GlcNAc Oligomers Research Articles

Comparison of the ability of partially N-acetylated chitosans and chitooligosaccharides to elicit resistance reactions in wheat leaves

Chitin, a linear polysaccharide composed of (1-->4)-linked 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc) residues, and chitosan, the fully or partially N-acetylated, water-soluble derivative of chitin composed of (1-->4)-linked GlcNAc and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN), have been proposed as elicitors of defense reactions in higher plants. We tested and compared the ability of purified (1-->4)-linked oligomers of GlcNAc (tetramer to decamer) and of GlcN (pentamer and heptamer) and partially N-acetylated chitosans with degrees of acetylation (DA) of 1%, 15%, 35%, 49%, and 60% and average degrees of polymerization between 540 and 1100 to elicit phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition, and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, nonwounded wheat (Triticum aestivum L.) leaves. Purified oligomers of (1-->4)-linked GlcN were not active as elicitors, whereas purified oligomers of (1-->4)-linked GlcNAc with a degree of polymerization >/= 7 strongly elicited POD activities but not PAL activities. Partially N-acetylated, polymeric chitosans elicited both PAL and POD activities, and maximum elicitation was observed with chitosans of intermediate DAs. All chitosans but not the chitin oligomers induced the deposition of lignin, the appearance of necrotic cells exhibiting yellow autofluorescence under ultraviolet light, and macroscopically visible necroses; those with intermediate DAs were most active. These results suggest that different mechanisms are involved in the elicitation of POD activities by GlcNAc oligomers, and of PAL and POD activities by partially N-acetylated chitosan polymers and that both enzymes have to be activated for lignin biosynthesis and ensuing necrosis to occur.

Effect of N-acetyl- d-glucosamine and d-glucosamine oligomers on canine polymorphonuclear cells in vitro

The effect of N-acetyl- d-glucosamine (GlcNAc) and d-glucosamine (GlcN) oligomers on the activation and viability of canine polymorphonuclear cells (PMNs) was evaluated. Checkerboard assays of both oligomers revealed that the GlcNAc oligomer induced chemotactic migration, while GlcN oligomer induced chemotactic and chemokinetic migration. The GlcN oligomer showed about twice the chemotactic activity of the GlcNAc oligomer, and induced slight enhancement of the chemiluminescence response (CL) of canine PMNs to zymosan, as well as shortening of the peak time of the CL response compared with the control and the GlcNAc oligomer. Neither oligomer showed cytotoxicity for canine PMNs at a concentration of 100 μg/ml. These results suggest that GlcNAc and GlcN oligomers might be activating factors for canine PMN in vivo.

Ｘｅｎｏｐｕｓの初期発生過程に発現する糖転移酵素 ヒアルロナンシンターゼそれともキチン（ＧｌｃＮＡｃオリゴマー）シンターゼ？

Ｘｅｎｏｐｕｓの初期発生過程に発現する糖転移酵素 ヒアルロナンシンターゼそれともキチン（ＧｌｃＮＡｃオリゴマー）シンターゼ？

Influence of chain length of N-acetyl- d-glucosamine and d-glucosamine residues on direct and complement-mediated chemotactic activities for canine polymorphonuclear cells

The direct and complement-mediated chemotactic activities (CA) of N-acetyl- d-glucosamine (GlcNAc), d-glucosamine (GlcN), GlcNAc and GlcN oligomers, chitin and chitosan for canine polymorphonuclear cells (PMNs) were investigated in vitro. GlcNAc6 and GlcN56̃ provided the most effective stimulation of CA, with GlcN6 having a seven-fold stronger effect than GlcNAc6. Serum incubated with chitin and chitosan (37 °C, 30min) had a similar CA to that of zymosan. The total complement activity (CH50) of serum mixed with chitin and chitosan was lower than control level, but monomers and oligomers of GlcNAc or GlcN did not decrease complement activity. Subcutaneous injection of GlcN6 and chitosan enhanced more PMN migration than GlcNAc6 and chitin in vivo. It was demonstrated that GlcN residues induced higher direct CA than GlcNAc residues, while complement-mediated CA was only induced by chitin and chitosan, but not by GlcNAc, GlcN and their oligomers.

