Hydroxyproline Glycosides in Secretory Arabinogalactan-Protein of Phaseolus vulgaris L

The carbohydrate moiety of secretory arabinogalactan protein in bean seedlings (Phaseolus vulgaris L. cv. Prélude) is attached to the peptide backbone through hydroxyproline, serine, and threonine. Hydroxyproline-linked side chains, consisting of arabinose, galactose, glucose, and rhamnose, comprise the major part of the sugar residues. These hydroxyproline glycosides differ from those in non-extractable cell wall protein but show similarities with those in wall protein of the alga Chlamydomonas.     

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.     

Enzymic analysis of cell wall structure in apple fruit cortical tissue

Galactanase from Phytophthora infestans and an arabinosidase isoenzyme from Sclerotinia fructigena attacked the cortical cell walls of apple fruits liberating galactose and arabinose residues, respectively. Other arabinosidase isoenzymes from S. fructigena attacked cell walls very slowly. A S. fructigena polygalacturonase isoenzyme liberated half of the uronic acid residues with few associated neutral residues, while a second polygalacturonase isoenzyme released more uronic acid with a substantial proportion of arabinose and galactose and lesser amounts of xylose, rhamnose and glucose; reaction products of this enzyme could be further degraded by the first isoenzyme to give high MW fragments, rich in arabinose with most of the xylose, rhamnose and glucose, and low MW fragments rich in galactose and uronic acid. Endoglucanase from Trichoderma viride released a small proportion of the glucose residues from cell walls together with uronic acid, arabinose, xylose and galactose; more extensive degradation occurred if walls were pre-treated with the second polygalacturonase isoenzyme. Endoglucanase reaction products were separated into a high MW fraction, rich in arabinose, and lower MW fractions rich in galactose and glucose residues. The high MW polygalacturonase and endoglucanase products could be degraded with an arabinosidase isoenzyme to release about 75% of their arabinose. Cell walls from ripe fruit showed similar susceptibility to arabinosidase and galactanase to those from unripe apples. Cell walls from fruit, ripened detached from the tree were more susceptible to degradation by polygalacturonase than walls from unripe fruit or fruit ripened on the tree. Endoglucanase released less carbohydrate from ripe fruit cell walls than from unripe fruit cell walls.     

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α-l-arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α-l-arabinofuranosidase did not affect the allergenic activity of the extract.     

Partial structure of a polysaccharide from leaves of Welwitschia mirabilis

Gum-tears from the leaves of Welwitschia mirabilis contain a polysaccharide composed of arabinose, galactose and glucuronic acid as main constituents with xylose, fucose and rhamnose in smaller quantities. Periodate oxidation and permethylation studies indicated that the gum could consist of a framework of glucuronic acid residues linked 1 → 4 and galactose residues linked 1 → 6 and of short chains of arabinose, xylose, fucose and rhamnose linked 1 → 3 to both residues. All rhamnose and fucose and part of arabinose were found as non-reducing terminal units.     

The purification and properties of the lectin from potato tubers, a hydroxyproline-containing glycoprotein

1. Potato lectin has been purified and shown to be a glycoprotein containing about 50% of carbohydrate. Most of the sugar residues (92%) are arabinose; small amounts of galactose, glucose and glucosamine are also present. 2. The most abundant amino acid is hydroxyproline (16% of the residues), 11.5% of the residues are half-cystine and phenylalanine is absent. The lectin also contains about one residue/molecule of a basic amino acid, not usually found in proteins, which has been tentatively identified as ornithine. There is indirect evidence that the components of the glycoprotein are linked through hydroxyproline and arabinose. 3. By gel filtration in 6m-guanidine-HCl on Sepharose 4B, it was found that both the native glycoprotein and its S-carboxymethylated derivative had subunit molecular weights of 46000 (+/-5000). In a non-denaturing solution, two of these units appear to be associated. 4. The lectin is specifically inhibited in its agglutination reaction by oligosaccharides that contain N-acetylglucosamine. Its specificity is similar to, but not identical with, that of wheat-germ agglutinin.     

Structural studies of water-soluble glycoproteins from Cannabis sativa L

Two carbohydrate-protein fractions, isolated from Cannabis sativa L. by extraction with water and chromatography on DEAE-cellulose, contained arabinose, galactose, glucose, mannose, galacturonic acid, 2-acetamido-2-deoxyglucose, and 2-acetamido-2-deoxygalactose. The structure of the carbohydrate moieties was investigated by methylation analysis and Smith degradation. A high percentage of end-groups indicates a large degree of branching, glucose and galactose being the main branch-points, linked at C-3 and C-6. The hexoses are also present as unbranched residues in the chain, largely as (1→3)- and (1→4)-linked units and as end-groups. Arabinofuranosyl units constitute the main part of the non-reducing end-groups, and are also present as part of the chain. The polysaccharide chains are probably linked to protein through the hydroxyl group of hydroxyproline.     

