Light and electron microscopic detection of (3 Gal beta 1,4 GlcNAc beta 1) sequences in asparagine-linked oligosaccharides with the Datura stramonium lectin

The Datura stramonium lectin recognizes with high affinity the disaccharide N-acetyllactosamine (Gal beta 1,4 GlcNAc). We have developed a highly specific cytochemical affinity technique in which an ovomucoid-gold complex serves as second step reagent for the visualization of this lectin bound to reactive sequences present in tissue sections. The lectin binding sites were detected in semithin and ultrathin sections of aldehyde-fixed and low temperature Lowicryl K4M embedded tissues. For light microscopical labeling the photochemical silver reaction for signal amplification was required. The application of this technique for the detection of N-acetyllactosamine containing asparagine-linked oligosaccharides in various intracellular organelles and the plasma membrane is demonstrated.     

An enzyme-linked lectin binding assay for quantitative determination of lectin receptors

An enzyme-linked lectin binding assay (ELBA) has been developed for the detection of soluble lectin binding substances (receptors) and the determination of their relative affinity for the lectin. The assay is based on competitive binding to enzyme-labeled lectin of a known lectin receptor, bound to a solid phase, and unknown sample receptors. In this paper the assay is exemplified with the mannose/glucose-specific pea lectin, with the glycoprotein ovalbumin as its receptor, and with horseradish peroxidase (EC 1.11.1.7) as the enzyme used for labeling. Also a method was developed for the preparation of peroxidase-labeled lectin. Labeling was started by mixing equimolar amounts of lectin and periodate-oxidized enzyme at pH 4.5 at a final concentration of 10(-4)M, after which conjugation was started by raising the pH to 9.5. This resulted in complete conjugation, after which the product could be diluted 50-500 times for application in ELBA. For the ELBA ovalbumin was adsorbed onto polystyrene microtiter plates. Sample receptors, added together with the enzyme-labeled lectin, inhibited binding of the latter to ovalbumin. Bound enzyme activity was colorimetrically determined after addition of o-phenylenediamine. Relative lectin affinity (KL) was expressed as (formula; see text) in which [X]50% is the concentration of sample receptor necessary to inhibit 50% of the binding of a certain amount of lectin, and [M]50% is the concentration of D-mannose necessary to inhibit 50% binding of the same amount of lectin. With this technique lectin affinity of both monovalent and polyvalent lectin binding substances can be estimated: low KL values mean high lectin affinity.     

Heterogenous Degradation of Myofibrillar Proteins

Pectic substances were extracted from the vegetables with oxalate buffer of pH 4.25 and, after saponification, fractionated into two components, weakly acidic pectic polysaccharide (WAP) and pectic acid, by DEAE-cellulose and Sephadex G-100 chromatographies. The galacturonic acid content (17.3~25.8%) of WAPs was much lower than that of pectic acids, though the neutral sugar compositions of both pectic substances were almost the same. The arabinose-galactose side chains were found to be very long or highly branched in WAPs compared with those in pectic acids.

All the WAPs were appreciably hydrolyzed by exo- and endopolygalacturonases. The limited-degradation products (the residual polysaccharides; i.e., the rhamnogalacturonan segments) obtained by endopolygalacturonase from both WAPs and pectic acids showed a similar behavior on Sephadex G-100 and Sepharose CL-4B gel filtrations; each of the rhamnogalacturonan segments was eluted in the void volume of the Sephadex G-100 column. From these results, we concluded that WAPs are probably an inherent pectic component of the cell walls of the vegetables.     

Ultrastructural study of galacturonic acid distribution in some pathogenic fungi using gold-complexed Aplysia depilans gonad lectin

Aplysia gonad lectin, isolated from the mollusc Aplysia depilans, was successfully conjugated to colloidal gold and used for ultrastructural detection of galacturonic acids in some pathogenic fungi. These sugar residues were found to occur in the fibrillar sheath surrounding hyphal cells of Ascocalyx abietina and in intravacuolar dense inclusions of this fungus spores. In hyphae and spores of Ophiostoma ulmi, galacturonic acids were detected mainly in the outermost wall layers. In contrast, these saccharides appeared associated with the innermost wall layers and especially the plasma membrane of Verticillium albo-atrum cells. Galacturonic acids were found to be absent in cells of Fusarium oxysporum f.sp. radicis-lycopersici and Candida albicans. These cytochemical data indicate therefore that a heterogeneity in wall composition exists between ascomycete fungi. The significance of the presence of galacturonic acids in the cell walls of certain fungi is still open to question.     

