GlcNAc Polymers Research Articles

Trichomonas vaginalisandTritrichomonas foetus:Expression of Chitin at the Cell Surface

Kneipp, L. F., Andrade, A.F.B., de Souza, W., Angluster, J., Alviano, C. S., and Travassos, L. R. 1998.Trichomonas vaginalisandTritrichomonas foetus:Expression of chitin at the cell surface.Experimental Parasitology89, 195–204. The expression of chitin as a structural component ofTrichomonas vaginalisandTritrichomonas foetuswas demonstrated by using enzymatic hydrolysis by recombinant (rec-) chitinase, chemical analysis, lectin, fluorescent Calcofluor and antibody binding, glycosidases of known specificity, high-performance liquid chromatography (HPLC), and flow cytometry. Chitinous structures were characterized by their insolubility in hot alkali and by releasing glucosamine on hydrolysis with 6 N HCl.N,N′-Diacetylchitobiose andN,N,′N″-triacetylchitotriose were identified by HPLC as enzymatic hydrolysis products of the alkali-resistant polysaccharide. The location of chitin on the surface ofT. vaginalisandT. foetuswas inferred from the decreased reactivity with whole parasites of ligands such asLycopersicon esculentum(TOL) andSolanum tuberosumlectins, fluorescent Calcofluor, and anti-chitin antibody, after cell treatment with rec-chitinase. Binding of [125I]TOL showed that, inT. vaginalisandT. foetus, the numbers of lectin receptors per cell were 4.2 × 105and 3.0 × 105, respectively. Binding of the lectin to the trichomonad surface was markedly decreased by treatment with rec-chitinase. TOL interaction with the parasites was not affected byN-acetyl-β-D-gluco-saminidase treatment, showing that the lectin receptors consisted of β-linked GlcNAc polymers and not of terminal β-linked GlcNAc residues.

Substrate specificities of tobacco chitinases.

Ten tobacco chitinases (1,4-N-acetyl-beta-D-glucosaminide glycanhydrolase, EC 3.2.1.14) were purified from tobacco leaves hypersensitively reacting to tobacco mosaic virus. The 10 enzymes, which belong to five distinct structural classes of plant chitinases, were incubated with several potential substrates such as chitin, a beta-1,4 N-acetyl-D-glucosamine (GlcNAc) polymer, chitosan (partially deacetylated chitin), chitin oligomers of variable length and bacterial cell wall. Tobacco chitinases are all endotype enzymes that liberate oligomers from chitin and are capable of processing the chito-oligomers further at differential rates. Chitin reaction products were separated and quantified by HPLC and differential kinetics of oligomer accumulation and degradation were observed with the distinct classes of chitinases. Depending on the substrate to be hydrolysed, each isoform displayed a different spectrum of activity. For example, class I isoforms were the most active on chitin and (GlcNAc)4-6 whereas class III basic isoforms were the most efficient in inducing bacterial lysis. Class V and class VI chitinases were shown to more readily hydrolyse chitin oligomers than the chitin polymer itself. Together, these data indicate that the 10 tobacco chitinases represent complementary enzymes which may have synergistic effects on their substrates. This paper discusses their implication in plant defense by attacking pathogen's structural components and in plant development by maturing signal molecules.

N-glycosylated proteins are involved in efficient internalization of Klebsiella pneumoniae by cultured human epithelial cells.

Klebsiella pneumoniae obtained from patients with urinary tract infections is able to invade cultured human epithelial cells. The internalization process is dependent upon both microfilaments and microtubules. To better understand the interaction of these invasive bacteria with the host cell receptor(s), bladder, lung, and ileocecal epithelial cells were infected with K. pneumoniae in the presence of various lectins possessing multiple glycan specificities. It was found that the N-acetylglucosamine (GlcNAc)-specific lectins concanavalin A, Datura stramonium agglutinin, and wheat germ agglutinin significantly inhibited the invasion of K. pneumoniae into these cells but did not interfere with the internalization of an invasive strain of Salmonella typhimurium. Conversely, internalization of K. pneumoniae but not S. typhimurium was also significantly inhibited when the bacteria were pretreated with GlcNAc or chitin hydrolysate, a GlcNAc polymer, prior to the gentamicin invasion assay. Other carbohydrates such as glucose, galactose, mannose, fucose, and N-acetylneuraminic acid had no inhibitory effects on K. pneumoniae uptake. Furthermore, internalization of K. pneumoniae but not S. typhimurium by HCT8 cells was also significantly inhibited when eukaryotic protein glycosylation was interrupted by tunicamycin or when host N-linked surface glycans were removed by pretreatment with N-glycosidase F. These studies suggest that a N-glycosylated protein receptor is involved in the internalization of K. pneumoniae by human epithelial cells in vitro. The results also indicate that internal GlcNAc residues might be a carbohydrate component of the receptor.

Beauveria bassiana submerged conidia production in a defined medium containing chitin, two hexosamines or glucose

Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH4Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 105/ ml after two days in GlcNAc medium as compared to 8.9 × 105/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 106/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.

Insect and fungal chitin synthetase activity: Specificity of lectins as enhancers and nucleoside peptides as inhibitors

Tribolium castaneum chitin synthetase (CS) activity is stimulated by chitooligosaccharide-binding lectins by up to 2.5-fold in the presence of N-acetyl- d-glucosamine (GlcNAc) and up to 3.5-fold in the absence of GlcNAc. Lectins of particularly high stimulating activity are those obtained from Triticum vulgaris and Phytolacca americana, but not those derived from eight other species with no specificity for GlcNAc. T. castaneum CS is inhibited 50% by 0.2 μ M nikkomycin Z (NZ) and the inhibitory effect is not alleviated by stimulating lectins. The CS of Neurospora crassa differs from that of T. castaneum by its lower sensitivity to inhibition by NZ and lesser stimulatory effects of lectins. Apparently the lectins do not act at the active site of Tribolum CS but may instead facilitate the detachment of GlcNAc polymers, thereby enhancing incorporation of label from uridine 5′-diphospho-[ 3H]GlcNAc.