Polysaccharides and glycoproteins of apple fruit cell walls

A proportion of the polysaccharides and glycoproteins of apple fruit cell walls can be readily extracted in neutral buffer at or below 20°. Removal of more material was not achieved with a wide range of dissociative aqueous reagents or non-aqueous solvents. Thus traditional degradative extractants were used to obtain soluble components for further characterization. Polysaccharides and glycoproteins were separated and purified by chromatography on DEAE-cellulose columns and by gel filtration. Purified components were hydrolysed and analysed for neutral sugar and uronic acid content and for their amino acid and hydroxyproline content. The possibility of linkages existing in the cell wall between polyuronide and glycoproteins containing hydroxyproline, arabinose and galactose residues is discussed. Because of aggregation between these components, which occurs after extraction, the presence of such linkages in vivo is difficult to establish. Other cell wall glycoproteins containing xylose and glucose residues are thought to have a possible role in stabilizing hemicellulose structure.     

Some properties of the lectin from Datura stramonium (thorn-apple) and the nature of its glycoprotein linkages

The lectin from Datura stramonium (thorn-apple; Solanaceae) has been purified by affinity chromatography and shown to be a glycoprotein containing about 40% (w/w) of carbohydrate. The most abundant amino acids are hydroxyproline, cystine, glycine and serine. Results obtained by gel filtration in 6m-guanidinium chloride on Sepharose 4B suggest that it has a subunit mol.wt. of about 30000 and that it probably associates into dimers. The lectin is inhibited specifically by chitin oligosaccharides and bacterial-cell-wall oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean (Glycine max) lectin and fetuin are also strong inhibitors of Datura lectin, indicating that it interacts with internal N-acetylglucosamine residues. Its specificity is similar to, but not identical with, that of potato (Solanum tuberosum) lectin. After prolonged proteolytic digestion of reduced and S-carboxymethylated or S-aminoethylated derivatives of the lectin, glycopeptides of mol.wt. of about 18000 were isolated. The glycopeptides contained all the carbohydrate and hydroxyproline of the original glycoprotein, and lesser amounts of serine, S-carboxymethylcysteine and other amino acids. The arabinose residues of the glycoprotein are present as β-l-arabinofuranosides linked to the polypeptide chain through the hydroxyproline residues, and can be removed by mild acid treatment; the ratio of arabinose to hydroxyproline is 3.4:1. Some of the serine residues of the polypeptide chain are substituted with one or two α-galactopyranoside residues, most of which can be removed by the action of α-galactosidase. The galactose residues are more easily removed from the acid-treated glycopeptide (from which arabinose has been removed) than from the complete glycopeptide, indicating a steric hindrance of the galactosidase action by the adjacent chains of arabinosides. There is a slow release of galactose residues by a process of β-elimination in 0.5m-NaOH (pH13.7) from the complete glycopeptide, and a fairly rapid release of galactose by this process from the acid-treated glycopeptide, which lacks arabinose. This is probably due to the inhibitory effect of the negative charge on the adjacent arabinofuranoside residues. The similarities and differences between the lectins from Datura and potato are discussed, as are their structural resemblance to glycopeptides that have been isolated from plant cell walls.     

Properties of potato lectin and the nature of its glycoprotein linkages

1. Potato lectin is a glycoprotein that contains about 47% (by weight) l-arabinose, 3% d-galactose and 11% hydroxyproline. It has a monomeric molecular weight of about 50000 and probably exists as a monomer-dimer system in aqueous solution, with the monomer predominating. It has a very high viscosity, which would indicate either that the molecule is very expanded or that it is an elongated ellipsoid. 2. After prolonged proteolytic digestion of a reduced and carboxymethylated derivative of the lectin, a glycopeptide was isolated (of mol.wt. 32000-34000) that included all the carbohydrate and hydroxyproline of the original glycoprotein but less than 30% of the total original amino acid residues. 3. The arabinose of the glycoprotein is present exclusively as the beta-arabinofuranoside and this includes those residues that are directly linked to the hydroxyproline residues of the polypeptide chain. All the arabinose of the glycoprotein is linked to the polypeptide chain through the hydroxyproline residues; the ratio of arabinose to hydroxyproline is 3.4:1. Although alpha-arabinofuranosides are known to be present in arabinans and arabinogalactans, the natural occurrence of beta-arabinofuranosides has not previously been reported. 4. Nine or ten serine residues of the polypeptide chain are substituted with single alpha-galactopyranoside residues that can be removed by the action of alpha-galactosidase from coffee beans but not by a beta-galactosidase. This is the first report of an alpha-galactoside linkage to serine. The effect of alpha-galactosidase is much greater on a glycopeptide from which the arabinose has been already removed, which indicates a steric hindrance of the galactosidase action by adjacent chains of arabinosides. 5. In 0.5m-NaOH (pH13.7), galactose residues were removed from the serine residues of the glycopeptide by a process of beta-elimination. This reaction took place very slowly in the intact glycopeptide but much more rapidly when the arabinofuranoside residues had been removed. This inhibitory effect of the arabinofuranoside residues on the beta-elimination reaction is likely to be due to a negative charge on the hydroxy groups of the adjacent arabinofuranoside residues, which would be ionized at this high pH value. 6. It is suggested that potato lectin may be representative of a class of soluble plant glycoproteins that would include precursors of the cell-wall glycoprotein extensin. If this is the case, extensin should also contain beta-l-arabinofuranosides linked to hydroxyproline and alpha-d-galactopyranosides linked to serine residues of the polypeptide chain.     

