Core Richness of N‑Glycans of Caenorhabditis elegans: A Case Study on Chemical and Enzymatic Release

Despite years of research, the glycome of the model nematode Caenorhabditis elegans is still not fully understood. Certainly, data over the years have indicated that this organism synthesizes unusual N-glycans with a range of galactose and fucose modifications on the Man_2–3GlcNAc₂ core region. Previously, up to four fucose residues were detected on its N-glycans, despite these lacking the fucosylated antennae typical of many other eukaryotes; some of these fucose residues are capped with hexose residues as shown by the studies of us and others. There have, though, been contrasting reports regarding the maximal number of fucose substitutions in C. elegans, which in part may be due to different methodological approaches, including use of either peptide:N-glycosidases F and A (PNGase F and A) or anhydrous hydrazine to cleave the N-glycans from glycopeptides. Here we compare the use of hydrazine with that of a new enzyme (rice PNGase Ar) and show that both enable release of glycans with more sugar residues on the proximal GlcNAc than previously resolved. By use of exoglycosidase sequencing, in conjunction with high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), we now reveal that actually up to five fucose residues modify the core region of C. elegans N-glycans and that the α1,3-fucose on the reducing terminus can be substituted by an α-linked galactose. Thus, traditional PNGase F and A release may be insufficient for release of the more highly core-modified N-glycans, especially those occurring in C. elegans, but novel enzymes can compete against chemical methods in terms of safety, ease of cleanup, and quality of resulting glycomic data

Mapping the Expressed Glycome and Glycosyltransferases of Zebrafish Liver Cells as a Relevant Model System for Glycosylation Studies

The emergence of zebrafish as a model organism for human diseases was accompanied by the development of cellular model systems that extended the possibilities for <i>in vitro</i> manipulation and <i>in vivo</i> studies after cell implantation. The exploitation of zebrafish cell systems is, however, still hampered by the lack of genomic and biochemical data. Here, we lay a path toward the efficient use of ZFL, a zebrafish liver-derived cell system, as a platform for studying glycosylation. To achieve this, we established the glycomic profile of ZFL by a combination of mass spectrometry and NMR. We demonstrated that glycoproteins were substituted by highly sialylated multiantennary <i>N</i>-glycans, some of them comprising the unusual zebrafish epitope Galβ1–4­[Neu5Ac­(α2,3)]­Galβ1–4­[Fuc­(α1,3)]­GlcNAc, and core 1 multisialylated <i>O</i>-glycans. Similarly, these analyses established that glycolipids were dominated by sialylated gangliosides. In parallel, analyzing the expression patterns of all putative sialyl- and fucosyltransferases, we directly correlated the identified structures to the set of enzymes involved in ZFL glycome. Finally, we demonstrated that this cell system was amenable to metabolic labeling using functionalized monosaccharides that permit <i>in vivo</i> imaging of glycosylation processes. Altogether, glycomics, genomics, and functional studies established ZFL as a relevant cellular model for the study of glycosylation

Glycolipids from <i>T. rangeli</i> and <i>T. cruzi</i> suppress NOS expression.

<p><i>Rhodnius</i> were injected with either Tr GIPL or Tc GIPL and three days later salivary glands were dissected, homogenized and NOS expression evaluated by Western blotting. A. Western blotting against NOS in salivary glands obtained from control and insects injected with Tr GIPLs. B. Western blotting against NOS in salivary glands obtained from control and insects injected with Tc GIPLs.</p

Chemical composition of GIPL purified from <i>T</i>. <i>rangeli</i> (Tr GIPL).

a<p>Determined by GC as trimethylsilyl derivatives of methylglycosides.</p>b<p>Determined by GC and GC-MS as fatty acid methyl esters (FAMEs).</p>c<p>Determined by GC and GC-MS after N-acetylation and trimethylsilylation.</p

Infection with <i>T. rangeli</i> reduces the NOS activity and the levels of NOS protein in the salivary glands of <i>R. prolixus</i>.

