Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility.
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S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S4-RNase carries various types of sugar chains, including plant-specific ones with beta1-->2-linked xylose and alpha1-->3-linked fucose residues. More than 70% of the total N-glycans of S4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAcbeta1-->4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s). (+info)
N-acetylneuraminic acid transport by Streptococcus oralis strain AR3.
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Streptococcus oralis has emerged as one of the most important organisms of the viridans streptococcus group in terms of infections and is recognised as an agent of infective endocarditis and, in immunocompromised patients, septicaemia. The mechanisms by which this organism proliferates in vivo are unknown. However, host-derived sialic acids -- including N-acetylneuraminic acid (NeuNAc) which is present in serum and cell-associated glycoproteins -- are a potential source of fermentable carbohydrate for bacterial proliferation, especially for sialidase-producing bacteria, including S. oralis. To further elucidate the role of NeuNAc in supporting growth, this study determined the ability of S. oralis strain AR3 (isolated from a patient with infective endocarditis) to transport NeuNAc and characterised the transport system. The transport of [14C]-labelled NeuNAc into S. oralis was monitored and this transport system was induced by growth of the bacteria in the presence of the N-acetylated sugars NeuNAc, N-acetylglucosamine and N-acetylmannosamine. The transport system followed typical Michaelis-Menten kinetics, with a Km of 21.0 microM and a Vmax of 2.65 nmoles of NeuNAc transported/min/mg of dry cell mass. NeuNAc transport was inhibited by the presence of exogenous N-glycolylneuraminic acid, a related sialic acid. Chlorhexidine, NaF and 2,4-dinitrophenol were potent inhibitors of the transport system, suggesting that the uptake of NeuNAc occurs via a proton motive force-dependent permease system. This is the first report of the mechanism by which NeuNAc transport occurs in pathogenic streptococci. This transport process may have relevance to the acquisition of a source of fermentable carbohydrate and thus bacterial proliferation in vivo. (+info)
Resting and cytokine-stimulated human small airway epithelial cells recognize and engulf apoptotic eosinophils.
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Eosinophils, which are prominent cells in asthmatic inflammation, undergo apoptosis and are recognized and engulfed by phagocytic macrophages in vitro. We have examined the ability of human small airway epithelial cells (SAEC) to recognize and ingest apoptotic human eosinophils. Cultured SAEC ingested apoptotic eosinophils but not freshly isolated eosinophils or opsonized erythrocytes. The ability of SAEC to ingest apoptotic eosinophils was enhanced by interleukin-1alpha (IL-1alpha) or tumor necrosis factor alpha (TNFalpha) in a time- and concentration-dependent fashion. IL-1alpha was found to be more potent than TNFalpha and each was optimal at 10(-10) mol/L, with a significant (P <.05) effect observed at 1 hour postcytokine incubation that was maximal at 5 hours. IL-1alpha stimulation not only increased the number of SAEC engulfing apoptotic eosinophils, but also enhanced their capacity for ingestion. The amino sugars glucosamine, n-acetyl glucosamine, and galactosamine significantly inhibited uptake of apoptotic eosinophils by both resting and IL-1alpha-stimulated SAEC, in contrast to the parent sugars glucose, galactose, mannose, and fucose. Incubation of apoptotic eosinophils with the tetrapeptide RGDS, but not RGES, significantly inhibited their uptake by both resting and IL-1alpha-stimulated SAEC, as did monoclonal antibody against alphavbeta3 and CD36. Thus, SAEC recognize apoptotic eosinophils via lectin- and integrin-dependent mechanisms. These data demonstrate a novel function for human bronchial epithelial cells that might represent an important mechanism in the resolution of eosinophil-induced asthmatic inflammation. (+info)
Sequential biosynthesis of sulfated and/or sialylated Lewis x determinants by transferases of the human bronchial mucosa.
