Chemoenzymatic Synthesis of Complex N-Glycans of the Parasite S. mansoni to Examine the Importance of Epitope Presentation on DC-SIGN recognition.

The importance of multivalency for N-glycan-protein interactions has primarily been studied by attachment of minimal epitopes to artificial multivalent scaffold and not in the context of multi-antennary glycans. N-glycans can be modified by bisecting GlcNAc, core xylosides and fucosides, and extended N-acetyl lactosamine moieties. The impact of such modifications on glycan recognition are also not well understood. We describe here a chemoenzymatic methodology that can provide N-glycans expressed by the parasitic worm S. mansoni having unique epitopes at each antenna and containing core xyloside. NMR, computational and electron microscopy were employed to investigate recognition of the glycans by the human lectin DC-SIGN. It revealed that core xyloside does not influence terminal epitope recognition. The multi-antennary glycans bound with higher affinity to DC-SIGN compared to mono-valent counterparts, which was attributed to proximity-induced effective concentration. The multi-antennary glycans cross-linked DC-SIGN into a dense network, which likely is relevant for antigen uptake and intracellular routing.

Mono- and Di-Fucosylated Glycans of the Parasitic Worm S. mansoni are Recognized Differently by the Innate Immune Receptor DC-SIGN.

The parasitic worm, Schistosoma mansoni, expresses unusual fucosylated glycans in a stage-dependent manner that can be recognized by the human innate immune receptor DC-SIGN, thereby shaping host immune responses. We have developed a synthetic approach for mono- and bis-fucosylated LacdiNAc (LDN-F and LDN-DF, respectively), which are epitopes expressed on glycolipids and glycoproteins of S. mansoni. It is based on the use of monosaccharide building blocks having carefully selected amino-protecting groups, facilitating high yielding and stereoselective glycosylations. The molecular interaction between the synthetic glycans and DC-SIGN was studied by NMR and molecular modeling, which demonstrated that the α1,3-fucoside of LDN-F can coordinate with the Ca2+ -ion of the canonical binding site of DC-SIGN allowing for additional interactions with the underlying LDN backbone. The 1,2-fucoside of LDN-DF can be complexed in a similar manner, however, in this binding mode GlcNAc and GalNAc of the LDN backbone are placed away from the protein surface resulting in a substantially lower binding affinity. Glycan microarray binding studies showed that the avidity and selectivity of binding is greatly enhanced when the glycans are presented multivalently, and in this format Lex and LDN-F gave strong responsiveness, whereas no binding was detected for LDN-DF. The data indicates that S. mansoni has developed a strategy to avoid detection by DC-SIGN in a stage-dependent manner by the addition of a fucoside to a number of its ligands.

Ion-Mobility Spectrometry Can Assign Exact Fucosyl Positions in Glycans and Prevent Misinterpretation of Mass-Spectrometry Data After Gas-Phase Rearrangement.

The fucosylation of glycans leads to diverse structures and is associated with many biological and disease processes. The exact determination of fucoside positions by tandem mass spectrometry (MS/MS) is complicated because rearrangements in the gas phase lead to erroneous structural assignments. Here, we demonstrate that the combined use of ion-mobility MS and well-defined synthetic glycan standards can prevent misinterpretation of MS/MS spectra and incorrect structural assignments of fucosylated glycans. We show that fucosyl residues do not migrate to hydroxyl groups but to acetamido moieties of N-acetylneuraminic acid as well as N-acetylglucosamine residues and nucleophilic sites of an anomeric tag, yielding specific isomeric fragment ions. This mechanistic insight enables the characterization of unique IMS arrival-time distributions of the isomers which can be used to accurately determine fucosyl positions in glycans.

Chemoenzymatic Approach for the Preparation of Asymmetric Bi-, Tri-, and Tetra-Antennary N-Glycans from a Common Precursor.

Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and unnatural Galα(1,4)-GlcNAc and Manß(1,4)-GlcNAc appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a substrate, and thus this structure can be uniquely extended. Next, the ß-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural α-Gal and ß-Man terminating antennae can sequentially be decaged by an appropriate glycosidase to liberate a terminal ß-GlcNAc moiety, which can be converted into LacNAc and then elaborated by a panel of glycosyltransferases. Asymmetric bi- and triantennary glycans could be obtained by removal of a terminal ß-GlcNAc moiety by treatment with ß-N-acetylglucosaminidase and selective extension of the other arms. The power of the methodology is demonstrated by the preparation of an asymmetric tetra-antennary N-glycan found in human breast carcinoma tissue, which represents the most complex N-glycan ever synthesized. Multistage mass spectrometry of the two isomeric triantennary glycans uncovered unique fragment ions that will facilitate identification of exact structures of glycans in biological samples.

Synthesis of Staphylococcus aureus Type 5 trisaccharide repeating unit: solving the problem of lactamization.

The chemical synthesis of an orthogonally protected trisaccharide derived from the polysaccharide of Staphylococcus aureus Type 5, which is an attractive candidate for the development of immunotherapies, is described. The challenging α-fucosylation and ß-mannosylation are addressed through the careful choice of protecting groups. Lactamization of a ß-D-ManpNAcA moiety during deprotection was avoided by a late stage oxidation approach. Versatility of the trisaccharide was demonstrated by its transformation into a spacer-containing repeating unit suitable for immunological investigations.