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Sun F.,Virginia Polytechnic Institute and State University | Kale S.D.,Virginia Polytechnic Institute and State University | Azurmendi H.F.,Virginia Polytechnic Institute and State University | Azurmendi H.F.,Laboratory of Bacterial Polysaccharides | And 4 more authors.
Molecular Plant-Microbe Interactions

Oomycetes such as Phytophthora sojae employ effector proteins that enter plant cells to facilitate infection. Entry of some effector proteins is mediated by RxLR motifs in the effectors and phosphoinositides (PIP) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effectors involved in PIP recognition. We have structurally and functionally characterized the P. s o j a e effector, avirulence homolog-5 (Avh5). Using nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that Avh5 is helical in nature, with a long N-terminal disordered region. NMR titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate, directly identified the ligand-binding residues. A C-terminal lysine-rich helical region (helix 2) was the principal lipid-binding site, with the N-terminal RxLR (RFLR) motif playing a more minor role. Mutations in the RFLR motif affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Mutations in the RFLR motif or in the basic region both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. Based on these findings, we propose a model where Avh5 interacts with PtdIns(3)P through its C terminus, and by binding of the RFLR motif, which promotes host cell entry. © 2013 The American Phytopathological Society. Source

Bush C.A.,University of Maryland Baltimore County | Yang J.,U.S. National Institutes of Health | Yang J.,CAS Institute of Microbiology | Yu B.,Laboratory of Bacterial Polysaccharides | And 2 more authors.
Journal of Bacteriology

Structural characterization of Streptococcus pneumoniae capsular polysaccharides (CPS) is a prerequisite for unraveling both antigenic and genetic relationships that exist between different serotypes. In the current study, comparative structural studies of S. pneumoniae CPS serogroup 10 (CPS10) were extended to include genetically related S. pneumoniae CPS34, CPS39, and CPS47F. High-resolution heteronuclear nuclear magnetic resonance (NMR) spectroscopy confirmed the published structure of CPS34 and, in conjunction with glycosyl composition analyses, revealed the following repeat unit structures of the other serotypes, which have not been previously characterized: Common and unique structural features of these polysaccharides, including different positions of O-acetylation, were unambiguously associated with specific genes in each corresponding cps locus. The only exception involved the gene designated wcrC, which is associated with the α1-2 transfer of Gal pyranoside (Galp) to ribitol-5-phosphate in the synthesis of CPS10A, CPS47F, and CPS34 but with α1-1 transfer of Gal to ribitol-5-phosphate in the synthesis of CPS39. The corresponding gene in the cps39 locus, although related to wcrC, more closely resembled a previously identified gene (i.e., wefM) of Streptococcus oralis that is associated with α1-1 transfer of Galp to ribitol-5-phosphate. These and other recent findings identify linkages from α-Galp to ribitol-5-phosphate and from this residue to adjacent Gal furanoside (Galf) as important sites of CPS structural and genetic diversity. © 2014, American Society for Microbiology. Source

Yu B.,Laboratory of Bacterial Polysaccharides | Van Ingen H.,University Utrecht | Freedberg D.I.,Laboratory of Bacterial Polysaccharides
Journal of Magnetic Resonance

Strong 1H-1H coupling can significantly reduce the accuracy of 1JCH measured from frequency differences in coupled HSQC spectra. Although accurate 1JCH values can be extracted from spectral simulation, it would be more convenient if the same accurate 1JCH values can be obtained experimentally. Furthermore, simulations reach their limit for residual dipolar coupling (RDC) measurement, as many significant, but immeasurable RDCs are introduced into the spin system when a molecule is weakly aligned, thus it is impossible to have a model spin system that truly represents the real spin system. Here we report a new J modulated method, constant-time INEPT CT-HSQC (CTi-CT-HSQC), to accurately measure one-bond scalar coupling constant and RDCs without strong coupling interference. In this method, changing the spacing between the two 180° pulses during a constant time INEPT period selectively modulates heteronuclear coupling in quantitative J fashion. Since the INEPT delays for measuring one-bond carbon-proton spectra are short compared to 3JHH, evolution due to (strong) 1H-1H coupling is marginal. The resulting curve shape is practically independent of 1H-1H coupling and only correlated to the heteronuclear coupling evolution. Consequently, an accurate 1JCH can be measured even in the presence of strong coupling. We tested this method on N-acetyl-glucosamine and mannose whose apparent isotropic 1JCH values are significantly affected by strong coupling with other methods. Agreement to within 0.5 Hz or better is found between 1JCH measured by this method and previously published simulation data. We further examined the strong coupling effects on RDC measurements and observed an error up to 100% for one bond RDCs using coupled HSQC in carbohydrates. We demonstrate that RDCs can be obtained with higher accuracy by CTi-CT-HSQC, which compensates the limitation of simulation method. © 2013 Elsevier Inc. All rights reserved. Source

Yu B.,Laboratory of Bacterial Polysaccharides | Van Ingen H.,University of Toronto | Van Ingen H.,University Utrecht | Vivekanandan S.,Laboratory of Bacterial Polysaccharides | And 4 more authors.
Journal of Magnetic Resonance

J couplings are essential for measuring RDCs (residual dipolar couplings), now routinely used to deduce molecular structure and dynamics of glycans and proteins. Accurate measurement of 1J CH is critical for RDCs to reflect the true structure and dynamics in the molecule of interest. We report noticeable discrepancies between 1J CH values measured with HSQC type pulse sequences in the 1H dimension from those measured in the 13C dimension for 17 sugars and show that these discrepancies arise from strong scalar coupling. In order to determine how to minimize errors in measuring 1J CH, we analyze the strong coupling effects in detail using the product operator-formalism and spectral simulations based on the solution of the Liouville equation (not considering relaxation effects) in the presence of strong coupling. We report that the apparent 1J CH measured with 2D HSQC-based sequences in either dimension can be in error by up to 4 Hz and that the values measured in the 1H dimension can disagree with those in the 13C dimension by up to 7 Hz. We demonstrate that spectral simulations can reproduce the errors induced by strong coupling and that these can be used to extract true 1J CH values. We find that the 1J CH values measured using a modified Z-filtered coupled HSQC are still affected by strong coupling. We conclude that spectral simulation yields accurate 1J CH with errors as low as 1% in the presence of strong coupling. © 2011 Elsevier Inc. All rights reserved. Source

McCarthy P.C.,Laboratory of Bacterial Polysaccharides | Saksena R.,Laboratory of Bacterial Polysaccharides | Peterson D.C.,Laboratory of Bacterial Polysaccharides | Lee C.-H.,Laboratory of Bacterial Polysaccharides | And 3 more authors.
Glycoconjugate Journal

Vaccination with meningococcal glycoconjugate vaccines has decreased the incidence of invasive meningitis worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures. Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined vaccine candidates by chemoenzymatic synthesis. Chemically synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. These resulting oligosialic acids have the same α(2-9) sialic acid repeat structure as Neisseria polysaccharide capsule with the addition of a conjugatable azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor CMP-9-deoxy-NeuNAc. Polymers with estimated DP < 47 (median DP 25) and DP < 100 (median DP 51) were produced. The receptor binding domain of the tetanus toxin protein (TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MSE analysis of peptides. Oligosialic acid azides were conjugated to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP < 47 and DP < 100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates. © 2013 Springer Science+Business Media New York (outside the USA). Source

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