Receptor-Transduction-Functions-Sites-Glycoprotein-s
The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase.Rice BGlu1 β-glucosidase nucleophile mutant E386G is a glycosynthase that can synthesize p-nitrophenyl (pNP)-cellooligosaccharides of up to 11 residues. lacto-n-neotetraose -ray crystal structures of the E386G glycosynthase with and without α-glucosyl fluoride were solved and the α-glucosyl fluoride complex was found to contain an ordered water molecule near the position of the nucleophile of the BGlu1 native structure, which is likely to stabilize the departing fluoride. The structures of E386G glycosynthase in complexes with cellotetraose and cellopentaose confirmed that the side chains of N245, S334, and Y341 interact with glucosyl residues in cellooligosaccharide binding subsites +2, +3, and +4. Mutants in which these residues were replaced in BGlu1 β-glucosidase hydrolyzed cellotetraose and cellopentaose with k(cat) K(m) values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNP-cellooligosaccharides than do the E386G glycosynthase and its S334A mutant, suggesting that Y341 and N245 play important roles in the synthesis of long oligosaccharides.
X-ray structural studies revealed that cellotetraose binds to the Y341A mutant of the glycosynthase in a very different, alternative mode not seen in complexes with the E386G glycosynthase, possibly explaining the similar hydrolysis, but poorer synthesis of longer oligosaccharides by Y341 mutants.Pentavalent pneumococcal oligosaccharide conjugate vaccine PncCRM is well-tolerated and able to induce an antibody response in infants.BACKGROUND The emergence of resistant pneumococci makes the treatment of pneumococcal diseases difficult. The currently available polysaccharide vaccines have very limited efficacy in young children. The immunogenicity can be improved by covalent coupling to protein carriers as has been shown with Haemophilus METHODS Thirty healthy infants were immunized with a pneumococcal conjugate vaccine at 2, 4 and 6 months of age. Oligosaccharides were derived from capsular polysaccharides of types 6B, 14, 18C, 19F and 23F and conjugated to the nontoxic mutant diphtheria toxin CRM197. The final vaccine was a mixture of these conjugates, containing micrograms of each oligosaccharide.
The infants received simultaneously H. influenzae type b oligosaccharide-CRM197 conjugate vaccine. Serum samples were taken before each dose and 1 month after the third dose. Control material was composed of 25 serum samples taken from children of the same age without pneumococcal vaccination. Snag it now -linked immunosorbent assay was used to measure serum IgG anti-pneumococcal polysaccharide concentrations and radioimmunoassay for the serum Ig anti-H. influenzae type b concentrations.RESULTS PncCRM vaccine was well-tolerated.
Pneumococcal type 18C induced a significant antibody increase after the first dose, whereas the other five oligosaccharides, including H. influenzae type b oligosaccharides, induced an increase after the second or third dose. The specific IgG concentrations at 7 months of age were significantly higher among the vaccinated infants than in the controls for all the five pneumococcal types.CONCLUSIONS Pneumococcal oligosaccharide-CRM197 conjugate vaccine is able to induce an IgG serum response in infants and anti-pneumococcal antibody concentrations were significantly higher than in controls of same age.Lectin affinity high-performance liquid chromatography interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins.
The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides.
