Analysis of gene mutation in plant cell wall by dielectric relaxation

Arabidopsis Thaliana is a plant composed mainly of cellulose and lignin. Geneticists need techniques able to make differences at the molecular level between modified plants (DML6, CAD C/D) and non-modified ones. Thermo-stimulated current (TSC) analysis is a promising route to identify gene mutations. For the non-modified plant, at low temperatures, TSC thermograms highlight three dielectric relaxation modes. From −150 to −110 ◦C, γCellulose is attributed to CH2OH and –OH groups of cellulose. Between −110 and −80 ◦C, βLignin is detected. From −80 to −40 ◦C, βCellulose is characteristic of the molecular mobility of glycosidic linkages. For the CAD C/D modified plants, only γCellulose and βLignin are observed; due to analogous enthalpy values, those modes have the same molecular origin as in the non-modified plant. So, the βLignin mode is associated with the molecular mobility of the lignin-OH groups. The CAD C/D gene mutation changes the chemical structure of lignin, which promotes hydrogen bonds in the network and inhibits molecular mobility of glucosidic rings. It is also interesting to note that the DML6 gene mutation induces a higher cooperativity of this βCellulose relaxation than in wild vegetal composites. In fact, this mutation promotes molecular mobility of glycosidic rings thanks to β1–4 glycosidic linkages

Remote participation during glycosylation reactions of galactose building blocks: Direct evidence from cryogenic vibrational spectroscopy

The stereoselective formation of 1,2‐cis‐glycosidic bonds is challenging. However, 1,2‐cis‐selectivity can be induced by remote participation of C4 or C6 ester groups. Reactions involving remote participation are believed to proceed via a key ionic intermediate, the glycosyl cation. Although mechanistic pathways were postulated many years ago, the structure of the reaction intermediates remained elusive owing to their short‐lived nature. Herein, we unravel the structure of glycosyl cations involved in remote participation reactions via cryogenic vibrational spectroscopy and first principles theory. Acetyl groups at C4 ensure α‐selective galactosylations by forming a covalent bond to the anomeric carbon in dioxolenium‐type ions. Unexpectedly, also benzyl ether protecting groups can engage in remote participation and promote the stereoselective formation of 1,2‐cis‐glycosidic bonds

Validation of the 3-under-2 principle of cell wall growth in Gram-positive bacteria by simulation of a simple coarse-grained model

The aim of this work is to propose a first coarse-grained model of Bacillus subtilis cell wall, handling explicitly the existence of multiple layers of peptidoglycans. In this first work, we aim at the validation of the recently proposed "three under two" principle.Comment: Revised introduction, results unchange

Differential Dynamics at Glycosidic Linkages of an Oligosaccharide as Revealed by 13C NMR Spin Relaxation and Stochastic Modeling

Among biomolecules, carbohydrates are unique in that not only can linkages be formed through different positions but the structures may also be branched. The trisaccharide \uf062-D-Glcp-(1\uf0ae3)[\uf062-D-Glcp-(1\uf0ae2)]-\uf061-D-Manp-OMe represents a model of a branched vicinally disubstituted structure. A 13C site-specific isotopologue with labeling in each of the two terminal glucosyl residues enabled acquisition of high-quality 13C NMR relaxation parameters T1, T2 and heteronuclear NOE, with standard deviations of \uf0a3 0.5%. For interpretation of the experimental NMR data a diffusive chain model was used in which the dynamics of the glycosidic linkages is coupled to the global reorientation motion of the trisaccharide. Brownian dynamics simulations relying on the potential of mean force at the glycosidic linkages were employed to evaluate spectral densities of the spin probes. Calculated NMR relaxation parameters showed very good agreement with experimental data, deviating < 3%. The resulting dynamics is described by correlation times of 196 ps and 174 ps for the \uf062-(1\uf0ae2)- and \uf062-(1\uf0ae3)-linked glucosyl residues, respectively, i.e., different and linkage dependent. Notably, the devised computational protocol was performed without any fitting of parameters

