Glycoprotein detection and localisation on <i>F</i>. <i>hepatica</i> NEJTeg by lectin blot and lectin fluorescence staining.

<p>The total protein profile of <i>F</i>. <i>hepatica</i> NEJTeg (Teg) was revealed after SDS-PAGE fractionation and visualised in-gel by silver staining (<b>A</b>). Fixed NEJ and nitrocellulose membranes were incubated with FITC-labelled or biotinylated-conjugate lectins respectively. Specific lectin binding to glycans with terminal Fuc- (AAL, LCA, PSA), Man- (ConA, GNL), Galβ1-3GalNAc- (Jacalin, PNA), Gal/GalNAc- (RCA-120, GSL-I, SBA, DBA), Lac- (ECL), oligosaccharides- (PHA-E, PHA-L) or GlcNAc- (WGA, sWGA, GSL-II) motifs is shown. Oral sucker (or) and ventral sucker (vs) are identified with arrows (<b>B</b>). Negative controls consisted of nitrocellulose membranes without lectin incubation (1), nitrocellulose membranes incubated with biotinylated-conjugate lectins that were previously incubated with their specific inhibitors (2) and fixed NEJ incubated with FITC-labelled lectins that were previously incubated with their specific inhibitors (3). The list of lectins used and their corresponding inhibitors are detailed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004688#pntd.0004688.t001" target="_blank">Table 1</a> (<b>C</b>). Nitrocellulose membranes were exposed to IRDye-labelled streptavidin to reveal positive lectin binding (green) at various molecular weights (markers in red). Glycoprotein profiles were visualised by IR system. Epifluorescence microscope was employed to detect glycan localisation (green) and DNA was counterstained with DAPI (blue). Merged image of both micrographs is included for each lectin. Scale bar = 100 μm</p

Identification of short <i>N-</i>glycans occupying an <i>N-</i>glycosylation site of FhCB1.

<p>Nano-RP-LC-ESI-ion trap-MS/MS with collision-induced dissociation (<b>A</b> and <b>B</b>) and with electron transfer dissociation (<b>C</b>) of the tryptic glycopeptide Y₇₉NVSENDLPESFDAR₉₃ from FhCB1 (BN1106_s6570B000050). The [M+3H]³⁺ parent ions (blue diamonds) at <i>m/z</i> 829.50 (<b>A</b>) and 878.70 (<b>B</b>) of the glycopeptide carrying a glycan of composition H2N2 and F1H2N2 respectively were selected. Fragment ions are indicated. The residual signals at the <i>m/z</i> corresponding to [H+3H]³⁺ (<b>Δ</b>), to the doubly charged ions that result from capture of 1 electron without dissociation [H+3H]^2+· (<b>X</b>) and to the singly charged ions that result from capture of 2 electrons without dissociation [H+3H]^+·· (*) are indicated (<b>C</b>). Monoisotopic masses are given. Man (green circle), GlcNAc (blue square) and Fuc (red triangle), pep (peptide moiety).</p

Characterisation of <i>F</i>. <i>hepatica</i> A-BB-NEJTeg glycoprotein composition detected following glycoprotein biotinylation, affinity chromatography isolation and LC-MS/MS analysis.

<p>Characterisation of <i>F</i>. <i>hepatica</i> A-BB-NEJTeg glycoprotein composition detected following glycoprotein biotinylation, affinity chromatography isolation and LC-MS/MS analysis.</p

List of the fragment ions after CID-MS/MS of parent ions of tryptic peptide of FhCL3 (BN1106_s10139B000014).

<p>List of the fragment ions after CID-MS/MS of parent ions of tryptic peptide of FhCL3 (BN1106_s10139B000014).</p

Glycosylation of <i>F</i>. <i>hepatica</i> NEJSom.