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.     

Gold-conjugated arabinogalactan-protein and other lectins as ultrastructural probes for the wheat/stem rust complex

Arabinogalactan-protein (AGP, "beta-lectin") was isolated from leek seeds, tested for specificity, conjugated with gold colloids, and used as a cytochemical probe to detect beta-linked bound sugars in ultrathin sections of wheat leaves infected with a compatible race of stem rust fungus. Similar sections were probed with other gold-labeled lectins to detect specific sugars. AGP-gold detected beta-glycosyl in all fungal walls and in the extrahaustorial matrix. Other lectin gold conjugates localized galactose in all fungal walls except in walls of the haustorial body. Limulus polyphemus lectin bound only to the outermost layer of intercellular hyphal walls of the fungus. Binding of these lectins was inhibited by their appropriate haptens and was diminished or abolished in specimens pretreated with protease, indicating that the target substances in the tissue were proteinaceous or that polysaccharides possessing affinity to the lectin probes had been removed by the enzyme from a proteinaceous matrix by passive escape. Binding of Lotus tetragonolobus lectin was limited to the two outermost fungal wall layers but was not hapten-inhibitable. Limax flavus lectin, specific for sialic acids, had no affinity to any structure in the sections. In the fungus, the most complex structure was the outermost wall layer of intercellular hyphal cells; it had affinity to all lectins tried so far, except to Limax flavus lectin and to wheat germ lectin included in an earlier study. In the host, AGP and the galactose-specific lectins bound to the inner domain of the wall in areas not in contact with the fungus. At host cell penetration sites, affinity to these lectins often extended throughout the host wall, confirming that it is modified at these sites. Pre-treatment with protease had no effect on lectin binding to the host wall. After protease treatment, host starch granules retained affinity to galactose-specific lectins, but lost affinity for AGP.     

Defining the carbohydrate specificities of aplysia gonad lectin exhibiting a peculiar D-galacturonic acid affinity

Aplysia gonad lectin (AGL), which has been shown to stimulate mitogenesis in human peripheral lymphocytes, to suppress tumor cells, and to induce neurite outgrowth and improve cell viability in cultured Aplysia neurons, exhibits a peculiar galacturonic acid/galactose specificity. The carbohydrate binding site of this lectin was characterized by enzyme-linked lectino-sorbent assay and by inhibition of AGL-glycan interactions. Examination of the lectin binding with 34 glycans revealed that it reacted strongly with the following glycoforms: most human blood group precursor (equivalent) glycoproteins (gps), two Galalpha1-->4Gal-containing gps, and two d-galacturonic acid (GalUA)-containing polysaccharides (pectins from apple and citrus fruits), but poorly with most human blood group A and H active and sialylated gps. Among the GalUA and mammalian saccharides tested for inhibition of AGL-glycan binding, GalUA mono- to trisaccharides were the most potent ones. They were 8.5 x 10(4) times more active than Gal and about 1.5 x 10(3) more active than the human blood group P(k) active disaccharide (E, Galalpha1-->4Gal). This disaccharide was 6, 28, and 120 times more efficient than Galbeta1-->3GlcNAc(I), Galbeta1-->3GalNAc(T), and Galbeta1--> 4GlcNAc (II), respectively, and 35 and 80 times more active than melibiose (Galalpha1-->6Glc) and human blood group B active disaccharide (Galalpha1-->3Gal), respectively, showing that the decreasing order of the lectin affinity toward alpha-anomers of Gal is alpha1-->4 > alpha1-->6 > alpha1-->3. From the data provided, the carbohydrate specificity of AGL can be defined as GalUAalpha1-->4 trisaccharides to mono GalUA > branched or cluster forms of E, I, and II monomeric E, I, and II, whereas GalNAc is inactive.     

Light and electron microscopic detection of (3 Gal β1,4 GlcNAc β1) sequences in asparagine-linked oligosaccharides with the Datura stramonium lectin

Summary The Datura stramonium lectin recognizes with high affinity the disaccharide N-acetyllactosamine (Gal 1,4 GlcNAc). We have developed a highly specific cytochemical affinity technique in which an ovomucoid-gold complex serves as second step reagent for the visualization of this lectin bound to reactive sequences present in tissue sections. The lectin binding sites were detected in semithin and ultrathin sections of aldehyde-fixed and low temperature Lowicryl K4M embedded tissues. For light microscopical labeling the photochemical silver reaction for signal amplification was required. The application of this technique for the detection of N-acetyllactosamine containing asparagine-linked oligosaccharides in various intracellular organelles and the plasma membrane is demonstrated.This study was supported by the Swiss National Science Foundation grant nr. 31-26273.89 (to J.R.) and GM 29470 from the National Institutes of Health (to I.J.G.). Dr. G. Egea was a recipient of a European Molecular Biology Organization long term fellowship.     