ApuA, a multifunctional x-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus

We have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved -amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal -amylase domain of ApuA was shown to have -(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase -(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host

Fragmentation characteristics of glycopeptides

Mass spectrometric analysis of glycopeptides is an emerging strategy for analysis of glycosylation patterns. Here we present an approach using energy resolved collision induced decomposition (CID) spectra to determine structural features of glycopeptides. Fragmentation of multiply protonated glycopeptides proceeds by a series of competing charge separation processes by cleavage of a glycosidic bond, each producing two charged products: a singly charged, “B” type sugar (oxonium) ion, and a complementary high mass fragment. Energy requirements (activation energies) of these processes are similar to each other, and are far less, than that required for peptide fragmentation. At higher collision energies these first generation products fragment further, yielding a complex fragmentation pattern. Analysis of low energy spectra (those corresponding to ca. 50% survival yield) are straightforward; the ions observed correspond to structural features present in the oligosaccharide, and are not complicated by consecutive reactions. This makes it feasible to identify and distinguish antenna- and core-fucosylated isomers; antenna fucosylation usually suggests presence of the Lewis-X antigen. In general, analysis of the triply protonated molecules are most advantageous, where neutral losses and monosaccharide oxonium ion formation are less abundant

Substrate-Assisted Catalysis Unifies Two Families of Chitinolytic Enzymes

Hen egg-white lysozyme has long been the paradigm for enzymatic glycosyl hydrolysis with retention of configuration, with a protonated carboxylic acid and a deprotonated carboxylate participating in general acid-base catalysis. In marked contrast, the retaining chitin degrading enzymes from glycosyl hydrolase families 18 and 20 all have a single glutamic acid as the catalytic acid but lack a nucleophile on the enzyme. Both families have a catalytic (βα)8-barrel domain in common. X-ray structures of three different chitinolytic enzymes complexed with substrates or inhibitors identify a retaining mechanism involving a protein acid and the carbonyl oxygen atom of the substrate’s C2 N-acetyl group as the nucleophile. These studies unambiguously demonstrate the distortion of the sugar ring toward a sofa conformation, long postulated as being close to that of the transition state in glycosyl hydrolysis.

Huisgen-based conjugation of water-soluble porphyrins to deprotected sugars: Towards mild strategies for the labelling of glycans

Fully deprotected alkynyl-functionalised mono- and oligosaccharides undergo CuAAC-based conjugation with water-soluble porphyrin azides in aqueous environments. The mild reaction conditions are fully compatible with the presence of labile glycosidic bonds. This approach provides an ideal strategy to conjugate tetrapyrroles to complex carbohydrates

From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals (sugar coding). Here, we test the hypothesis that the common N-glycan modifications by core fucosylation and introduction of the bisecting N-acetylglucosamine moiety have long-range effects with functional consequences. Molecular dynamics simulations indicate a shift in conformational equilibria between linear extension or backfolding of the glycan antennae upon substitution. We also present a new fingerprint-like mode of presentation for this multi-parameter system. In order to delineate definite structure-function relationships, we strategically combined chemoenzymatic synthesis with bioassaying cell binding and the distribution of radioiodinated neoglycoproteins in vivo. Of clinical relevance, tailoring the core region affects serum clearance markedly, e. g., prolonging circulation time for the neoglycoprotein presenting the N-glycan with both substitutions. alpha 2,3-Sialylation is another means toward this end, similarly seen for type II branching in triantennary N-glycans. This discovery signifies that rational glycoengineering along the given lines is an attractive perspective to optimize pharmacokinetic behavior of glycosylated pharmaproteins. Of general importance for the concept of the sugar code, the presented results teach the fundamental lesson that N-glycan core substitutions convey distinct characteristics to the concerned oligosaccharide relevant for cis and trans biorecognition processes. These modifications are thus molecular switches