Inhibitory Effects of Sialic Acid- or N-Acetylglucosamine-Specific Lectins on Histamine Release Induced by Compound 48/80, Bradykinin and a Polyethylenimine in Rat Peritoneal Mast Cells

The effects of seven lectins with various sugar-specificities on histamine release from rat peritoneal mast cells induced by non-immunologic stimuli were studied. The non-immunologic stimuli used were three basic secretagogues, compound 48/80, bradykinin and PEI6 (polyethylenimine with a molecular weight of 600). In this study, we observed inhibition of the histamine release by Macckia amurensis mitogen and Solanum tuberosum agglutinin (100 micrograms/ml at 37 degrees C for 10 min), which are specific for sialic acid-alpha 2,3-N-acetyl galactosamine (Sia alpha 2,3GalNAc) and N-acetyl glucosamine (GlcNAc) oligomers, respectively. The effects of Phytolacca americana mitogen and Sambucus sieboldiana agglutinin were different. Three lectins specific for mucin type oligosaccharides inhibited the histamine release induced by compound 48/80 but not that induced by bradykinin or PEI6. Since bradykinin and PEI6 additively enhanced the histamine release induced by compound 48/80, they partially shared the same signalling pathways. Glycoproteins with bisecting GlcNAc and Sia residues, as described previously (Jpn. J. Pharmacol. 57, 79-90, 1991), seemed to be one of the action sites for compound 48/80, bradykinin and PEI6. In addition to the direct activation of the pertussis toxin-sensitive G proteins, we propose another mechanism of non-immunologic stimuli via specific glycoproteins on rat peritoneal mast cells. The apparent sugar residues involved were asparagine-linked oligosaccharides with Sia (especially Sia alpha 2,3Gal), GlcNAc oligomers and/or bisecting GlcNAc.

Induction of Phytoalexin Formation in Suspension-cultured Rice Cells byN-Acetyl-chitooligosaccharides

Induction of phytoalexin formation in suspension-cultured rice cells by a series of N-acetylchitooligosaccharides and chitooligosaccharides was studied. N-acetylchitooligosaccharides larger than hexaose induced the formation of momilactones A and B as well as oryzalexins A, B, and D at very low concentrations like 10− 9–10− 6 M (N-acetylchitoheptaose). GlcNAc oligomers smaller than trimers had almost no activity and a series of deacetylated chitooligosaccharides were also inactive. Strict requirement for the size and structure of GlcNAc oligomers as well as the sensitivity to them strongly indicates the presence of recognition systems specific for these compounds in rice cells. The level of momilactone A produced reached 100–500 μg/g of cultured cells, which appeared to be enough to prevent the growth of pathogenic fungi such as Pyricularia oryzae, thus indicating the importance of this phenomenon in the defense systems of rice plants. Suspension-cultured cells obtained only from a suitable period of cultivation, mainly those from lag phase, could respond to the elicitor and produce phytoalexins.

Chitin utilization by marine bacteria. Chemotaxis to chitin oligosaccharides by Vibrio furnissii.

The adhesion/deadhesion apparatus of the marine bacterium Vibrio furnissii (Yu, C., Lee, A., Bassler, B. L., and Roseman, S. (1991) J. Biol. Chem. 266, 24260-24267) probably catalyzes the first step in colonizing chitin. Evidence is presented here for a second step, chemotaxis to chitin hydrolysis products. V. furnissii swarms toward chitin oligomers (GlcNAc)n, n = 1-6, at initial concentrations as low as 10 microM. A modified capillary assay was used for quantitation; the cells exhibit low level constitutive taxis to GlcNAc but not to the oligosaccharides. A mutant defective in the GlcNAc receptor (IINag of the phosphotransferase system) showed inducible taxis to the oligosaccharides. Two (or more) independently inducible receptors with overlapping specificities recognize (GlcNAc)n, n = 2-4. (GlcNAc)5 and (GlcNAc)6 were inactive in the capillary assay; expression of this receptor(s) apparently require special induction conditions. The (GlcNAc)n, n = 1-4, chemoreceptors of V. furnissii may be the most potent reported for bacteria. L-Amino acids were weak, constitutive attractants; glutamine, not known to be an attractant in other bacteria, was the most effective amino acid. The most potent receptor in Escherichia coli, Tar (aspartate), is not expressed in V. furnissii. The chemotactic responses were greatly affected by growth and induction conditions and the presence of nutrients in the assay media. Taxis to GlcNAc and GlcNAc oligomers was optimally induced by growth in lactate medium containing 0.6 mM sugar, while growth on the sugar per se resulted in poor taxis. Chemotaxis to the sugars increased 2- to 3-fold when the cells were starved. Nutrients in the assay medium, especially compounds that feed into or are part of the Krebs cycle, were potent inhibitors of taxis to the sugars and Gln. With the exception of isocitrate, inhibition of taxis correlated with the rate of oxidation of these compounds. The results suggest a link between catabolism and taxis in this organism, i.e. interactions or "cross-talk" between systems that are regulated by protein phosphorylation (Stock, J. A., Ninfa, A. J., and Stock, A. M. (1989) Microbiol. Rev. 53, 450-490).

