Free N-glycans already occur at an early stage of seed development. (73/1610)

As a part of our studies to elucidate the physiological significance of free N-glycans in differentiating or growing plant cells, we first demonstrate that two kinds of free N-glycans already occur at an early stage of seed development. In this report, we used the developing Ginkgo biloba seeds as a model plant, since we have already revealed a functional feature of the Ginkgo endo-beta-N-acetylglucosaminidase and structural features of N-glycans linked to storage glycoproteins in the developing seeds [Kimura, Y. et al. (1998) Biosci. Biotechnol. Biochem. 62, 253-261; Kimura, Y. and Matsuo, S. (2000) Biosci. Biotechnol. Biochem. 64, 562-568]. The structures of free N-glycans, which were determined by a combination of ESI-MS, sequential a-mannosidase digestions, partial acetolysis, and two dimensional sugar chain map, fell into two categories. One dominant species is a high-mannose type structure having one GlcNAc residue at the reducing end (Man(9-5)GlcNAc(1)). The concentration of this type of free glycan (as the pyridylaminated derivatives) is about 2.2 nmol in 1 g fresh weight. The detailed structural analysis revealed that the high-mannose type structures have a common core unit; Manalpha1-6(Man1-3)Manalpha1-6(Manalpha1-3)Ma nbeta1-4GlcNAc. The other minor species of free N-glycans is the plant complex type structure having an N-acetylchitobiose unit at the reducing end (Man(3)Xyl(1)Fuc(1)GlcNAc(2)). The concentration of this type of free glycan (as the pyridylaminated derivative) was about 75 pmol in 1 g fresh weight.  (+info)

Role of vacA and cagA in Helicobacter pylori inhibition of mucin synthesis in gastric mucous cells. (74/1610)

The aim of this study was to investigate the effect of Helicobacter pylori on the function of gastric mucous cells. H. pylori (10(4) to 10(7) CFU/well) was incubated with the mucin-producing gastric cell line HM02 for 12 and 24 h. Mucin synthesis and secretion were determined by the incorporation of D-N-[acetyl-(14)C]glucosamine into intracellular and released high-molecular-weight glycoproteins. cagA-positive, cytotoxin-producing and non-cytotoxin-producing H. pylori strains impaired the incorporation of D-N-[acetyl-(14)C]glucosamine into intracellular glycoproteins. Significant inhibition of mucin synthesis was noted after 12 and 24 h of cocultivation with a bacterial load of >/=10(5) bacteria (bacterium/cell ratio = 0.25). The cagA-positive, cytotoxin-producing strains (HP64, HP57, and HP87) caused significantly stronger inhibition of intracellular mucin synthesis than the cagA-positive, non-cytotoxin-producing strains (HP05, HP83, and HP84). The cagA-negative, non-cytotoxin-producing strains (HP01, HP04, and HP85) did not affect intracellular mucin synthesis. The results indicate that H. pylori directly impairs mucin synthesis in gastric mucous cells and that cytotoxic cagA-positive strains cause more profound inhibition of mucin synthesis. We suggest that the increased inhibitory effect of cagA-positive, cytotoxin-producing strains on mucin synthesis can be considered one possible factor responsible for the increased risk of developing peptic ulceration with these H. pylori strains.  (+info)

Location of the glucuronosyltransferase domain in the heparan sulfate copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants. (75/1610)

Heparan sulfate formation occurs by the copolymerization of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues. Recent studies have shown that these reactions are catalyzed by a copolymerase encoded by EXT1 and EXT2, members of the exostosin family of putative tumor suppressors linked to hereditary multiple exostoses. Previously, we identified a collection of Chinese hamster ovary cell mutants (pgsD) that failed to make heparan sulfate (Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massague, J., Lindahl, U., and Esko, J. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2267-2271). Here, we show that pgsD mutants contain mutations that either alter GlcA transferase activity selectively or that affect both GlcNAc and GlcA transferase activities. Expression of EXT1 corrects the deficiencies in the mutants, whereas EXT2 and the related EXT-like cDNAs do not. Analysis of the EXT1 mutant alleles revealed clustered missense mutations in a domain that included a (D/E)X(D/E) motif thought to bind the nucleotide sugar from studies of other transferases. These findings provide insight into the location of the GlcA transferase subdomain of the enzyme and indicate that loss of the GlcA transferase domain may be sufficient to cause hereditary multiple exostoses.  (+info)

Ligand-mediated retargeting of recombinant adenovirus for gene transfer in vivo. (76/1610)

The development of efficient and safe methods for in vivo gene transfer is central to the success of gene therapy. Recombinant adenoviral vectors, although highly efficient, are limited by the host immune response, potential safety hazards due to obligatory cotransfer of viral proteins, and their broad tissue tropism. Here, we demonstrate in an animal model that host range and tissue tropism of a recombinant adenovirus from a distant species can be modified by complexing adenovirus with a cell-specific ligand. Thus, a replication-deficient lacZ recombinant human adenovirus, which naturally does not infect avian cells, allowed highly efficient and specific gene transfer to the liver of ducks in vivo when complexed with N-acetylglucosamine, a ligand for the chicken hepatic lectin. This combination of ligand-mediated receptor targeting with adenoviral uptake and intracellular processing of a given gene represents a novel approach to gene therapy of inherited and acquired liver diseases.  (+info)

Cytotoxic mAb from rheumatic carditis recognizes heart valves and laminin. (77/1610)

