10 research outputs found
Lectin-Array Blotting: Profiling Protein Glycosylation in Complex Mixtures
By combining electrophoretic protein separation with
lectin-array-based glycan profiling into a single experiment, we have
developed a high-throughput method for the rapid analysis of protein
glycosylation in biofluids. Fluorescently tagged proteins are separated
by SDS-PAGE and transferred by diffusion to a microscope slide covered
with multiple copies of 20 different lectins, where they are trapped
by specific carbohydrate protein interactions while retaining their
relative locations on the gel. A fluorescence scan of the slide then
provides an affinity profile with each of the 20 lectins containing
a wealth of structural information regarding the present glycans.
The affinity of the employed lectins toward <i>N</i>-glycans
was verified on a glycan array of 76 structures. While current lectin-based
methods for glycan analysis provide only a picture of the bulk glycosylation
in complex protein mixtures or are focused on a few specific known
biomarkers, our array-based glycoproteomics method can be used as
a biomarker discovery tool for the qualitative exploration of protein
glycosylation in an unbiased fashion
Toward the Solid-Phase Synthesis of Heparan Sulfate Oligosaccharides: Evaluation of Iduronic Acid and Idose Building Blocks
Glycan
arrays have been established as the premier technical platform
for assessing the specificity of carbohydrate binding proteins, an
important step in functional glycomics research. Access to large libraries
of well-characterized oligosaccharides remains a major bottleneck
of glycan array research, and this is particularly true for glycosaminoglycans
(GAGs), a class of linear sulfated polysaccharides which are present
on most animal cells. Solid-supported synthesis is a potentially powerful
tool for the accelerated synthesis of relevant GAG libraries with
variations in glycan sequence and sulfation pattern. We have evaluated
a series of iduronic acid and idose donors, including a couple of
novel <i>n</i>-pentenyl orthoester donors in the sequential
assembly of heparan sulfate precursors from monosaccharide building
blocks in solution and on a polystyrene resin. The systematic study
of donor and acceptor performance up to the trisaccharide stage in
solution and on the solid support have resulted in a general strategy
for the solid-phase assembly of this important class of glycans
NMR and Molecular Recognition of N‑Glycans: Remote Modifications of the Saccharide Chain Modulate Binding Features
Glycans
play a key role as recognition elements in the communication
of cells and other organisms. Thus, the analysis of carbohydrate–protein
interactions has gained significant importance. In particular, nuclear
magnetic resonance (NMR) techniques are considered powerful tools
to detect relevant features in the interaction between sugars and
their natural receptors. Here, we present the results obtained in
the study on the molecular recognition of different mannose-containing
glycans by <i>Pisum sativum</i> agglutinin. NMR experiments
supported by Corcema-ST analysis, isothermal titration calorimetry
(ITC) experiments, and molecular dynamics (MD) protocols have been
successfully applied to unmask important binding features and especially
to determine how a remote branching substituent significantly alters
the binding mode of the sugar entity. These results highlight the
key influence of common structural modifications in natural glycans
on molecular recognition processes and underscore their importance
for the development of biomedical applications
Chemo-Enzymatic Synthesis of <sup>13</sup>C Labeled Complex N‑Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry
Methods for the absolute quantification
of glycans are needed in
glycoproteomics, during development and production of biopharmaceuticals
and for the clinical analysis of glycan disease markers. Here we present
a strategy for the chemo-enzymatic synthesis of <sup>13</sup>C labeled
N-glycan libraries and provide an example for their use as internal
standards in the profiling and absolute quantification of mAb glycans
by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)
mass spectrometry. A synthetic biantennary glycan precursor was <sup>13</sup>C-labeled on all four amino sugar residues and enzymatically
derivatized to produce a library of 15 glycan isotopologues with a
mass increment of 8 Da over the natural products. Asymmetrically elongated
glycans were accessible by performing enzymatic reactions on partially
protected UV-absorbing intermediates, subsequent fractionation by
preparative HPLC, and final hydrogenation. Using a preformulated mixture
of eight internal standards, we quantified the glycans in a monoclonal
therapeutic antibody with excellent precision and speed
Chemo-Enzymatic Synthesis of <sup>13</sup>C Labeled Complex N‑Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry
Methods for the absolute quantification
of glycans are needed in
glycoproteomics, during development and production of biopharmaceuticals
and for the clinical analysis of glycan disease markers. Here we present
a strategy for the chemo-enzymatic synthesis of <sup>13</sup>C labeled
N-glycan libraries and provide an example for their use as internal
standards in the profiling and absolute quantification of mAb glycans
by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)