<p>A. <i>Rhodnius</i> were dissected 7 days after control injection of water or <i>T. rangeli</i> and assayed for NADPH-diaphorase activity. Results from three experiments were evaluated statistically using the Student t test (* p<0.05). B. Salivary gland extracts from control or <i>T. rangeli-</i>injected insects were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with primary antibody anti-NOS and then with an anti-rabbit antibody conjugated to alkaline phosphatase. This experiment was performed three times. Tr, <i>Trypanosoma rangeli</i> cells evalutated for NOS blotting. N, salivary glands from non-injected insects. C, control salivary glands from insects injected with water. I, Salivary glands from <i>T. rangeli</i>-injected insects. C. NADPH-diaphorase activity was measured in salivary gland extracts of salivary glands three days after injection with 100 ng of glycolipids from either <i>T. rangeli</i> (Tr GIPL), <i>P. serpens (</i>Ps GIPL<i>)</i> or <i>T. cruzi</i> eGPI-mucin (Tc Mucin). The experiment was performed three times and analyzed by ANOVA (* p<0.05).</p

NADPH-diaphorase activity of NOS in <i>Rhodnius prolixus</i> salivary glands after a blood meal and the expression of NOS.

<p>A. Salivary glands were dissected in different days after blood feeding and evaluated for NOS NAPDH-diaphorase activity. Salivary glands were assayed in 10 mM Tris-HCl pH 8,0, 0,05 M NaCl, 0,1%, Triton X-100, 1 mM CaCl₂, 5 µM FAD, 1 mM NADPH and 0,5 mg/mL MTT. MTT reduction was followed at 540 nm for 30 min at 37°C. Also samples were obtained and NOS content evaluated by Western blotting. Each point is the average and SE of 05 different experiments. B. Immunoblotting using an anti-NOS antibody. Blottings were developed with the use of a secondary antibody conjugated to alkaline phosphatase in the presence of the substrate Western Blue. Molecular mass markers are indicated at the left. C. Upper panel<b>,</b> total RNA from the salivary glands at different days after feeding was isolated and cDNA was synthesized. Samples were then analyzed by semi-quantitative PCR with temperatures of 55, 72 and 94°C for 27 cycles with primers for NOS. Lower panel, analysis of 18 S RNA levels. In this case reaction occurred for 25 cycles. The products of reactions shown on panels C were separated on agarose gel 1.4% stained with ethidium bromide and photographed under ultraviolet light. Molecular mass standards are indicated at the left.</p

Tc GIPL does not affect regular blood feeding, anti-clotting and apyrase activity.

<p><i>Rhodnius</i> injected or not with Tc GIPLs were evaluated for their ability to feed on blood. Parallel controls in each panel were obtained in insects inject with GIPL solvent. Three days after the injection insects were either allowed to feed on a rabbit ear or their salivary glands were dissected and evaluated for anti-hemostatic activities. A. Weight gain after blood feeding. B. Apyrase activity. C. aPTT activity. Data is the mean ± S.E. of three different experiments.</p

<i>T. rangeli</i> infection downregulates NOS production.

<p><i>Rhodnius</i> were infected with <i>T. rangeli</i> and three days later salivary glands were dissected and analyzed by immunocytochemistry using anti-NOS. A. no antibodies. B. Control salivary glands developed with anti-NOS and a secondary antibody. C. Infected salivary glands developed with both antibodies. (E), salivary gland epithelia, (L), salivary gland lumen, (N), nucleus of salivary gland epithelial cells.</p

Glycolipid-mediated suppression of NO synthesis occurs through the manipulation of intracellular phosphorylation-dephosphorylation circuits.

<p>Intracellular circuits of protein-phosphorylation and dephosphorylation were evaluated through different assays. A. Salivary glands obtained from either control or <i>T. rangeli</i>-infected insects were dissected three days after the injection, homogeneized and phosphorylated in the presence of ³²P-ATP followed by SDS-gel electrophoresis and autoradiograph. B. A similar experiment was conducted with salivary glands isolated from insects injected with Tr GIPL or Tc GIPL. C. Following a blood meal on rabbit ear salivary glands were dissected at different points in time. Total protein phosphatase activity was followed during the refilling cycle of salivary glands using pNPP as substrate. Data is the mean ± S.E. of three different experiments. D. Insects were injected with Tr GIPL and evaluated for protein phosphatase activity in the presence and in the absence of SO. The fraction of enzyme activity inhibited by SO in control and Tr GIPL-injected insects is shown. Data is the mean ± S.E. of three different experiments.</p

<i>T. rangeli</i> infection downregulates NOS synthesis.

<p><i>Rhodnius</i> were infected with <i>T. rangeli</i> and three days later salivary glands were dissected and incubated in the presence of the NO fluorescent probe DAF-FM. A, C are contrast-phase imaging of B and D, respectively. A. Control salivary gland. B. DAF-FM fluorescent image of a control salivary gland shown on A. C. Infected salivary gland. D. DAF-FM fluorescence image of an infected salivary gland shown on C.</p