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

Molecular Recognition of Complex-Type Biantennary <i>N</i>‑Glycans by Protein Receptors: a Three-Dimensional View on Epitope Selection by NMR

The current surge in defining glycobiomarkers by applying lectins rekindles interest in definition of the sugar-binding sites of lectins at high resolution. Natural complex-type <i>N</i>-glycans can present more than one potential binding motif, posing the question of the actual mode of interaction when interpreting, for example, lectin array data. By strategically combining <i>N</i>-glycan preparation with saturation-transfer difference NMR and modeling, we illustrate that epitope recognition depends on the structural context of both the sugar and the lectin (here, wheat germ agglutinin and a single hevein domain) and cannot always be predicted from simplified model systems studied in the solid state. We also monitor branch-end substitutions by this strategy and describe a three-dimensional structure that accounts for the accommodation of the α2,6-sialyl­ated terminus of a biantennary <i>N</i>-glycan by viscumin. In addition, we provide a structural explanation for the role of terminal α2,6-sialyl­ation in precluding the interaction of natural <i>N</i>-glycans with lectin from Maackia amurensis. The approach described is thus capable of pinpointing lectin-binding motifs in natural <i>N</i>-glycans and providing detailed structural explanations for lectin selectivity

PVL staining of breast tumoral tissues.

<p>A. A Breast tumor TMA (formalin-fixed) was stained and imaged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128190#pone.0128190.g008" target="_blank">Fig 8A</a>. Tumors were all positive with a vast majority of cancer cells stained. Labeling intensity was estimated on a scale ranging from 1 to 4. Representative examples of tumors of each staining level are shown. B. Staining level distributions according to tumor molecular characteristics. (HR = Hormone Receptors, TN = Triple Negative).</p

PVL staining of healthy tissues.

<p>A tissue microarray (TMA) comprising healthy tissues from respiratory, digestive and genital origin was stained with 0.7 μg ml^<b>-1</b> rPVL-biot in presence of 0.1 M Fucose or 0.1 M GlcNAc followed by Streptavidin-HRP. AEC was used as a peroxidase substrate to reveal the PVL staining and counterstaining was performed using hematoxylin. Slides were imaged using a NanoZoomer slide scanner with a 20x magnification. 40x digital magnifications are also shown as insets.</p

PVL binding of tumor cell lines.

<p>A. Flow cytometry histograms show rPVL-Alexa 488 binding to a lung immortalized cell line (HBEC-3KT), two lung tumor cell lines (H358 and A549) as well as on a breast tumor cell line (MCF-7). The x axis indicates fluorescence intensity. The y axis indicates cell number. Black line: untreated control cells; blue line rPVL-Alexa 488 5 μg ml^-1 for 30 mn; red line: rPVL-alexa 488 5 μg ml^-1 in the presence of GlcNAc 100 mM; green line: rPVL-Alexa 488 5 μg ml^-1 after sialidase pretreatment. B. Microscopy images of A549 NSCLC cells treated for 30 min at 37°C with 5 μg ml^-1 rPVL labeled with Alexa 488 in the presence or absence of 100 mM GlcNAc. Green channel shows rPVL-Alexa 488, blue channel shows nuclei labeled with DAPI staining.</p

Representation of glycoconjugates and binding of rPVL to glycans on chips.

<p>A. Examples of normal and truncated oligosaccharides that can be found on normal or cancer tissues. Coding for schematic representation of monosaccharides is in the lower part of the figure. The heptasaccharide used in binding experiments is indicated as “hepta”. B. Synthesis of heptasaccharide azide 2 corresponding to oligosaccharide “hepta” in panel A.</p

Determination of affinity characteristics and thermodynamic contributions for the binding of rPVL with different oligosaccharides.

<p>^a only one experiment by ITC</p><p>^b stoichiometry value fixed during fitting procedure.</p><p>Standard deviation lower than 20% were obtained for ITC experiments.</p><p>Determination of affinity characteristics and thermodynamic contributions for the binding of rPVL with different oligosaccharides.</p

PVL binds to tumor tissue with a mixed Neu5Ac and GlcNAc specificity.