Glycoconjugates of the lateral prostate of the guinea pig: A lectin histochemical study

The present study examined the glycoconjugates of the lateral prostate using a battery of lectins. The results indicated that the secretory epithelium was rich in mannose (Man), N-acetylglucosamine (GlcNAc), galactose (Gal), N-acetylgalactosamine (GalNAc), and complex oligosaccharides. Con A (concanavalin A), LCA (Lens culinaris agglutinin), PSA (Pisum sativum agglutinin), WGA (wheat germ agglutinin), PWM (pokeweed mitogen), RCA-I (Racinus communis isolectin I), and PHA-P (Phaseolus vulgaris agglutinin-P) reacted intensely with both epithelia and stroma, while SBA (soybean agglutinin) and PNA (peanut agglutinin), which bind to terminal Gal, GalNAc, and Gal beta 1,3 GalNAc appeared to be specific to the secretory epithelium. SBA and PNA were useful as markers in the study of the secretory function of the prostate gland. The present study has shown that the Golgi apparatus of prostatic epithelial cells was rich in fucose (Fuc), oligomers of GlcNAc, Gal beta 1,3 GalNAc, Gal, and Man containing glycoconjugates, indicating that the gland was actively involved in glycosylation. The present study has also shown that LTA (Lotus tetragonolobus agglutinin) is a good marker for the epithelial Golgi. PNA also bound to the epithelial basement membrane and the connective tissue in the lamina propria. The present study has thus established, for the first time, the glycoconjugate patterns in the lateral prostate of the guinea pig.

Toxicity of protease-resistant domains from the delta-endotoxin of Bacillus thuringiensis subsp. israelensis in Culex quinquefasciatus and Aedes aegypti bioassays

The mosquitocidal glycoprotein endotoxin of Bacillus thuringiensis subsp. israelensis was digested with chymotrypsin to yield protease-resistant domains which were then separated from smaller protease digestion products by high-performance liquid chromatography. Once purified, the domains no longer bound wheat germ agglutinin, a lectin which binds N-acetylglucosamine (GlcNAc) and GlcNAc oligomers. Purified protease-resistant domains were as toxic for Culex quinquefasciatus larvae as intact solubilized toxin. In separate experiments, the toxicity of chymotrypsin-digested endotoxin for Aedes aegypti larvae was reduced fivefold or more. A model is presented in which GlcNAc-containing oligosaccharides are required for toxicity for A. aegypti larvae but not C. quinquefasciatus larvae.

Characterization of the Adhesive Holdfast of Marine and Freshwater Caulobacters

Caulobacters are prosthecate (stalked) bacteria that elaborate an attachment organelle called a holdfast at the tip of the cellular stalk. We examined the binding of lectins to the holdfasts of 16 marine Caulobacter strains and 10 freshwater species or strains by using a panel of fluorescein-conjugated lectins and fluorescence microscopy. The holdfasts of all the marine isolates bound to only wheat germ agglutinin (WGA) and other lectins that bind N-acetylglucosamine (GlcNac) residues. The freshwater caulobacters showed more variability in holdfast composition. Some bound only to WGA and comparable lectins as the marine strains did. Others bound additional or other lectins, and some did not bind to the lectins tested. The binding of WGA appeared to involve the regions of the holdfast involved with adhesion; a holdfast bound to WGA was significantly less adhesive to glass. Competition experiments with WGA-binding holdfasts and oligomers of GlcNac demonstrated that trimers of GlcNac (the preferred substrate for WGA binding) were more effective than dimers or monomers in preventing WGA binding to holdfasts, suggesting that stretches of contiguous GlcNac residues occur in the WGA-binding holdfasts. In addition, differences between freshwater and marine holdfasts in the strength of WGA binding were noted. The effect of a number of proteolytic and glycolytic enzymes on holdfast integrity was examined; the proteases had no effect for all caulobacters. None of the glycolytic enzymes had an effect on marine caulobacter holdfasts, but chitinase and lysozyme (both attack oligomers of GlcNac) disrupted the holdfasts of those freshwater caulobacters that bound WGA. Despite some similarity to chitin, holdfasts did not bind Calcofluor and no measurable effects on holdfast production were detectable after cell growth in the presence of diflubenzuron or polyoxin D, inhibitors of chitin synthesis in other systems. Finally, the holdfasts of all caulobacters bound to colloidal gold particles, without regard to the coating used to stabilize the gold particles. This binding was stronger or more specific than WGA binding; treatment with colloidal gold particles prevented WGA binding, but the reverse was not the case.