Anti-streptococcal antibodies cross-reactive with N-acetyl-betaD-glucosamine (GlcNAc) and myosin are present in the sera of patients with rheumatic fever (RF). However, their role in tissue injury is not clear. In this study, we show that anti-GlcNAc/anti-myosin mAb 3.B6 from a rheumatic carditis patient was cytotoxic for human endothelial cell lines and reacted with human valvular endothelium and underlying basement membrane. Reactivity of mAb 3.B6 with the valve was inhibited by human cardiac myosin > laminin > GlcNAc. The mAb 3.B6 epitopes were localized in fragments of human cardiac myosin, including heavy meromyosin (HMM), the S1 subfragment, and two light meromyosin (LMM) peptides containing amino acid sequences KEALISSLTRGKLTYTQQ (LMM 1) and SERVQLLHSQNTSLINQK (LMM 33). A novel feature of mAb 3.B6 was its reactivity with the extracellular matrix protein laminin, which may explain its reactivity with the valve surface. A laminin A-chain peptide (HTQNT) that includes homology to LMM33 inhibited the reactivity of mAb 3.B6 with human valve. These data support the hypothesis that cross-reactive antibodies in rheumatic carditis cause injury at the endothelium and underlying matrix of the valve.  (+info)

Structural basis for chitin recognition by defense proteins: GlcNAc residues are bound in a multivalent fashion by extended binding sites in hevein domains. (78/1610)

BACKGROUND: Many plants respond to pathogenic attack by producing defense proteins that are capable of reversible binding to chitin, a polysaccharide present in the cell wall of fungi and the exoskeleton of insects. Most of these chitin-binding proteins include a common structural motif of 30 to 43 residues organized around a conserved four-disulfide core, known as the 'hevein domain' or 'chitin-binding' motif. Although a number of structural and thermodynamic studies on hevein-type domains have been reported, these studies do not clarify how chitin recognition is achieved. RESULTS: The specific interaction of hevein with several (GlcNAc)(n) oligomers has been studied using nuclear magnetic resonance (NMR), analytical ultracentrifugation and isothermal titration microcalorimetry (ITC). The data demonstrate that hevein binds (GlcNAc)(2-4) in 1:1 stoichiometry with millimolar affinity. In contrast, for (GlcNAc)(5), a significant increase in binding affinity is observed. Analytical ultracentrifugation studies on the hevein-(GlcNAc)(5,8) interaction allowed detection of protein-carbohydrate complexes with a ratio of 2:1 in solution. NMR structural studies on the hevein-(GlcNAc)(5) complex showed the existence of an extended binding site with at least five GlcNAc units directly involved in protein-sugar contacts. CONCLUSIONS: The first detailed structural model for the hevein-chitin complex is presented on the basis of the analysis of NMR data. The resulting model, in combination with ITC and analytical ultracentrifugation data, conclusively shows that recognition of chitin by hevein domains is a dynamic process, which is not exclusively restricted to the binding of the nonreducing end of the polymer as previously thought. This allows chitin to bind with high affinity to a variable number of protein molecules, depending on the polysaccharide chain length. The biological process is multivalent.  (+info)

Evidence for the participation of beta-hexosaminidase in human sperm-zona pellucida interaction in vitro. (79/1610)

Mammalian sperm-zona pellucida (ZP) interaction is mediated by sperm lectin-like proteins and ZP glycoproteins. We have previously reported the participation of binding sites for N-acetylglucosamine (GlcNAc) residues in human sperm function, including sperm interaction with the ZP. Additionally, previous results from our laboratory suggested that some of these events may be mediated by the glycosidase N-acetylglucosaminidase (beta-hexosaminidase, Hex, in mammals). In this study, we report the possible participation of Hex in human sperm-ZP interaction. Human recombinant Hex (hrHex) was obtained by expression in a stable transfected CHO cell line. When the recombinant enzyme was present during hemizona (HZ) assays, the number of sperm bound per HZ was significantly reduced. The same result was obtained when HZ were preincubated with hrHex. Additionally, the presence of a Hex-specific substrate during the HZ assay produced the same inhibitory effect. These results suggest the participation of a sperm Hex in the interaction with human ZP in vitro.  (+info)

Chitin catabolism in the marine bacterium Vibrio furnissii. Identification, molecular cloning, and characterization of A N, N'-diacetylchitobiose phosphorylase. (80/1610)

The major product of bacterial chitinases is N,N'-diacetylchitobiose or (GlcNAc)(2). We have previously demonstrated that (GlcNAc)(2) is taken up unchanged by a specific permease in Vibrio furnissii (unlike Escherichia coli). It is generally held that marine Vibrios further metabolize cytoplasmic (GlcNAc)(2) by hydrolyzing it to two GlcNAcs (i.e. a "chitobiase "). Here we report instead that V. furnissii expresses a novel phosphorylase. The gene, chbP, was cloned into E. coli; the enzyme, ChbP, was purified to apparent homogeneity, and characterized kinetically. The DNA sequence indicates that chbP encodes an 89-kDa protein. The enzymatic reaction was characterized as follows. (GlcNAc)(2)+P(i) GlcNAc-alpha-1-P+GlcNAc K'(cq)=1.0+/-0.2 Reaction 1 The K(m) values for the four substrates were in the range 0.3-1 mm. p-Nitrophenyl-(GlcNAc)(2) was cleaved at 8.5% the rate of (GlcNAc)(2), and p-nitrophenyl (PNP)-GlcNAc was 36% as active as GlcNAc in the reverse direction. All other compounds tested displayed +info)