<p>A. Sections from ethanol-fixed colon carcinoma and adjacent healthy tissue from 2 different patients (# 5345 and 5378) were stained with 1 μg ml^<b>-1</b> rPVL-biot in presence of 0.1 M Fucose or 0.1 M GlcNAc followed by Streptavidin-HRP. AEC was used as a peroxidase substrate to reveal the rPVL staining and counterstaining was performed using hematoxylin. Slides were imaged using a NanoZoomer slide scanner with a 20x magnification.40x digital magnifications are also shown as insets. B. Canine breast tumor sections (formalin fixed) were treated or not with glycosidases and then stained with 2 μg ml^<b>-1</b> rPVL-biot and imaged as in A.</p

PVL binding in the presence of glycosylation inhibitors or after treatment with glycosidases.

<p>A. Biotinylated rPVL (1 μg ml^<b>-1</b>) or MAH (5 μg ml^<b>-1</b>) were incubated with A549 cells grown for 4 days in presence of DMSO (green line) or of either 400 μM 2-Fluoro-Fucose or 100 μM Fluoro-Neu5Ac (pink line). Lectin binding was revealed by PE conjugated Streptavidin. Percentage of inhibitions are indicated according to the mean fluorescence intensities. B. Similar experiment than in A but with A549 cells grown in the presence of 5 μM Kifunensine for 4 days or in the presence of either 6 mM Benzyl-GalNAc or 10 μM PPMP for 48h. C. Similar experiment than in A and B but with cells treated with 2.5 U (pink line) or 12.5 U ß-D-N-acetyl-hexosaminidase (blue line).</p

Mutation of Val-356 is sufficient to block proteolysis of the IL-6R and the Asp358Ala variant.

<p>(A) Amino-acid residues from Asp-340 to Ala-370 of the human IL-6R. The identified cleavage site between Pro-355 (P1) and Val-356 (P1′) is indicated. (B) Overview of the six different IL-6R cleavage site mutants. Mutations are colored in either green or blue; the amino-acid residues of the wild-type cleavage site are shown in red. (C) HEK293 cells were transiently transfected with expression plasmids encoding the wild-type IL-6R (PV) or the double mutants (IE, DG) as indicated. Cells were either treated with 100 nM PMA for 2 h or DMSO as vehicle control. sIL-6R was precipitated from the supernatant with concanavalin A-covered sepharose beads, and the cells were lysed. Both were analyzed via western blot, and β-actin served as the loading control. One out of three experiments with similar outcomes is shown. (D, E) The experiment was performed as described under (C), but sIL-6R generation was analyzed via ELISA. In (D), the amount of sIL-6R generated after PMA stimulation of the wild-type IL-6R (PV) was set to 100%, and all other values were calculated accordingly. In (E), the amount of sIL-6R without stimulation was considered as constitutive shedding and set to 1, and the increase of sIL-6R was calculated. Data shown are the mean ± SD from at least three independent experiments (*<i>p</i> < 0.05, ns = no significant difference). (F–H) HEK293 cells were transiently transfected with expression plasmids encoding the wild-type IL-6R (PV) or the single mutants (DV, IV, PE, PG) as indicated. The experiments were performed as described in (C) to (E). (I) Overview of the four different IL-6R cleavage site mutants of the IL-6R Asp358Ala variant. Mutations are colored in either green or blue, the amino-acid residues of the wild-type cleavage site are shown in red, and the Asp358Ala single nucleotide polymorphism (SNP) is colored in orange. (J) ADAM17-mediated proteolysis of the IL-6R variants depicted in (I) were analyzed as described in (D). (K–M) Equal numbers of Ba/F3-gp130 cells were incubated for 48 h with increasing amounts (0–100 ng/ml) of either IL-6 or Hyper-IL-6. The stably transduced IL-6R variant is indicated above the diagram. One representative experiment out of three performed is shown (mean ± SD, biological triplicates).</p