Mechanism of neutrophil chemiluminescence induced by wheat germ agglutinin: partial characterization of the antigens recognized by wheat germ agglutinin

Wheat germ agglutinin (WGA) stimulated neutrophils to produce significant levels of luminol-dependent chemiluminescence (CL). Since WGA is known to bind N-acetylglucosamine (GlcNAc) oligomers and N- acetylneuraminic acid (NANA), we attempted to determine which binding property of WGA is essential for induction of CL. The succinylated form of WGA (SuWGA), which is no longer able to bind NANA, was still able to induce CL. N-Acetylglucosamine at a concentration of 20 mmol/L almost completely inhibited WGA-induced CL production by neutrophils, whereas bovine submaxillary gland mucin, a potent blocker of NANA binding of WGA, failed to inhibit CL production. Lectins with the GlcNAc-binding property were examined for their ability to induce CL. Those that have higher valences and have a tendency to bind GlcNAc oligomers in the internal portion of glycoconjugates were able to induce CL, whereas those that have low valences and bind terminal GlcNAc of glycoconjugates failed to induce CL even at high concentrations. Attempts were made to characterize the neutrophil membrane proteins recognized by WGA. Glycoproteins with a molecular weight of 25,000 daltons were identified by a 50 mmol/L GlcNAc elution of WGA gels loaded with 125I-labeled neutrophil membrane proteins. Elution with 500 mumol/L GlcNAc trimer produced several glycoproteins of different molecular weights in addition to the glycoproteins of 25,000 daltons. 125I-labeled WGA and SuWGA were used for autoradiographic analysis of cell extracts of the neutrophils separated on sodium dodecyl sulfate polyacrylamide gels. WGA recognized multiple glycoproteins of different molecular weights, whereas SuWGA bound only a few of them. Glycoproteins of 25,000 daltons, probably corresponding to those identified by 50 mmol/L GlcNAc elution, were also recognized.

Mechanism of Neutrophil Chemiluminescence Induced by Wheat Germ Agglutinin: Partial Characterization of the Antigens Recognized by Wheat Germ Agglutinin

Wheat germ agglutinin (WGA) stimulated neutrophils to produce significant levels of luminol-dependent chemiluminescence (CL). Since WGA is known to bind N-acetylglucosamine (GlcNAc) oligomers and N-acetylneuraminic acid (NANA), we attempted to determine which binding property of WGA is essential for induction of CL. The succinylated form of WGA (SuWGA), which is no longer able to bind NANA, was still able to induce CL. N-Acetylglucosamine at a concentration of 20 mmol/L almost completely inhibited WGA-induced CL production by neutrophils, whereas bovine submaxillary gland mucin, a potent blocker of NANA binding of WGA, failed to inhibit CL production. Lectins with the GlcNAc-binding property were examined for their ability to induce CL. Those that have higher valences and have a tendency to bind GlcNAc oligomers in the internal portion of glycoconjugates were able to induce CL, whereas those that have low valences and bind terminal GlcNAc of glycoconjugates failed to induce CL even at high concentrations. Attempts were made to characterize the neutrophil membrane proteins recognized by WGA. Glycoproteins with a molecular weight of 25,000 daltons were identified by a 50 mmol/L GlcNAc elution of WGA gels loaded with 125I-labeled neutrophil membrane proteins. Elution with 500 mumol/L GlcNAc trimer produced several glycoproteins of different molecular weights in addition to the glycoproteins of 25,000 daltons. 125I-labeled WGA and SuWGA were used for autoradiographic analysis of cell extracts of the neutrophils separated on sodium dodecyl sulfate polyacrylamide gels. WGA recognized multiple glycoproteins of different molecular weights, whereas SuWGA bound only a few of them. Glycoproteins of 25,000 daltons, probably corresponding to those identified by 50 mmol/L GlcNAc elution, were also recognized.