Fig. 9.

Glycan epitopes in D. immitis. a A summary of neutral and anionic N-glycan structures highlighting major antennal modifications; for a fuller set of defined structures refer to Supplementary Table 3. b The presence of HexNAc and phosphorylcholine residues in L3 larvae was probed using CGL3 (red) and TEPC 15 (green) by indirect fluorescence microscopy; also shown are the DAPI staining (for DNA) and DIC (differential interference contrast) images and the scale bar corresponds to 10 µm. c MALDI-TOF–MS of the AEAB-labelled neutral N-glycome before and after hydrofluoric acid treatment indicating loss of antennal fucose and phosphorylcholine residues. d The binding of Coprinopsis galectin (CGL3; 10 µg/ml), wheat germ agglutinin (WGA; 10 µg/ml) and human mannose binding lectin (MBL) to the Dirofilaria glycans increases after hydrofluoric acid treatment, while that to anti-phosphorylcholine (TEPC 15 IgA monoclonal) decreases. The charts indicate the uncorrected fluorescence values with the standard deviations (mean of 10 spots; analysed with an unpaired two-tailed parametric t-test with a 95% confidence level) as well as a negative control (spotting buffer only; example array scans are shown for the WGA and MBL data). Note that previous data indicate that CGL3 can recognise LacdiNAc, whereas WGA is known to bind HexNAc_n motifs and MBL to a wide range of glycans including those with terminal mannose or N-acetylglucosamine residues. Refer to Supplementary Figures 16–19 and 21 for (i) western blotting and further micrograph data (including controls) on epitopes recognised by CGL3 and TEPC 15, (ii) a summary of MS data on proteins affinity purified on CGL3- and TEPC 15-Sepharose, (iii) further MBL binding experiments (before and after endoglycosidase H treatment) to the immobilised glycome pools and fractionated immobilised glycans as well as data on other lectin interactions to the natural glycans and to defined di- and tri-saccharides