The fermentation of cell wall substances in grass silage and in potato pulp

Summary The amount of acid formed in grass silage was greater than could have been formed from the soluble sugars present, even when only a lactic fermentation took place. This seemed to point to fermentation of cell wall substances by lactic acid bacteria. Lactic acid fermentation in potato pulp always takes place with cell wall substances as substrates, as sugars are absent. It was found that galactose, probably occurring as galactan, and also some pectic acid were fermented in potato pulp. Some lactobacilli were isolated from potato pulp; streptobacteria which could ferment galactan but no pectic or galacturonic acid, and betabacteria which could ferment galacturonic acid but no galactan or pectic acid. A number of homofermentative lactobacilli were all found to belong to the speciesStreptobacterium casei. It was shown that a strain of this species could ferment galactan in potato pulp sterilised previously with ethylene oxide. Part of this work was carried out at the Netherlands Institute for Dairy Research, Ede, Netherlands.     

Evidence for glycoprotein components of the hepatocellular bile acid transporter

The hepatocellular transporter, responsible for the uptake of bile acids and some foreign substances, can be shown to contain carbohydrate moieties. The hepatocellular uptake of cholate and phallotoxin is immediately inhibited by addition of wheat-germ agglutinin. Concanavalin A and lentil lectin reduce the uptake in a time-dependent manner. Apparently sialic acids or N-acetylglucosamine residues are involved in the translocation process. Polypeptides (Mr 50,000, 54,000) of the above transport system, identified by affinity labeling with [3H]isothiocyanatobenzamido cholate and [3H2]diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid, are heterogenously glycosylated. Binding of 80-90% of the 54, 50 kDa polypeptides to all immobilized lectins tested suggests that both high-mannose and complex type oligosaccharides with fucose and terminal sialic acid residues occur as carbohydrate chains. A 67 kDa labeled polypeptide is not glycosylated. Pilot experiments for purification of the above glycosylated membrane proteins on concanavalin A, lentil lectin and wheat-germ lectin columns are described. However, lectin affinity chromatography is not suitable as a one-step purification procedure for the labeled polypeptides.     

Glycoproteins from the cell wall of Phaseolus coccineus.

1. The use of a modified sodium chlorite/acetic acid delignification procedure for the solubilization of a hydroxyproline-rich glycoprotein fraction from the depectinated cell walls of Phaseolus coccineus is described. 2. The crude glycoprotein was associated with some pectic material; hydroxyproline and serine were the most abundant amino acids, and arabinose, galactose and galacturonic acid the predominant monosaccharides. 3. The bulk of the hydroxyproline is O-glycosidically substituted with tetra- and tri-arabinofuranosides. From methylation analysis the linkages in these arabinosides could be inferred. 4. Ion-exchange chromatography of the crude glycoprotein gave one major and two minor hydroxyproline-rich fractions, with similar amino acid but different monosaccharide composition. 5. In the major fraction, serine appears to be O-glycosidically substituted with a single galactopyranoside residue that can be removed by the action of alpha-galactosidase but not beta-galactosidase. Removal of arabinofuranoside residues by partial acid hydrolysis greatly enhanced the action of alpha-galactosidase. 6. Methylation followed by carboxy reduction with LiAl2H4 has shown the presence of (1 leads to 4)-linked galacturonic acid in the crude glycoprotein fraction but not in the major fraction from the ion-exchange column. Hence the bulk of the pectic material is not associated with the major glycoprotein component. It is suggested that the glycoprotein is held in the wall by phenolic cross-links. 7. Similarities with the glycopeptide moiety of potato lectin provides further evidence for a class of hydroxyproline-rich glycoproteins with common features.     