Studies of the polysaccharides from sunflower heads

Extraction of sunflower heads with ammonium oxalate afforded water-soluble pectin material and water-insoluble glycoprotein material, the carbohydrate portion of which consisted of galacturonic acid and xylose residues; the pectin material defied fractionation with cetylpyridinium chloride. Extraction with hydrochloric acid (pH 1.5) afforded water-soluble and water-insoluble polysaccharide materials. The former, when fractionated with cetylpyridinium chloride, gave a glycoprotein, the carbohydrate moiety of which was composed of galacturonic acid, galactose (major), glucose, arabinose, and xylose, and also a rhamnan. The latter was a glycoprotein, the carbohydrate portion of which consisted of galactose (major), glucose, xylose, and rhamnose residues. Extraction of the sunflower heads with water also gave glycoprotein material, which was fractionated by paper electrophoresis into a glyco-protein, the carbohydrate moiety ofwhich was composed of galacturonic acid (minor), galactose, glucose, xylose, arabinose, and rhamnose (major) residues, and a heteropolysaccharide composed of galactose (major), glucose, xylose, and arabinose residues.     

Analytical and structural features of the gum exudate from Combretum hartmannianum schweinf.

The gum exudate from Combretum hartmannianum is water-soluble, forms very viscous solutions, and contains galactose (22%), arabinose (43%), mannose (10%), xylose (6%), rhamnose (4%), glucuronic acid (6%), 4-O-methylglucuronic acid (2%), and galacturonic acid (7%). The acidic components produced on hydrolysis of the gum were 6-O-(β-D-glucopyranosyluronic acid)-D-galactose, and two saccharides that had the same chromatographic mobility, and contained mannose and galacturonic acid, and galactose and 4-O-methylglucuronic acid, respectively. Methylation and methanolysis of the gum indicated the presence of terminal uronic acid, rhamnose, xylose, galactose, arabinofuranose, and arabinopyranose. Controlled, acid hydrolysis indicated the presence of (1→3)-linked arabinopyranose side-chains and (1→6)-linked galactose residues. C. hartmannianum gum, when subjected to two Smith-degradations, yielded Polysaccharides I and II, both of which contained galactose, arabinose, and mannose. Insufficient crude gum was available for a complete structural study, but the molecule was shown to contain long, sparsely branched chains of (1→6)-linked galactose residues, to which are attached (1→3)-linked arabinose and (1→3)-linked mannose side-chains.     

Isolation of portulaca oleracea (regla) mucilage and identification of its structure

Portulaca oleracea leaves were found to contain 0.42% of a mucilage mixture. The mucilage was fractionated into an acidic and a neutral fraction. The acidic fraction consists of galacturonic acid residues joined by α-(1→4)-linkages; 60% of these residues are present as the calcium salt, and esterified galacturonic acid residues are absent. The neutral fraction is composed of 41% of arabinose and 43% of galactose residues, besides traces of rhamnose residues.     

Extraction and purification of pectins from Alcohol Insoluble Solids from ripe and unripe apples

Pectins are extracted from Alcohol Insoluble Solids of ripe and unripe apples and fractionated by ion exchange chromatography and gelfiltration. In the extracts mainly pectins with neutral sugar contents of 0·15, 0·24 and 0·53 mol neutral sugar residues/mole galacturonate residues are present. The pectin molecules contain rhamnose, arabinose, xylose, galactose, glucose and galacturonic acid residues. No mannose could be detected. The neutral sugar composition of the glycans bound to the galacturonan was found to be constant, except for the relative amount of galactose. During ripening the neutral sugar composition of the extractable pectin does not change.     

A simple procedure for the synthesis of alpha-32P-nucleoside triphosphates.

