Phenoloxidase (PO) activity in haemolymph of <i>G</i>. <i>mellonella</i>.

<p>(A) Haemolymph samples were incubated with PBS or different amounts of PHL and the melanisation catalysed by PO was measured at 492 nm after mixing with 3,4-dihydroxy-dl-phenylalanine substrate solution. (B) Haemolymph was incubated with PBS, BSA (475 μg), PLL (475 μg) or PHL (475 μg; active or inhibited by 0.2M l-Fuc and Me-α-l-Fuc) and subsequently mixed with the substrate 3,4-dihydroxy-dl-phenylalanine. Products of melanisation catalysed by PO were measured at 492 nm. Data presented as means ± SD; * indicates significant difference p < 0.05, ** p < 0.01 (Dunnett's and Tukey’s test, respectively).</p

Data collection and refinement statistics.

<p>Values in parentheses correspond to highest resolution shell.</p

Box and whisker plot for glycan array screening for PHL (200 μg/ml) labeled with DyLight 488 NHS Ester.

<p>The top 19 saccharides and control trehalose (sample 20) were selected for display. The 7 saccharides giving the highest average signals are depicted. Linker formula—sp2: -O-CH₂CH₂NH₂; sp3: -O-(CH₂)₃NH₂. The bottom and top of the box are the first and third quartiles; the band inside the box is the second quartile (the median); the ends of the whiskers represent the minimum and maximum values of the data and the small squares inside the boxes represent the mean. RFU, relative fluorescence units. Complete glycan array results and the raw data are given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006564#ppat.1006564.s001" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006564#ppat.1006564.s002" target="_blank">S2</a> Tables, respectively.</p

Localization of Fuc- and Gal-specific sites with respect to the PHL dimer.

<p>Residues closer than 4Å from the observed ligands are coloured.</p

Determination of carbohydrate specificity/affinity of PHL towards l-fucose and d-galactose.

<p><b>(A)</b> SPR sensorgrams (differential curves) displaying PHL binding (0.25 μM) to CM5 sensor chip with immobilized l-fucoside in the presence of competing saccharides. Response decreases with increasing concentration of the inhibitor–l-fucose (0–50 mM), d-galactose (0–250 mM). <b>(B)</b> A logarithmic plot of inhibition curves calculated from SPR measurements <b>(C)</b> ITC curves of PHL (50 μM) titration by l-fucose (20 mM) and d-galactose (50 mM). 20 injections of 2.0 μl of sugars were added every 240 s to a PHL-containing cell. Lower plots show the total heat released as a function of total ligand concentration for the titration shown in the upper panels.</p

Equilibrium dissociation binding constants for interaction between PHL and carbohydrate ligands determined by isothermal titration calorimetry at 25°C (standard deviations were calculated from three independent measurements).

<p>Equilibrium dissociation binding constants for interaction between PHL and carbohydrate ligands determined by isothermal titration calorimetry at 25°C (standard deviations were calculated from three independent measurements).</p

Comparison of PHL/Me-α-l-Fuc and PLL/l-Fuc (PDB ID: 5C9P) structure.

<p>Top (A) and bottom (B) view of PHL (yellow/green) and PLL (magenta/pink) monomer. Differences are mainly visible in loop regions. (C) Sequence alignment of PHL/PLL repetitions. Residues responsible for l-Fuc or d-Gal binding are highlighted with a magenta and orange background, respectively. Different residues in the binding sites shown in bold.</p

Sequence alignment of PHL (NCBI Reference Sequence: WP_012776886.1) and PLL (Sequence ID: 5C9L_A) proteins.

<p>The asterisk indicates fully conserved residues, the colon a strong conservation between groups of amino acids and the dot is for weak similarity.</p

Haemagglutination inhibition assay with PHL using microscopy.

<p>Minimal inhibitory concentrations (MIC) of carbohydrate ligands and their potency towards l-fucose were determined.</p

Characterization of novel bangle lectin from <i>Photorhabdus asymbiotica</i> with dual sugar-binding specificity and its effect on host immunity - Fig 5

<p><b>(A) PHL monomer (chain A) overall architecture with individual blades labelled.</b> BGH trisaccharide molecules in sites 1F (between blades W1 and W2), 3F (between W3 and W4) and 6F (between W6 and W7) are shown as sticks. <b>(B) Side view of PHL dimer with intermonomer contacts shown</b>. Participating residues coloured in green for one monomer and cyan for the second monomer. Disulfide bridge formed by Cys279 is highlighted in magenta. <b>(C) Alignment of PHL sequence repeats.</b> Full arrows indicate individual β-sheets. Boxes highlight the conserved residues participating in ligand binding, the colours of the boxes correspond to the residue colouring in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006564#ppat.1006564.g006" target="_blank">Fig 6A</a>. Residues stabilizing Fuc ligands are indicated with an empty pentagon under the column, residues binding Gal ligands are indicated with a full hexagon under the column. Coloured residues in bold bind the corresponding monosaccharide, additional interactions with the oligosaccharide are shown in coloured regular font. The underlined residues only participate in binding through the water bridge. The colour of the residue stands for individual binding sites: site 1F (blue), site 2G (cyan), site 3F (green), site 4G (dark yellow), site 6G (orange), site 6F (red). Residues in italics were not resolved in any structure.</p