<p>The total protein profile of <i>F</i>. <i>hepatica</i> NEJSom (Som) was revealed after SDS-PAGE fractionation and visualised in-gel by silver staining <b>(A).</b> NEJSom preparations were SDS-PAGE fractioned, transferred to nitrocellulose membranes and incubated with the biotinylated-labelled lectins GSL-I, SBA, PNA and Jacalin (<b>B</b>). Negative controls consisted of nitrocellulose membranes without lectin incubation (1) and nitrocellulose membranes incubated with biotinylated-conjugate lectins that were previously incubated with their specific inhibitors (2). The list of lectins used and their corresponding inhibitors are detailed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004688#pntd.0004688.t001" target="_blank">Table 1</a> (<b>C</b>). An additional incubation with IRDye-labelled streptavidin was used to detect positive lectin binding at different molecular weights (red markers). Glycoproteins were revealed by infrared imaging.</p

Identification of short <i>N-</i>glycans occupying an <i>N-</i>glycosylation site of on the YNVSENDLPESFDAR tryptic peptide.

<p>Nano-RP-LC-ESI-ion trap-MS/MS with collision-induced dissociation (<b>A</b> and <b>B</b>) and with electron transfer dissociation (<b>C</b>) of the tryptic glycopeptide YNVSENDLPESFDAR from FhProCB2 (gi|27526823). The [M+3H]³⁺ parent ions (blue diamonds) at <i>m/z</i> 824.70 (<b>A</b>) and 873.70 (<b>B</b>) of the glycopeptide carrying a glycan of composition H2N2 and F1H2N2 respectively were selected. Fragment ions are indicated. The residual signals at the <i>m/z</i> corresponding to [H+3H]³⁺ (<b>Δ</b>), to the doubly charged ions that result from capture of 1 electron without dissociation [H+3H]^2+· (<b>X</b>) and to the singly charged ions that result from capture of 2 electrons without dissociation [H+3H]^+·· (*) are indicated (<b>C</b>). Monoisotopic masses are given. Man (green circle), GlcNAc (blue square) and Fuc (red triangle).pep, peptide moiety.</p

MALDI-TOF-MS spectra of AA-labelled <i>N-</i>linked glycans of <i>F</i>. <i>hepatica</i> NEJTeg before and after exoglycosidase treatments.

<p>N-glycans released from NEJTeg using PNG-F were labelled with fluorophore 2-aminobenzoic acid (2-AA). MS spectra of AA-labelled <i>N-</i>glycans were acquired by MALDI-TOF-MS without exoglycosidase treatment (<b>A</b>) and after α-mannosidase (<b>B</b>), β-<i>N</i>-acetylglucosaminidase (<b>C</b>) and β-galactosidase (<b>D</b>) treatments. Unidentified peaks are represented with an asterisk (*). Green circle, Man; blue square, GlcNAc; red triangle, Fuc.</p

Identification of <i>F</i>. <i>hepatica</i> cathepsins in NEJTeg.

<p>NEJTeg was loaded in SDS-PAGE and stained with Coomassie Blue in order to stain protein bands (<b>A</b>). Bands 1 (B1), 2 (B2), 3 (B3) and 4 (B4), were excised, tryptic digested and identified with LC-MS/MS analysis. Potential <i>N-</i>glycosylation sites (Pot <i>N-</i>gly) and monoisotopic mass of the tryptic peptides containing glycosylation were predicted (<b>B</b>). UP (unique peptide).</p

List of the characterised <i>F</i>. <i>hepatica</i> NEJTeg <i>N-</i>glycans identified by MALDI-TOF-MS.

<p>Composition of glycan subsets expressed in terms of hexose (H), <i>N-</i>acetylhexosamine (N) and deoxyhexose (F). The five most prominent peaks shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004688#pntd.0004688.g003" target="_blank">Fig 3A</a> are indicated (<b>#</b>). Red triangle, Fuc; green circle, Man; yellow circle, Gal; blue square, GlcNAc; yellow square, GalNAc.</p