mass spectrometry. A synthetic biantennary glycan precursor was <sup>13</sup>C-labeled on all four amino sugar residues and enzymatically
derivatized to produce a library of 15 glycan isotopologues with a
mass increment of 8 Da over the natural products. Asymmetrically elongated
glycans were accessible by performing enzymatic reactions on partially
protected UV-absorbing intermediates, subsequent fractionation by
preparative HPLC, and final hydrogenation. Using a preformulated mixture
of eight internal standards, we quantified the glycans in a monoclonal
therapeutic antibody with excellent precision and speed
Specific anti-glycan antibodies are sustained during and after parasite clearance in <i>Schistosoma japonicum</i>-infected rhesus macaques
<div><p>Background</p><p>Human immunity to <i>Schistosoma</i> infection requires many years of exposure, and multiple infections and treatments to develop. Unlike humans, rhesus macaques clear an established schistosome infection naturally at the same time acquiring immunity towards re-infection. In macaques, schistosome egg production decreases after 8 weeks post-infection and by week 22, physiological impairment of the worm caused by unclarified antibody-mediated processes is observed. Since strong antibody responses have been observed against schistosome glycan antigens in human and animal infections, we here investigate if anti-glycan antibodies are associated with immunity against schistosome infections in macaques.</p><p>Methods</p><p>We used a microarray containing a large repertoire of glycoprotein- and glycolipid-derived glycans from different schistosome life stages to analyse anti-glycan serum IgG and IgM from <i>S</i>. <i>japonicum</i>-infected macaques during the course of infection and self-cure. We also used an <i>in vitro</i> schistosomula assay to investigate whether macaque sera containing anti-glycan antibodies can kill schistosomula.</p><p>Conclusions/significance</p><p>Antibody responses towards schistosome glycans at week 4 post-infection were dominated by IgM while IgG was high at week 8. The profound increase in IgG was observed mainly for antibodies towards a large subset of glycans that contain (multi-)fucosylated terminal GalNAcβ1-4GlcNAc (LDN), and Galβ1-4(Fucα1–3)GlcNAc (LeX) motifs. In general, glycans with a higher degree of fucosylation gave rise to stronger antibody responses than non-fucosylated glycans. Interestingly, even though many IgG and IgM responses had declined by week 22 post-infection, IgG towards O-glycans with highly fucosylated LDN motifs remained. When incubating macaque serum with schistosomula <i>in vitro</i>, schistosomula death was positively correlated with the duration of infection of macaques; macaque serum taken 22 weeks post-infection caused most schistosomula to die, suggesting the presence of potentially protective antibodies. We hypothesize that IgGs against highly fucosylated LDN motifs that remain when the worms deteriorate are associated with infection clearance and the resistance to re-infection in macaques.</p></div
Averaged serum IgG responses from <i>S</i>. <i>japonicum</i>-infected rhesus macaques to synthetic core modified N-glycan fractions.
<p>The horizontal axis depicts core modified N-glycan fractions that have been synthesized and described by Brzezicka et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005339#pntd.0005339.ref035" target="_blank">35</a>]. Average median fluorescence intensities are shown for <i>S</i>. <i>japonicum</i>-infected macaque serum IgG over a time course of 22 weeks. Each peak along the vertical axis corresponds to each individual glycan fraction printed on the glycan microarray. Core xylosylated, core α6-fucosylated and core α3-fucosylated structures are indicated. Within core xylosylated structures, those that have additional monosaccharides on the α3-mannose (Δ) and those that miss the core α6-mannose (✧) are indicated.</p
<i>In vitro</i> schistosomula incubation with <i>S</i>. <i>japonicum</i>-infected rhesus macaque sera collected at different infection time points.
<p>Macaque sera were incubated with 3 hour (h) transformed schistosomula. Loss of Schistosomula integrity was visualized by PI positivity. A) Gross morphology of schistosomula after 24 h and 48 h of incubation with macaque sera. B) Percentage of PI-positive schistosomula after 24 h and 48 h of incubation with macaque sera at different infection time points.</p
Averaged serum IgG and IgM response from schistosome-infected macaques to glycans isolated from different life stages of schistosomes.
<p>The horizontal axis indicates N- and O-glycan fractions from <i>Schistosoma</i> cercariae, adult worms, and eggs. Schistosome GSL glycans are shown as a group irrespective of the life stage. Average background-subtracted median fluorescence intensities are shown for IgG and IgM over a time course of 22 weeks. Each bar corresponds to antibody binding to individual glycan fractions printed on the glycan microarray. N: N-glycans. O: O-glycans and L: GSL glycans.</p
Glycan fractions that have statistically different serum IgG and IgM responses at week 22 post-infection.
<p>(A) 37 glycan fractions have a significant difference in IgG and IgM binding (p<0.05). Represented glycans are marked with Δ. (B) The glycan composition and most likely motif of representative fractions of IgG dominant and (C) IgM dominant groups are depicted.</p