Comparisons of <i>Caenorhabditis</i> Fucosyltransferase Mutants Reveal a Multiplicity of Isomeric N‑Glycan Structures

Recent studies have shown a remarkable degree of plasticity in the N-glycome of the model nematode <i>Caenorhabditis elegans</i>; ablation of glycosylation-relevant genes can result in radically altered N-glycan profiles despite only minor biological phenotypic effects. Up to four fucose residues and five different linkages of fucose are known on the N-glycans of <i>C. elegans</i>. Due to the complexity in the wild type, we established three mutant strains defective in two core fucosyltransferases each (<i>fut-1;fut-6</i>, <i>fut-1;fut-8</i>, and <i>fut-6;fut-8</i>). Enzymatically released N-glycans were subject to HPLC and MALDI-TOF MS/MS, in combination with various treatments, to verify structural details. The N-glycome of the <i>fut-1;fut-6</i> mutant was the most complex of the three double-mutant strains due to the extension of the core α1,6-fucose as well as the presence of fucose on the bisecting galactose. In contrast, maximally two fucoses were found on N-glycans of the <i>fut-1;fut-8</i> and <i>fut-6;fut-8</i> strains. The different locations and capping of fucose meant that up to 13 isomeric structures, many highly galactosylated, were determined for some single masses. These data not only show the high variability of the N-glycomic capacity of a “simple” nematode but also exemplify the need for multiple approaches to reveal individual glycan structures within complex invertebrate glycomes

Mass Spectrometric Analysis of Neutral and Anionic N‑Glycans from a <i>Dictyostelium discoideum</i> Model for Human Congenital Disorder of Glycosylation CDG IL

The HL241 mutant strain of the cellular slime mold <i>Dictyostelium discoideum</i> is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its <i>alg9</i> mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting <i>N-</i>acetylglucosamine, bisecting <i>N-</i>acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism

Zwitterionic Phosphodiester-Substituted Neoglycoconjugates as Ligands for Antibodies and Acute Phase Proteins

Zwitterionic modifications of glycans, such as phosphorylcholine and phosphoethanolamine, are known from a range of prokaryotic and eukaryotic species and are recognized by mammalian antibodies and pentraxins; however, defined saccharide ligands modified with these zwitterionic moieties for high-throughput studies are lacking. In this study, we prepared and tested example mono- and disaccharides 6-substituted with either phosphorylcholine or phosphoethanolamine as bovine serum albumin neoglycoconjugates or printed in a microarray format for subsequent assessment of their binding to lectins, pentraxins, and antibodies. C-Reactive protein and anti-phosphorylcholine antibodies bound specifically to ligands with phosphorylcholine, but recognition by concanavalin A was abolished or decreased as compared with that to the corresponding nonzwitterionic compounds. Furthermore, in array format, the phosphorylcholine-modified ligands were recognized by IgG and IgM in sera of either non-infected or nematode-infected dogs and pigs. Thereby, these new compounds are defined ligands which allow the assessment of glycan-bound phosphorylcholine as a target of both the innate and adaptive immune systems in mammals