Chemical analysis of grapefruit (Citrus paradisi) pectic polysaccharides demonstrated that galacturonic acid constitutes 78% by weight of the total carbohydrates found. The remaining 22% was accounted for by a number of sugars which include galactose, glucose, arabinose, xylose, and mannose and, by weight, galactose accounted for almost 50% of the total neutral sugar components found in these pectic polysaccharides. Treatment of pectic polysaccharides with galactose oxidase followed by reduction of oxidized galactose residues with tritiated potassium borohydride resulted in the labeling of pectic polysaccharides. Analysis of the labeled polysaccharides demonstrated that of the total radioactivity incorporated more than 90% was recovered in the galactose residues. These results clearly demonstrate the successful utilization of the galactose oxidase/tritiated potassium borohydride method in labeling plant pectic polysaccharide.     

A developmentally regulated hydroxyproline-rich glycoprotein from the cell walls of soybean seed coats

In soybean seeds the level of hydroxyproline is regulated in a developmental and tissue-specific manner. The seed coat contains approximately 77% of the total hydroxyproline in the seed at all stages of development. We determined the ratio of hydroxyproline to dry weight in a number of tissues within the seed; however, only the seed coat shows an increase in this ratio during development. Within the many cell layers of the seed coat, hydroxyproline is most abundant in the external layer. The hydroxyproline is present as an hydroxyproline-rich cell wall glycoprotein. The protein is rich in hydroxyproline (36%), lysine (11%), proline (10%), histidine (9%), tyrosine (9%), and serine (8%). The carbohydrate portion is 90 mole% arabinose and 10 mole% galactose. The arabinose residues are attached to hydroxyproline mostly in the form of trisaccharides. The apparent molecular weight of this glycoprotein is 100,000 daltons.     

Structure of the arabinogalactan from zea shoots

The structure of the arabinogalactan obtained from the buffer-homogenate of Zea mays L. (hybrid B73 × Mo17) shoots has been studied. The purified polysaccharide was investigated by methylation analysis before and after controlled acid hydrolysis. Arabinogalactan-1 consists of arabinose, galactose, xylose, uronic acid, and glucose in the molar ratio of 37.1:55.8:3.0:4.1:trace, and arabinogalactan-2 consists of the same sugars in the ratio of 35.4:53.9:1.6:9.2:trace. A trace of protein was detected in arabinogalactan-1 and about 0.2% was present in 2. About 20% of the galactose residues in arabinogalactan-1 constitute a (1 → 3)-linked galactan chain and approximately 60% constitute a (1 → 6)-linked galactan sequence. About 15% of the galactose residues in arabinogalactan-1 are substituted by galactose in the 3- and 6-positions, thereby constituting branch points of the galactan framework. The remainder (5%) of the galactose residues in arabinogalactan-1 are located at nonreducing terminal positions. About 85% of the (1 → 6)-galactosyl sequence is substituted, mostly by single arabinose residues. Nonreducing terminal glucuronic acid is attached to C-6 of galactose residues. The basic structure of arabinogalactan-2 is similar to that of arabinogalactan-1.     

Polysaccharides of cell cultures of <Emphasis Type="Italic">Silene vulgaris</Emphasis>

Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3–6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5–1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures.     

Characterization of a biologically active arabinogalactan from the leaves of Plantago major L.

PMIa is a Type II arabinogalactan with anti-complementary activity isolated from the leaves of Plantago major L. It has a molecular weight of 77000–80000 Da and consists of arabinose (38%), galactose (49%), rhamnose (6%), galacturonic acid (7%) and 1.5% protein with hydroxyproline, alanine and serine as the main amino acids. Characterization of PMIa by methylation and GC-MS, methanolysis and GC, Smith degradation, weak acid hydrolysis, ¹³C-NMR, ¹H-NMR, two-dimensional heteronuclear NMR and DEPT show that it consists of 1,3-linked galactan chains with 1,6-linked galactan side chains attached to position 6. The side chains are further branched in position 3 with 1,3-linked galactose residues which have 1,6-linked galactose attached to position 6; these 1,3- and 1,6-linked galactose chains altogether probably form a network. Terminal and 1,5-linked arabinose in furanose form are attached to the galactan mainly through position 3 of the 1,6-linked galactose side chains.     

Cell-Wall Carbohydrates of the Endosperm of the Seed of Gleditsia triacanthos

The endosperm of the seed of Gleditsia triacanthos L. contains 18.55% of its dry weight as nonreserve, cell-wall carbohydrates. Of this carbohydrate material, comprising mainly mannose, galactose, and glucose, 76.1% was of low-molecular weight or highly hydrophilic. Mannose, galactose, and glucose were also the major sugar components of the polysaccharides extracted with alkali (23.1% of the cell-wall), while the same sugars, with minor amounts of arabinose, form the residues. Methylation analysis of the polysaccharides and the borate-sodium hydroxide residue indicate that the cell walls are built up on a network of galactomannans, with high Man/Gal ratios, reinforced with minor amounts of cellulose.