<i>Fasciola hepatica</i> Surface Coat Glycoproteins Contain Mannosylated and Phosphorylated N-glycans and Exhibit Immune Modulatory Properties Independent of the Mannose Receptor

<div><p>Fascioliasis, caused by the liver fluke <i>Fasciola hepatica</i>, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, <i>F</i>. <i>hepatica</i> employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of <i>F</i>. <i>hepatica’s</i> tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg’s binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg’s ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that <i>F</i>. <i>hepatica</i> contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host’s immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.</p></div

MALDI-FT-ICR-MS analysis of FhTeg N-glycans indicates the presence of phosphorylated glycan species.

<p><b>A:</b> FT-ICR-MS spectrum indicating the phosphorylated glycans identified by accurate mass determination with external calibration and additional comparison with the internal confirmed oligomannose glycans. <b>B:</b> Zoom region showing the high resolution separation between the Man₆GlcNAc₂ glycan and a phosphorylated glycan of almost identical mass. <b>C:</b> The scatter plot indicates the deviation of the measured <i>m/z</i> from the calculated <i>m/z</i> of phosphorylated glycans in comparison with hypothetical sulphated glycans and the oligomannosyl glycans also present in the spectrum.</p

FhTeg binding to dendritic cells is mediated by MR and is carbohydrate and calcium dependent.

<p><b>A-B:</b> MR-transfected CHO cells (A) and BMDCs (B) were stimulated with and without inhibitors, i.e. EGTA (10mM), anti-MR (1 μg ml^-1), mannan (A: 100 μg ml^-1;B: 1 mg/mL), GalNAc-4S (A: 1mM; B: 25 mM), for 45 min prior to stimulation with fluorescently labelled FhTeg (A: 1–10 μg ml^-1; B: 5 μg/mL) for 45 min. Fluorescently labelled BSA was also used as control. FhTeg binding to cells was assessed by flow cytometry and reported in bar chart format. Data shown is the mean ± SD of one representative experiment; the experiment was repeated 2–3 times, **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001 compared to FhTeg. <b>C-D:</b> BMDCs were stimulated with fluorescently labelled FhTeg (10μg ml^-1, green) or BSA (<u>10g</u> ml^-1, green)) for 45 min prior to paraformaldehyde fixation and mounting with DAPI (blue); Scale bar: 25μm.</p

The immune properties of FhTeg are independent of the MR receptor.

<p><b>A</b>: BMDCs were stimulated with mannan for 30 min prior to incubation with FhTeg for 2.5 h. Total RNA was extracted, and after reverse transcription cDNA was analyzed with qPCR for SOCS3. RNA expression was normalized to GAPDH and actin control genes. <b>B:</b> BMDCs were pre-incubated with mannan prior stimulation with PBS or FhTeg (10μg) before addition of LPS (100ng ml^-1) for 18 h. IL12p70 levels were measured with commercial ELISA kits. Data are presented as the mean ± SEM of two independent experiments. ***<i>p</i> ≤ 0.001; ****<i>p</i> ≤ 0.0001 compared to LPS group. <b>C,D:</b> BMDCs isolated from MR-knockout mice were stimulated with fluorescently labelled FhTeg or BSA (10μg ml^-1, green) for 45 min prior to paraformaldehyde fixation. FhTeg binding to cells was assessed by flow cytometry and reported in bar chart format. <b>E:</b> BMDCs isolated from MR-knockout mice were stimulated with FhTeg (10μg) for 2.5 h. Total RNA was extracted, and after reverse transcription cDNA was analyzed with qPCR for SOCS3. RNA expression was normalized to GAPDH and actin control genes. <b>F.</b> BMDCs derived from MR knockout mice were stimulated with <b>PBS</b> or FhTeg (10μg) before addition of LPS (100ng ml^-1) for 18 h. IL12p70 levels were measured with commercial ELISA kits. Data are presented as the mean ± SEM of two independent experiments. *<i>p</i> ≤ 0.05, **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001.</p

FhTeg preparation is rich in oligomannose and truncated complex type <i>N-</i>glycans carrying fucose or sulfate/phosphate moieties.

<p>Fh tegumental antigens were digested with trypsin followed by PNGase F treatment. Released N-glycans were subsequently labelled with 2-AA and analysed by MALDI-TOF-MS in the negative ion-reflector mode. Signals are labelled with monoisotopic masses. Most abundant <i>N</i>-glycan structures are annotated in the spectrum while minor peaks are reported in the supplementing material (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004601#pntd.0004601.s004" target="_blank">S1 Table</a>). The signal at <i>m/z</i> 1582.8 [M-H]^- is annotated according to MALDI-TOF/TOF-MS analysis.</p

ATP:ADP antiporter mimics turbo-state.

<p>(<i>A</i>) Overview of the models used in this figure. Model A and D are from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi-1003371-g001" target="_blank">Figure 1</a>, model A–glyc is model A without glycosomal localization, as described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi.1003371-Haanstra2" target="_blank">[31]</a>, model A+AAT is model A with an ATP:ADP antiporter. (<i>B–C</i>) Steady-state concentrations of glycosomal Glc-6-P and Fru-1,6-BP are depicted in the various models. (<i>D</i>) Increasing the activity of the ATP:ADP antiporter (V_{max,ATP:ADP antiporter}) in model D leads to a high risk of accumulation of hexose phosphates. The green line indicates the concentration of Fru-1,6-BP in the original model of glycolysis (17.2 mM, panel C, model A). Glc_e in this simulation is 25 mM. (<i>E</i>) Time course simulation of model D at 25 mM Glc_e and various values for the V_{max,ATP:ADP antiporter} parameter. Plotted is the concentration of glycosomal phosphates (ΣP similar as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi-1003371-g002" target="_blank">Figure 2</a>, moiety 5 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi-1003371-t002" target="_blank">Table 2</a>). ATP:ADP antiporter activity values below 1 nmol·min⁻¹·mg protein⁻¹ result in depletion of glycosomal phosphates (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi-1003371-g002" target="_blank">Figure 2</a>). <i>k_TOX</i> = 2 µl·min⁻¹·mg protein⁻¹ in all models. Solid lines indicate medians, shaded areas and error bars show interquartile ranges, as derived from the uncertainty modeling.</p

Simulations of 6PGDH inhibition and 6-PG accumulation.

<p>(<i>A–B</i>) The effects of inhibition of 6PGDH on 6-PG concentrations and metabolic fluxes were simulated by reducing <i>V</i>_max,6PGDH in model C and D at high oxidative stress (<i>k_TOX</i> = 200 µl·min⁻¹·mg protein⁻¹). Simulations at low oxidative stress (<i>k_TOX</i> = 2 µl·min⁻¹·mg protein⁻¹) are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi.1003371.s009" target="_blank">Figure S6</a>. ATP production flux as steady-state flux through PFK is indicated in red, while trypanothione reductase steady-state flux is indicated in yellow, both plotted on the left y-axis. Steady-state concentration of cytosolic (blue) and glycosomal (green) 6-phosphogluconate are plotted on the right y-axis. Shaded areas indicate interquartile ranges. (<i>C</i>) Steady-state flux through glycolysis as a function of the glycosomal 6-PG concentration in model A. A glycosomal 6-PG concentration of around 500 mM reduces the glycolytic flux by 50%.</p

Model stoichiometry by modules and their subsequent reactions.

<p>Bold characters indicate external metabolites which are kept fixed. Other metabolites are internal variables of the system.</p