微生物果胶酶研究进展

果胶酶是一类分解果胶质的酶的总称,它能将复杂的果胶分解为半乳糖醛酸等小分子。目前果胶酶在食品、纺织、医药、造纸、环境、生物技术、饲料等领域得到广泛应用。果胶酶主要来自微生物。综述了微生物果胶酶生产菌的菌种、选育、鉴定、发酵方法和发酵条件优化,酶的分离纯化、酶学性质和分子生物学方面的研究进展,并介绍了果胶酶的应用进展,最后展望了微生物果胶酶研究的广阔前景。     

Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki

A pectic polysaccharide DL-2A with a molar mass of 8.5 x 10(5), was obtained from the boiling water extract of Diospyros kaki leaves. It had [alpha]20D -21.8 degrees (c 0.22, H2O) and consisted of rhamnose, arabinose, galactose, xylose and galacturonic acid units in the molar ratio of 0.4:3.4:2.4:1.0:0.8, along with traces of glucuronic acid. About 16.7% of galacturonic acid existed as the methyl ester. A combination of linkage analyses, periodate oxidation, partial acid hydrolysis, selective lithium-degraded reaction, ESIMS, 1H- and 13C- NMR spectral analyses revealed its structural features. It was found that DL-2A possessed an alpha-(1-->4)-galacturonan backbone with some insertions of alpha-1,2-Rhap residues. The side-chains of arabino-3,6-galactan were attached to the backbone via O-4 of Rhap residues and O-3 of GalAp residues, while 4-linked xylose residues (forming short linear chains) were directly linked to O-4 of rhamnose residues, not as part of the xylogalacturonan. These novel structural features enlarge the knowledge on the fine structure of pectic substances in the plant kingdom.     

Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus: a cytochemical affinity technique with the use of fetuin-gold complexes

The development of a cytochemical affinity technique for the demonstration of sialic acid residues by light and electron microscopy is reported. The lectin from the slug Limax flavus, with its narrow specificity for N-acetyl- and N-glycolylneuraminic acid, was applied to tissue sections. Subsequently fetuin-gold complexes were used to visualize the tissue-bound lectin. Different cytochemical controls, including sugar inhibition tests, neuraminidase digestion, the use of fetuin-gold complexes alone, or acid hydrolysis of sections, proved the specificity of the technique. Postembedding staining was performed on frozen, paraffin, or semithin resin sections for light microscopy and on thin sections from low temperature Lowicryl K4M-embedded material for electron microscopy. The distribution of sialic acid residues in rat pancreas, liver, and colonic mucosa was investigated.     

Microfluorometry of pectic materials in the dehiscence zone of almond (Prunus dulcis [Mill.] DA Webb) fruits

We examined the middle lamella and the primary and secondary walls in almond pericarp dehiscence zone cells using a fluorescent cytochemical method which permitted specific, quantitative detection of pectic cell wall materials. Glycol methacrylate-embedded sections were stained with coriphosphine and pectin-specific fluorescent emissions at 630 nm were quantified using green excitation (546 nm). Examination of sectioned material extracted with purified pecto-lytic enzyme preparations was used to demonstrate the relative specificity of the staining reaction for pectic substances.     

A complement fixing polysaccharide from Biophytum petersianum Klotzsch, a medicinal plant from Mali, West Africa

Biophytum petersianum Klotzsch (syn. Biophytum sensitivum (L.) DC) is a medicinal plant having a traditional use, among others, as a wound healing remedy in Mali and other countries. As a water extract of the aerial parts of the plant is a frequently used preparation, we decided to look for a bioactive polysaccharide in this extract. One of the obtained polysaccharide fractions, BP100 III, isolated from a 100 degrees C water extract from the aerial parts of B. petersianum and having a monosaccharide composition typical for pectic substances, was shown to exhibit potent dose-dependent complement fixating activity. The BP100 III fraction was subjected to degradation by endo-alpha-d-(1-->4)-polygalacturonase, and three fractions were obtained by gel filtration. The highest molecular weight fraction, BP100 III.1, had a more potent activity in the complement test system than the native polymer, while the two lower molecular weight fractions were less active than the native polymer. The major part of BP100 III.1 consists of galacturonic acid and rhamnose, with branches being present on both the rhamnose and galacturonic acid residues. Arabinogalactan type II is also present in the polymer, indicating that BP100 III.1 has a structure typical of the hairy region of pectins. The major part of the two other fractions is a galacturonan, containing a strikingly high number of branch points, some to which xylose is attached. These results indicate that the pectic substance in B. petersianum contains both rhamnogalacturonan and xylogalacturonan regions.     