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The structural determination of sulfated carbohydrate chains from a cystic fibrosis patient respiratory mucins has shown that sulfation may occur either on the C-3 of the terminal Gal, or on the C-6 of the GlcNAc residue of a terminal N -acetyllactosamine unit. The two enzymes responsible for the transfer of sulfate from PAPS to the C-3 of Gal or to the C-6 of GlcNAc residues have been characterized in human respiratory mucosa. These two enzymes, in conjunction with fucosyl- and sialyltransferases, allow the synthesis of different sulfated epitopes such as 3-sulfo Lewis x (with a 3- O -sulfated Gal), 6-sulfo Lewis x and 6-sulfo-sialyl Lewis x (with a 6- O -sulfated GlcNAc). In the present study, the sequential biosynthesis of these epitopes has been investigated using microsomal fractions from human respiratory mucosa incubated with radiolabeled nucleotide-sugars or PAPS, and oligosaccharide acceptors, mostly prepared from human respiratory mucins. The structures of the radiolabeled products have been determined by their coelution in HPAEC with known oligosaccharidic standards. In the biosynthesis of 6- O -sulfated carbohydrate chains by the human respiratory mucosa, the 6- O -sulfation of a terminal nonreducing GlcNAc residue precedes beta1-4-galactosylation, alpha2-3-sialylation (to generate 6-sulfo-sialyl- N -acetyllactosamine), and alpha1-3-fucosylation (to generate the 6-sulfo-sialyl Lewis x determinant). The 3- O -sulfation of a terminal N -acetyllactosamine may occur if this carbohydrate unit is not substituted. Once an N -acetyllactosamine unit is synthesized, alpha1-3-fucosylation of the GlcNAc residue to generate a Lewis x structure blocks any further substitution. Therefore, the present study defines the pathways for the biosynthesis of Lewis x, sialyl Lewis x, sulfo Lewis x, and 6-sulfo-sialyl Lewis x determinants in the human bronchial mucosa. (+info)
Cloning and expression of a human gene encoding an N-acetylgalactosamine-alpha2,6-sialyltransferase (ST6GalNAc I): a candidate for synthesis of cancer-associated sialyl-Tn antigens.
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The sialyl-Tn (sTn) antigen is a well known cancer-associated antigen, the expression of which is related to the prognosis of cancer patients. We aimed to isolate a human gene encoding an N -acetylgalactosamine alpha2,6-sialyltransferase which synthesizes sTn antigen, and to characterize the enzyme. Degenerate primers encoding sialyl motifs were used for the polymerase chain reaction to amplify complementary DNAs prepared from RNAs of human pyloric mucosae with intestinal metaplasia, which abundantly expressed sTn antigen, followed by screening of full-length cDNAs using the amplified DNA fragment as a probe. We isolated two human cDNA clones, long-form (2.46 kb) and short-form (2.23 kb) cDNAs. The former encodes an active enzyme with a predicted 600 amino acid sequence. The latter, a splice-variant of the long-form, encodes an inactive enzyme. HCT15 human colorectal cancer cells stably expressing the long-form cDNA expressed sTn epitopes on O -glycans. The long form cDNA was considered to encode a human homologue of chick ST6GalNAc I for the following reasons: (1) the putative amino acid sequence showed greater homology to that of chick ST6GalNAc I (55%) compared to other sialyltransferases, (2) it encodes the extraordinarily long stem region that is a typical feature of chick ST6GalNAc I, and (3) the substrate specificity was very similar to that of chick ST6GalNAc I. In situ hybridization demonstrated that the localization of transcripts correlated well with that of sTn antigen in gastric cancer cells and Goblet cells in intestinal metaplastic glands. Thus, we determined that the long-form cDNA of the human ST6GalNAc I gene encodes the probable candidate for the human sTn synthase(s). (+info)
Transposon-induced mutations in two loci of Listeria monocytogenes serotype 1/2a result in phage resistance and lack of N-acetylglucosamine in the teichoic acid of the cell wall.
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Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916DeltaE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected. (+info)
Regulation of the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol, feedback and product inhibition.
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The assembly of the core oligosaccharide region of asparagine-linked glycoproteins proceeds by means of the dolichol pathway. The first step of this pathway, the reaction of dolichol phosphate with UDP-GlcNAc to form N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-dolichol), is under investigation as a possible site of metabolic regulation. This report describes feedback inhibition of this reaction by the second intermediate of the pathway, N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-GlcNAc-P-P-dolichol), and product inhibition by GlcNAc-P-P-dolichol itself. These influences were revealed when the reactions were carried out in the presence of showdomycin, a nucleoside antibiotic, present at concentrations that block the de novo formation of GlcNAc-GlcNAc-P-P-dolichol but not that of GlcNAc-P-P-dolichol. The apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol under basal conditions were 4.4 and 2.8 microM, respectively. Inhibition was also observed under conditions where mannosyl-P-dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-P-dolichol; the apparent K(i) values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol were 2.2 and 11 microM, respectively. Kinetic analysis of the types of inhibition indicated competitive inhibition by GlcNAc-P-P-dolichol toward the substrate UDP-GlcNAc and non-competitive inhibition toward dolichol phosphate. Inhibition by GlcNAc-GlcNAc-P-P-dolichol was uncompetitive toward UDP-GlcNAc and competitive toward dolichol phosphate. A model is presented for the kinetic mechanism of the synthesis of GlcNAc-P-P-dolichol. GlcNAc-P-P-dolichol also exerts a stimulatory effect on the biosynthesis of Man-P-dol, i.e. a reciprocal relationship to that previously observed between these two intermediates of the dolichol pathway. This network of inhibitory and stimulatory influences may be aspects of metabolic control of the pathway and thus of glycoprotein biosynthesis in general. (+info)
The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences.
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Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC(50) (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase. (+info)