepidoptera: Noctuidae), contains an electrophoretically distinct protein band on nondenaturing polyacrylamide gels. The protein, called the Busseola diapause protein (BDP), was purified by a combination of density gradient ultracentrifugation, gel permeation, and affinity chromatography. It is a high molecular weight protein (M_r ～5 × 10⁵; pl = 6.1) that is composed of two subunits, I (M_r ～88,000 ± 4,000) and II (M_r ～79,000 ± 1,000), which are not linked by disulfide bridges. The protein contains both lipids (2%) as well as covalently bound carbohydrates (1%). The inability to stain the fluorescein isothiocyanate-conjugated concanavalin A (FITC-Con A) suggests that the carbohydrate moiety of BDP is not of the high mannose type. Amino acid analysis showed a high tyrosine plus phenylalanine content (16 mol%). Labeling studies using [³⁵S]-methionine showed that de novo synthesis by the fat body tissue occurs only in diapausing larval insects. It is proposed that the BDP could serve a storage function by providing the amino acids needed for the synthesis of pupal and adult structures.     

Binding-site Analysis of the Ether Linkages between Lignin and Hemicelluloses in Lignin–Carbohydrate Complexes by DDQ-Oxidation

Lignin-carbohydrate complexes (LCC; nor-C-1-M, com-C-1-A) isolated from normal and compression woods of Pinus densiflora were hydrolyzed with two types of cellulase preparations, and the hydrolyzates formed were fractionated by adsorption chromatography on polyvinyl gel into water-soluble materials and LCC fragments. To elucidate the binding sites between the lignin and carbohydrate, the cellulase-degraded LCC fragments were subjected to acetylation, and then oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which was confirmed to oxidatively cleave the benzyl ether linkages between the lignin and carbohydrate. The DDQ-oxidized fraction was then methylated by the method of Prehm, hydrolyzed, reduced and acetylated. A GC-MS analysis of the methylated sugar revealed that alditol acetates from 6-O-methyl mannose, 6-O-methyl galactose, 6-O-methyl glucose and a small amount of their 2-O- or 3-O-methyl isomers existed in both methylated fractions. 2-O-Methyl xylose and 3-O-methyl xylose were also identified in the fraction from the acidic LCC (com-C-1-A). These results led to the conclusion that acetylglucomannan and β-1,4-galactan were preferably bound to the lignin at C-6 position of the hexoses, and that arabinoglucuronoxylan did likewise at the C-2 and C-3 positions of xylose units.     

Chemical and immunological properties of lipophorins from seven insect orders

The major insect hemolymph lipoprotein, lipophorin, was isolated from adults of eight insect species representing seven insect orders. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to compare their respective apoprotein components. In all species examined lipophorin was composed of at least two apoproteins, apolipophorin I (M_r ～ 250,000) and apolipophorin II (M_r ～ 78,000), and two species had a third apoprotein, apolipophorin III (M_r ～ 17,000). The density of each isolated lipophorin was determined from the refractive index of KBr following density gradient centrifugation. Immunoblotting with anti-larval Manduca sexta apolipophorin I and II of the apoproteins separated by SDS-PACE indicated cross reactivity between anti-M sexta apoLp-ll and apoLp-ll in all species tested. Anti-M sexta apoLp-l exhibited no cross reactivity for any species tested. Fluorescent lectin staining of the apoproteins separated on SDS-PAGE gels revealed the presence of covalently bound carbohydrate residues.     

EVIDENCE THAT THE MAJOR PROTEIN IN RAT SCIATIC NERVE MYELIN IS A GLYCOPROTEIN

Evidence is presented that the major protein of rat sciatic nerve myelin is a glycoprotein. When myelin proteins were separated by polyacrylamide gel electrophoresis, the major band which was stained with amido black–Coomassie blue was also stained with periodic acid-Schiff reagents for carbohydrate. Radioactive labelling of myelin in vivo with [³H]leucine and [¹⁴C]fucose, followed by electrophoresis of the proteins, indicated that with both isotopes the major labelled peak corresponded to the major stained band. In addition, a second smaller peak of [¹⁴C]fucose migrated ahead of the major peak. Delipidated myelin contained galactose, mannose, fucose and sialic acid.     

A cellulosic exopolysaccharide produced from sugarcane molasses by a Zoogloea sp.

An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated from an agro-industrial environment in the north-eastern region of Brazil. The extracellular polysaccharide produced from sugarcane molasses was hydrolysed with trifluoroacetic acid (mild and strong conditions) giving 88% of soluble material. The main monosaccharides present in the soluble fraction were glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0.1%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-methylhexitol (74.7%) and 2,3,-di-O-methylhexitol (17.7%). Enzyme hydrolysis of the polysaccharide with a cellulase confirmed the presence of (1→4)-β- -glucopyranosyl residues.     

