Density Variant Glycan Microarray for Evaluating Cross-Linking of Mucin-like Glycoconjugates by Lectins

Interactions of mucin glycoproteins with cognate receptors are dictated by the structures and spatial organization of glycans that decorate the mucin polypeptide backbone. The glycan-binding proteins, or lectins, that interact with mucins are often oligomeric receptors with multiple ligand binding domains. In this work, we employed a microarray platform comprising synthetic glycopolymers that emulate natural mucins arrayed at different surface densities to evaluate how glycan valency and spatial separation affect the preferential binding mode of a particular lectin. We evaluated a panel of four lectins (Soybean agglutinin (SBA), <i>Wisteria floribunda</i> lectin (WFL), <i>Vicia villosa</i>-B-4 agglutinin (VVA), and <i>Helix pomatia</i> agglutin (HPA)) with specificity for α-<i>N</i>-acetylgalactosamine (α-GalNAc), an epitope displayed on mucins overexpressed in many adenocarcinomas. While these lectins possess the ability to agglutinate A₁-blood cells carrying the α-GalNAc epitope and cross-link low valency glycoconjugates, only SBA showed a tendency to form intermolecular cross-links among the arrayed polyvalent mucin mimetics. These results suggest that glycopolymer microarrays can reveal discrete higher-order binding preferences beyond the recognition of individual glycan epitopes. Our findings indicate that glycan valency can set thresholds for cross-linking by lectins. More broadly, well-defined synthetic glycopolymers enable the integration of glycoconjugate structural and spatial diversity in a single microarray screening platform

Sulfur-containing metabolites from <i>Mtb</i>.

<p>(A) Sulfated compounds, clockwise from top: S881, Sulfolipid-1, SL₁₂₇₈. (B) Reduced sulfur compounds, from top to bottom: MSH, cysteine, methionine.</p

A Bioorthogonal Quadricyclane Ligation

New additions to the bioorthogonal chemistry compendium can advance biological research by enabling multiplexed analysis of biomolecules in complex systems. Here we introduce the quadricyclane ligation, a new bioorthogonal reaction between the highly strained hydrocarbon quadricyclane and Ni bis(dithiolene) reagents. This reaction has a second-order rate constant of 0.25 M^–1 s^–1, on par with fast bioorthogonal reactions of azides, and proceeds readily in aqueous environments. Ni bis(dithiolene) probes selectively labeled quadricyclane-modified bovine serum albumin, even in the presence of cell lysate. We have demonstrated that the quadricyclane ligation is compatible with, and orthogonal to, strain-promoted azide–alkyne cycloaddition and oxime ligation chemistries by performing all three reactions in one pot on differentially functionalized protein substrates. The quadricyclane ligation joins a small but growing list of tools for the selective covalent modification of biomolecules

New Aldehyde Tag Sequences Identified by Screening Formylglycine Generating Enzymes <i>in Vitro</i> and <i>in Vivo</i>

New Aldehyde Tag Sequences Identified by Screening Formylglycine Generating Enzymes <i>in Vitro</i> and <i>in Vivo</i

Venn diagram illustrating the convergent transcriptional regulation of <i>Mtb</i> sulfur metabolism genes by various conditions of environmental stress.

<p>Venn diagram illustrating the convergent transcriptional regulation of <i>Mtb</i> sulfur metabolism genes by various conditions of environmental stress.</p

<i>In Vivo</i> Imaging of <i>Caenorhabditis elegans</i> Glycans

The nematode <i>Caenorhabditis elegans</i> is an excellent model organism for studies of glycan dynamics, a goal that requires tools for imaging glycans <i>in vivo</i>. Here we applied the bioorthogonal chemical reporter technique for the molecular imaging of mucin-type O-glycans in live <i>C. elegans</i>. We treated worms with azidosugar variants of <i>N</i>-acetylglucosamine (GlcNAc), <i>N</i>-acetylgalactosamine (GalNAc), and <i>N</i>-acetylmannosamine (ManNAc), resulting in the metabolic labeling of their cell-surface glycans with azides. Subsequently, the worms were reacted <i>via</i> copper-free click reaction with fluorophore-conjugated difluorinated cyclooctyne (DIFO) reagents. We identified prominent localization of mucins in the pharynx of all four larval stages, in the adult hermaphrodite pharynx, vulva and anus, and in the tail of the adult male. Using a multicolor, time-resolved imaging strategy, we found that the distribution and dynamics of the glycans varied anatomically and with respect to developmental stage

Sulfatase biochemistry.

<p>(A) Sulfatase-catalyzed hydrolysis of a sulfated metabolite. (B) Modification of the active site cysteine of type I sulfatases by FGE.</p

Feedback regulation of RsrA activity and SigR (σ^R)-mediated transcription by MSH in <i>S. coelicolor</i>.

<p>The anti-sigma factor RsrA binds the alternative sigma factor σ^R in its reduced state (RsrA^red), preventing the association of σ^R with RNA polymerase (RNA Pol). Under conditions of oxidative stress (e.g., hydrogen peroxide or diamide treatment), RsrA is oxidized (RsrA^ox) and no longer binds σ^R, enabling it to form a complex with RNA polymerase, which induces the transcription of MSH biosynthetic genes. The reduction of oxidized RsrA by MSH facilitates sequestration of σ^R, which, in turn, suppresses MSH biosynthesis. Importantly, when oxidizing or alkylating agents deplete MSH levels, RsrA remains oxidized, which stimulates σ^R-mediated transcription and MSH biosynthesis <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002036#ppat.1002036-Park2" target="_blank">[45]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002036#ppat.1002036-Newton3" target="_blank">[67]</a>.</p

Sulfur metabolism genes from <i>M. tuberculosis</i> induced by various conditions of environmental stress.

a<p>The <i>cysN</i> and <i>cysC</i> genes of <i>Mtb</i> are fused into a single, bifunctional <i>cysNC</i> gene <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002036#ppat.1002036-Pinto1" target="_blank">[22]</a>. However, the transcript is often reported as <i>cysN</i> in microarray data.</p><p>SDS, sodium dodecyl sulfate.</p

A Hydrophilic Azacyclooctyne for Cu-Free Click Chemistry

Biomolecules labeled with azides can be detected through Cu-free click chemistry with cyclooctyne probes, but their intrinsic hydrophobicity can compromise bioavailability. Here, we report the synthesis and evaluation of a novel azacyclooctyne, 6,7-dimethoxyazacyclooct-4-yne (DIMAC). Generated in nine steps from a glucose analogue, DIMAC reacted with azide-labeled proteins and cells similarly to cyclooctynes. However, its superior polarity and water solubility reduced nonspecific binding, thereby improving the sensitivity of azide detection