Apple juice pectic substances

Apple juice pectic substances are characterised by a high content of galacturonic acids (75·6%), a high degree of esterification (83), a low content of neutral sugars (0·109 moles/mole of galacturonate residues) and a high molecular weight ($\text{M}{}_{\mathrm{v}}\text{= 95 000}$). Degradation by heat (β-elimination) and by endopolygalacturonase showed that the pectic macromolecules consist of long homogalacturonan regions (92% of total galacturonides) and of ‘hairy’ regions in which neutral sugars form long or short side-chains.     

Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger

A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a combination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the amount of non-esterified galacturonic acid units located between two other non-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducing end of the polymer. The difference between total- and exo-BS was calculated to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-ester pectin with either plant pectin methyl-esterase (p-PME, P-series), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esterification using base (B-series) were analysed and compared with a fully de-esterified pectic acid sample obtained from the same raw material. Clear differences for the increase of the amounts of blocksequence could be seen between de-esterification of the P- and F-series samples supporting a blockwise and a homogenous de-esterification mechanism, respectively. f-PME and base treatment showed only minor differences in the increase of galacturonic acid units in BS, despite differences seen in their methyl-esterification pattern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blocklength for p-PME to de-esterify blockwise.     

Purification and properties of a Ca2+-independent sialic acid-binding lectin from human placenta with preferential affinity to O-acetylsialic acids

A Ca2+-independent sialic acid-specific lectin from two developmental stages of human placenta was similarly purified to apparent homogeneity by DEAE-cellulose chromatography, affinity chromatography on bovine submaxillary mucin, and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration disclosed a molecular mass of 53 kDa. The specificity of the lectin for O-acetylsialic acids was substantiated by the dependence of hemagglutination on the presence of acetylated sialic acids on the surface of mammalian erythrocytes of various sources, by hapten inhibition in hemagglutination assays with protease-treated rabbit erythrocytes and by hapten inhibition of binding of labeled N-acetylneuraminic acid-bovine serum albumin to the lectin in a solid-phase assay. Bovine and equine submaxillary mucins that contain 9(7,8)-O-acetyl and 4-O-acetylsialic acids were potent inhibitors in contrast to the non-acetylated sialic acids of ovine submaxillary mucin. Absence of inhibitory efficiency of other negatively charged substances like phosphorylated sugars, glucuronic acid, heparin, or oligodeoxynucleotides emphasized the importance of structural features instead of simple ionic interaction. In the presence of acetylation, the pattern of inhibition by gangliosides in the solid-phase assay indicated a preference to alpha-2,8- or alpha-2,6-linked sialic acids in comparison to alpha-2,3-linked moieties. Chemical modification of the lectin by group-specific reagents allowed to emphasize the role of primarily lysine residues, but also, although less pronounced, arginine, tryptophan, and carboxyl groups for ligand binding and/or maintenance of the active conformational state. Application of reagents, specific for histidine or tyrosine residues, failed to affect lectin activity.     

Localization of Cell Wall Polysaccharides during Kiwifruit (Actinidia deliciosa) Ripening

The localization of pectin, cellulose, xyloglucan, and callose was compared in kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang and A. R. Ferguson var. deliciosa "Hayward") at harvest, at the end of the first phase of softening, and when ripe. Pectin was visualized using three different methods: labeling of galacturonic acid residues, labeling of negatively charged groups, and labeling with JIM 5 (nonesterified residues) and JIM 7 (methyl-esterified) monoclonal antibodies. Labeling of pectin gave different results depending on the detection system used. Differences related to patterns of change during ripening and to spatial distribution of label intensity. Cell wall pectin was available for labeling at all stages of fruit softening, but no clear differentiation of the middle lamella region was seen, although JIM 5 binding predominated where the middle lamellae joined the intercellular spaces in unripe fruit. Negatively charged groups (cationic gold labeling) and, to a lesser extent, galacturonic acid residues (Aplysia depilans gonad lectin labeling) were preferentially located near the cell wall/plasma membrane boundary. The lack of strong binding of the JIM antibodies indicated that the reactive groups were inaccessible. Cellulose remained intact and labeled densely across the wall at all stages of fruit ripening. Distribution of xyloglucan was patchy at harvest but was scattered throughout the wall later in ripening. Alterations to labeling of xyloglucan indicated that some epitopes were differentially exposed. Plasmodesmatal regions were clearly different in composition to other wall areas, showing an absence of cellulose labeling, specific pectin labeling, and callose presence. A similar predominance of pectin labeling compared with cellulose also occurred at the middle lamella wedge near intercellular spaces.