7 research outputs found
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