Sequence-Defined Glycopolymer Segments Presenting Mannose: Synthesis and Lectin Binding Affinity

We present for the first time the synthesis of sequence-defined monodisperse glycopolymer segments via solid-phase polymer synthesis. Functional building blocks displaying alkyne moieties and hydrophilic ethylenedioxy units were assembled stepwise on solid phase. The resulting polymer segments were conjugated with mannose sugars via 1,3-dipolar cycloaddition. The obtained mono-, di-, and trivalent mannose structures were then subject to Con A lectin binding. Surface plasmon resonance studies showed a nonlinear increase in binding regarding the number and spacing of sugar ligands. The results of Con A lectin binding assays indicate that the chemical composition of the polymeric scaffold strongly contributes to the binding activities as well as the spacing between the ligands and the number of presented mannose units. Our approach now allows for the synthesis of highly defined glycooligomers and glycopolymers with a diversity of properties to investigate systematically multivalent effects of polymeric ligands

Understanding Selectin Counter-Receptor Binding from Electrostatic Energy Computations and Experimental Binding Studies

Higher organisms defend themselves against invading micro-organisms and harmful substances with their immune system. Key players of the immune system are the white blood cells (WBC), which in case of infection move in an extravasation process from blood vessels toward infected tissue promoting inflammation. This process starts with the attachment of the WBC to the blood vessel wall, mediated by protein pair interactions of selectins and counter-receptors (C-R). Individual selectin C-R binding is weak and varies only moderately between the three selectin types. Multivalency enhances such small differences, rendering selectin-binding type specific. In this work, we study selectin C-R binding, the initial step of extravasation. We performed electrostatic energy computations based on the crystal structure of one selectin type co-crystallized with the ligating part of the C-R. The agreement with measured free energies of binding is satisfactory. Additionally, we modeled selectin mutant structures in order to explain differences in binding of the different selectin types. To verify our modeling procedures, surface plasmon resonance data were measured for several mutants and compared with computed binding affinities. Binding affinities computed with soaked rather than co-crystallized selectin C-R structures do not agree with measured data. Hence, these structures are inappropriate to describe the binding mode. The analysis of selectin/C-R binding unravels the role played by individual molecular components in the binding event. This opens new avenues to prevent immune system malfunction, designing drugs that can control inflammatory processes by moderating selectin C-R binding

Immunodetection of zona pellucida-like domain protein from salmon samples.

<p><b>A</b>, Coomassie stained SDS-polyacrylamide gel and B, corresponding Western blot of separated crude cupula extracts. Lane 1, untreated sample, lane 2, PNGase F treated sample with faster migration of zona pellucida-like domain protein. <b>C</b>, HE stained inner ear cross section, asterisk marks the cupula and the arrowhead the subcupulary region with sensory and supporting cells. <b>D</b>, immunostaining of inner ear cross section, the arrow probably indicates staining of supporting cells which produce the zona pellucida-like domain protein. This is even more pronounced in <b>E</b>.</p

The cupula.

<p><b>A</b>, localization of the cupula in the inner ear. <b>B</b>, dissected cupula from salmon stained with Evans blue.</p

Peptide sequences obtained from the 45 kDa gel band from salmon.

<p>Data base searches were performed using Mascot and annotation of the MS/MS spectra was done manually. Amino-acid residues that differ from the published salmon sequence (C0H9B6) are bold. Amino-acid modifications: pyroQ, pyroglutamate, (delta mass: −17); ox, oxidized methionine (delta mass: +16); cam, carbamidomethyl, (delta mass: +57).</p>a<p>peptide with additional amino-acid exchange.</p><p>*peptide with additional modification.</p><p>Peptide sequences obtained from the 45 kDa gel band from salmon.</p

Visualization of cupula proteins.

<p><b>A</b>, crude extracts from isolated cupulae from salmon, (lane 1) and chicken (lane 2) were separated on a 12% SDS-PAGE under reducing conditions and silver stained. The arrowhead highlights a dominant protein (∼45 kDa) chosen for further analyses. Lane 3, marker proteins. In the 60 kDa range additional yet unidentified protein components are visible. <b>B</b>, deglycosylation of salmon cupula protein extract. Lane 1, cupula extract untreated; lane 2, cupula extract+PNGase F (100 NEB units), lane 3, PNGase F control (500 NEB units). Arrowheads indicate molecular weight shift of the 45 kDa protein due to the <i>N</i>-deglycosylation.</p

Sulfated Hyperbranched and Linear Polyglycerols Modulate HMGB1 and Morphological Plasticity in Neural Cells

The objective of this study was to establish if polyglycerols with sulfate or sialic acid functional groups interact with high mobility group box 1 (HMGB1), and if so, which polyglycerol could prevent loss of morphological plasticity in excitatory neurons in the hippocampus. Considering that HMGB1 binds to heparan sulfate and that heparan sulfate has structural similarities with dendritic polyglycerol sulfates (dPGS), we performed the experiments to show if polyglycerols can mimic heparin functions by addressing the following questions: (1) do dendritic and linear polyglycerols interact with the alarmin molecule HMGB1? (2) Does dPGS interaction with HMGB1 influence the redox status of HMGB1? (3) Can dPGS prevent the loss of dendritic spines in organotypic cultures challenged with lipopolysaccharide (LPS)? LPS plays a critical role in infections with Gram-negative bacteria and is commonly used to test candidate therapeutic agents for inflammation and endotoxemia. Pathologically high LPS concentrations and other stressful stimuli cause HMGB1 release and post-translational modifications. We hypothesized that (i) electrostatic interactions of hyperbranched and linear polysulfated polyglycerols with HMGB1 will likely involve sites similar to those of heparan sulfate. (ii) dPGS can normalize HMGB1 compartmentalization in microglia exposed to LPS and prevent dendritic spine loss in the excitatory hippocampal neurons. We performed immunocytochemistry and biochemical analyses combined with confocal microscopy to determine cellular and extracellular locations of HMGB1 and morphological plasticity. Our results suggest that dPGS interacts with HMGB1 similarly to heparan sulfate. Hyperbranched dPGS and linear sulfated polymers prevent dendritic spine loss in hippocampal excitatory neurons. MS/MS analyses reveal that dPGS-HMGB1 interactions result in fully oxidized HMGB1 at critical cysteine residues (Cys23, Cys45, and Cys106). Triply oxidized HMGB1 leads to the loss of its pro-inflammatory action and could participate in dPGS-mediated spine loss prevention. LPG-Sia exposure to HMGB1 results in the oxidation of Cys23 and Cys106 but does not normalize spine density