Molecular characteristics of lipophorin,the juvenile hormone-binding protein in the hemolymph of the Colorado potato beetle

Lipophorin, the protein that specifically binds juvenile hormone in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, is a high-density lipoprotein of M_r ～ 574,000. Lipophorin contains 43% lipid and is composed of two apoproteins: apolipophorin I (M_r ～ 251,000) and apolipophorin II (M_r ～ 78,000). Both apoproteins contain mannose residues. Carotenoids make up a substantial part of the lipid fraction. Lipophorin constitutes about 25% of the total hemolymph proteins. Its concentration in the hemolymph (26 μM in 4-day-old long-day and 40 μM in 4-day-old short-day beetles) changes with different physiological conditions concomitant with changes in total protein content. Lipophorin specifically binds 10R-juvenile hormone III with high affinity. The dissociation constant for 10R-juvenile hormone III is 12 ± 2 nM. One lipophorin molecule contains one specific juvenile hormone-binding site. The concentration of binding sites therefore equals that of lipophorin in hemolymph.     

Studies on the structure of a phosphoglycoprotein from the parasitic protozoan Trypanosoma cruzi.

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.     

Protein-bound oligosaccharides of Semliki Forest virus

Semliki Forest virus was grown in BHK cells and labeled in vivo with radio-active monosaccharides. promnase digenst of the virus chromatographer on Bio-Gel P 6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson J. and Clamp J.R. (1971) Biochem. J. 123, 739–745) The former was labeled with [³H]fucose, [³H]galactose, [³H]mannose and [¹⁴C]glucosamine, the latter only with [³H]mannose and [¹⁴C]glucosamine. The three envelope glycoproteins E₁, E₂ and E₃ were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E₁ and E₃ revealed glycopeptides of A-type. E₂ revealed glycopeptides of B-type. E₂ yielded additionally a glycopeptide (M_r3100) which was heavily labeled from [³H]galactose, but only marginally from [¹⁴C]glucosamine, [³H]fucose and [³H]mannose. Wether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E₁ and 4000 in E₃; the B-type unit of E₂ had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggast that E₁ and E₃ contain on the average one A-type unit; E₂ probably contains one 3100 dalton unit plus one or two B-type units.     

Isolation and Characterization of a Neutral Glycosphingolipid from the Epimastigote Form of Trypanosoma mega1

ABSTRACT. A glycosphingolipid fraction from Trypanosoma mega was isolated after acetylation and was further purified on a silicic acid column. Final purification was by preparative thin-layer chromatography. The carbohydrate components of the glycolipid were fucose and galactose in approximately equimolar amounts. The neutral glycolipid of T. mega has a sphingosine base composition that consists of sphingosine and traces of dihydrosphingosine. Fatty acids forming amide groups with the sphingosine bases were analyzed by gas-liquid chromatography-mass spectrometry and are a mixture of normal and α-hydroxy fatty acids. Normal C_16:0, C_18:0, and 2-hydroxy C_18:0 are the predominant fatty acids.     

EXTRACELLULAR POLYSACCHARIDES FROM ANKISTRODESMUS DENSUS (CHLOROPHYCEAE)

The dissolved extracellular compounds produced by the Chlorococcalean Ankistrodesmus densus Kors. were investigated for their molecular masses and chemical composition. Gel permeation chromatography showed apparent relative molecular masses of 2 × 10⁶ and 10⁴, respectively, for two distinct fractions, termed A and B. The higher molecular weight fraction (A) contained mainly fucose and 3- O -methylgalactose, whereas galactose, glucose, mannose, and rhamnose were present in smaller amounts. Methylation analyses showed that the main structural features are fucose as a highly branched part of polymer A with substitutions in both position 2 and position 4, the substitutions on one of those positions being primarily terminal 3- O -methylgalactose and the other one involved as the linkage of the main chain of the polymer. Because of the presence of both fucose and 3- O -methylgalactose, this polymer is highly hydrophobic. In fraction B, mannose represented more than 60% of the carbohydrate material present, whereas the remaining part contained rhamnose, fucose, xylose, and glucose in almost equal amounts. 3- O -methylgalactose and galactose were present as minor elements. Fraction B is basically a mannose-containing polymer in which the mannose units are either 1→4 or 1→2 linked. Traces of glucuronic acid and protein were present in both fractions; neither sulfate nor phosphate was detected.